CD134/CD137 Dual Costimulation-Elicited IFN-γ Maximizes Effector T Cell Function but Limits Treg Expansion

PubMed Central

Rose, Marie-Clare St.; Taylor, Roslyn A.; Bandyopadhyay, Suman; Qui, Harry Z.; Hagymasi, Adam T.; Vella, Anthony T.; Adler, Adam J.

2012-01-01

T cell tolerance to tumor antigens represents a major hurdle in generating tumor immunity. Combined administration of agonistic monoclonal antibodies to the costimulatory receptors CD134 plus CD137 can program T cells responding to tolerogenic antigen to undergo expansion and effector T cell differentiation, and also elicits tumor immunity. Nevertheless, CD134 and CD137 agonists can also engage inhibitory immune components. To understand how immune stimulatory versus inhibitory components are regulated during CD134 plus CD137 dual costimulation, the current study utilized a model where dual costimulation programs T cells encountering a highly tolerogenic self-antigen to undergo effector differentiation. IFN-γ was found to play a pivotal role in maximizing the function of effector T cells while simultaneously limiting the expansion of CD4+CD25+Foxp3+ Tregs. In antigen-responding effector T cells, IFN-γ operates via a direct cell-intrinsic mechanism to cooperate with IL-2 to program maximal expression of granzyme B. Simultaneously, IFN-γ limits expression of the IL-2 receptor alpha chain (CD25) and IL-2 signaling through a mechanism that does not involve T-bet-mediated repression of IL-2. IFN-γ also limited CD25 and Foxp3 expression on bystanding CD4+Foxp3+ Tregs, and limited the potential of these Tregs to expand. These effects could not be explained by the ability of IFN-γ to limit IL-2 availability. Taken together, during dual costimulation IFN-γ interacts with IL-2 through distinct mechanisms to program maximal expression of effector molecules in antigen-responding T cells while simultaneously limiting Treg expansion. PMID:23295363

Structural Evolution of Differential Amino Acid Effector Regulation in Plant Chorismate Mutases*

PubMed Central

Westfall, Corey S.; Xu, Ang; Jez, Joseph M.

2014-01-01

Chorismate mutase converts chorismate into prephenate for aromatic amino acid biosynthesis. To understand the molecular basis of allosteric regulation in the plant chorismate mutases, we analyzed the three Arabidopsis thaliana chorismate mutase isoforms (AtCM1–3) and determined the x-ray crystal structures of AtCM1 in complex with phenylalanine and tyrosine. Functional analyses show a wider range of effector control in the Arabidopsis chorismate mutases than previously reported. AtCM1 is activated by tryptophan with phenylalanine and tyrosine acting as negative effectors; however, tryptophan, cysteine, and histidine activate AtCM3. AtCM2 is a nonallosteric form. The crystal structure of AtCM1 in complex with tyrosine and phenylalanine identifies differences in the effector sites of the allosterically regulated yeast enzyme and the other two Arabidopsis isoforms. Site-directed mutagenesis of residues in the effector site reveals key features leading to differential effector regulation in these enzymes. In AtCM1, mutations of Gly-213 abolish allosteric regulation, as observed in AtCM2. A second effector site position, Gly-149 in AtCM1 and Asp-132 in AtCM3, controls amino acid effector specificity in AtCM1 and AtCM3. Comparisons of chorismate mutases from multiple plants suggest that subtle differences in the effector site are conserved in different lineages and may lead to specialized regulation of this branch point enzyme. PMID:25160622

CD4+ T helper 2 cells – microbial triggers, differentiation requirements and effector functions

PubMed Central

Okoye, Isobel S; Wilson, Mark S

2011-01-01

Over the past 10 years we have made great strides in our understanding of T helper cell differentiation, expansion and effector functions. Within the context of T helper type 2 (Th2) cell development, novel innate-like cells with the capacity to secrete large amounts of interleukin-5 (IL-5), IL-13 and IL-9 as well as IL-4-producing and antigen-processing basophils have (re)-emerged onto the type 2 scene. To what extent these new players influence αβ+ CD4+ Th2 cell differentiation is discussed throughout this appraisal of the current literature. We highlight the unique features of Th2 cell development, highlighting the three necessary signals, T-cell receptor ligation, co-stimulation and cytokine receptor ligation. Finally, putting these into context, microbial and allergenic properties that trigger Th2 cell differentiation and how these influence Th2 effector function are discussed and questioned. PMID:22043920

A central role for Notch in effector CD8+ T cell differentiation

PubMed Central

Backer, Ronald A.; Helbig, Christina; Gentek, Rebecca; Kent, Andrew; Laidlaw, Brian J.; Dominguez, Claudia X.; de Souza, Yevan S.; van Trierum, Stella E.; van Beek, Ruud; Rimmelzwaan, Guus F.; ten Brinke, Anja; Willemsen, A. Marcel; van Kampen, Antoine H. C.; Kaech, Susan M.; Blander, J. Magarian; van Gisbergen, Klaas; Amsen, Derk

2014-01-01

Activated CD8+ T cells choose between terminal effector cell (TEC) or memory precursor cell (MPC) fates. We show that Notch controls this choice. Notch promoted differentiation of immediately protective TECs and was correspondingly required for clearance of an acute influenza virus infection. Notch activated a major portion of the TEC-specific gene expression program and suppressed the MPC-specific program. Expression of Notch receptors was induced on naïve CD8+ T cells by inflammatory mediators and interleukin 2 (IL-2) via mTOR and T-bet dependent pathways. These pathways were subsequently amplified downstream of Notch, creating a positive feedback loop. Notch thus functions as a central hub where information from different sources converges to match effector T cell differentiation to the demands of the infection. PMID:25344724

Primary Murine CD4+ T Cells Fail to Acquire the Ability to Produce Effector Cytokines When Active Ras Is Present during Th1/Th2 Differentiation

PubMed Central

Janardhan, Sujit V.; Marks, Reinhard; Gajewski, Thomas F.

2014-01-01

Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4+ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4+ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4+ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4+ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo. PMID:25397617

Direct anti-inflammatory effects of granulocyte colony-stimulating factor (G-CSF) on activation and functional properties of human T cell subpopulations in vitro.

PubMed

Malashchenko, Vladimir Vladimirovich; Meniailo, Maxsim Evgenievich; Shmarov, Viacheslav Anatolievich; Gazatova, Natalia Dinislamovna; Melashchenko, Olga Borisovna; Goncharov, Andrei Gennadievich; Seledtsova, Galina Victorovna; Seledtsov, Victor Ivanovich

2018-03-01

We investigated the direct effects of human granulocyte colony-stimulating factor (G-CSF) on functionality of human T-cell subsets. CD3 + T-lymphocytes were isolated from blood of healthy donors by positive magnetic separation. T cell activation with particles conjugated with antibodies (Abs) to human CD3, CD28 and CD2 molecules increased the proportion of cells expressing G-CSF receptor (G-CSFR, CD114) in all T cell subpopulations studied (CD45RA + /CD197 + naive T cells, CD45RA - /CD197 + central memory T cells, CD45RA - /CD197 - effector memory T cells and CD45RA + /CD197 - terminally differentiated effector T cells). Upon T-cell activation in vitro, G-CSF (10.0 ng/ml) significantly and specifically enhanced the proportion of CD114 + T cells in central memory CD4 + T cell compartment. A dilution series of G-CSF (range, 0.1-10.0 ng/ml) was tested, with no effect on the expression of CD25 (interleukin-2 receptor α-chain) on activated T cells. Meanwhile, G-CSF treatment enhanced the proportion of CD38 + T cells in CD4 + naïve T cell, effector memory T cell and terminally differentiated effector T cell subsets, as well as in CD4 - central memory T cells and terminally differentiated effector T cells. G-CSF did not affect IL-2 production by T cells; relatively low concentrations of G-CSF down-regulated INF-γ production, while high concentrations of this cytokine up-regulated IL-4 production in activated T cells. The data obtained suggests that G-CSF could play a significant role both in preventing the development of excessive and potentially damaging inflammatory reactivity, and in constraining the expansion of potentially cytodestructive T cells. Copyright © 2018 Elsevier Inc. All rights reserved.

α4β7+ CD4+ Effector/Effector Memory T Cells Differentiate into Productively and Latently Infected Central Memory T Cells by Transforming Growth Factor β1 during HIV-1 Infection.

PubMed

Cheung, Ka-Wai; Wu, Tongjin; Ho, Sai Fan; Wong, Yik Chun; Liu, Li; Wang, Hui; Chen, Zhiwei

2018-04-15

HIV-1 transmission occurs mainly through mucosal tissues. During mucosal transmission, HIV-1 preferentially infects α 4 β 7 + gut-homing CCR7 - CD4 + effector/effector memory T cells (T EM ) and results in massive depletion of these cells and other subsets of T EM in gut-associated lymphoid tissues. However, besides being eliminated by HIV-1, the role of T EM during the early stage of infection remains inconclusive. Here, using in vitro -induced α 4 β 7 + gut-homing T EM (α 4 β 7 + T EM ), we found that α 4 β 7 + T EM differentiated into CCR7 + CD4 + central memory T cells (T CM ). This differentiation was HIV-1 independent but was inhibited by SB431542, a specific transforming growth factor β (TGF-β) receptor I kinase inhibitor. Consistently, T EM -to-T CM differentiation was observed in α 4 β 7 + T EM stimulated with TGF-β1 (TGF-β). The T CM properties of the TGF-β-induced T EM -derived T CM (α 4 β 7 + T CM ) were confirmed by their enhanced CCL19 chemotaxis and the downregulation of surface CCR7 upon T cell activation in vitro Importantly, the effect of TGF-β on T CM differentiation also held in T EM directly isolated from peripheral blood. To investigate the significance of the TGF-β-dependent T EM -to-T CM differentiation in HIV/AIDS pathogenesis, we observed that both productively and latently infected α 4 β 7 + T CM could differentiate from α 4 β 7 + T EM in the presence of TGF-β during HIV-1 infection. Collectively, this study not only provides a new insight for the plasticity of T EM but also suggests that the TGF-β-dependent T EM -to-T CM differentiation is a previously unrecognized mechanism for the formation of latently infected T CM after HIV-1 infection. IMPORTANCE HIV-1 is the causative agent of HIV/AIDS, which has led to millions of deaths in the past 30 years. Although the implementation of highly active antiretroviral therapy has remarkably reduced the HIV-1-related morbidity and mortality, HIV-1 is not eradicated in treated patients due to the presence of latent reservoirs. Besides, the pathogenesis in CD4 T cells early after infection still remains elusive. Immediately after HIV-1 mucosal infection, CD4 T cells are preferentially infected and depleted. However, in addition to being depleted, the other roles of the CD4 T cells, especially the effector/effector memory T cells (T EM ), in disease progression are not completely understood. The significance of this study is in revealing a novel mechanism for the formation of latently HIV-1-infected central memory CD4 T cells, a major latent reservoir from CD4 T EM after infection. Our findings suggest previously unrecognized roles of CD4 T EM in HIV-1 pathogenesis. Copyright © 2018 American Society for Microbiology.

Effector and memory CD8+ T cell differentiation: toward a molecular understanding of fate determination.

PubMed

Belz, Gabrielle T; Kallies, Axel

2010-06-01

CD8(+) T cells play a key role in protecting the body against invading microorganisms. Their capacity to control infection relies on the development of peripheral effector and memory T cells. Much of our current knowledge has been gained by tracking alterations of the phenotype of CD8(+) T cells but the molecular understanding of the events that underpin the emergence of heterogeneous effector and memory CD8(+) T cells in response to infection has remained limited. This review focuses on the recent progress in our understanding of the molecular wiring of this differentiation process. Copyright 2010 Elsevier Ltd. All rights reserved.

Differential Effector Engagement by Oncogenic KRAS.

PubMed

Yuan, Tina L; Amzallag, Arnaud; Bagni, Rachel; Yi, Ming; Afghani, Shervin; Burgan, William; Fer, Nicole; Strathern, Leslie A; Powell, Katie; Smith, Brian; Waters, Andrew M; Drubin, David; Thomson, Ty; Liao, Rosy; Greninger, Patricia; Stein, Giovanna T; Murchie, Ellen; Cortez, Eliane; Egan, Regina K; Procter, Lauren; Bess, Matthew; Cheng, Kwong Tai; Lee, Chih-Shia; Lee, Liam Changwoo; Fellmann, Christof; Stephens, Robert; Luo, Ji; Lowe, Scott W; Benes, Cyril H; McCormick, Frank

2018-02-13

KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Autophagy is essential for effector CD8 T cell survival and memory formation

PubMed Central

Xu, Xiaojin; Araki, Koichi; Li, Shuzhao; Han, Jin-Hwan; Ye, Lilin; Tan, Wendy G.; Konieczny, Bogumila T.; Bruinsma, Monique W.; Martinez, Jennifer; Pearce, Erika L; Green, Douglas R.; Jones, Dean P.; Virgin, Herbert W.; Ahmed, Rafi

2014-01-01

The importance of autophagy in memory CD8 T cell differentiation in vivo is not well defined. We show here that autophagy is dynamically regulated in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection. Autophagy decreased in activated proliferating T cells, and was then upregulated at the peak of the effector T cell response. Consistent with this model, deletion of the key autophagy genes Atg7 or Atg5 in virus-specific CD8 T cells had minimal effect on generating effector cells but greatly enhanced their death during the contraction phase resulting in compromised memory formation. These findings provide insight into when autophagy is needed during effector and memory T cell differentiation in vivo and also warrant a re-examination of our current concepts about the relationship between T cell activation and autophagy. PMID:25362489

Phenotypic and Functional Characterization of Herpes Simplex Virus Glycoprotein B Epitope-Specific Effector and Memory CD8+ T Cells from Symptomatic and Asymptomatic Individuals with Ocular Herpes

PubMed Central

Khan, Arif A.; Srivastava, Ruchi; Spencer, Doran; Garg, Sumit; Fremgen, Daniel; Vahed, Hawa; Lopes, Patricia P.; Pham, Thanh T.; Hewett, Charlie; Kuang, Jasmine; Ong, Nicolas; Huang, Lei; Scarfone, Vanessa M.; Nesburn, Anthony B.

2015-01-01

ABSTRACT Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8+ T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8+ T cells play a key role in the “natural” protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8+ T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found the following. (i) Healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8+ T cells (TEM cells) (CD45RAlow CCR7low CD44high CD62Llow). In contrast, SYMP patients had frequent less-differentiated central memory CD8+ T cells (TCM cells) (CD45RAlow CCR7high CD44low CD62Lhigh). (ii) ASYMP individuals had significantly higher proportions of multifunctional effector CD8+ T cells which responded mainly to gB342–350 and gB561–569 “ASYMP” epitopes, and simultaneously produced IFN-γ, CD107a/b, granzyme B, and perforin. In contrast, effector CD8+ T cells from SYMP individuals were mostly monofunctional and were directed mainly against nonoverlapping gB17–25 and gB183–191 “SYMP” epitopes. (iii) Immunization of an HLA-A*02:01 transgenic mouse model of ocular herpes with “ASYMP” CD8+ TEM cell epitopes, but not with “SYMP” CD8+ TCM cell epitopes, induced a strong CD8+ T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8+ TEM cells in protection against herpes and should be considered in the development of an effective vaccine. IMPORTANCE A significantly higher proportion of differentiated and multifunctional HSV-1 gB-specific effector memory CD8+ T cells (TEM cells) (CD45RAlow CCR7low CD44high CD62Llow) were found in healthy ASYMP individuals who are seropositive for HSV-1 but never had any recurrent herpetic disease, while there were frequent less-differentiated and monofunctional central memory CD8+ T cells (TCM cells) (CD45RAlow CCR7high CD44low CD62Lhigh) in SYMP patients. Immunization with “ASYMP” CD8+ TEM cell epitopes, but not with “SYMP” CD8+ TCM cell epitopes, induced a strong protective HSV-specific CD8+ T cell response in HLA-A*02:01 transgenic mice. These findings are important for the development of a safe and effective T cell-based herpes vaccine. PMID:25609800

Phenotypic and functional characterization of herpes simplex virus glycoprotein B epitope-specific effector and memory CD8+ T cells from symptomatic and asymptomatic individuals with ocular herpes.

PubMed

Khan, Arif A; Srivastava, Ruchi; Spencer, Doran; Garg, Sumit; Fremgen, Daniel; Vahed, Hawa; Lopes, Patricia P; Pham, Thanh T; Hewett, Charlie; Kuang, Jasmine; Ong, Nicolas; Huang, Lei; Scarfone, Vanessa M; Nesburn, Anthony B; Wechsler, Steven L; BenMohamed, Lbachir

2015-04-01

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8(+) T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8(+) T cells play a key role in the "natural" protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8(+) T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found the following. (i) Healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8(+) T cells (TEM cells) (CD45RA(low) CCR7(low) CD44(high) CD62L(low)). In contrast, SYMP patients had frequent less-differentiated central memory CD8(+) T cells (TCM cells) (CD45RA(low) CCR7(high) CD44(low) CD62L(high)). (ii) ASYMP individuals had significantly higher proportions of multifunctional effector CD8(+) T cells which responded mainly to gB342-350 and gB561-569 "ASYMP" epitopes, and simultaneously produced IFN-γ, CD107(a/b), granzyme B, and perforin. In contrast, effector CD8(+) T cells from SYMP individuals were mostly monofunctional and were directed mainly against nonoverlapping gB17-25 and gB183-191 "SYMP" epitopes. (iii) Immunization of an HLA-A*02:01 transgenic mouse model of ocular herpes with "ASYMP" CD8(+) TEM cell epitopes, but not with "SYMP" CD8(+) TCM cell epitopes, induced a strong CD8(+) T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8(+) TEM cells in protection against herpes and should be considered in the development of an effective vaccine. A significantly higher proportion of differentiated and multifunctional HSV-1 gB-specific effector memory CD8(+) T cells (TEM cells) (CD45RA(low) CCR7(low) CD44(high) CD62L(low)) were found in healthy ASYMP individuals who are seropositive for HSV-1 but never had any recurrent herpetic disease, while there were frequent less-differentiated and monofunctional central memory CD8(+) T cells (TCM cells) (CD45RA(low) CCR7(high) CD44(low) CD62L(high)) in SYMP patients. Immunization with "ASYMP" CD8(+) TEM cell epitopes, but not with "SYMP" CD8(+) TCM cell epitopes, induced a strong protective HSV-specific CD8(+) T cell response in HLA-A*02:01 transgenic mice. These findings are important for the development of a safe and effective T cell-based herpes vaccine. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Enhanced Requirement for TNFR2 in Graft Rejection Mediated by Low Affinity Memory CD8+ T Cells During Heterologous Immunity

PubMed Central

Krummey, Scott M.; Chen, Ching-Wen; Guasch, Sara A.; Liu, Danya; Wagener, Maylene; Larsen, Christian P; Ford, Mandy L.

2016-01-01

The affinity of a T cell receptor (TCR) binding to peptide:MHC profoundly impacts the phenotype and function of effector and memory cell differentiation. Little is known about the effect of low affinity priming on memory cell generation and function, which is particularly important in heterologous immunity, when microbe-specific T cells cross-react with allogeneic antigen and mediate graft rejection. We found that low affinity primed memory CD8+ T cells produced high levels of TNF ex vivo in response to heterologous rechallenge compared to high affinity primed memory T cells. Low affinity secondary effectors significantly upregulated TNFR2 on the cell surface and contained a higher frequency of TNFR2hi proliferating cells. Low affinity primed secondary effectors concurrently downregulated TNF production. Importantly, blockade of TNFR2 attenuated graft rejection in low but not high affinity primed animals. These data establish a functional connection between TNF signaling and TCR priming affinity and have implications for the immunomodulation of pathogenic T cell responses during transplantation. PMID:27481849

Distinct Roles for CXCR6+ and CXCR6− CD4+ T Cells in the Pathogenesis of Chronic Colitis

PubMed Central

Hase, Koji; Obata, Yuuki; Furusawa, Yukihiro; Ebisawa, Masashi; Nakagawa, Tomoo; Sato, Toru; Katsuno, Tatsuro; Saito, Yasushi; Shimaoka, Takeshi; Yokosuka, Osamu; Yokote, Kotaro; Ohno, Hiroshi

2013-01-01

CD4+ T cells play a central role in the development of inflammatory bowel disease (IBD) via high-level production of effector cytokines such as IFN-γ and TNF-α. To better characterize the colitogenic CD4+ T cells, we examined their expression of CXCR6, a chemokine receptor that is expressed by T cells upon activation and is upregulated in several inflammatory diseases. We found that 80% of colonic lamina propria CD4+ T cells expressed CXCR6 in the CD45RBhigh T cell-transferred colitis model. CXCR6 expression was similarly upregulated in inflamed mucosa of patients with Crohn’s disease. Although surface marker analysis demonstrated that both CXCR6+ and CXCR6− CD4+ T-cell subsets consist of the cells with effector and effector-memory cells, the more cells in the CXCR6+ subset produced IFN-γ and TNF-α compared to CXCR6− subset, and only the CXCR6+ subset produced IL-17A. Nevertheless, adoptive retransfer of lamina propria CXCR6+ T cells into Rag1 −/− recipients failed to induce the disease due to limited expansion of the transferred cells. By contrast, retransfer of CXCR6− cells evoked colitis similar to that observed in CD4+CD45RBhigh T cell-transferred mice, and resulted in their conversion into CXCR6+ cells. Collectively, these observations suggest that the CXCR6+CD4+ T-cell subset consists of terminally differentiated effector cells that serve as the major source of effector cytokines in the inflamed tissue, whereas CXCR6−CD4+ T-cell subset serves as a colitogenic memory compartment that retains the ability to proliferate and differentiate into CXCR6+CD4+ T cells. PMID:23840334

Bim controls IL-15 availability and limits engagement of multiple BH3-only proteins

PubMed Central

Kurtulus, S; Sholl, A; Toe, J; Tripathi, P; Raynor, J; Li, K-P; Pellegrini, M; Hildeman, D A

2015-01-01

During the effector CD8+ T-cell response, transcriptional differentiation programs are engaged that promote effector T cells with varying memory potential. Although these differentiation programs have been used to explain which cells die as effectors and which cells survive and become memory cells, it is unclear if the lack of cell death enhances memory. Here, we investigated effector CD8+ T-cell fate in mice whose death program has been largely disabled because of the loss of Bim. Interestingly, the absence of Bim resulted in a significant enhancement of effector CD8+ T cells with more memory potential. Bim-driven control of memory T-cell development required T-cell-specific, but not dendritic cell-specific, expression of Bim. Both total and T-cell-specific loss of Bim promoted skewing toward memory precursors, by enhancing the survival of memory precursors, and limiting the availability of IL-15. Decreased IL-15 availability in Bim-deficient mice facilitated the elimination of cells with less memory potential via the additional pro-apoptotic molecules Noxa and Puma. Combined, these data show that Bim controls memory development by limiting the survival of pre-memory effector cells. Further, by preventing the consumption of IL-15, Bim limits the role of Noxa and Puma in causing the death of effector cells with less memory potential. PMID:25124553

Bim controls IL-15 availability and limits engagement of multiple BH3-only proteins.

PubMed

Kurtulus, S; Sholl, A; Toe, J; Tripathi, P; Raynor, J; Li, K-P; Pellegrini, M; Hildeman, D A

2015-01-01

During the effector CD8+ T-cell response, transcriptional differentiation programs are engaged that promote effector T cells with varying memory potential. Although these differentiation programs have been used to explain which cells die as effectors and which cells survive and become memory cells, it is unclear if the lack of cell death enhances memory. Here, we investigated effector CD8+ T-cell fate in mice whose death program has been largely disabled because of the loss of Bim. Interestingly, the absence of Bim resulted in a significant enhancement of effector CD8+ T cells with more memory potential. Bim-driven control of memory T-cell development required T-cell-specific, but not dendritic cell-specific, expression of Bim. Both total and T-cell-specific loss of Bim promoted skewing toward memory precursors, by enhancing the survival of memory precursors, and limiting the availability of IL-15. Decreased IL-15 availability in Bim-deficient mice facilitated the elimination of cells with less memory potential via the additional pro-apoptotic molecules Noxa and Puma. Combined, these data show that Bim controls memory development by limiting the survival of pre-memory effector cells. Further, by preventing the consumption of IL-15, Bim limits the role of Noxa and Puma in causing the death of effector cells with less memory potential.

Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

PubMed Central

Imberg, Keren; Mercer, Frances; Zhong, Shi; Krogsgaard, Michelle; Unutmaz, Derya

2013-01-01

Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8+ and CD4+ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression. PMID:23437112

Profiling calcium signals of in vitro polarized human effector CD4+ T cells.

PubMed

Kircher, Sarah; Merino-Wong, Maylin; Niemeyer, Barbara A; Alansary, Dalia

2018-06-01

Differentiation of naïve CD4 + T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca 2+ signals, mainly mediated by the store operated Ca 2+ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4 + T cell subsets show differential Ca 2+ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4 + effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca 2+ release activated Ca 2+ currents (I CRAC ) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca 2+ profiles of helper CD4 + Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4 + T cells. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Effector CD8 T cells dedifferentiate into long-lived memory cells.

PubMed

Youngblood, Ben; Hale, J Scott; Kissick, Haydn T; Ahn, Eunseon; Xu, Xiaojin; Wieland, Andreas; Araki, Koichi; West, Erin E; Ghoneim, Hazem E; Fan, Yiping; Dogra, Pranay; Davis, Carl W; Konieczny, Bogumila T; Antia, Rustom; Cheng, Xiaodong; Ahmed, Rafi

2017-12-21

Memory CD8 T cells that circulate in the blood and are present in lymphoid organs are an essential component of long-lived T cell immunity. These memory CD8 T cells remain poised to rapidly elaborate effector functions upon re-exposure to pathogens, but also have many properties in common with naive cells, including pluripotency and the ability to migrate to the lymph nodes and spleen. Thus, memory cells embody features of both naive and effector cells, fuelling a long-standing debate centred on whether memory T cells develop from effector cells or directly from naive cells. Here we show that long-lived memory CD8 T cells are derived from a subset of effector T cells through a process of dedifferentiation. To assess the developmental origin of memory CD8 T cells, we investigated changes in DNA methylation programming at naive and effector cell-associated genes in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection in mice. Methylation profiling of terminal effector versus memory-precursor CD8 T cell subsets showed that, rather than retaining a naive epigenetic state, the subset of cells that gives rise to memory cells acquired de novo DNA methylation programs at naive-associated genes and became demethylated at the loci of classically defined effector molecules. Conditional deletion of the de novo methyltransferase Dnmt3a at an early stage of effector differentiation resulted in reduced methylation and faster re-expression of naive-associated genes, thereby accelerating the development of memory cells. Longitudinal phenotypic and epigenetic characterization of the memory-precursor effector subset of virus-specific CD8 T cells transferred into antigen-free mice revealed that differentiation to memory cells was coupled to erasure of de novo methylation programs and re-expression of naive-associated genes. Thus, epigenetic repression of naive-associated genes in effector CD8 T cells can be reversed in cells that develop into long-lived memory CD8 T cells while key effector genes remain demethylated, demonstrating that memory T cells arise from a subset of fate-permissive effector T cells.

TCR Signal Strength Alters T–DC Activation and Interaction Times and Directs the Outcome of Differentiation

PubMed Central

van Panhuys, Nicholas

2016-01-01

The ability of CD4+ T cells to differentiate into effector subsets underpins their ability to shape the immune response and mediate host protection. During T cell receptor-induced activation of CD4+ T cells, both the quality and quantity of specific activatory peptide/MHC ligands have been shown to control the polarization of naive CD4+ T cells in addition to co-stimulatory and cytokine-based signals. Recently, advances in two-­photon microscopy and tetramer-based cell tracking methods have allowed investigators to greatly extend the study of the role of TCR signaling in effector differentiation under in vivo conditions. In this review, we consider data from recent in vivo studies analyzing the role of TCR signal strength in controlling the outcome of CD4+ T cell differentiation and discuss the role of TCR in controlling the critical nature of CD4+ T cell interactions with dendritic cells during activation. We further propose a model whereby TCR signal strength controls the temporal aspects of T–DC interactions and the implications for this in mediating the downstream signaling events, which influence the transcriptional and epigenetic regulation of effector differentiation. PMID:26834747

Increased numbers of preexisting memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells.

PubMed

Joshi, Nikhil S; Cui, Weiguo; Dominguez, Claudia X; Chen, Jonathan H; Hand, Timothy W; Kaech, Susan M

2011-10-15

Memory CD8 T cells acquire effector memory cell properties after reinfection and may reach terminally differentiated, senescent states ("Hayflick limit") after multiple infections. The signals controlling this process are not well understood, but we found that the degree of secondary effector and memory CD8 T cell differentiation was intimately linked to the amount of T-bet expressed upon reactivation and preexisting memory CD8 T cell number (i.e., primary memory CD8 T cell precursor frequency) present during secondary infection. Compared with naive cells, memory CD8 T cells were predisposed toward terminal effector (TE) cell differentiation because they could immediately respond to IL-12 and induce T-bet, even in the absence of Ag. TE cell formation after secondary (2°) or tertiary infections was dependent on increased T-bet expression because T-bet(+/-) cells were resistant to these phenotypic changes. Larger numbers of preexisting memory CD8 T cells limited the duration of 2° infection and the amount of IL-12 produced, and consequently, this reduced T-bet expression and the proportion of 2° TE CD8 T cells that formed. Together, these data show that over repeated infections, memory CD8 T cell quality and proliferative fitness is not strictly determined by the number of serial encounters with Ag or cell divisions, but is a function of the CD8 T cell differentiation state, which is genetically controlled in a T-bet-dependent manner. This differentiation state can be modulated by preexisting memory CD8 T cell number and the intensity of inflammation during reinfection. These results have important implications for vaccinations involving prime-boost strategies.

Controlling transcription in human pluripotent stem cells using CRISPR-effectors.

PubMed

Genga, Ryan M; Kearns, Nicola A; Maehr, René

2016-05-15

The ability to manipulate transcription in human pluripotent stem cells (hPSCs) is fundamental for the discovery of key genes and mechanisms governing cellular state and differentiation. Recently developed CRISPR-effector systems provide a systematic approach to rapidly test gene function in mammalian cells, including hPSCs. In this review, we discuss recent advances in CRISPR-effector technologies that have been employed to control transcription through gene activation, gene repression, and epigenome engineering. We describe an application of CRISPR-effector mediated transcriptional regulation in hPSCs by targeting a synthetic promoter driving a GFP transgene, demonstrating the ease and effectiveness of CRISPR-effector mediated transcriptional regulation in hPSCs. Copyright © 2015 Elsevier Inc. All rights reserved.

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors*

PubMed Central

Aurass, Philipp; Gerlach, Thomas; Becher, Dörte; Voigt, Birgit; Karste, Susanne; Bernhardt, Jörg; Riedel, Katharina; Hecker, Michael; Flieger, Antje

2016-01-01

Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phase-specific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests their phase specific function. The distinct temporal or spatial presence of such proteins might have important implications for functional assignments in the future or for use as life-stage specific markers for pathogen analysis. PMID:26545400

A gene expression signature that correlates with CD8+T cell expansion in acute Epstein Barr virus infection1

PubMed Central

Greenough, Thomas C.; Straubhaar, Juerg R.; Kamga, Larisa; Weiss, Eric R.; Brody, Robin M.; McManus, Margaret M.; Lambrecht, Linda K.; Somasundaran, Mohan; Luzuriaga, Katherine F.

2015-01-01

Virus specific CD8+ T cells expand dramatically during acute Epstein Barr virus (EBV) infection, and their persistence is important for lifelong control of EBV-related disease. To better define the generation and maintenance of these effective CD8+ T cell responses, we used microarrays to characterize gene expression in total and EBV-specific CD8+ T cells isolated from the peripheral blood of ten individuals followed from acute infectious mononucleosis (AIM) into convalescence (CONV). In total CD8+ T cells, differential expression of genes in AIM and CONV was most pronounced among those encoding proteins important in T cell activation/differentiation, cell division/metabolism, chemokines/cytokines and receptors, signaling and transcription factors (TF), immune effector functions, and negative regulators. Within these categories, we identified 28 genes that correlated with CD8+ T cell expansion in response to an acute EBV infection. In EBV-specific CD8+ T cells, we identified 33 genes that were differentially expressed in AIM and CONV. Two important TF, T-bet and Eomesodermin (Eomes), were upregulated and maintained at similar levels in both AIM and CONV; by contrast, protein expression declined from AIM to CONV. Expression of these TF varied among cells with different epitope specificities. Altogether, gene and protein expression patterns suggest that a large proportion, if not a majority of CD8+ T cells in AIM are virus-specific, activated, dividing, and primed to exert effector activities. High expression of T-bet and Eomes may help to maintain effector mechanisms in activated cells, and to enable proliferation and transition to earlier differentiation states in CONV. PMID:26416268

Peripheral tissues reprogram CD8+ T cells for pathogenicity during graft-versus-host disease

PubMed Central

Conlan, Thomas; Jardine, Laura; Tkacz, Claire; Ferrer, Ivana R.; Lomas, Cara; Ward, Sophie; West, Heather; Dertschnig, Simone; Means, Terry K.; Kaplan, Daniel H.; Bennett, Clare L.

2018-01-01

Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic stem cell transplantation induced by the influx of donor-derived effector T cells (TE) into peripheral tissues. Current treatment strategies rely on targeting systemic T cells; however, the precise location and nature of instructions that program TE to become pathogenic and trigger injury are unknown. We therefore used weighted gene coexpression network analysis to construct an unbiased spatial map of TE differentiation during the evolution of GVHD and identified wide variation in effector programs in mice and humans according to location. Idiosyncrasy of effector programming in affected organs did not result from variation in T cell receptor repertoire or the selection of optimally activated TE. Instead, TE were reprogrammed by tissue-autonomous mechanisms in target organs for site-specific proinflammatory functions that were highly divergent from those primed in lymph nodes. In the skin, we combined the correlation-based network with a module-based differential expression analysis and showed that Langerhans cells provided in situ instructions for a Notch-dependent T cell gene cluster critical for triggering local injury. Thus, the principal determinant of TE pathogenicity in GVHD is the final destination, highlighting the need for target organ–specific approaches to block immunopathology while avoiding global immune suppression. PMID:29515032

Ezh2 phosphorylation state determines its capacity to maintain CD8+ T memory precursors for antitumor immunity.

PubMed

He, Shan; Liu, Yongnian; Meng, Lijun; Sun, Hongxing; Wang, Ying; Ji, Yun; Purushe, Janaki; Chen, Pan; Li, Changhong; Madzo, Jozef; Issa, Jean-Pierre; Soboloff, Jonathan; Reshef, Ran; Moore, Bethany; Gattinoni, Luca; Zhang, Yi

2017-12-14

Memory T cells sustain effector T-cell production while self-renewing in reaction to persistent antigen; yet, excessive expansion reduces memory potential and impairs antitumor immunity. Epigenetic mechanisms are thought to be important for balancing effector and memory differentiation; however, the epigenetic regulator(s) underpinning this process remains unknown. Herein, we show that the histone methyltransferase Ezh2 controls CD8 + T memory precursor formation and antitumor activity. Ezh2 activates Id3 while silencing Id2, Prdm1 and Eomes, promoting the expansion of memory precursor cells and their differentiation into functional memory cells. Akt activation phosphorylates Ezh2 and decreases its control of these transcriptional programs, causing enhanced effector differentiation at the expense of T memory precursors. Engineering T cells with an Akt-insensitive Ezh2 mutant markedly improves their memory potential and capability of controlling tumor growth compared to transiently inhibiting Akt. These findings establish Akt-mediated phosphorylation of Ezh2 as a critical target to potentiate antitumor immunotherapeutic strategies.

Non-parent of Origin Expression of Numerous Effector Genes Indicates a Role of Gene Regulation in Host Adaption of the Hybrid Triticale Powdery Mildew Pathogen.

PubMed

Praz, Coraline R; Menardo, Fabrizio; Robinson, Mark D; Müller, Marion C; Wicker, Thomas; Bourras, Salim; Keller, Beat

2018-01-01

Powdery mildew is an important disease of cereals. It is caused by one species, Blumeria graminis , which is divided into formae speciales each of which is highly specialized to one host. Recently, a new form capable of growing on triticale ( B.g. triticale ) has emerged through hybridization between wheat and rye mildews ( B.g. tritici and B.g. secalis , respectively). In this work, we used RNA sequencing to study the molecular basis of host adaptation in B.g. triticale . We analyzed gene expression in three B.g. tritici isolates, two B.g. secalis isolates and two B.g. triticale isolates and identified a core set of putative effector genes that are highly expressed in all formae speciales . We also found that the genes differentially expressed between isolates of the same form as well as between different formae speciales were enriched in putative effectors. Their coding genes belong to several families including some which contain known members of mildew avirulence ( Avr ) and suppressor ( Svr ) genes. Based on these findings we propose that effectors play an important role in host adaptation that is mechanistically based on Avr-Resistance gene-Svr interactions. We also found that gene expression in the B.g. triticale hybrid is mostly conserved with the parent-of-origin, but some genes inherited from B.g. tritici showed a B.g. secalis -like expression. Finally, we identified 11 unambiguous cases of putative effector genes with hybrid-specific, non-parent of origin gene expression, and we propose that they are possible determinants of host specialization in triticale mildew. These data suggest that altered expression of multiple effector genes, in particular Avr and Svr related factors, might play a role in mildew host adaptation based on hybridization.

Network Analysis Reveals a Common Host-Pathogen Interaction Pattern in Arabidopsis Immune Responses.

PubMed

Li, Hong; Zhou, Yuan; Zhang, Ziding

2017-01-01

Many plant pathogens secrete virulence effectors into host cells to target important proteins in host cellular network. However, the dynamic interactions between effectors and host cellular network have not been fully understood. Here, an integrative network analysis was conducted by combining Arabidopsis thaliana protein-protein interaction network, known targets of Pseudomonas syringae and Hyaloperonospora arabidopsidis effectors, and gene expression profiles in the immune response. In particular, we focused on the characteristic network topology of the effector targets and differentially expressed genes (DEGs). We found that effectors tended to manipulate key network positions with higher betweenness centrality. The effector targets, especially those that are common targets of an individual effector, tended to be clustered together in the network. Moreover, the distances between the effector targets and DEGs increased over time during infection. In line with this observation, pathogen-susceptible mutants tended to have more DEGs surrounding the effector targets compared with resistant mutants. Our results suggest a common plant-pathogen interaction pattern at the cellular network level, where pathogens employ potent local impact mode to interfere with key positions in the host network, and plant organizes an in-depth defense by sequentially activating genes distal to the effector targets.

Developmental Regulation of Effector and Resident Memory T Cell Generation during Pediatric Viral Respiratory Tract Infection.

PubMed

Connors, Thomas J; Baird, J Scott; Yopes, Margot C; Zens, Kyra D; Pethe, Kalpana; Ravindranath, Thyyar M; Ho, Siu-Hong; Farber, Donna L

2018-05-30

Viral respiratory tract infections (VRTI) remain a leading cause of morbidity and mortality among infants and young children. In mice, optimal protection to VRTI is mediated by recruitment of effector T cells to the lungs and respiratory tract, and subsequent establishment of tissue resident memory T cells (Trm), which provide long-term protection. These critical processes of T cell recruitment to the respiratory tract, their role in disease pathogenesis, and establishment of local protective immunity remain undefined in pediatric VRTI. In this study, we investigated T cell responses in the upper respiratory tract (URT) and lower respiratory tract (LRT) of infants and young children with VRTI, revealing developmental regulation of T cell differentiation and Trm generation in situ. We show a direct concurrence between T cell responses in the URT and LRT, including a preponderance of effector CD8 + T cells that was associated with disease severity. During infant VRTI, there was an accumulation of terminally differentiated effector cells (effector memory RA + T cells) in the URT and LRT with reduced Trm in the early neonatal period, and decreased effector memory RA + T cell and increased Trm formation with age during the early years of childhood. Moreover, human infant T cells exhibit increased expression of the transcription factor T-bet compared with adult T cells, suggesting a mechanism for preferential generation of effector over Trm. The developmental regulation of respiratory T cell responses as revealed in the present study is important for diagnosing, monitoring, and treating VRTI in the critical early life stages. Copyright © 2018 by The American Association of Immunologists, Inc.

GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

PubMed

Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

2015-01-01

Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

Translocation of Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell movement.

PubMed

Khang, Chang Hyun; Berruyer, Romain; Giraldo, Martha C; Kankanala, Prasanna; Park, Sook-Young; Czymmek, Kirk; Kang, Seogchan; Valent, Barbara

2010-04-01

Knowledge remains limited about how fungal pathogens that colonize living plant cells translocate effector proteins inside host cells to regulate cellular processes and neutralize defense responses. To cause the globally important rice blast disease, specialized invasive hyphae (IH) invade successive living rice (Oryza sativa) cells while enclosed in host-derived extrainvasive hyphal membrane. Using live-cell imaging, we identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH. In each newly entered rice cell, effectors were first secreted into BICs at the tips of the initially filamentous hyphae in the cell. These tip BICs were left behind beside the first-differentiated bulbous IH cells as the fungus continued to colonize the host cell. Fluorescence recovery after photobleaching experiments showed that the effector protein PWL2 (for prevents pathogenicity toward weeping lovegrass [Eragrostis curvula]) continued to accumulate in BICs after IH were growing elsewhere. PWL2 and BAS1 (for biotrophy-associated secreted protein 1), BIC-localized secreted proteins, were translocated into the rice cytoplasm. By contrast, BAS4, which uniformly outlines the IH, was not translocated into the host cytoplasm. Fluorescent PWL2 and BAS1 proteins that reached the rice cytoplasm moved into uninvaded neighbors, presumably preparing host cells before invasion. We report robust assays for elucidating the molecular mechanisms that underpin effector secretion into BICs, translocation to the rice cytoplasm, and cell-to-cell movement in rice.

Pseudomonas syringae pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or Suppress Innate Immune Responses in Nicotiana benthamiana.

PubMed

Choi, Sera; Jayaraman, Jay; Segonzac, Cécile; Park, Hye-Jee; Park, Hanbi; Han, Sang-Wook; Sohn, Kee Hoon

2017-01-01

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium -mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa , Psa -NZ V13 and Psa -NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana . Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium -mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1 , NDR1 , or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana . In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta .

Pseudomonas syringae pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or Suppress Innate Immune Responses in Nicotiana benthamiana

PubMed Central

Choi, Sera; Jayaraman, Jay; Segonzac, Cécile; Park, Hye-Jee; Park, Hanbi; Han, Sang-Wook; Sohn, Kee Hoon

2017-01-01

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium-mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa, Psa-NZ V13 and Psa-NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana. Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium-mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1, NDR1, or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana. In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta. PMID:29326748

Tailored immune responses: novel effector helper T cell subsets in protective immunity.

PubMed

Kara, Ervin E; Comerford, Iain; Fenix, Kevin A; Bastow, Cameron R; Gregor, Carly E; McKenzie, Duncan R; McColl, Shaun R

2014-02-01

Differentiation of naïve CD4⁺ cells into functionally distinct effector helper T cell subsets, characterised by distinct "cytokine signatures," is a cardinal strategy employed by the mammalian immune system to efficiently deal with the rapidly evolving array of pathogenic microorganisms encountered by the host. Since the T(H)1/T(H)2 paradigm was first described by Mosmann and Coffman, research in the field of helper T cell biology has grown exponentially with seven functionally unique subsets having now been described. In this review, recent insights into the molecular mechanisms that govern differentiation and function of effector helper T cell subsets will be discussed in the context of microbial infections, with a focus on how these different helper T cell subsets orchestrate immune responses tailored to combat the nature of the pathogenic threat encountered.

White matter microstructure alterations correlate with terminally differentiated CD8+ effector T cell depletion in the peripheral blood in mania: Combined DTI and immunological investigation in the different phases of bipolar disorder.

PubMed

Magioncalda, Paola; Martino, Matteo; Tardito, Samuele; Sterlini, Bruno; Conio, Benedetta; Marozzi, Valentina; Adavastro, Giulia; Capobianco, Laura; Russo, Daniel; Parodi, Alessia; Kalli, Francesca; Nasi, Giorgia; Altosole, Tiziana; Piaggio, Niccolò; Northoff, Georg; Fenoglio, Daniela; Inglese, Matilde; Filaci, Gilberto; Amore, Mario

2018-05-01

White matter (WM) microstructural abnormalities and, independently, signs of immunological activation were consistently demonstrated in bipolar disorder (BD). However, the relationship between WM and immunological alterations as well as their occurrence in the various phases of BD remain unclear. In 60 type I BD patients - 20 in manic, 20 in depressive, 20 in euthymic phases - and 20 controls we investigated: (i) diffusion tensor imaging (DTI)-derived fractional anisotropy (FA), radial diffusivity (RD) and axial diffusivity (AD) using a tract-based spatial statistics (TBSS) approach; (ii) circulating T cell subpopulations frequencies, as well as plasma levels of different cytokines; (iii) potential relationships between WM and immunological data. We found: (i) a significant widespread combined FA-RD alteration mainly in mania, with involvement of the body of corpus callosum (BCC) and superior corona radiata (SCR); (ii) significant increase in CD4+ T cells as well as significant decrease in CD8+ T cells and their subpopulations effector memory (CD8+ CD28-CD45RA-), terminal effector memory (CD8+ CD28-CD45RA+) and CD8+ IFNγ+ in mania; (iii) a significant relationship between WM and immunological alterations in the whole cohort, and a significant correlation of FA-RD abnormalities in the BCC and SCR with reduced frequencies of CD8+ terminal effector memory and CD8+ IFNγ+ T cells in mania only. Our data show a combined occurrence of WM and immunological alterations in mania. WM abnormalities highly correlated with reduction in circulating CD8+ T cell subpopulations that are terminally differentiated effector cells prone to tissue migration, suggesting that these T cells could play a role in WM alteration in BD. Copyright © 2018 Elsevier Inc. All rights reserved.

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation.

PubMed

Montoya, Misty M; Ansel, K Mark

2017-04-13

CD4 + T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from naïve CD4 + T cells in vitro by activating them in the presence of the polarizing cytokines IL-1β, IL-6, IL-23, and TGFβ. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, RORγt, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Developmentally determined reduction in CD31 during gestation is associated with CD8+ T cell effector differentiation in preterm infants

PubMed Central

Scheible, Kristin M.; Emo, Jason; Yang, Hongmei; Holden-Wiltse, Jeanne; Straw, Andrew; Huyck, Heidie; Misra, Sara; Topham, David J.; Ryan, Rita M.; Reynolds, Anne Marie; Mariani, Thomas J.; Pryhuber, Gloria S.

2015-01-01

Homeostatic T cell proliferation is more robust during human fetal development. In order to understand the relative effect of normal fetal homeostasis and perinatal exposures on CD8+ T cell behavior in PT infants, we characterized umbilical cord blood CD8+ T cells from infants born between 23–42 weeks gestation. Subjects were recruited as part of the NHLBI-sponsored Prematurity and Respiratory Outcomes Program. Cord blood from PT infants had fewer naïve CD8+ T cells and lower regulatory CD31 expression on both naïve and effector, independent of prenatal exposures. CD8+ T cell in vitro effector function was greater at younger gestational ages, an effect that was exaggerated in infants with prior inflammatory exposures. These results suggest that CD8+ T cells earlier in gestation have loss of regulatory co-receptor CD31 and greater effector differentiation, which may place PT neonates at unique risk for CD8+ T cell-mediated inflammation and impaired T cell memory formation. PMID:26232733

Neural control of the kidney: functionally specific renal sympathetic nerve fibers.

PubMed

DiBona, G F

2000-11-01

The sympathetic nervous system provides differentiated regulation of the functions of various organs. This differentiated regulation occurs via mechanisms that operate at multiple sites within the classic reflex arc: peripherally at the level of afferent input stimuli to various reflex pathways, centrally at the level of interconnections between various central neuron pools, and peripherally at the level of efferent fibers targeted to various effectors within the organ. In the kidney, increased renal sympathetic nerve activity regulates the functions of the intrarenal effectors: the tubules, the blood vessels, and the juxtaglomerular granular cells. This enables a physiologically appropriate coordination between the circulatory, filtration, reabsorptive, excretory, and renin secretory contributions to overall renal function. Anatomically, each of these effectors has a dual pattern of innervation consisting of a specific and selective innervation by unmyelinated slowly conducting C-type renal sympathetic nerve fibers in addition to an innervation that is shared among all the effectors. This arrangement permits the maximum flexibility in the coordination of physiologically appropriate responses of the tubules, the blood vessels, and the juxtaglomerular granular cells to a variety of homeostatic requirements.

Functionally specific renal sympathetic nerve fibers: role in cardiovascular regulation.

PubMed

DiBona, G F

2001-06-01

The sympathetic nervous system provides differentiated regulation of the functions of various organs. This differentiated regulation occurs through mechanisms that operate at multiple sites within the classic reflex arc: peripherally at the level of afferent input stimuli to various reflex pathways, centrally at the level of interconnections between various central neuron pools, and peripherally at the level of efferent fibers targeted to various effectors within the organ. In the kidney, increased renal sympathetic nerve activity regulates the functions of the intrarenal effectors: the tubules, the blood vessels, and the juxtaglomerular granular cells. This enables a physiologically appropriate coordination between the circulatory, filtration, reabsorptive, excretory, and renin secretory contributions to overall renal function. Anatomically, each of these effectors has a dual pattern of innervation consisting of a specific and selective innervation by unmyelinated slowly conducting C-type renal sympathetic nerve fibers and an innervation that is shared among all the effectors. This arrangement facilitates maximum flexibility in the coordination of the tubules, the blood vessels, and the juxtaglomerular granular cells so as to produce physiologically appropriate responses to a variety of homeostatic requirements.

α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko

We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blotmore » and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated.« less

Regulatory T cells inhibit acute IFN-γ synthesis without blocking T-helper cell type 1 (Th1) differentiation via a compartmentalized requirement for IL-10

PubMed Central

Sojka, Dorothy K.; Fowell, Deborah J.

2011-01-01

CD4+CD25+Forkhead box P3 (Foxp3)+ regulatory T cells (Tregs) control immune responses to self and foreign antigens in secondary lymphoid organs and at tissue sites of inflammation. Tregs can modify the function of many immune cells and have been proposed to block early proliferation, differentiation, and effector function. Acute ablation of Tregs has revealed rapid cytokine production immediately after Treg removal, suggesting that Tregs may regulate effector function acutely rather than regulating the programming for immune function. We developed in vitro and in vivo models that enabled the direct test of Treg regulation of T-helper cell type 1 (Th1) differentiation. CD28 signaling is known to abrogate Treg suppression of IL-2 secretion and proliferation, but our studies show that Treg suppression of IFN-γ during Th1 priming proceeds despite enhanced CD28 signaling. Importantly, during Th1 differentiation, Tregs inhibited early IFN-γ transcription without disrupting expression of Th1-specific T-box transcription factor (Tbet) and Th1 programming. Acute shutoff of effector cytokine production by Tregs was selective for IFN-γ but not TNF-α and was independent of TGF-β and Epstein-Barr virus-induced gene 3. In vivo, Tregs potently controlled CD4 IFN-γ and CD4 effector cell expansion in the lymph node (four- to fivefold reduction) but not Th1 programming, independent of IL-10. Tregs additionally reduced CD4 IFN-γ in the inflamed dermis (twofold reduction) dependent on their production of IL-10. We propose a model for Treg inhibition of effector function based on acute cytokine regulation. Interestingly, Tregs used different regulatory mechanisms to regulate IFN-γ (IL-10–dependent or –independent) subject to the target T-cell stage of activation and its tissue location. PMID:22025707

RhoA orchestrates glycolysis for Th2 cell differentiation and allergic airway inflammation

PubMed Central

Yang, Jun-Qi; Kalim, Khalid W.; Li, Yuan; Zhang, Shuangmin; Hinge, Ashwini; Filippi, Marie-Dominique; Zheng, Yi; Guo, Fukun

2015-01-01

Background Mitochondrial metabolism is known to be important for T cell activation. However, its involvement in effector T cell differentiation has just begun to gain attention. Importantly, how metabolic pathways are integrated with T cell activation and effector cell differentiation and function remains largely unknown. Objective We sought to test our hypothesis that RhoA GTPase orchestrates glycolysis for Th2 cell differentiation and Th2-mediated allergic airway inflammation. Methods Conditional RhoA-deficient mice were generated by crossing RhoAflox/flox mice with CD2-Cre transgenic mice. Effects of RhoA on Th2 differentiation were evaluated by in vitro Th2-polarized culture conditions, and in vivo in ovalbumin (OVA)-induced allergic airway inflammation. Cytokines were measured by intracellular staining and ELISA. T cell metabolism was measured by Seahorse XF24 Analyzer and flow cytometry. Results Disruption of RhoA inhibited T cell activation and Th2 differentiation in vitro and prevented the development of allergic airway inflammation in vivo, with no effect on Th1 cells. RhoA deficiency in activated T cells led to multiple defects in metabolic pathways such as glycolysis and oxidative phosphorylation. Importantly, RhoA couples glycolysis to Th2 cell differentiation and allergic airway inflammation via regulating IL-4 receptor mRNA expression and Th2-specific signaling events. Finally, inhibition of Rho-associated protein kinase (ROCK), an immediate downstream effector of RhoA, blocked Th2 differentiation and allergic airway inflammation. Conclusion RhoA is a key component of the signaling cascades leading to Th2-differentiation and allergic airway inflammation, at least in part, through the control of T cell metabolism and via ROCK pathway. PMID:26100081

Trichloroethylene-induced alterations in DNA methylation were enriched in polycomb protein binding sites in effector/memory CD4+ T cells

PubMed Central

Gilbert, Kathleen M.; Blossom, Sarah J.; Reisfeld, Brad; Erickson, Stephen W.; Vyas, Kanan; Maher, Mary; Broadfoot, Brannon; West, Kirk; Bai, Shasha; Cooney, Craig A.; Bhattacharyya, Sudeepa

2017-01-01

Abstract Exposure to industrial solvent and water pollutant trichloroethylene (TCE) can promote autoimmunity, and expand effector/memory (CD62L) CD4+ T cells. In order to better understand etiology reduced representation bisulfite sequencing was used to study how a 40-week exposure to TCE in drinking water altered methylation of ∼337 770 CpG sites across the entire genome of effector/memory CD4+ T cells from MRL+/+ mice. Regardless of TCE exposure, 62% of CpG sites in autosomal chromosomes were hypomethylated (0–15% methylation), and 25% were hypermethylated (85–100% methylation). In contrast, only 6% of the CpGs on the X chromosome were hypomethylated, and 51% had mid-range methylation levels. In terms of TCE impact, TCE altered (≥ 10%) the methylation of 233 CpG sites in effector/memory CD4+ T cells. Approximately 31.7% of these differentially methylated sites occurred in regions known to bind one or more Polycomb group (PcG) proteins, namely Ezh2, Suz12, Mtf2 or Jarid2. In comparison, only 23.3% of CpG sites not differentially methylated by TCE were found in PcG protein binding regions. Transcriptomics revealed that TCE altered the expression of ∼560 genes in the same effector/memory CD4+ T cells. At least 80% of the immune genes altered by TCE had binding sites for PcG proteins flanking their transcription start site, or were regulated by other transcription factors that were in turn ordered by PcG proteins at their own transcription start site. Thus, PcG proteins, and the differential methylation of their binding sites, may represent a new mechanism by which TCE could alter the function of effector/memory CD4+ T cells. PMID:29129997

New insights into Blimp-1 in T lymphocytes: a divergent regulator of cell destiny and effector function.

PubMed

Fu, Shin-Huei; Yeh, Li-Tzu; Chu, Chin-Chen; Yen, B Lin-Ju; Sytwu, Huey-Kang

2017-07-21

B lymphocyte-induced maturation protein-1 (Blimp-1) serves as a master regulator of the development and function of antibody-producing B cells. Given that its function in T lymphocytes has been identified within the past decade, we review recent findings with emphasis on its role in coordinated control of gene expression during the development, differentiation, and function of T cells. Expression of Blimp-1 is mainly confined to activated T cells and is essential for the production of interleukin (IL)-10 by a subset of forkhead box (Fox)p3 + regulatory T cells with an effector phenotype. Blimp-1 is also required to induce cell elimination in the thymus and critically modulates peripheral T cell activation and proliferation. In addition, Blimp-1 promotes T helper (Th) 2 lineage commitment and limits Th1, Th17 and follicular helper T cell differentiation. Furthermore, Blimp-1 coordinates with other transcription factors to regulate expression of IL-2, IL-21 and IL-10 in effector T lymphocytes. In CD8 + T cells, Blimp-1 expression is distinct in heterogeneous populations at the stages of clonal expansion, differentiation, contraction and memory formation when they encounter antigens. Moreover, Blimp-1 plays a fundamental role in coordinating cytokine receptor signaling networks and transcriptional programs to regulate diverse aspects of the formation and function of effector and memory CD8 + T cells and their exhaustion. Blimp-1 also functions as a gatekeeper of T cell activation and suppression to prevent or dampen autoimmune disease, antiviral responses and antitumor immunity. In this review, we discuss the emerging roles of Blimp-1 in the complex regulation of gene networks that regulate the destiny and effector function of T cells and provide a Blimp-1-dominated transcriptional framework for T lymphocyte homeostasis.

Constitutive Lck Activity Drives Sensitivity Differences between CD8+ Memory T Cell Subsets.

PubMed

Moogk, Duane; Zhong, Shi; Yu, Zhiya; Liadi, Ivan; Rittase, William; Fang, Victoria; Dougherty, Janna; Perez-Garcia, Arianne; Osman, Iman; Zhu, Cheng; Varadarajan, Navin; Restifo, Nicholas P; Frey, Alan B; Krogsgaard, Michelle

2016-07-15

CD8(+) T cells develop increased sensitivity following Ag experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that >50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with <20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore, we observed superior cytotoxic effector function in TEM compared with TCM, and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8(+) TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase, and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM Collectively, this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity, and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications. Copyright © 2016 by The American Association of Immunologists, Inc.

Early exposure to interleukin-21 limits rapidly generated anti-Epstein-Barr virus T-cell line differentiation.

PubMed

Orio, Julie; Carli, Cédric; Janelle, Valérie; Giroux, Martin; Taillefer, Julie; Goupil, Mathieu; Richaud, Manon; Roy, Denis-Claude; Delisle, Jean-Sébastien

2015-04-01

The adoptive transfer of ex vivo-expanded Epstein-Barr virus (EBV)-specific T-cell lines is an attractive strategy to treat EBV-related neoplasms. Current evidence suggests that for adoptive immunotherapy in general, clinical responses are superior if the transferred cells have not reached a late or terminal effector differentiation phenotype before infusion. The cytokine interleukin (IL)-21 has shown great promise at limiting late T-cell differentiation in vitro, but this remains to be demonstrated in anti-viral T-cell lines. We adapted a clinically validated protocol to rapidly generate EBV-specific T-cell lines in 12 to 14 days and tested whether the addition of IL-21 at the initiation of the culture would affect T-cell expansion and differentiation. We generated clinical-scale EBV-restricted T-cell line expansion with balanced T-cell subset ratios. The addition of IL-21 at the beginning of the culture decreased both T-cell expansion and effector memory T-cell accumulation, with a relative increase in less-differentiated T cells. Within CD4 T-cell subsets, exogenous IL-21 was notably associated with the cell surface expression of CD27 and high KLF2 transcript levels, further arguing for a role of IL-21 in the control of late T-cell differentiation. Our results show that IL-21 has profound effects on T-cell differentiation in a rapid T-cell line generation protocol and as such should be further explored as a novel approach to program anti-viral T cells with features associated with early differentiation and optimal therapeutic efficacy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

N-ras couples antigen receptor signaling to Eomesodermin and to functional CD8+ T cell memory but not to effector differentiation

PubMed Central

Iborra, Salvador; Ramos, Manuel; Arana, David M.; Lázaro, Silvia; Aguilar, Francisco; Santos, Eugenio; López, Daniel

2013-01-01

Signals from the TCR that specifically contribute to effector versus memory CD8+ T cell differentiation are poorly understood. Using mice and adoptively transferred T lymphocytes lacking the small GTPase N-ras, we found that N-ras–deficient CD8+ T cells differentiate efficiently into antiviral primary effectors but have a severe defect in generating protective memory cells. This defect was rescued, although only partly, by rapamycin-mediated inhibition of mammalian target of rapamycin (mTOR) in vivo. The memory defect correlated with a marked impairment in vitro and in vivo of the antigen-mediated early induction of T-box transcription factor Eomesodermin (Eomes), whereas T-bet was unaffected. Besides N-ras, early Eomes induction in vitro required phosphoinositide 3-kinase (PI3K)–AKT but not extracellular signal-regulated kinase (ERK) activation, and it was largely insensitive to rapamycin. Consistent with N-ras coupling Eomes to T cell memory, retrovirally enforced expression of Eomes in N-ras–deficient CD8+ T cells effectively rescued their memory differentiation. Thus, our study identifies a critical role for N-ras as a TCR-proximal regulator of Eomes for early determination of the CD8+ T cell memory fate. PMID:23776078

Effectors of root sedentary nematodes target diverse plant cell compartments to manipulate plant functions and promote infection.

PubMed

Jaouannet, Maëlle; Rosso, Marie-Noëlle

2013-09-01

Sedentary plant-parasitic nematodes maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors, which are synthesized in the esophageal glands and injected into the plant tissue through the syringe-like stylet, play a central role in these processes. Previous work on nematode effectors has shown that the apoplasm is targeted during invasion of the host while the cytoplasm is targeted during the induction and the maintenance of the feeding site. A large number of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection have now been identified. This work has shown that the targeting and the role of effectors are more complex than previously thought. This review will not cover the prolific recent findings in nematode effector function but will instead focus on recent selected examples that illustrate the variety of plant cell compartments that effectors are addressed to in order reach their plant targets.

Cellular and Molecular Dynamics of Th17 Differentiation and its Developmental Plasticity in the Intestinal Immune Response

PubMed Central

Bhaumik, Suniti; Basu, Rajatava

2017-01-01

After emerging from the thymus, naive CD4 T cells circulate through secondary lymphoid tissues, including gut-associated lymphoid tissue of the intestine. The activation of naïve CD4 T cells by antigen-presenting cells offering cognate antigen initiate differentiation programs that lead to the development of highly specialized T helper (Th) cell lineages. Although initially believed that developmental programing of effector T cells such as T helper 1 (Th1) or T helper 2 (Th2) resulted in irreversible commitment to a fixed fate, subsequent studies have demonstrated greater flexibility, or plasticity, in effector T cell stability than originally conceived. This is particularly so for the Th17 subset, differentiation of which is a highly dynamic process with overlapping developmental axes with inducible regulatory T (iTreg), T helper 22 (Th22), and Th1 cells. Accordingly, intermediary stages of Th17 cells are found in various tissues, which co-express lineage-specific transcription factor(s) or cytokine(s) of developmentally related CD4 T cell subsets. A highly specialized tissue like that of the intestine, which harbors the largest immune compartment of the body, adds several layers of complexity to the intricate process of Th differentiation. Due to constant exposure to millions of commensal microbes and periodic exposure to pathogens, the intestinal mucosa maintains a delicate balance between regulatory and effector T cells. It is becoming increasingly clear that equilibrium between tolerogenic and inflammatory axes is maintained in the intestine by shuttling the flexible genetic programming of a developing CD4 T cell along the developmental axis of iTreg, Th17, Th22, and Th1 subsets. Currently, Th17 plasticity remains an unresolved concern in the field of clinical research as targeting Th17 cells to cure immune-mediated disease might also target its related subsets. In this review, we discuss the expanding sphere of Th17 plasticity through its shared developmental axes with related cellular subsets such as Th22, Th1, and iTreg in the context of intestinal inflammation and also examine the molecular and epigenetic features of Th17 cells that mediate these overlapping developmental programs. PMID:28408906

The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.

PubMed

Akondy, Rama S; Monson, Nathan D; Miller, Joseph D; Edupuganti, Srilatha; Teuwen, Dirk; Wu, Hong; Quyyumi, Farah; Garg, Seema; Altman, John D; Del Rio, Carlos; Keyserling, Harry L; Ploss, Alexander; Rice, Charles M; Orenstein, Walter A; Mulligan, Mark J; Ahmed, Rafi

2009-12-15

The live yellow fever vaccine (YF-17D) offers a unique opportunity to study memory CD8(+) T cell differentiation in humans following an acute viral infection. We have performed a comprehensive analysis of the virus-specific CD8(+) T cell response using overlapping peptides spanning the entire viral genome. Our results showed that the YF-17D vaccine induces a broad CD8(+) T cell response targeting several epitopes within each viral protein. We identified a dominant HLA-A2-restricted epitope in the NS4B protein and used tetramers specific for this epitope to track the CD8(+) T cell response over a 2 year period. This longitudinal analysis showed the following. 1) Memory CD8(+) T cells appear to pass through an effector phase and then gradually down-regulate expression of activation markers and effector molecules. 2) This effector phase was characterized by down-regulation of CD127, Bcl-2, CCR7, and CD45RA and was followed by a substantial contraction resulting in a pool of memory T cells that re-expressed CD127, Bcl-2, and CD45RA. 3) These memory cells were polyfunctional in terms of degranulation and production of the cytokines IFN-gamma, TNF-alpha, IL-2, and MIP-1beta. 4) The YF-17D-specific memory CD8(+) T cells had a phenotype (CCR7(-)CD45RA(+)) that is typically associated with terminally differentiated cells with limited proliferative capacity (T(EMRA)). However, these cells exhibited robust proliferative potential showing that expression of CD45RA may not always associate with terminal differentiation and, in fact, may be an indicator of highly functional memory CD8(+) T cells generated after acute viral infections.

The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules.

PubMed

Simon, Katharina; Hennen, Stephanie; Merten, Nicole; Blättermann, Stefanie; Gillard, Michel; Kostenis, Evi; Gomeza, Jesus

2016-01-08

Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gαi/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Developmental plasticity of murine and human Foxp3(+) regulatory T cells.

PubMed

Liston, Adrian; Piccirillo, Ciriaco A

2013-01-01

Murine and human CD4(+) regulatory T (Treg) cells expressing the Forkhead box p3 (Foxp3) transcription factor represent a distinct, highly differentiated CD4(+) T cell lineage that is programmed for dominant self-tolerance and control of immune responses against a variety of foreign antigens. Sustained Foxp3 expression in these cells drives the differentiation of a regulatory phenotype and ensures the stability of their suppressive functions under a variety of inflammatory settings. Some recent studies have challenged this premise and advanced the notion that Foxp3(+) Treg cells manifest a high degree of functional plasticity that enables them to adapt and reprogram into effector-like T cells in response to various inflammatory stimuli. The concept of Treg cell plasticity remains highly contentious, with a high degree of variation in measured plasticity potential observed under different experimental conditions. In this chapter, we propose a unifying model of Treg cell plasticity, which hypothesizes that the stable fates of regulatory and effector T (Teff) cell lineages allow transient plasticity into the alternative lineage under a discrete set of microenvironmental influences associated with, respectively, the initiation and resolution phases of infection. This model utilizes a theoretical framework consistent with the requirements for effective immune regulation and accounts for both the extraordinary long-term stability of Treg cells and the observed fate plasticity. Copyright © 2013 Elsevier Inc. All rights reserved.

IFN-γ regulates CD8+ memory T cell differentiation and survival in response to weak, but not strong, TCR signals.

PubMed

Stoycheva, Diana; Deiser, Katrin; Stärck, Lilian; Nishanth, Gopala; Schlüter, Dirk; Uckert, Wolfgang; Schüler, Thomas

2015-01-15

In response to primary Ag contact, naive mouse CD8(+) T cells undergo clonal expansion and differentiate into effector T cells. After pathogen clearance, most effector T cells die, and only a small number of memory T cell precursors (TMPs) survive to form a pool of long-lived memory T cells (TMs). Although high- and low-affinity CD8(+) T cell clones are recruited into the primary response, the TM pool consists mainly of high-affinity clones. It remains unclear whether the more efficient expansion of high-affinity clones and/or cell-intrinsic processes exclude low-affinity T cells from the TM pool. In this article, we show that the lack of IFN-γR signaling in CD8(+) T cells promotes TM formation in response to weak, but not strong, TCR agonists. The IFN-γ-sensitive accumulation of TMs correlates with reduced mammalian target of rapamycin activation and the accumulation of long-lived CD62L(hi)Bcl-2(hi)Eomes(hi) TMPs. Reconstitution of mammalian target of rapamycin or IFN-γR signaling is sufficient to block this process. Hence, our data suggest that IFN-γR signaling actively blocks the formation of TMPs responding to weak TCR agonists, thereby promoting the accumulation of high-affinity T cells finally dominating the TM pool. Copyright © 2015 by The American Association of Immunologists, Inc.

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

PubMed

Gupta, Bhawna; Iancu, Emanuela M; Gannon, Philippe O; Wieckowski, Sébastien; Baitsch, Lukas; Speiser, Daniel E; Rufer, Nathalie

2012-07-01

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

Non-parent of Origin Expression of Numerous Effector Genes Indicates a Role of Gene Regulation in Host Adaption of the Hybrid Triticale Powdery Mildew Pathogen

PubMed Central

Praz, Coraline R.; Menardo, Fabrizio; Robinson, Mark D.; Müller, Marion C.; Wicker, Thomas; Bourras, Salim; Keller, Beat

2018-01-01

Powdery mildew is an important disease of cereals. It is caused by one species, Blumeria graminis, which is divided into formae speciales each of which is highly specialized to one host. Recently, a new form capable of growing on triticale (B.g. triticale) has emerged through hybridization between wheat and rye mildews (B.g. tritici and B.g. secalis, respectively). In this work, we used RNA sequencing to study the molecular basis of host adaptation in B.g. triticale. We analyzed gene expression in three B.g. tritici isolates, two B.g. secalis isolates and two B.g. triticale isolates and identified a core set of putative effector genes that are highly expressed in all formae speciales. We also found that the genes differentially expressed between isolates of the same form as well as between different formae speciales were enriched in putative effectors. Their coding genes belong to several families including some which contain known members of mildew avirulence (Avr) and suppressor (Svr) genes. Based on these findings we propose that effectors play an important role in host adaptation that is mechanistically based on Avr-Resistance gene-Svr interactions. We also found that gene expression in the B.g. triticale hybrid is mostly conserved with the parent-of-origin, but some genes inherited from B.g. tritici showed a B.g. secalis-like expression. Finally, we identified 11 unambiguous cases of putative effector genes with hybrid-specific, non-parent of origin gene expression, and we propose that they are possible determinants of host specialization in triticale mildew. These data suggest that altered expression of multiple effector genes, in particular Avr and Svr related factors, might play a role in mildew host adaptation based on hybridization. PMID:29441081

Fucosyltransferase Induction during Influenza Virus Infection Is Required for the Generation of Functional Memory CD4+ T Cells

PubMed Central

Carrette, Florent; Henriquez, Monique L.; Fujita, Yu

2018-01-01

T cells mediating influenza viral control are instructed in lymphoid and nonlymphoid tissues to differentiate into memory T cells that confer protective immunity. The mechanisms by which influenza virus–specific memory CD4+ T cells arise have been attributed to changes in transcription factors, cytokines and cytokine receptors, and metabolic programming. The molecules involved in these biosynthetic pathways, including proteins and lipids, are modified to varying degrees of glycosylation, fucosylation, sialation, and sulfation, which can alter their function. It is currently unknown how the glycome enzymatic machinery regulates CD4+ T cell effector and memory differentiation. In a murine model of influenza virus infection, we found that fucosyltransferase enzymatic activity was induced in effector and memory CD4+ T cells. Using CD4+ T cells deficient in the Fut4/7 enzymes that are expressed only in hematopoietic cells, we found decreased frequencies of effector cells with reduced expression of T-bet and NKG2A/C/E in the lungs during primary infection. Furthermore, Fut4/7−/− effector CD4+ T cells had reduced survival with no difference in proliferation or capacity for effector function. Although Fut4/7−/− CD4+ T cells seeded the memory pool after primary infection, they failed to form tissue-resident cells, were dysfunctional, and were unable to re-expand after secondary infection. Our findings highlight an important regulatory axis mediated by cell-intrinsic fucosyltransferase activity in CD4+ T cell effectors that ensure the development of functional memory CD4+ T cells. PMID:29491007

A systems biology perspective on plant-microbe interactions: biochemical and structural targets of pathogen effectors.

PubMed

Pritchard, Leighton; Birch, Paul

2011-04-01

Plants have biochemical defences against stresses from predators, parasites and pathogens. In this review we discuss the interaction of plant defences with microbial pathogens such as bacteria, fungi and oomycetes, and viruses. We examine principles of complex dynamic networks that allow identification of network components that are differentially and predictably sensitive to perturbation, thus making them likely effector targets. We relate these principles to recent developments in our understanding of known effector targets in plant-pathogen systems, and propose a systems-level framework for the interpretation and modelling of host-microbe interactions mediated by effectors. We describe this framework briefly, and conclude by discussing useful experimental approaches for populating this framework. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Origin and differentiation of human memory CD8 T cells after vaccination.

PubMed

Akondy, Rama S; Fitch, Mark; Edupuganti, Srilatha; Yang, Shu; Kissick, Haydn T; Li, Kelvin W; Youngblood, Ben A; Abdelsamed, Hossam A; McGuire, Donald J; Cohen, Kristen W; Alexe, Gabriela; Nagar, Shashi; McCausland, Megan M; Gupta, Satish; Tata, Pramila; Haining, W Nicholas; McElrath, M Juliana; Zhang, David; Hu, Bin; Greenleaf, William J; Goronzy, Jorg J; Mulligan, Mark J; Hellerstein, Marc; Ahmed, Rafi

2017-12-21

The differentiation of human memory CD8 T cells is not well understood. Here we address this issue using the live yellow fever virus (YFV) vaccine, which induces long-term immunity in humans. We used in vivo deuterium labelling to mark CD8 T cells that proliferated in response to the virus and then assessed cellular turnover and longevity by quantifying deuterium dilution kinetics in YFV-specific CD8 T cells using mass spectrometry. This longitudinal analysis showed that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is maintained by quiescent cells that divide less than once every year (doubling time of over 450 days). Although these long-lived YFV-specific memory CD8 T cells did not express effector molecules, their epigenetic landscape resembled that of effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen.

Regulation of vesicular trafficking and leukocyte function by Rab27 GTPases and their effectors

PubMed Central

Catz, Sergio Daniel

2013-01-01

The Rab27 family of GTPases regulates the efficiency and specificity of exocytosis in hematopoietic cells, including neutrophils, CTLs, NK cells, and mast cells. However, the mechanisms regulated by Rab27 GTPases are cell-specific, as they depend on the differential expression and function of particular effector molecules that are recruited by the GTPases. In addition, Rab27 GTPases participate in multiple steps of the regulation of the secretory process, including priming, tethering, docking, and fusion through sequential interaction with multiple effector molecules. Finally, recent reports suggest that Rab27 GTPases and their effectors regulate vesicular trafficking mechanisms other than exocytosis, including endocytosis and phagocytosis. This review focuses on the latest discoveries on the function of Rab27 GTPases and their effectors Munc13-4 and Slp1 in neutrophil function comparatively to their functions in other leukocytes. PMID:23378593

Effector T-cells are expanded in systemic lupus erythematosus patients with high disease activity and damage indexes.

PubMed

Piantoni, S; Regola, F; Zanola, A; Andreoli, L; Dall'Ara, F; Tincani, A; Airo', P

2018-01-01

Background and objectives T-cell activation may be one of the pathogenic mechanisms of systemic lupus erythematosus (SLE). After repeated antigenic stimulation, T-cells undergo different modifications, leading to the differentiation into effector memory T-cells (CCR7-CD45RA-) and terminally differentiated effector memory (TDEM) T-cells (CCR7-CD45RA+). Similarly, down-modulation of CD28 may lead to the expansion of the CD28- T-cells, a subpopulation with peculiar effector activities. The aim of this study was the characterization of T-cell phenotype in a cohort of patients with SLE according to disease activity and damage index. Materials and methods Phenotypic analysis of peripheral blood T lymphocytes of 51 SLE patients and 21 healthy controls was done by flow-cytometry. SLE disease activity was evaluated by SLE Disease Activity Index-2000 (SLEDAI-2K) and damage by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index (SDI). The variations between different groups were evaluated by Mann-Whitney test. Bonferroni correction was applied to adjust for multiple comparisons ( p adj ). Spearman rank test was used to evaluate the correlations between quantitative variables. Results CD4+ lymphopenia was found among SLE patients. Patients showed a trend for a higher percentage of TDEM among the CD4+ T-cell subpopulation in comparison with healthy controls ( p = .04). SLE patients were divided into two groups according to disease activity: patients with SLEDAI-2K ≥ 6 ( n = 13) had a higher percentage of circulating CD4+ T-cells with CD28- phenotype ( p adj  = .005) as well as those with an effector memory ( p adj  = .004) and TDEM ( p adj  = .002) phenotype and a trend of decrease of regulatory T-cells (TREGs) ( p = .02), in comparison with patients with low disease activity ( n = 38). Patients with damage (SDI ≥ 1) tended to show an expansion of TDEM among CD4+ T-cells as compared with patients with no damage ( p = .01). In SLE patients an inverse correlation was found between the percentages of TREGs and those of TDEM ( p < .01) or CD4 + CD28- ( p < .01) T-cells. Conclusions CD4+ T-cell subpopulations displaying phenotype characteristics of effector lymphocytes are proportionally expanded in patients with active SLE and a higher damage index. These findings may suggest a role of effector T-cells in the pathogenesis of the disease and in the mechanisms of damage in SLE.

Functional heterogeneity of human effector CD8+ T cells.

PubMed

Takata, Hiroshi; Naruto, Takuya; Takiguchi, Masafumi

2012-02-09

Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

A zebrafish larval model reveals early tissue-specific innate immune responses to Mucor circinelloides.

PubMed

Voelz, Kerstin; Gratacap, Remi L; Wheeler, Robert T

2015-11-01

Mucormycosis is an emerging fungal infection that is clinically difficult to manage, with increasing incidence and extremely high mortality rates. Individuals with diabetes, suppressed immunity or traumatic injury are at increased risk of developing disease. These individuals often present with defects in phagocytic effector cell function. Research using mammalian models and phagocytic effector cell lines has attempted to decipher the importance of the innate immune system in host defence against mucormycosis. However, these model systems have not been satisfactory for direct analysis of the interaction between innate immune effector cells and infectious sporangiospores in vivo. Here, we report the first real-time in vivo analysis of the early innate immune response to mucormycete infection using a whole-animal zebrafish larval model system. We identified differential host susceptibility, dependent on the site of infection (hindbrain ventricle and swim bladder), as well as differential functions of the two major phagocyte effector cell types in response to viable and non-viable spores. Larval susceptibility to mucormycete spore infection was increased upon immunosuppressant treatment. We showed for the first time that macrophages and neutrophils were readily recruited in vivo to the site of infection in an intact host and that spore phagocytosis can be observed in real-time in vivo. While exploring innate immune effector recruitment dynamics, we discovered the formation of phagocyte clusters in response to fungal spores that potentially play a role in fungal spore dissemination. Spores failed to activate pro-inflammatory gene expression by 6 h post-infection in both infection models. After 24 h, induction of a pro-inflammatory response was observed only in hindbrain ventricle infections. Only a weak pro-inflammatory response was initiated after spore injection into the swim bladder during the same time frame. In the future, the zebrafish larva as a live whole-animal model system will contribute greatly to the study of molecular mechanisms involved in the interaction of the host innate immune system with fungal spores during mucormycosis. © 2015. Published by The Company of Biologists Ltd.

Blockade of PD-1/PD-L1 Promotes Adoptive T-Cell Immunotherapy in a Tolerogenic Environment

PubMed Central

Kenna, Tony J.; Galea, Ryan; Large, Justin; Yagita, Hideo; Steptoe, Raymond J.

2015-01-01

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The co-inhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PD-L1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcm-like cells. PMID:25741704

Blockade of PD-1/PD-L1 promotes adoptive T-cell immunotherapy in a tolerogenic environment.

PubMed

Blake, Stephen J P; Ching, Alan L H; Kenna, Tony J; Galea, Ryan; Large, Justin; Yagita, Hideo; Steptoe, Raymond J

2015-01-01

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The co-inhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PD-L1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcm-like cells.

Network-based expression analyses and experimental validations revealed high co-expression between Yap1 and stem cell markers compared to differentiated cells.

PubMed

Dehghanian, Fariba; Hojati, Zohreh; Esmaeili, Fariba; Masoudi-Nejad, Ali

2018-05-21

The Hippo signaling pathway is identified as a potential regulatory pathway which plays critical roles in differentiation and stem cell self-renewal. Yap1 is a primary transcriptional effector of this pathway. The importance of Yap1 in embryonic stem cells (ESCs) and differentiation procedure remains a challenging question, since two different observations have been reported. To answer this question we used co-expression network and differential co-expression analyses followed by experimental validations. Our results indicate that Yap1 is highly co-expressed with stem cell markers in ESCs but not in differentiated cells (DCs). The significant Yap1 down-regulation and also translocation of Yap1 into the cytoplasm during P19 differentiation was also detected. Moreover, our results suggest the E2f7, Lin28a and Dppa4 genes as possible regulatory nuclear factors of Hippo pathway in stem cells. The present findings are actively consistent with studies that suggested Yap1 as an essential factor for stem cell self-renewal. Copyright © 2018 Elsevier Inc. All rights reserved.

Notch regulates Th17 differentiation and controls trafficking of IL-17 and metabolic regulators within Th17 cells in a context-dependent manner

PubMed Central

Coutaz, Manuel; Hurrell, Benjamin P.; Auderset, Floriane; Wang, Haiping; Siegert, Stefanie; Eberl, Gerard; Ho, Ping-Chih; Radtke, Freddy; Tacchini-Cottier, Fabienne

2016-01-01

Th17 cells play critical roles in host defense and autoimmunity. Emerging data support a role for Notch signaling in Th17 cell differentiation but whether it is a positive or negative regulator remains unclear. We report here that T cell-specific deletion of Notch receptors enhances Th17 cell differentiation in the gut, with a corresponding increase in IL-17 secretion. An increase in Th17 cell frequency was similarly observed following immunization of T cell specific Notch mutant mice with OVA/CFA. However, in this setting, Th17 cytokine secretion was impaired, and increased intracellular retention of IL-17 was observed. Intracellular IL-17 co-localized with the CD71 iron transporter in the draining lymph node of both control and Notch-deficient Th17 cells. Immunization induced CD71 surface expression in control, but not in Notch-deficient Th17 cells, revealing defective CD71 intracellular transport in absence of Notch signaling. Moreover, Notch receptor deficient Th17 cells had impaired mTORC2 activity. These data reveal a context-dependent impact of Notch on vesicular transport during high metabolic demand suggesting a role for Notch signaling in the bridging of T cell metabolic demands and effector functions. Collectively, our findings indicate a prominent regulatory role for Notch signaling in the fine-tuning of Th17 cell differentiation and effector function. PMID:27974744

Control of regulatory T cell lineage commitment and maintenance.

PubMed

Josefowicz, Steven Z; Rudensky, Alexander

2009-05-01

Foxp3-expressing regulatory T (Treg) cells suppress pathology mediated by immune responses against self and foreign antigens and commensal microorganisms. Sustained expression of the transcription factor Foxp3, a key distinguishing feature of Treg cells, is required for their differentiation and suppressor function. In addition, Foxp3 expression prevents deviation of Treg cells into effector T cell lineages and confers dependence of Treg cell survival and expansion on growth factors, foremost interleukin-2, provided by activated effector T cells. In this review we discuss Treg cell differentiation and maintenance with a particular emphasis on molecular regulation of Foxp3 expression, arguably a key to mechanistic understanding of biology of regulatory T cells.

Cellular Factors Targeting APCs to Modulate Adaptive T Cell Immunity

PubMed Central

Do, Jeongsu; Min, Booki

2014-01-01

The fate of adaptive T cell immunity is determined by multiple cellular and molecular factors, among which the cytokine milieu plays the most important role in this process. Depending on the cytokines present during the initial T cell activation, T cells become effector cells that produce different effector molecules and execute adaptive immune functions. Studies thus far have primarily focused on defining how these factors control T cell differentiation by targeting T cells themselves. However, other non-T cells, particularly APCs, also express receptors for the factors and are capable of responding to them. In this review, we will discuss how APCs, by responding to those cytokines, influence T cell differentiation and adaptive immunity. PMID:25126585

HIV-TB coinfection impairs CD8(+) T-cell differentiation and function while dehydroepiandrosterone improves cytotoxic antitubercular immune responses.

PubMed

Suarez, Guadalupe V; Angerami, Matías T; Vecchione, María B; Laufer, Natalia; Turk, Gabriela; Ruiz, Maria J; Mesch, Viviana; Fabre, Bibiana; Maidana, Patricia; Ameri, Diego; Cahn, Pedro; Sued, Omar; Salomón, Horacio; Bottasso, Oscar A; Quiroga, María F

2015-09-01

Tuberculosis (TB) is the leading cause of death among HIV-positive patients. The decreasing frequencies of terminal effector (TTE ) CD8(+) T cells may increase reactivation risk in persons latently infected with Mycobacterium tuberculosis (Mtb). We have previously shown that dehydroepiandrosterone (DHEA) increases the protective antitubercular immune responses in HIV-TB patients. Here, we aimed to study Mtb-specific cytotoxicity, IFN-γ secretion, memory status of CD8(+) T cells, and their modulation by DHEA during HIV-TB coinfection. CD8(+) T cells from HIV-TB patients showed a more differentiated phenotype with diminished naïve and higher effector memory and TTE T-cell frequencies compared to healthy donors both in total and Mtb-specific CD8(+) T cells. Notably, CD8(+) T cells from HIV-TB patients displayed higher Terminal Effector (TTE ) CD45RA(dim) proportions with lower CD45RA expression levels, suggesting a not fully differentiated phenotype. Also, PD-1 expression levels on CD8(+) T cells from HIV-TB patients increased although restricted to the CD27(+) population. Interestingly, DHEA plasma levels positively correlated with TTE in CD8(+) T cells and in vitro DHEA treatment enhanced Mtb-specific cytotoxic responses and terminal differentiation in CD8(+) T cells from HIV-TB patients. Our data suggest that HIV-TB coinfection promotes a deficient CD8(+) T-cell differentiation, whereas DHEA may contribute to improving antitubercular immunity by enhancing CD8(+) T-cell functions during HIV-TB coinfection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular basis for allosteric specificity regulation in class Ia ribonucleotide reductase from Escherichia coli

PubMed Central

Zimanyi, Christina M; Chen, Percival Yang-Ting; Kang, Gyunghoon; Funk, Michael A; Drennan, Catherine L

2016-01-01

Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively. Here, we have determined structures of Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound (CDP/dATP, UDP/dATP, ADP/dGTP, GDP/TTP) that reveal the conformational rearrangements responsible for this remarkable allostery. These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell. DOI: http://dx.doi.org/10.7554/eLife.07141.001 PMID:26754917

Plant parasitic nematode effectors target host defense and nuclear functions to establish feeding cells.

PubMed

Quentin, Michaëel; Abad, Pierre; Favery, Bruno

2013-01-01

Plant parasitic nematodes are microscopic worms, the most damaging species of which have adopted a sedentary lifestyle within their hosts. These obligate endoparasites have a biotrophic relationship with plants, in which they induce the differentiation of root cells into hypertrophied, multinucleate feeding cells (FCs). Effectors synthesized in the esophageal glands of the nematode are injected into the plant cells via the syringe-like stylet and play a key role in manipulating the host machinery. The establishment of specialized FCs requires these effectors to modulate many aspects of plant cell morphogenesis and physiology, including defense responses. This cell reprogramming requires changes to host nuclear processes. Some proteins encoded by parasitism genes target host nuclei. Several of these proteins were immunolocalized within FC nuclei or shown to interact with host nuclear proteins. Comparative genomics and functional analyses are gradually revealing the roles of nematode effectors. We describe here these effectors and their hypothesized roles in the unique feeding behavior of these pests.

Ubiquitin Ligases and Deubiquitinating Enzymes in CD4+ T Cell Effector Fate Choice and Function.

PubMed

Layman, Awo A K; Oliver, Paula M

2016-05-15

The human body is exposed to potentially pathogenic microorganisms at barrier sites such as the skin, lungs, and gastrointestinal tract. To mount an effective response against these pathogens, the immune system must recruit the right cells with effector responses that are appropriate for the task at hand. Several types of CD4(+) T cells can be recruited, including Th cells (Th1, Th2, and Th17), T follicular helper cells, and regulatory T cells. These cells help to maintain normal immune homeostasis in the face of constantly changing microbes in the environment. Because these cells differentiate from a common progenitor, the composition of their intracellular milieu of proteins changes to appropriately guide their effector function. One underappreciated process that impacts the levels and functions of effector fate-determining factors is ubiquitylation. This review details our current understanding of how ubiquitylation regulates CD4(+) T cell effector identity and function. Copyright © 2016 by The American Association of Immunologists, Inc.

Host protein BSL1 associates with Phytophthora infestans RXLR effector AVR2 and the Solanum demissum Immune receptor R2 to mediate disease resistance.

PubMed

Saunders, Diane G O; Breen, Susan; Win, Joe; Schornack, Sebastian; Hein, Ingo; Bozkurt, Tolga O; Champouret, Nicolas; Vleeshouwers, Vivianne G A A; Birch, Paul R J; Gilroy, Eleanor M; Kamoun, Sophien

2012-08-01

Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor.

T-bet Down-Modulation in Tolerized Th1 Effector CD4 Cells Confers a TCR-Distal Signaling Defect That Selectively Impairs IFN-γ Expression1

PubMed Central

Long, Meixiao; Slaiby, Aaron M.; Hagymasi, Adam T.; Mihalyo, Marianne A.; Lichtler, Alexander C.; Reiner, Steven L.; Adler, Adam J.

2010-01-01

When Th1 effector CD4 cells encounter tolerizing Ag in vivo, their capacity to express the effector cytokines IFN-γ and TNF-α is lost more rapidly than noneffector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with Ag vs agents that bypass TCR-proximal signaling. IFN-γ and TNF-α expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and -distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-γ -expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-γ, but not TNF-α. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-γ, but not TNF-α. Analysis of histone acetylation at the IFN-γ promoter further suggested that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN-γ expression potential through alterations in chromatin structure. PMID:16393991

Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity.

PubMed

Wang, Dongrui; Aguilar, Brenda; Starr, Renate; Alizadeh, Darya; Brito, Alfonso; Sarkissian, Aniee; Ostberg, Julie R; Forman, Stephen J; Brown, Christine E

2018-05-17

Chimeric antigen receptor-modified (CAR-modified) T cells have shown promising therapeutic effects for hematological malignancies, yet limited and inconsistent efficacy against solid tumors. The refinement of CAR therapy requires an understanding of the optimal characteristics of the cellular products, including the appropriate composition of CD4+ and CD8+ subsets. Here, we investigated the differential antitumor effect of CD4+ and CD8+ CAR T cells targeting glioblastoma-associated (GBM-associated) antigen IL-13 receptor α2 (IL13Rα2). Upon stimulation with IL13Rα2+ GBM cells, the CD8+ CAR T cells exhibited robust short-term effector function but became rapidly exhausted. By comparison, the CD4+ CAR T cells persisted after tumor challenge and sustained their effector potency. Mixing with CD4+ CAR T cells failed to ameliorate the effector dysfunction of CD8+ CAR T cells, while surprisingly, CD4+ CAR T cell effector potency was impaired when coapplied with CD8+ T cells. In orthotopic GBM models, CD4+ outperformed CD8+ CAR T cells, especially for long-term antitumor response. Further, maintenance of the CD4+ subset was positively correlated with the recursive killing ability of CAR T cell products derived from GBM patients. These findings identify CD4+ CAR T cells as a highly potent and clinically important T cell subset for effective CAR therapy.

Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity

PubMed Central

Wang, Dongrui; Starr, Renate; Alizadeh, Darya; Brito, Alfonso; Sarkissian, Aniee; Ostberg, Julie R.; Forman, Stephen J.; Brown, Christine E.

2018-01-01

Chimeric antigen receptor–modified (CAR-modified) T cells have shown promising therapeutic effects for hematological malignancies, yet limited and inconsistent efficacy against solid tumors. The refinement of CAR therapy requires an understanding of the optimal characteristics of the cellular products, including the appropriate composition of CD4+ and CD8+ subsets. Here, we investigated the differential antitumor effect of CD4+ and CD8+ CAR T cells targeting glioblastoma-associated (GBM-associated) antigen IL-13 receptor α2 (IL13Rα2). Upon stimulation with IL13Rα2+ GBM cells, the CD8+ CAR T cells exhibited robust short-term effector function but became rapidly exhausted. By comparison, the CD4+ CAR T cells persisted after tumor challenge and sustained their effector potency. Mixing with CD4+ CAR T cells failed to ameliorate the effector dysfunction of CD8+ CAR T cells, while surprisingly, CD4+ CAR T cell effector potency was impaired when coapplied with CD8+ T cells. In orthotopic GBM models, CD4+ outperformed CD8+ CAR T cells, especially for long-term antitumor response. Further, maintenance of the CD4+ subset was positively correlated with the recursive killing ability of CAR T cell products derived from GBM patients. These findings identify CD4+ CAR T cells as a highly potent and clinically important T cell subset for effective CAR therapy. PMID:29769444

CD4 T Cell Responses in Latent and Chronic Viral Infections

PubMed Central

Walton, Senta; Mandaric, Sanja; Oxenius, Annette

2013-01-01

The spectrum of tasks which is fulfilled by CD4 T cells in the setting of viral infections is large, ranging from support of CD8 T cells and humoral immunity to exertion of direct antiviral effector functions. While our knowledge about the differentiation pathways, plasticity, and memory of CD4 T cell responses upon acute infections or immunizations has significantly increased during the past years, much less is still known about CD4 T cell differentiation and their beneficial or pathological functions during persistent viral infections. In this review we summarize current knowledge about the differentiation, direct or indirect antiviral effector functions, and the regulation of virus-specific CD4 T cells in the setting of persistent latent or active chronic viral infections with a particular emphasis on herpes virus infections for the former and chronic lymphocytic choriomeningitis virus infection for the latter. PMID:23717308

Soluble GARP has potent antiinflammatory and immunomodulatory impact on human CD4⁺ T cells.

PubMed

Hahn, Susanne A; Stahl, Heiko F; Becker, Christian; Correll, Anita; Schneider, Franz-Joseph; Tuettenberg, Andrea; Jonuleit, Helmut

2013-08-15

Glycoprotein A repetitions predominant (GARP) is expressed on the surface of activated human regulatory T cells (Treg) and regulates the bioavailability of transforming growth factor-β (TGF-β). GARP has been assumed to require membrane anchoring. To investigate the function of GARP in more detail, we generated a soluble GARP protein (sGARP) and analyzed its impact on differentiation and activation of human CD4⁺ T cells. We demonstrate that sGARP efficiently represses proliferation and differentiation of naïve CD4⁺ T cells into T effector cells. Exposure to sGARP induces Foxp3, decreases proliferation and represses interleukin (IL)-2 and interferon-γ production, resulting in differentiation of naïve T cells into induced Treg. This is associated with Smad2/3 phosphorylation and partially inhibited by blockade of TGF-β signaling. Furthermore, in the presence of the proinflammatory cytokines IL-6 and IL-23, sGARP facilitates the differentiation of naïve T cells into Th17 cells. More important, in a preclinical humanized mouse model of xenogeneic graft-versus-host disease (GVHD), sGARP prevents T cell-mediated destructive inflammation by enhancing Treg and inhibiting T effector cell activity. These results demonstrate a crucial role of sGARP in modulation of peripheral tolerance and T effector cell function, opening the possibility to use sGARP as a potent immunomodulator of inflammatory diseases including transplant rejection, autoimmunity, and allergy.

Acute virus control mediated by licensed NK cells sets primary CD8+ T cell dependence on CD27 costimulation1,2,3

PubMed Central

Teoh, Jeffrey J.; Gamache, Awndre E.; Gillespie, Alyssa L.; Stadnisky, Michael D.; Yagita, Hideo; Bullock, Timothy N.J.; Brown, Michael G.

2016-01-01

Natural killer (NK) cells represent a critical first-line of immune defense against a bevy of viral pathogens, and infection can provoke them to mediate both supportive and suppressive effects on virus-specific adaptive immunity. In mice expressing MHC I Dk, a major MCMV resistance factor and self-ligand of the inhibitory Ly49G2 (G2) receptor, licensed G2+ NK cells provide essential host resistance against murine (M)CMV infection. Additionally G2+ NK cell responses to MCMV increase the rate and extent of dendritic cell (DC) recovery, as well as early priming of CD8+ T-cell effectors in response to MCMV. However, relatively little is known about the NK-cell effect on co-stimulatory ligand patterns displayed by DCs, or ensuing effector and memory T-cell responses. Here we found that CD27-dependent CD8+ T-cell priming and differentiation is shaped by the efficiency of NK responses to virus infection. Surprisingly, differences in specific NK responses to MCMV in Dk-disparate mice failed to distinguish early DC co-stimulatory patterns. Nonetheless, while CD27 deficiency did not impede licensed NK-mediated resistance, both CD70 and CD27 were required to efficiently prime and regulate effector CD8+ T-cell differentiation in response to MCMV, which eventually resulted in biased memory T-cell precursor formation in Dk mice. In contrast, CD8+ T-cells accrued more slowly in non-Dk mice, and eventually differentiated into terminal effector cells regardless of CD27 stimulation. Disparity in this requirement for CD27 signaling indicates that specific virus control mediated by NK cells can shape DC co-stimulatory signals needed to prime CD8+ T cells and eventual T-cell fate decisions. PMID:27798162

KHYG-1 and NK-92 represent different subtypes of LFA-1-mediated NK cell adhesiveness.

PubMed

Suck, Garnet; Tan, Suet-Mien; Chu, Sixian; Niam, Madelaine; Vararattanavech, Ardcharaporn; Lim, Tsyr Jong; Koh, Mickey B C

2011-01-01

Novel cancer cellular therapy approaches involving long-term ex vivo IL-2 stimulated highly cytotoxic natural killer (NK) cells are emerging. However, adhesion properties of such NK cells are not very well understood. Herein, we describe the novel observation of permanently activated alphaLbeta2 integrin leukocyte function-associated antigen (LFA)-1 adhesion receptor in long-term IL-2 activated NK cells and the permanent NK cell lines KHYG-1 and NK-92. We show that such cytokine activated NK effectors constitutively adhered to the LFA-1-ligand ICAM-1, whereas binding to the lower affinity ligand ICAM-3 required additional exogenous activating conditions. The results demonstrate an extended conformation and an intermediate affinity state for the LFA-1 population expressed by the NK cells. Interestingly, adhesion to ICAM-1 or K562 induced pronounced cell spreading in KHYG-1, but not in NK-92, and partially in long-term IL-2 stimulated primary NK cells. It is conceivable that such differential adhesion characteristics may impact motility potential of such NK effectors with relevance to clinical tumor targeting. KHYG-1 could be a useful model in planning future targeted therapeutic approaches involving NK effectors with augmented functions.

The alpha subunit of Go modulates cell proliferation and differentiation through interactions with Necdin.

PubMed

Ju, Hyunhee; Lee, Sujin; Kang, Sunghak; Kim, Sung-Soo; Ghil, Sungho

2014-07-10

Heterotrimeric GTP-binding proteins (G-proteins) play an important role in mediating signal transduction generated by neurotransmitters or hormones. Go, a member of the Gi/Go subfamily, is the most abundant G-protein found in the brain. Recently, the alpha subunit of Go (Gαo) was characterized as an inducer of neuronal differentiation. However, its underlying molecular mechanisms have remained unclear to date, since the downstream effectors of Gαo are ambiguous. A neurally differentiated embryonal carcinoma-derived protein (Necdin) was isolated as an interacting partner for Gαo from a mouse brain cDNA library using yeast two-hybrid screening. Interactions between the proteins were confirmed with several affinity binding assays, both in vitro and in vivo. Necdin interacted directly and preferentially with activated Gαo, compared to wild-type protein. Interestingly, Gαo did not interact with Gαi, despite high sequence homology between the two proteins. We subsequently analyzed whether Gαo modulates the cellular activities of Necdin. Notably, expression of Gαo significantly augmented Necdin-mediated cellular responses, such as proliferation and differentiation. Moreover, activation of type 1 cannabinoid receptor (CB1R), a Gi/oα-coupled receptor, augmented cell growth suppression, which was mediated by Gαo and Necdin in U87MG cells containing CB1R, Gαo, and Necdin as normal components. These results collectively suggest that Necdin is a candidate downstream effector for Gαo. Our findings provide novel insights into the cellular roles of Gαo and its coupled receptor.

Somatostatin/somatostatin receptor signalling: phosphotyrosine phosphatases.

PubMed

Florio, Tullio

2008-05-14

Activation of phosphotyrosine phosphatases (PTPs) by somatostatin receptor (SSTR) represents one of the main intracellular mechanisms involved in the antiproliferative effect of somatostatin (SST) and analogues. Since their molecular cloning, the role of PTPs is emerging as a major regulator of different cell functions including cell proliferation, differentiation, cell to cell interactions, cell matrix adhesion and cell migration. It was demonstrated that PTPs possess high substrate specificity and their activity is tightly regulated. Importantly, different G protein-coupled receptors transduce their biological activities through PTPs. PTPs were identified as down-stream effectors of SSTRs to transduce antiproliferative signals, and so far, three family members (SHP-1, SHP-2 and DEP-1/PTPeta) have been identified as selective SSTR intracellular effectors. Here, the molecular mechanisms leading SSTRs to regulate PTP activity are discussed, focusing on recent data showing a close interplay between PTPs and tyrosine kinases to transduce tumoral cell growth arrest following SST analogs administration.

Identification of Nascent Memory CD8 T Cells and Modeling of Their Ontogeny.

PubMed

Crauste, Fabien; Mafille, Julien; Boucinha, Lilia; Djebali, Sophia; Gandrillon, Olivier; Marvel, Jacqueline; Arpin, Christophe

2017-03-22

Primary immune responses generate short-term effectors and long-term protective memory cells. The delineation of the genealogy linking naive, effector, and memory cells has been complicated by the lack of phenotypes discriminating effector from memory differentiation stages. Using transcriptomics and phenotypic analyses, we identify Bcl2 and Mki67 as a marker combination that enables the tracking of nascent memory cells within the effector phase. We then use a formal approach based on mathematical models describing the dynamics of population size evolution to test potential progeny links and demonstrate that most cells follow a linear naive→early effector→late effector→memory pathway. Moreover, our mathematical model allows long-term prediction of memory cell numbers from a few early experimental measurements. Our work thus provides a phenotypic means to identify effector and memory cells, as well as a mathematical framework to investigate their genealogy and to predict the outcome of immunization regimens in terms of memory cell numbers generated. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Stepwise cytoskeletal polarization as a series of checkpoints in innate but not adaptive cytolytic killing

NASA Astrophysics Data System (ADS)

Wülfing, Christoph; Purtic, Bozidar; Klem, Jennifer; Schatzle, John D.

2003-06-01

Cytolytic killing is a major effector mechanism in the elimination of virally infected and tumor cells. The innate cytolytic effectors, natural killer (NK) cells, and the adaptive effectors, cytotoxic T cells (CTL), despite differential immune recognition, both use the same lytic mechanism, cytolytic granule release. Using live cell video fluorescence microscopy in various primary cell models of NK cell and CTL killing, we show here that on tight target cell contact, a majority of the NK cells established cytoskeletal polarity required for effective lytic function slowly or incompletely. In contrast, CTLs established cytoskeletal polarity rapidly. In addition, NK cell killing was uniquely sensitive to minor interference with cytoskeletal dynamics. We propose that the stepwise NK cell cytoskeletal polarization constitutes a series of checkpoints in NK cell killing. In addition, the use of more deliberate progression to effector function to compensate for inferior immune recognition specificity provides a mechanistic explanation for how the same effector function can be used in the different functional contexts of the innate and adaptive immune response.

Transition of late-stage effector T cells to CD27+ CD28+ tumor-reactive effector memory T cells in humans after adoptive cell transfer therapy

PubMed Central

Powell, Daniel J.; Dudley, Mark E.; Robbins, Paul F.; Rosenberg, Steven A.

2007-01-01

In humans, the pathways of memory T-cell differentiation remain poorly defined. Recently, adoptive cell transfer (ACT) of tumor-reactive T lymphocytes to metastatic melanoma patients after nonmyeloablative chemotherapy has resulted in persistence of functional, tumor-reactive lymphocytes, regression of disease, and induction of melanocyte-directed autoimmunity in some responding patients. In the current study, longitudinal phenotypic analysis was performed on melanoma antigen–specific CD8+ T cells during their transition from in vitro cultured effector cells to long-term persistent memory cells following ACT to 6 responding patients. Tumor-reactive T cells used for therapy were generally late-stage effector cells with a CD27Lo CD28Lo CD45RA− CD62 ligand− (CD62L−) CC chemokine receptor 7− (CCR7−) interleukin-7 receptor αLo (IL-7RαLo) phenotype. After transfer, rapid up-regulation and continued expression of IL-7Rα in vivo suggested an important role for IL-7R in immediate and long-term T-cell survival. Although the tumor antigen–specific T-cell population contracted between 1 and 4 weeks after transfer, stable numbers of CD27+ CD28+ tumor-reactive T cells were maintained, demonstrating their contribution to the development of long-term, melanoma-reactive memory CD8+ T cells in vivo. At 2 months after transfer, melanoma-reactive T cells persisted at high levels and displayed an effector memory phenotype, including a CD27+ CD28+ CD62L− CCR7− profile, which may explain in part their ability to mediate tumor destruction. PMID:15345595

Persistent viral infection in humans can drive high frequency low-affinity T-cell expansions

PubMed Central

Khan, Naeem; Cobbold, Mark; Cummerson, Joanne; Moss, Paul A H

2010-01-01

CD8 T cells that recognize cytomegalovirus (CMV) -encoded peptides can be readily detected by staining with human leucocyte antigen (HLA) –peptide tetramers. These cells are invariably highly differentiated effector memory cells with high avidity T-cell receptors (TCR). In this report we demonstrate an HLA-A*0201 restricted CMV-specific CD8 T-cell response (designated YVL) that represents several percent of the CD8 T-cell subset, yet fails to bind tetrameric major histocompatibility complex (MHC) ligands. However, these tetramer-negative cells are both phenotypically and functionally similar to other CMV-specific CD8 T cells. YVL peptide-specific CD8 T-cell clones were generated and found to be of high avidity in both cytotoxicity and interferon-γ (IFN-γ) assays, and comparable with other CMV peptide-specific CD8 T-cell clones. However, under conditions of CD8 blockade, the response was almost nullified even at very high ligand concentrations. This was also the case in IFN-γ experiments using peripheral blood mononuclear cells stimulated with peptide ex vivo. In contrast, all other CMV specificities (tetramer-positive) displayed minimal or only partial CD8 dependence. This suggests that YVL-specific responses depict a low-affinity TCR–MHC–peptide interaction, that is compensated by substantial CD8 involvement for functional purposes, yet cannot engage multivalent soluble ligands for ex vivo analysis. It is interesting that such a phenomenon is apparent in the face of a persistent virus infection such as CMV, where the responding cells represent an immunodominant response in that individual and may present a highly differentiated effector phenotype. PMID:20722762

Zeb2 recruits HDAC-NuRD to inhibit Notch and controls Schwann cell differentiation and remyelination.

PubMed

Wu, Lai Man Natalie; Wang, Jincheng; Conidi, Andrea; Zhao, Chuntao; Wang, Haibo; Ford, Zachary; Zhang, Liguo; Zweier, Christiane; Ayee, Brian G; Maurel, Patrice; Zwijsen, An; Chan, Jonah R; Jankowski, Michael P; Huylebroeck, Danny; Lu, Q Richard

2016-08-01

The mechanisms that coordinate and balance a complex network of opposing regulators to control Schwann cell (SC) differentiation remain elusive. Here we demonstrate that zinc-finger E-box-binding homeobox 2 (Zeb2, also called Sip1) transcription factor is a critical intrinsic timer that controls the onset of SC differentiation by recruiting histone deacetylases HDAC 1 and 2 (HDAC1/2) and nucleosome remodeling and deacetylase complex (NuRD) co-repressor complexes in mice. Zeb2 deletion arrests SCs at an undifferentiated state during peripheral nerve development and inhibits remyelination after injury. Zeb2 antagonizes inhibitory effectors including Notch and Sox2. Importantly, genome-wide transcriptome analysis reveals a Zeb2 target gene encoding the Notch effector Hey2 as a potent inhibitor for Schwann cell differentiation. Strikingly, a genetic Zeb2 variant associated with Mowat-Wilson syndrome disrupts the interaction with HDAC1/2-NuRD and abolishes Zeb2 activity for SC differentiation. Therefore, Zeb2 controls SC maturation by recruiting HDAC1/2-NuRD complexes and inhibiting a Notch-Hey2 signaling axis, pointing to the critical role of HDAC1/2-NuRD activity in peripheral neuropathies caused by ZEB2 mutations.

Three Antagonistic Cyclic di-GMP-Catabolizing Enzymes Promote Differential Dot/Icm Effector Delivery and Intracellular Survival at the Early Steps of Legionella pneumophila Infection

PubMed Central

Allombert, Julie; Lazzaroni, Jean-Claude; Baïlo, Nathalie; Gilbert, Christophe; Charpentier, Xavier; Doublet, Patricia

2014-01-01

Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole. PMID:24379287

T cell fates ‘zipped up’: how the Bach2 basic leucine zipper transcriptional repressor directs T cell differentiation and function1

PubMed Central

Richer, Martin J.; Lang, Mark L.; Butler, Noah S.

2016-01-01

Recent data illustrate a key role for the transcriptional regulator Bach2 in orchestrating T cell differentiation and function. Although Bach2 has a well-described role in B cell differentiation, emerging data show that Bach2 is a prototypical member of a novel class of transcription factors that regulates transcriptional activity in T cells at super enhancers, or regions of high transcriptional activity. Accumulating data demonstrate specific roles for Bach2 in favoring regulatory T cell generation, restraining effector T cell differentiation and potentiating memory T cell development. Evidence suggests that Bach2 regulates various facets of T cell function by repressing other key transcriptional regulator such as Blimp-1. This review examines our current understanding of the role of Bach2 in T cell function and highlights the growing evidence that this transcriptional repressor functions as a key regulator involved in maintenance of T cell quiescence, T cell subset differentiation and memory T cell generation. PMID:27496973

Increased numbers of pre-existing memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells1

PubMed Central

Joshi, Nikhil S.; Cui, Weiguo; Dominguez, Claudia; Chen, Jonathan H.; Hand, Timothy W.; Kaech, Susan M.

2011-01-01

Memory CD8 T cells acquire TEM properties following reinfection, and may reach terminally differentiated, senescent states (“Hayflick limit”) after multiple infections. The signals controlling this process are not well understood, but we found that the degree of 2o effector and memory CD8 T cell differentiation was intimately linked to the amount of T-bet expressed upon reactivation and pre-existing memory CD8 T cell number (i.e., 1o memory CD8 T cell precursor frequency) present during secondary infection. Compared to naïve cells, memory CD8 T cells were predisposed towards terminal effector (TE) cell differentiation because they could immediately respond to IL-12 and induce T-bet, even in the absence of antigen. TE cell formation following 2o or 3o infections was dependent on increased T-bet expression because T-bet+/− cells were resistant to these phenotypic changes. Larger numbers of pre-existing memory CD8 T cells limited the duration of 2o infection and the amount of IL-12 produced, and consequently, this reduced T-bet expression and the proportion of 2o TE CD8 T cells that formed. Together, these data show that, over repeated infections, memory CD8 T cell quality and proliferative fitness is not strictly determined by the number of serial encounters with antigen or cell divisions, but is a function of the CD8 T cell differentiation state, which is genetically controlled in a T-bet-dependent manner. This differentiation state can be modulated by pre-existing memory CD8 T cell number and the intensity of inflammation during reinfection. These results have important implications for vaccinations involving prime-boost strategies. PMID:21930973

mTOR at the Transmitting and Receiving Ends in Tumor Immunity

PubMed Central

Guri, Yakir; Nordmann, Thierry M.; Roszik, Jason

2018-01-01

Cancer is a complex disease and a leading cause of death worldwide. Immunity is critical for cancer control. Cancer cells exhibit high mutational rates and therefore altered self or neo-antigens, eliciting an immune response to promote tumor eradication. Failure to mount a proper immune response leads to cancer progression. mTOR signaling controls cellular metabolism, immune cell differentiation, and effector function. Deregulated mTOR signaling in cancer cells modulates the tumor microenvironment, thereby affecting tumor immunity and possibly promoting carcinogenesis. PMID:29662490

mTOR at the Transmitting and Receiving Ends in Tumor Immunity.

PubMed

Guri, Yakir; Nordmann, Thierry M; Roszik, Jason

2018-01-01

Cancer is a complex disease and a leading cause of death worldwide. Immunity is critical for cancer control. Cancer cells exhibit high mutational rates and therefore altered self or neo-antigens, eliciting an immune response to promote tumor eradication. Failure to mount a proper immune response leads to cancer progression. mTOR signaling controls cellular metabolism, immune cell differentiation, and effector function. Deregulated mTOR signaling in cancer cells modulates the tumor microenvironment, thereby affecting tumor immunity and possibly promoting carcinogenesis.

Genome-scale identification of Legionella pneumophila effectors using a machine learning approach.

PubMed

Burstein, David; Zusman, Tal; Degtyar, Elena; Viner, Ram; Segal, Gil; Pupko, Tal

2009-07-01

A large number of highly pathogenic bacteria utilize secretion systems to translocate effector proteins into host cells. Using these effectors, the bacteria subvert host cell processes during infection. Legionella pneumophila translocates effectors via the Icm/Dot type-IV secretion system and to date, approximately 100 effectors have been identified by various experimental and computational techniques. Effector identification is a critical first step towards the understanding of the pathogenesis system in L. pneumophila as well as in other bacterial pathogens. Here, we formulate the task of effector identification as a classification problem: each L. pneumophila open reading frame (ORF) was classified as either effector or not. We computationally defined a set of features that best distinguish effectors from non-effectors. These features cover a wide range of characteristics including taxonomical dispersion, regulatory data, genomic organization, similarity to eukaryotic proteomes and more. Machine learning algorithms utilizing these features were then applied to classify all the ORFs within the L. pneumophila genome. Using this approach we were able to predict and experimentally validate 40 new effectors, reaching a success rate of above 90%. Increasing the number of validated effectors to around 140, we were able to gain novel insights into their characteristics. Effectors were found to have low G+C content, supporting the hypothesis that a large number of effectors originate via horizontal gene transfer, probably from their protozoan host. In addition, effectors were found to cluster in specific genomic regions. Finally, we were able to provide a novel description of the C-terminal translocation signal required for effector translocation by the Icm/Dot secretion system. To conclude, we have discovered 40 novel L. pneumophila effectors, predicted over a hundred additional highly probable effectors, and shown the applicability of machine learning algorithms for the identification and characterization of bacterial pathogenesis determinants.

HIV-specific CD8+ T cells: serial killers condemned to die?

PubMed

Petrovas, Constantinos; Mueller, Yvonne M; Katsikis, Peter D

2004-04-01

An increasing body of evidence supports a key role for cytotoxic CD8+ T cells (CTL) in controlling HIV infection. Although a vigorous HIV-specific CD8+ T cell response is raised during the primary infection, these cells ultimately fail to control virus and prevent disease progression. The failure of CTL to control HIV infection has been attributed to a number of strategies HIV employs to evade the immune system. Recently, intrinsic defects in the CTL themselves have been proposed to contribute to the failure of CTL to control HIV. HIV-specific CD8+ T cells differ in their effector/memory phenotype from other virus-specific CD8+ T cells indicating that their differentiation status differs. This altered differentiation may affect effector functions as well as homing properties of these cells. Other studies have indicated that activation of HIV-specific CTL may be impaired and this contributes to their dysfunction. The effector function of these CTL may also be affected. There are conflicting reports about their ability to kill, whereas IFNgamma production does not appear to be impaired in these cells. In this review we focus on recent work indicating that apoptosis may be an important mechanism through which HIV evades the CTL response. In particular, HIV-specific CD8+ T cells are highly susceptible to CD95/Fas-induced apoptosis. This leads to the hypothesis that virus-specific cytotoxic T cells can be eliminated upon binding CD95L/FasL on HIV-infected cells. Understanding the intrinsic defects of CTL in HIV infection could lead to new therapeutic strategies and optimized vaccination protocols that enhance the HIV-specific cytotoxic response.

Intervention of PKC-θ as an immunosuppressive regimen

PubMed Central

Sun, Zuoming

2012-01-01

PKC-θ is selectively enriched in T cells and specifically translocates to immunological synapse where it mediates critical T cell receptor signals required for T cell activation, differentiation, and survival. T cells deficient in PKC-θ are defective in their ability to differentiate into inflammatory effector cells that mediate actual immune responses whereas, their differentiation into regulatory T cells (Treg) that inhibits the inflammatory T cells is enhanced. Therefore, the manipulation of PKC-θ activity can shift the ratio between inflammatory effector T cells and inhibitory Tregs, to control T cell-mediated immune responses that are responsible for autoimmunity and allograft rejection. Indeed, PKC-θ-deficient mice are resistant to the development of several Th2 and Th17-dependent autoimmune diseases and are defective in mounting alloimmune responses required for rejection of transplanted allografts and graft-versus-host disease. Selective inhibition of PKC-θ is therefore considered as a potential treatment for prevention of autoimmune diseases and allograft rejection. PMID:22876242

The transcription factors Thpok and LRF are necessary and partly redundant for T helper cell differentiation

PubMed Central

Carpenter, Andrea C.; Grainger, John R.; Xiong, Yumei; Kanno, Yuka; Chu, H. Hamlet; Wang, Lie; Naik, Shruti; dos Santos, Liliane; Wei, Lai; Jenkins, Marc K.; O’Shea, John J.; Belkaid, Yasmine; Bosselut, Rémy

2014-01-01

Summary T helper (Th) cells are critical for defenses against infection and recognize peptides bound to Class II Major Histocompatibility Complex (MHC-II) molecules. Although transcription factors have been identified that direct helper cells into specific effector fates, whether a ‘master’ regulator controls the developmental program common to all Th cells remains unclear. Here we showed that the two transcription factors Thpok and LRF share this function. Although disruption of both factors did not prevent the generation of MHC II-specific T cells, these cells failed to express Th cell genes or undergo Th cell differentiation in vivo. In contrast, T cells lacking Thpok only displayed LRF-dependent functions and contributed to multiple effector responses, both in vitro and in vivo, with the notable exception of Th2 cell responses that control extra-cellular parasites. These findings identify the Thpok-LRF pair as a core node of Th cell differentiation and function. PMID:23041065

Bcl-2 Allows Effector and Memory CD8+ T Cells To Tolerate Higher Expression of Bim

PubMed Central

Kurtulus, Sema; Tripathi, Pulak; Moreno-Fernandez, Maria E.; Sholl, Allyson; Katz, Jonathan D.; Grimes, H. Leighton; Hildeman, David A.

2014-01-01

As acute infections resolve, most effector CD8+ T cells die, whereas some persist and become memory T cells. Recent work showed that subsets of effector CD8+ T cells, identified by reciprocal expression of killer cell lectin-like receptor G1 (KLRG1) and CD127, have different lifespans. Similar to previous reports, we found that effector CD8+ T cells reported to have a longer lifespan (i.e., KLRG1lowCD127high) have increased levels of Bcl-2 compared with their shorter-lived KLRG1highCD127low counterparts. Surprisingly, we found that these effector KLRG1lowCD127high CD8+ T cells also had increased levels of Bim compared with KLRG1highCD127low cells. Similar effects were observed in memory cells, in which CD8+ central memory T cells expressed higher levels of Bim and Bcl-2 than did CD8+ effector memory T cells. Using both pharmacologic and genetic approaches, we found that survival of both subsets of effector and memory CD8+ T cells required Bcl-2 to combat the proapoptotic activity of Bim. Interestingly, inhibition or absence of Bcl-2 led to significantly decreased expression of Bim in surviving effector and memory T cells. In addition, manipulation of Bcl-2 levels by IL-7 or IL-15 also affected expression of Bim in effector CD8+ T cells. Finally, we found that Bim levels were significantly increased in effector CD8+ T cells lacking Bax and Bak. Together, these data indicate that cells having the highest levels of Bim are selected against during contraction of the response and that Bcl-2 determines the level of Bim that effector and memory T cells can tolerate. PMID:21451108

Multiple layers of transcriptional regulation by PLZF in NKT-cell development.

PubMed

Mao, Ai-Ping; Constantinides, Michael G; Mathew, Rebecca; Zuo, Zhixiang; Chen, Xiaoting; Weirauch, Matthew T; Bendelac, Albert

2016-07-05

The transcription factor PLZF [promyelocytic leukemia zinc finger, encoded by zinc finger BTB domain containing 16 (Zbtb16)] is induced during the development of innate and innate-like lymphocytes to direct their acquisition of a T-helper effector program, but the molecular mechanisms involved are poorly understood. Using biotinylation-based ChIP-seq and microarray analysis of both natural killer T (NKT) cells and PLZF-transgenic thymocytes, we identified several layers of regulation of the innate-like NKT effector program. First, PLZF bound and regulated genes encoding cytokine receptors as well as homing and adhesion receptors; second, PLZF bound and activated T-helper-specific transcription factor genes that in turn control T-helper-specific programs; finally, PLZF bound and suppressed the transcription of Bach2, a potent general repressor of effector differentiation in naive T cells. These findings reveal the multilayered architecture of the transcriptional program recruited by PLZF and elucidate how a single transcription factor can drive the developmental acquisition of a broad effector program.

Leptin Directly Promotes T Cell Glycolytic Metabolism to Drive Effector T cell Differentiation in Autoimmunity

PubMed Central

Gerriets, Valerie A.; Danzaki, Keiko; Kishton, Rigel J.; Eisner, William; Nichols, Amanda G.; Saucillo, Donte C.; Shinohara, Mari L.; MacIver, Nancie J.

2016-01-01

Upon activation, T cells require energy for growth, proliferation and function. Effector T cells (Teff), such as Th1 and Th17, utilize high levels of glucose uptake and glycolysis to fuel proliferation and function. In contrast, Treg instead require oxidative metabolism to fuel suppressive function. It remains unknown how Teff/Treg metabolism is altered in settings of malnutrition, when nutrients are limited and circulating leptin levels are low. We therefore examined the role of malnutrition and associated hypoleptinemia on Teff versus Treg. We found that both malnutrition-associated hypoleptinemia and T cell-specific leptin receptor knockout suppressed Teff number, function, and glucose metabolism, but did not alter Treg metabolism or suppressive function. Using the autoimmune model EAE, we confirmed that fasting-induced hypoleptinemia altered Teff, but not Treg, glucose metabolism and function in vivo, leading to decreased disease severity. To explore potential mechanisms, we examined HIF-1α, a key regulator of Th17 differentiation and Teff glucose metabolism, and found HIF-1α expression was decreased in T cell-specific leptin receptor knockout Th17 cells, and in Teff cells from fasted EAE mice, but was unchanged in Treg. Altogether, these data demonstrate a selective, cell-intrinsic requirement for leptin to upregulate glucose metabolism and maintain function in Teff, but not Treg. PMID:27222115

Retinoic acid-induced differentiation increases the rate of oxygen consumption and enhances the spare respiratory capacity of mitochondria in SH-SY5Y cells.

PubMed

Xun, Zhiyin; Lee, Do-Yup; Lim, James; Canaria, Christie A; Barnebey, Adam; Yanonne, Steven M; McMurray, Cynthia T

2012-04-01

Retinoic acid (RA) is used in differentiation therapy to treat a variety of cancers including neuroblastoma. The contributing factors for its therapeutic efficacy are poorly understood. However, mitochondria (MT) have been implicated as key effectors in RA-mediated differentiation process. Here we utilize the SH-SY5Y human neuroblastoma cell line as a model to examine how RA influences MT during the differentiation process. We find that RA confers an approximately sixfold increase in the oxygen consumption rate while the rate of glycolysis modestly increases. RA treatment does not increase the number of MT or cause measurable changes in the composition of the electron transport chain. Rather, RA treatment significantly increases the mitochondrial spare respiratory capacity. We propose a competition model for the therapeutic effects of RA. Specifically, the high metabolic rate in differentiated cells limits the availability of metabolic nutrients for use by the undifferentiated cells and suppresses their growth. Thus, RA treatment provides a selective advantage for the differentiated state. Published by Elsevier Ireland Ltd.

Retinoic acid-induced differentiation increases the rate of oxygen consumption and enhances the spare respiratory capacity of mitochondria in SH-SY5Y cells

PubMed Central

Xun, Zhiyin; Lee, Do-Yup; Lim, James; Canaria, Christie A.; Barnebey, Adam; Yanonne, Steven M.; McMurray, Cynthia T.

2012-01-01

Retinoic acid (RA) is used in differentiation therapy to treat a variety of cancers including neuroblastoma. The contributing factors for its therapeutic efficacy are poorly understood. However, mitochondria (MT) have been implicated as key effectors in RA-mediated differentiation process. Here we utilize the SH-SY5Y human neuroblastoma cell line as a model to examine how RA influences MT during the differentiation process. We find that RA confers an approximately 6-fold increase in the oxygen consumption rate while the rate of glycolysis modestly increases. RA treatment does not increase the number of MT or cause measurable changes in the composition of the electron transport chain. Rather, RA treatment significantly increases the mitochondrial spare respiratory capacity. We propose a competition model for the therapeutic effects of RA. Specifically, the high metabolic rate in differentiated cells limits the availability of metabolic nutrients for use by the undifferentiated cells and suppresses their growth. Thus, RA treatment provides a selective advantage for the differentiated state. PMID:22336883

Genome-wide Analysis of the H3K4 Histone Demethylase RBP2 Reveals a Transcriptional Program Controlling Differentiation

PubMed Central

Lopez-Bigas, Nuria; Kisiel, Tomasz A.; DeWaal, Dannielle C.; Holmes, Katie B.; Volkert, Tom L.; Gupta, Sumeet; Love, Jennifer; Murray, Heather L.; Young, Richard A.; Benevolenskaya, Elizaveta V.

2010-01-01

SUMMARY Retinoblastoma protein (pRB) mediates cell-cycle withdrawal and differentiation by interacting with a variety of proteins. RB-Binding Protein 2 (RBP2) has been shown to be a key effector. We sought to determine transcriptional regulation by RBP2 genome-wide by using location analysis and gene expression profiling experiments. We describe that RBP2 shows high correlation with the presence of H3K4me3 and its target genes are separated into two functionally distinct classes: differentiation-independent and differentiation-dependent genes. The former class is enriched by genes that encode mitochondrial proteins, while the latter is represented by cell-cycle genes. We demonstrate the role of RBP2 in mitochondrial biogenesis, which involves regulation of H3K4me3-modified nucleosomes. Analysis of expression changes upon RBP2 depletion depicted genes with a signature of differentiation control, analogous to the changes seen upon reintroduction of pRB. We conclude that, during differentiation, RBP2 exerts inhibitory effects on multiple genes through direct interaction with their promoters. PMID:18722178

Recruitment of bone marrow CD11b+Gr-1+ cells by polymeric nanoparticles for antigen cross-presentation

NASA Astrophysics Data System (ADS)

Yang, Ya-Wun; Luo, Wen-Hui

2017-03-01

The objective of this study was to investigate the function of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on the activation of antigen-specific CD8+ T cell responses via the CD11b+Gr-1+ myeloid subpopulations in murine bone marrow (BM). PLGA NPs containing ovalbumin (OVA) were fabricated by the double-emulsion method. The CD11b+Gr-1lowLy-6Chigh and CD11b+Gr-1highLy-6Clow subsets from mice bone marrow were sorted and treated with the PLGA/OVA NPs, followed by co-culture with the carboxyfluorescein succinimidyl ester (CFSE)-labelled OT-I CD8+ cells. Co-culture of OT-I CD8+ T cells with PLGA/OVA NPs-primed CD11b+Gr-1+ subsets upregulated the expression of IL-2, TNF-α, INF-γ, granzyme B, and perforin, resulting in proliferation of CD8+ T cells and differentiation into effector cytotoxic T lymphocytes (CTLs). In vivo proliferation of CFSE-labelled OT-I CD8+ cells in response to OVA was also obtained in the animals immunized with PLGA/OVA NPs. The results presented in this study demonstrate the ability of polymeric NPs to recruit two CD11b+Gr-1+ myeloid subsets for effective presentation of exogenous antigen to OT-I CD8+ T cells in the context of major histocompatibility complex (MHC) class I, leading to an induction of antigen-specific cell proliferation and differentiation into effector cells.

Characterization of naïve, memory and effector T cells in progressive multiple sclerosis.

PubMed

Nielsen, Birgitte Romme; Ratzer, Rikke; Börnsen, Lars; von Essen, Marina Rode; Christensen, Jeppe Romme; Sellebjerg, Finn

2017-09-15

We characterized naïve, central memory (CM), effector memory (EM) and terminally differentiated effector memory (TEMRA) CD4 + and CD8 + T cells and their expression of CD49d and CD26 in peripheral blood in patients with multiple sclerosis (MS) and healthy controls. CD26 + CD28 + CD4 + TEMRA T cells were increased in all subtypes of MS, and CD26 + CD28 + CD8 + TEMRA T cells were increased in relapsing-remitting and secondary progressive MS. Conversely, in progressive MS, CD49d + CM T cells were decreased and natalizumab increased the circulating number of all six subsets but reduced the frequency of most subsets expressing CD49d and CD26. Copyright © 2017 Elsevier B.V. All rights reserved.

Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling

PubMed Central

Linnemann, Carsten; Schildberg, Frank A; Schurich, Anna; Diehl, Linda; Hegenbarth, Silke I; Endl, Elmar; Lacher, Svenja; Müller, Christa E; Frey, Jürgen; Simeoni, Luca; Schraven, Burkhart; Stabenow, Dirk; Knolle, Percy A

2009-01-01

Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after αCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve αCD3/CD28-stimulated CD8 cells. Consequently, αCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs. PMID:19740334

Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling.

PubMed

Linnemann, Carsten; Schildberg, Frank A; Schurich, Anna; Diehl, Linda; Hegenbarth, Silke I; Endl, Elmar; Lacher, Svenja; Müller, Christa E; Frey, Jürgen; Simeoni, Luca; Schraven, Burkhart; Stabenow, Dirk; Knolle, Percy A

2009-09-01

Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after alphaCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve alphaCD3/CD28-stimulated CD8 cells. Consequently, alphaCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, In-Hyun; Erbay, Ebru; Nuzzi, Paul

The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added atmore » a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector.« less

Autoreactive T effector memory differentiation mirrors β-cell function in type 1 diabetes.

PubMed

Yeo, Lorraine; Woodwyk, Alyssa; Sood, Sanjana; Lorenc, Anna; Eichmann, Martin; Pujol-Autonell, Irma; Melchiotti, Rossella; Skowera, Ania; Fidanis, Efthymios; Dolton, Garry M; Tungatt, Katie; Sewell, Andrew K; Heck, Susanne; Saxena, Alka; Beam, Craig A; Peakman, Mark

2018-05-31

In type 1 diabetes, cytotoxic CD8 T cells with specificity for β-cell autoantigens are found in the pancreatic islets where they are implicated in the destruction of insulin-secreting β cells. In contrast, the disease relevance of β-cell-reactive CD8 T cells that are detectable in the circulation, and their relationship to β-cell function, are not known. Here, we tracked multiple, circulating β-cell-reactive CD8 T cell subsets and measured β-cell function longitudinally for two years, starting immediately after diagnosis of type 1 diabetes. We found that change in β-cell-specific effector memory CD8 T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8 T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer specific protein 37 and CD16, and reduced expression of CD28) compared with their CD57-negative counterparts, and network association modelling indicated that the dynamics of β-cell-reactive CD57+ effector memory CD8 T cell subsets were strongly linked. Thus, coordinated changes in circulating β-cell-specific CD8 T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.

Structure of the effector-binding domain of the arabinose repressor AraR from Bacillus subtilis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Procházková, Kateřina; Čermáková, Kateřina; Pachl, Petr

2012-02-01

The crystal structure of the effector-binding domain of the transcriptional repressor AraR from B. subtilis in complex with the effector molecule (l-arabinose) was determined at 2.2 Å resolution. A detailed analysis of the crystal identified a dimer organization that is distinctive from that of other members of the GalR/LacI family. In Bacillus subtilis, the arabinose repressor AraR negatively controls the expression of genes in the metabolic pathway of arabinose-containing polysaccharides. The protein is composed of two domains of different phylogenetic origin and function: an N-terminal DNA-binding domain belonging to the GntR family and a C-terminal effector-binding domain that shows similaritymore » to members of the GalR/LacI family. The crystal structure of the C-terminal effector-binding domain of AraR in complex with the effector l-arabinose has been determined at 2.2 Å resolution. The l-arabinose binding affinity was characterized by isothermal titration calorimetry and differential scanning fluorimetry; the K{sub d} value was 8.4 ± 0.4 µM. The effect of l-arabinose on the protein oligomeric state was investigated in solution and detailed analysis of the crystal identified a dimer organization which is distinctive from that of other members of the GalR/LacI family.« less

Determination of Stability and Control Derivatives using Computational Fluid Dynamics and Automatic Differentiation

NASA Technical Reports Server (NTRS)

Park, Michael A.; Green, Lawrence L.; Montgomery, Raymond C.; Raney, David L.

1999-01-01

With the recent interest in novel control effectors there is a need to determine the stability and control derivatives of new aircraft configurations early in the design process. These derivatives are central to most control law design methods and would allow the determination of closed-loop control performance of the vehicle. Early determination of the static and dynamic behavior of an aircraft may permit significant improvement in configuration weight, cost, stealth, and performance through multidisciplinary design. The classical method of determining static stability and control derivatives - constructing and testing wind tunnel models - is expensive and requires a long lead time for the resultant data. Wind tunnel tests are also limited to the preselected control effectors of the model. To overcome these shortcomings, computational fluid dynamics (CFD) solvers are augmented via automatic differentiation, to directly calculate the stability and control derivatives. The CFD forces and moments are differentiated with respect to angle of attack, angle of sideslip, and aircraft shape parameters to form these derivatives. A subset of static stability and control derivatives of a tailless aircraft concept have been computed by two differentiated inviscid CFD codes and verified for accuracy with central finite-difference approximations and favorable comparisons to a simulation database.

EffectorP: predicting fungal effector proteins from secretomes using machine learning.

PubMed

Sperschneider, Jana; Gardiner, Donald M; Dodds, Peter N; Tini, Francesco; Covarelli, Lorenzo; Singh, Karam B; Manners, John M; Taylor, Jennifer M

2016-04-01

Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au. © 2015 CSIRO New Phytologist © 2015 New Phytologist Trust.

Effect of Cytomegalovirus (CMV) and Ageing on T-Bet and Eomes Expression on T-Cell Subsets.

PubMed

Hassouneh, Fakhri; Lopez-Sejas, Nelson; Campos, Carmen; Sanchez-Correa, Beatriz; Tarazona, Raquel; Pera, Alejandra; Solana, Rafael

2017-06-29

The differential impact of ageing and cytomegalovirus (CMV) latent infection on human T-cell subsets remains to some extent controversial. The purpose of this study was to analyse the expression of the transcription factors T-bet and Eomes and CD57 on CD4+, CD4 hi CD8 lo and CD8+ T-cell subsets in healthy individuals, stratified by age and CMV serostatus. The percentage of CD4+ T-cells expressing T-bet or Eomes was very low, in particular in CD4+ T-cells from young CMV-seronegative individuals, and were higher in CMV-seropositive older individuals, in both CD57- and CD57+ CD4+ T-cells. The study of the minor peripheral blood double-positive CD4 hi CD8 lo T-cells showed that the percentage of these T-cells expressing both Eomes and T-bet was higher compared to CD4+ T-cells. The percentage of CD4 hi CD8 lo T-cells expressing T-bet was also associated with CMV seropositivity and the coexpression of Eomes, T-bet and CD57 on CD4 hi CD8 lo T-cells was only observed in CMV-seropositive donors, supporting the hypothesis that these cells are mature effector memory cells. The percentage of T-cells expressing Eomes and T-bet was higher in CD8+ T-cells than in CD4+ T-cells. The percentages of CD8+ T-cells expressing Eomes and T-bet increased with age in CMV-seronegative and -seropositive individuals and the percentages of CD57- CD8+ and CD57+ CD8+ T-cells coexpressing both transcription factors were similar in the different groups studied. These results support that CMV chronic infection and/or ageing are associated to the expansion of highly differentiated CD4+, CD4 hi CD8 lo and CD8+ T-cells that differentially express T-bet and Eomes suggesting that the expression of these transcription factors is essential for the generation and development of an effector-memory and effector T lymphocytes involved in conferring protection against chronic CMV infection.

Antigen expression level threshold tunes the fate of CD8 T cells during primary hepatic immune responses.

PubMed

Tay, Szun Szun; Wong, Yik Chun; McDonald, David M; Wood, Nicole A W; Roediger, Ben; Sierro, Frederic; Mcguffog, Claire; Alexander, Ian E; Bishop, G Alex; Gamble, Jennifer R; Weninger, Wolfgang; McCaughan, Geoffrey W; Bertolino, Patrick; Bowen, David G

2014-06-24

CD8 T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity. The reasons for these heterogeneous outcomes are not well understood. To identify factors that govern the fate of CD8 T cells activated by hepatocyte-expressed antigen, we exploited recombinant adenoassociated viral vectors that enabled us to vary potential parameters determining these outcomes in vivo. Our findings reveal a threshold of antigen expression within the liver as the dominant factor determining T-cell fate, irrespective of T-cell receptor affinity or antigen cross-presentation. Thus, when a low percentage of hepatocytes expressed cognate antigen, high-affinity T cells developed and maintained effector function, whereas, at a high percentage, they became functionally exhausted and silenced. Exhaustion was not irreversibly determined by initial activation, but was maintained by high intrahepatic antigen load during the early phase of the response; cytolytic function was restored when T cells primed under high antigen load conditions were transferred into an environment of low-level antigen expression. Our study reveals a hierarchy of factors dictating the fate of CD8 T cells during hepatic immune responses, and provides an explanation for the different immune outcomes observed in a variety of immune-mediated liver pathologic conditions.

In vitro effects of 4-hydroperoxycyclophosphamide on human immunoregulatory T subset function. I. Selective effects on lymphocyte function in T-B cell collaboration.

PubMed

Ozer, H; Cowens, J W; Colvin, M; Nussbaum-Blumenson, A; Sheedy, D

1982-01-01

The alkylating agent cyclophosphamide may suppress or enhance immune responses in vivo but is inactive in vitro unless metabolized by microsomal enzyme activation. 4-hydroperoxycyclophosphamide (4-HC) is a synthetic compound that is spontaneously converted in aqueous solution to the active metabolites. In this report, we examined the in vitro sensitivity of functional human T cell subsets to 4-HC in a polyclonal B cell differentiation assay and in the generation of mitogen-induced suppressor cells for effector B cell function. Con A-induced T suppression of B cell differentiation is completely abrogated by a 1-h pretreatment of T cells at very low concentrations of between 10(-2) and 20 nmol/ml, whereas inducer T cell function is sensitive only to concentrations in greater than 40 nmol/ml. The effects of 4-HC on suppressor T cells appear to occur at concentrations that do not result in DNA cross-linking or decreased blastogenesis. Con A-induced T suppressors are generated from within the OKT4+, OKT8- subset and are sensitive to low-dose 4-HC only before activation, whereas differentiated suppressor cells are resistant to concentrations in greater than 80 nmol/ml. Low-dose 4-HC pretreatment of the B cell population results in abrogation of immunoglobulin secretion when treated B cells are cocultured with unfractionated T cells, however, this effect is completely reversible if pretreated B cells are cocultured with T cells devoid of suppressor activity. These results demonstrate that human presuppressor cells for B-effector function differentiate in response to Con A from the OKT4+, OKT8- subset and are exquisitely sensitive to low concentrations of CYP whereas mature suppressor and inducer functions are resistant to all but very high concentrations in vitro. The differential sensitivity of functional T and B cell subsets to 4-HC in vitro can be a very useful probe in dissecting immunoregulatory interactions with man.

Double lead spiral platen parallel jaw end effector

NASA Technical Reports Server (NTRS)

Beals, David C.

1989-01-01

The double lead spiral platen parallel jaw end effector is an extremely powerful, compact, and highly controllable end effector that represents a significant improvement in gripping force and efficiency over the LaRC Puma (LP) end effector. The spiral end effector is very simple in its design and has relatively few parts. The jaw openings are highly predictable and linear, making it an ideal candidate for remote control. The finger speed is within acceptable working limits and can be modified to meet the user needs; for instance, greater finger speed could be obtained by increasing the pitch of the spiral. The force relaxation is comparable to the other tested units. Optimization of the end effector design would involve a compromise of force and speed for a given application.

Y4lO of Rhizobium sp. strain NGR234 is a symbiotic determinant required for symbiosome differentiation.

PubMed

Yang, Feng-Juan; Cheng, Li-Li; Zhang, Ling; Dai, Wei-Jun; Liu, Zhe; Yao, Nan; Xie, Zhi-Ping; Staehelin, Christian

2009-02-01

Type 3 (T3) effector proteins, secreted by nitrogen-fixing rhizobia with a bacterial T3 secretion system, affect the nodulation of certain host legumes. The open reading frame y4lO of Rhizobium sp. strain NGR234 encodes a protein with sequence similarities to T3 effectors from pathogenic bacteria (the YopJ effector family). Transcription studies showed that the promoter activity of y4lO depended on the transcriptional activator TtsI. Recombinant Y4lO protein expressed in Escherichia coli did not acetylate two representative mitogen-activated protein kinase kinases (human MKK6 and MKK1 from Medicago truncatula), indicating that YopJ-like proteins differ with respect to their substrate specificities. The y4lO gene was mutated in NGR234 (strain NGROmegay4lO) and in NGR Omega nopL, a mutant that does not produce the T3 effector NopL (strain NGR Omega nopLOmegay4lO). When used as inoculants, the symbiotic properties of the mutants differed. Tephrosia vogelii, Phaseolus vulgaris cv. Yudou No. 1, and Vigna unguiculata cv. Sui Qing Dou Jiao formed pink effective nodules with NGR234 and NGR Omega nopL Omega y4lO. Nodules induced by NGR Omega y4lO were first pink but rapidly turned greenish (ineffective nodules), indicating premature senescence. An ultrastructural analysis of the nodules induced by NGR Omega y4lO revealed abnormal formation of enlarged infection droplets in ineffective nodules, whereas symbiosomes harboring a single bacteroid were frequently observed in effective nodules induced by NGR234 or NGR Omega nopL Omega y4lO. It is concluded that Y4lO is a symbiotic determinant involved in the differentiation of symbiosomes. Y4lO mitigated senescence-inducing effects caused by the T3 effector NopL, suggesting synergistic effects for Y4lO and NopL in nitrogen-fixing nodules.

Comprehensive Approach for Identifying the T Cell Subset Origin of CD3 and CD28 Antibody-Activated Chimeric Antigen Receptor-Modified T Cells.

PubMed

Schmueck-Henneresse, Michael; Omer, Bilal; Shum, Thomas; Tashiro, Haruko; Mamonkin, Maksim; Lapteva, Natalia; Sharma, Sandhya; Rollins, Lisa; Dotti, Gianpietro; Reinke, Petra; Volk, Hans-Dieter; Rooney, Cliona M

2017-07-01

The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45RO + CCR7 - effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28-activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell-derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro-expanded T cells. Copyright © 2017 by The American Association of Immunologists, Inc.

Differential requirements of CD4(+) T-cell signals for effector cytotoxic T-lymphocyte (CTL) priming and functional memory CTL development at higher CD8(+) T-cell precursor frequency.

PubMed

Umeshappa, Channakeshava S; Nanjundappa, Roopa H; Xie, Yufeng; Freywald, Andrew; Xu, Qingyong; Xiang, Jim

2013-04-01

Increased CD8(+) T-cell precursor frequency (PF) precludes the requirement of CD4(+) helper T (Th) cells for primary CD8(+) cytotoxic T-lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) -pulsed dendritic cells (DC(OVA)) derived from C57BL/6, CD40 knockout (CD40(-/-)) or CD40 ligand knockout (CD40L(-/-)) mice were used to immunize C57BL/6, Ia(b-/-), CD40(-/-) or CD40L(-/-) mice, whose PF was previously increased with transfer of 1 × 10(6) CD8(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTI, OTI(CD40(-/-)) or OTI(CD40L(-/-)) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4(+) T-cell help and Th-provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA-expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4(+) T-cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4(+) T-cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4(+) T-cell functions. © 2012 Blackwell Publishing Ltd.

Bcl11b is essential for licensing Th2 differentiation during helminth infection and allergic asthma

USDA-ARS?s Scientific Manuscript database

Naïve CD4+ T-helper cells differentiate into Th2 effector cells during asthma and helminth (worm) infection. Here we report that mice lacking the transcription factor Bcl11b in mature CD4+ T-cells are incapable of mounting an effective Th2 response in asthma and worm infection with a major reductio...

Periodic and chaotic oscillations in a tumor and immune system interaction model with three delays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bi, Ping; Center for Partial Differential Equations, East China Normal University, 500 Dongchuan Rd., Shanghai 200241; Ruan, Shigui, E-mail: ruan@math.miami.edu

2014-06-15

In this paper, a tumor and immune system interaction model consisted of two differential equations with three time delays is considered in which the delays describe the proliferation of tumor cells, the process of effector cells growth stimulated by tumor cells, and the differentiation of immune effector cells, respectively. Conditions for the asymptotic stability of equilibria and existence of Hopf bifurcations are obtained by analyzing the roots of a second degree exponential polynomial characteristic equation with delay dependent coefficients. It is shown that the positive equilibrium is asymptotically stable if all three delays are less than their corresponding critical valuesmore » and Hopf bifurcations occur if any one of these delays passes through its critical value. Numerical simulations are carried out to illustrate the rich dynamical behavior of the model with different delay values including the existence of regular and irregular long periodic oscillations.« less

Molecular regulation of effector and memory T cell differentiation

PubMed Central

Chang, John T; Wherry, E John; Goldrath, Ananda W

2015-01-01

Immunological memory is a cardinal feature of adaptive immunity and an important goal of vaccination strategies. Here we highlight advances in the understanding of the diverse T lymphocyte subsets that provide acute and long-term protection from infection. These include new insights into the transcription factors, and the upstream ‘pioneering’ factors that regulate their accessibility to key sites of gene regulation, as well as metabolic regulators that contribute to the differentiation of effector and memory subsets; ontogeny and defining characteristics of tissue-resident memory lymphocytes; and origins of the remarkable heterogeneity exhibited by activated T cells. Collectively, these findings underscore progress in delineating the underlying pathways that control diversification in T cell responses but also reveal gaps in the knowledge, as well as the challenges that arise in the application of this knowledge to rationally elicit desired T cell responses through vaccination and immunotherapy. PMID:25396352

Modification of Hematopoietic Stem/Progenitor Cells with CD19-Specific Chimeric Antigen Receptors as a Novel Approach for Cancer Immunotherapy

PubMed Central

Ryan, Christine; Giannoni, Francesca; Hardee, Cinnamon L.; Tremcinska, Irena; Katebian, Behrod; Wherley, Jennifer; Sahaghian, Arineh; Tu, Andy; Grogan, Tristan; Elashoff, David; Cooper, Laurence J.N.; Hollis, Roger P.; Kohn, Donald B.

2013-01-01

Abstract Chimeric antigen receptors (CARs) against CD19 have been shown to direct T-cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. However, in some cases, there has been insufficient persistence of effector cells, limiting clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to deliver the CD19-specific CAR, with potential for ensuring persistent production of effector cells of multiple lineages targeting B-lineage malignant cells. Assessments were performed using in vitro myeloid or natural killer (NK) cell differentiation of human HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. Gene transfer did not impair hematopoietic differentiation and cell proliferation when transduced at 1–2 copies/cell. CAR-bearing myeloid and NK cells specifically lysed CD19-positive cells, with second-generation CAR including CD28 domains being more efficient in NK cells. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a strategy for generating multiple lineages of effector cells for immunotherapy against B-lineage malignancies to augment graft-versus-leukemia activity. PMID:23978226

Long-term live-cell imaging reveals new roles for Salmonella effector proteins SseG and SteA.

PubMed

McQuate, Sarah E; Young, Alexandra M; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S; Palmer, Amy E

2017-01-01

Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single-cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here, we establish a pipeline for long-term (17 h) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyper-replication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. © 2016 John Wiley & Sons Ltd.

Long-Term Live Cell Imaging Reveals New Roles For Salmonella Effector Proteins SseG and SteA

PubMed Central

McQuate, Sarah E.; Young, Alexandra M.; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S.; Palmer, Amy E.

2016-01-01

Summary Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here we establish a pipeline for long-term (16 hours) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages, and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyperreplication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. PMID:27376507

Regulators and effectors of bone morphogenetic protein signalling in the cardiovascular system.

PubMed

Luo, Jiang-Yun; Zhang, Yang; Wang, Li; Huang, Yu

2015-07-15

Bone morphogenetic proteins (BMPs) play key roles in the regulation of cell proliferation, differentiation and apoptosis in various tissues and organs, including the cardiovascular system. BMPs signal through both Smad-dependent and -independent cascades to exert a wide spectrum of biological activities. Cardiovascular disorders such as abnormal angiogenesis, atherosclerosis, pulmonary hypertension and cardiac hypertrophy have been linked to aberrant BMP signalling. To correct the dysregulated BMP signalling in cardiovascular pathogenesis, it is essential to get a better understanding of how the regulators and effectors of BMP signalling control cardiovascular function and how the dysregulated BMP signalling contributes to cardiovascular dysfunction. We hence highlight several key regulators of BMP signalling such as extracellular regulators of ligands, mechanical forces, microRNAs and small molecule drugs as well as typical BMP effectors like direct downstream target genes, mitogen-activated protein kinases, reactive oxygen species and microRNAs. The insights into these molecular processes will help target both the regulators and important effectors to reverse BMP-associated cardiovascular pathogenesis. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Independently evolved virulence effectors converge onto hubs in a plant immune system network.

PubMed

Mukhtar, M Shahid; Carvunis, Anne-Ruxandra; Dreze, Matija; Epple, Petra; Steinbrenner, Jens; Moore, Jonathan; Tasan, Murat; Galli, Mary; Hao, Tong; Nishimura, Marc T; Pevzner, Samuel J; Donovan, Susan E; Ghamsari, Lila; Santhanam, Balaji; Romero, Viviana; Poulin, Matthew M; Gebreab, Fana; Gutierrez, Bryan J; Tam, Stanley; Monachello, Dario; Boxem, Mike; Harbort, Christopher J; McDonald, Nathan; Gai, Lantian; Chen, Huaming; He, Yijian; Vandenhaute, Jean; Roth, Frederick P; Hill, David E; Ecker, Joseph R; Vidal, Marc; Beynon, Jim; Braun, Pascal; Dangl, Jeffery L

2011-07-29

Plants generate effective responses to infection by recognizing both conserved and variable pathogen-encoded molecules. Pathogens deploy virulence effector proteins into host cells, where they interact physically with host proteins to modulate defense. We generated an interaction network of plant-pathogen effectors from two pathogens spanning the eukaryote-eubacteria divergence, three classes of Arabidopsis immune system proteins, and ~8000 other Arabidopsis proteins. We noted convergence of effectors onto highly interconnected host proteins and indirect, rather than direct, connections between effectors and plant immune receptors. We demonstrated plant immune system functions for 15 of 17 tested host proteins that interact with effectors from both pathogens. Thus, pathogens from different kingdoms deploy independently evolved virulence proteins that interact with a limited set of highly connected cellular hubs to facilitate their diverse life-cycle strategies.

Blocking Virus Replication during Acute Murine Cytomegalovirus Infection Paradoxically Prolongs Antigen Presentation and Increases the CD8+ T Cell Response by Preventing Type I IFN-Dependent Depletion of Dendritic Cells.

PubMed

Loo, Christopher P; Snyder, Christopher M; Hill, Ann B

2017-01-01

Increasing amounts of pathogen replication usually lead to a proportionate increase in size and effector differentiation of the CD8 + T cell response, which is attributed to increased Ag and inflammation. Using a murine CMV that is highly sensitive to the antiviral drug famciclovir to modulate virus replication, we found that increased virus replication drove increased effector CD8 + T cell differentiation, as expected. Paradoxically, however, increased virus replication dramatically decreased the size of the CD8 + T cell response to two immunodominant epitopes. The decreased response was due to type I IFN-dependent depletion of conventional dendritic cells and could be reproduced by specific depletion of dendritic cells from day 2 postinfection or by sterile induction of type I IFN. Increased virus replication and type I IFN specifically inhibited the response to two immunodominant epitopes that are known to be dependent on Ag cross-presented by DCs, but they did not inhibit the response to "inflationary" epitopes whose responses can be sustained by infected nonhematopoietic cells. Our results show that type I IFN can suppress CD8 + T cell responses to cross-presented Ag by depleting cross-presenting conventional dendritic cells. Copyright © 2016 by The American Association of Immunologists, Inc.

RORC2 is involved in T cell polarization through interaction with the FOXP3 promoter.

PubMed

Burgler, Simone; Mantel, Pierre-Yves; Bassin, Claudio; Ouaked, Nadia; Akdis, Cezmi A; Schmidt-Weber, Carsten B

2010-06-01

The process of Th cell differentiation toward polarized effector T cells tailors specific immunity against invading pathogens while allowing tolerance against commensal microorganisms, harmless allergens, or autologous Ags. Identification of the mechanisms underlying this polarization process is therefore central to understand how the immune system confers immunity and tolerance. The present study demonstrates that retinoic acid receptor-related orphan receptor C2 (RORC2), a key transcription factor in Th17 cell development, inhibits FOXP3 expression in human T cells. Although overexpression of RORC2 in naive T cells reduces levels of FOXP3, small interfering RNA-mediated knockdown of RORC2 enhances its expression. RORC2 mediates this inhibition at least partially by binding to two out of four ROR-responsive elements on the FOXP3 promoter. Knockdown of RORC2 promotes high FOXP3 levels and decreased expression of proinflammatory cytokines beta form of pro-IL-1, IL-6, IL-17A, IFN-gamma, and TNF-alpha in differentiating naive T cells, suggesting that the role of RORC2 in Th17 cell development involves not only induction of Th17-characteristic genes, but also suppression of regulatory T cell-specific programs. Together, this study identifies RORC2 as a polarizing factor in transcriptional cross-regulation and provides novel viewpoints on the control of immune tolerance versus effector immune responses.

Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

PubMed

Kobayashi, Michihiro; Nabinger, Sarah C; Bai, Yunpeng; Yoshimoto, Momoko; Gao, Rui; Chen, Sisi; Yao, Chonghua; Dong, Yuanshu; Zhang, Lujuan; Rodriguez, Sonia; Yashiro-Ohtani, Yumi; Pear, Warren S; Carlesso, Nadia; Yoder, Mervin C; Kapur, Reuben; Kaplan, Mark H; Daniel Lacorazza, Hugo; Zhang, Zhong-Yin; Liu, Yan

2017-04-01

The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064. © 2016 AlphaMed Press.

T inflammatory memory CD8 T cells participate to antiviral response and generate secondary memory cells with an advantage in XCL1 production.

PubMed

Jubin, Virginie; Ventre, Erwan; Leverrier, Yann; Djebali, Sophia; Mayol, Katia; Tomkowiak, Martine; Mafille, Julien; Teixeira, Marie; Teoh, Denise Y-L; Lina, Bruno; Walzer, Thierry; Arpin, Christophe; Marvel, Jacqueline

2012-06-01

Besides the classically described subsets of memory CD8 T cells generated under infectious conditions, are T inflammatory memory cells generated under sterile priming conditions, such as sensitization to allergens. Although not fully differentiated as pathogen-induced memory cells, they display memory properties that distinguish them from naive CD8 T cells. Given these memory cells are generated in an antigen-specific context that is devoid of pathogen-derived danger signals and CD4 T cell help, we herein questioned whether they maintained their activation and differentiation potential, could be recruited in an immune response directed against a pathogen expressing their cognate antigen and further differentiate in fully competent secondary memory cells. We show that T inflammatory memory cells can indeed take part to the immune response triggered by a viral infection, differentiate into secondary effectors and further generate typical central memory CD8 T cells and effector memory CD8 T cells. Furthermore, the secondary memory cells they generate display a functional advantage over primary memory cells in their capacity to produce TNF-α and the XCL1 chemokine. These results suggest that cross-reactive stimulations and differentiation of cells directed against allergens or self into fully competent pathogen-induced memory cells might have incidences in inflammatory immuno-pathologies.

T-cell differentiation and CD56+ levels in polypoidal choroidal vasculopathy and neovascular age-related macular degeneration.

PubMed

Subhi, Yousif; Nielsen, Marie Krogh; Molbech, Christopher Rue; Oishi, Akio; Singh, Amardeep; Nissen, Mogens Holst; Sørensen, Torben Lykke

2017-11-20

Polypoidal choroidal vasculopathy (PCV) and neovascular age-related macular degeneration (AMD) are prevalent age-related diseases characterized by exudative changes in the macula. Although they share anatomical and clinical similarities, they are also distinctly characterized by their own features, e.g. vascular abnormalities in PCV and drusen-mediated progression in neovascular AMD. PCV remains etiologically uncharacterized, and ongoing discussion is whether PCV and neovascular AMD share the same etiology or constitute two substantially different diseases. In this study, we investigated T-cell differentiation and aging profile in human patients with PCV, patients with neovascular AMD, and age-matched healthy control individuals. Fresh venous blood was prepared for flow cytometry to investigate CD4 + and CD8 + T-cell differentiation (naïve, central memory, effector memory, effector memory CD45ra + ), loss of differentiation markers CD27 and CD28, and expression of aging marker CD56. Patients with PCV were similar to the healthy controls in all aspects. In patients with neovascular AMD we found significantly accelerated T-cell differentiation (more CD28 - CD27 - cells) and aging (more CD56 + cells) in the CD8 + T-cell compartment. These findings suggest that PCV and neovascular AMD are etiologically different in terms of T cell immunity, and that neovascular AMD is associated with T-cell immunosenescence.

Efficient production of erythroid, megakaryocytic and myeloid cells, using single cell-derived iPSC colony differentiation.

PubMed

Hansen, Marten; Varga, Eszter; Aarts, Cathelijn; Wust, Tatjana; Kuijpers, Taco; von Lindern, Marieke; van den Akker, Emile

2018-04-28

Hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) provide opportunities not only for fundamental research and disease modelling/drug testing but also for large-scale production of blood effector cells for future clinical application. Although there are multiple ways to differentiate human iPSCs towards hematopoietic lineages, there is a need to develop reproducible and robust protocols. Here we introduce an efficient way to produce three major blood cell types using a standardized differentiation protocol that starts with a single hematopoietic initiation step. This system is feeder-free, avoids EB-formation, starts with a hematopoietic initiation step based on a novel single cell-derived iPSC colony differentiation and produces multi-potential progenitors within 8-10 days. Followed by lineage-specific growth factor supplementation these cells can be matured into well characterized erythroid, megakaryocytic and myeloid cells with high-purity, without transcription factor overexpression or any kind of pre-purification step. This standardized differentiation system provides a simple platform to produce specific blood cells in a reproducible manner for hematopoietic development studies, disease modelling, drug testing and the potential for future therapeutic applications. Copyright © 2018. Published by Elsevier B.V.

Advanced Aerodynamic Design of Passive Porosity Control Effectors

NASA Technical Reports Server (NTRS)

Hunter, Craig A.; Viken, Sally A.; Wood, Richard M.; Bauer, Steven X. S.

2001-01-01

This paper describes aerodynamic design work aimed at developing a passive porosity control effector system for a generic tailless fighter aircraft. As part of this work, a computational design tool was developed and used to layout passive porosity effector systems for longitudinal and lateral-directional control at a low-speed, high angle of attack condition. Aerodynamic analysis was conducted using the NASA Langley computational fluid dynamics code USM3D, in conjunction with a newly formulated surface boundary condition for passive porosity. Results indicate that passive porosity effectors can provide maneuver control increments that equal and exceed those of conventional aerodynamic effectors for low-speed, high-alpha flight, with control levels that are a linear function of porous area. This work demonstrates the tremendous potential of passive porosity to yield simple control effector systems that have no external moving parts and will preserve an aircraft's fixed outer mold line.

Redirection to the bone marrow improves T cell persistence and antitumor functions.

PubMed

Khan, Anjum B; Carpenter, Ben; Santos E Sousa, Pedro; Pospori, Constandina; Khorshed, Reema; Griffin, James; Velica, Pedro; Zech, Mathias; Ghorashian, Sara; Forrest, Calum; Thomas, Sharyn; Gonzalez Anton, Sara; Ahmadi, Maryam; Holler, Angelika; Flutter, Barry; Ramirez-Ortiz, Zaida; Means, Terry K; Bennett, Clare L; Stauss, Hans; Morris, Emma; Lo Celso, Cristina; Chakraverty, Ronjon

2018-05-01

A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that redirecting T cells to specialized niches in the BM that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration toward vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15-dependent homeostatic expansion and promoted the differentiation of memory precursor-like cells with low expression of programmed death-1, resistance to apoptosis, and a heightened capacity to generate polyfunctional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, redirected homing of T cells to the BM confers increased memory differentiation and antitumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells.

Scleroderma pathogenesis: a pivotal role for fibroblasts as effector cells

PubMed Central

2013-01-01

Scleroderma (systemic sclerosis; SSc) is characterised by fibrosis of the skin and internal organs in the context of autoimmunity and vascular perturbation. Overproduction of extracellular matrix components and loss of specialised epithelial structures are analogous to the process of scar formation after tissue injury. Fibroblasts are the resident cells of connective tissue that become activated at sites of damage and are likely to be important effector cells in SSc. Differentiation into myofibroblasts is a hallmark process, although the mechanisms and cellular origins of this important fibroblastic cell are still unclear. This article reviews fibroblast biology in the context of SSc and highlights the potentially important place of fibroblast effector cells in fibrosis. Moreover, the heterogeneity of fibroblast properties, multiplicity of regulatory pathways and diversity of origin for myofibroblasts may underpin clinical diversity in SSc, and provide novel avenues for targeted therapy. PMID:23796020

Vegetative compatibility groups partition variation in the virulence of Verticillium dahliae on strawberry

PubMed Central

Fan, Rong; Cockerton, Helen M.; Armitage, Andrew D.; Bates, Helen; Cascant-Lopez, Emma; Antanaviciute, Laima; Xu, Xiangming; Hu, Xiaoping

2018-01-01

Verticillium dahliae infection of strawberry (Fragaria x ananassa) is a major cause of disease-induced wilting in soil-grown strawberries across the world. To understand what components of the pathogen are affecting disease expression, the presence of the known effector VdAve1 was screened in a sample of Verticillium dahliae isolates. Isolates from strawberry were found to contain VdAve1 and were divided into two major clades, based upon their vegetative compatibility groups (VCG); no UK strawberry isolates contained VdAve1. VC clade was strongly related to their virulence levels. VdAve1-containing isolates pathogenic on strawberry were found in both clades, in contrast to some recently published findings. On strawberry, VdAve1-containing isolates had significantly higher virulence during early infection, which diminished in significance as the infection progressed. Transformation of a virulent non-VdAve1 containing isolate, with VdAve1 was found neither to increase nor decrease virulence when inoculated on a susceptible strawberry cultivar. There are therefore virulence factors that are epistatic to VdAve1 and potentially multiple independent routes to high virulence on strawberry in V. dahliae lineages. Genome sequencing a subset of isolates across the two VCGs revealed that isolates were differentiated at the whole genome level and contained multiple changes in putative effector content, indicating that different clonal VCGs may have evolved different strategies for infecting strawberry, leading to different virulence levels in pathogenicity tests. It is therefore important to consider both clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same race determining effector. PMID:29451893

The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae.

PubMed

Deng, Meng-Ying; Sun, Yun-Hao; Li, Pai; Fu, Bei; Shen, Dong; Lu, Yong-Jun

2016-10-01

Virulent protein toxins secreted by the bacterial pathogens can cause cytotoxicity by various molecular mechanisms to combat host cell defense. On the other hand, these proteins can also be used as probes to investigate the defense pathway of host innate immunity. Ralstonia solanacearum, one of the most virulent bacterial phytopathogens, translocates more than 70 effector proteins via type III secretion system during infection. Here, we characterized the cytotoxicity of effector RipI in budding yeast Saccharomyce scerevisiae, an alternative host model. We found that over-expression of RipI resulted in severe growth defect and arginine (R) 117 within the predicted integrase motif was required for inhibition of yeast growth. The phenotype of death manifested the hallmarks of apoptosis. Our data also revealed that RipI-induced apoptosis was independent of Yca1 and mitochondria-mediated apoptotic pathways because Δyca1 and Δaif1 were both sensitive to RipI as compared with the wild type. We further demonstrated that RipI was localized in the yeast nucleus and the N-terminal 1-174aa was required for the localization. High-throughput RNA sequencing analysis showed that upon RipI over-expression, 101 unigenes of yeast ribosome presented lower expression level, and 42 GO classes related to the nucleus or recombination were enriched with differential expression levels. Taken together, our data showed that a nuclear-targeting effector RipI triggers yeast apoptosis, potentially dependent on its integrase function. Our results also provided an alternative strategy to dissect the signaling pathway of cytotoxicity induced by the protein toxins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Vegetative compatibility groups partition variation in the virulence of Verticillium dahliae on strawberry.

PubMed

Fan, Rong; Cockerton, Helen M; Armitage, Andrew D; Bates, Helen; Cascant-Lopez, Emma; Antanaviciute, Laima; Xu, Xiangming; Hu, Xiaoping; Harrison, Richard J

2018-01-01

Verticillium dahliae infection of strawberry (Fragaria x ananassa) is a major cause of disease-induced wilting in soil-grown strawberries across the world. To understand what components of the pathogen are affecting disease expression, the presence of the known effector VdAve1 was screened in a sample of Verticillium dahliae isolates. Isolates from strawberry were found to contain VdAve1 and were divided into two major clades, based upon their vegetative compatibility groups (VCG); no UK strawberry isolates contained VdAve1. VC clade was strongly related to their virulence levels. VdAve1-containing isolates pathogenic on strawberry were found in both clades, in contrast to some recently published findings. On strawberry, VdAve1-containing isolates had significantly higher virulence during early infection, which diminished in significance as the infection progressed. Transformation of a virulent non-VdAve1 containing isolate, with VdAve1 was found neither to increase nor decrease virulence when inoculated on a susceptible strawberry cultivar. There are therefore virulence factors that are epistatic to VdAve1 and potentially multiple independent routes to high virulence on strawberry in V. dahliae lineages. Genome sequencing a subset of isolates across the two VCGs revealed that isolates were differentiated at the whole genome level and contained multiple changes in putative effector content, indicating that different clonal VCGs may have evolved different strategies for infecting strawberry, leading to different virulence levels in pathogenicity tests. It is therefore important to consider both clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same race determining effector.

Two-axis angular effector

DOEpatents

Vaughn, Mark R.; Robinett, III, Rush D.; Phelan, John R.; Van Zuiden, Don M.

1997-01-21

A new class of coplanar two-axis angular effectors. These effectors combine a two-axis rotational joint analogous to a Cardan joint with linear actuators in a manner to produce a wider range of rotational motion about both axes defined by the joint. This new class of effectors also allows design of robotic manipulators having very high strength and efficiency. These effectors are particularly suited for remote operation in unknown surroundings, because of their extraordinary versatility. An immediate application is to the problems which arise in nuclear waste remediation.

T3SEdb: data warehousing of virulence effectors secreted by the bacterial Type III Secretion System.

PubMed

Tay, Daniel Ming Ming; Govindarajan, Kunde Ramamoorthy; Khan, Asif M; Ong, Terenze Yao Rui; Samad, Hanif M; Soh, Wei Wei; Tong, Minyan; Zhang, Fan; Tan, Tin Wee

2010-10-15

Effectors of Type III Secretion System (T3SS) play a pivotal role in establishing and maintaining pathogenicity in the host and therefore the identification of these effectors is important in understanding virulence. However, the effectors display high level of sequence diversity, therefore making the identification a difficult process. There is a need to collate and annotate existing effector sequences in public databases to enable systematic analyses of these sequences for development of models for screening and selection of putative novel effectors from bacterial genomes that can be validated by a smaller number of key experiments. Herein, we present T3SEdb http://effectors.bic.nus.edu.sg/T3SEdb, a specialized database of annotated T3SS effector (T3SE) sequences containing 1089 records from 46 bacterial species compiled from the literature and public protein databases. Procedures have been defined for i) comprehensive annotation of experimental status of effectors, ii) submission and curation review of records by users of the database, and iii) the regular update of T3SEdb existing and new records. Keyword fielded and sequence searches (BLAST, regular expression) are supported for both experimentally verified and hypothetical T3SEs. More than 171 clusters of T3SEs were detected based on sequence identity comparisons (intra-cluster difference up to ~60%). Owing to this high level of sequence diversity of T3SEs, the T3SEdb provides a large number of experimentally known effector sequences with wide species representation for creation of effector predictors. We created a reliable effector prediction tool, integrated into the database, to demonstrate the application of the database for such endeavours. T3SEdb is the first specialised database reported for T3SS effectors, enriched with manual annotations that facilitated systematic construction of a reliable prediction model for identification of novel effectors. The T3SEdb represents a platform for inclusion of additional annotations of metadata for future developments of sophisticated effector prediction models for screening and selection of putative novel effectors from bacterial genomes/proteomes that can be validated by a small number of key experiments.

Delayed Expansion and Contraction of CD8+ T Cell Response during Infection with Virulent Salmonella typhimurium1

PubMed Central

Luu, Rachel A.; Gurnani, Komal; Dudani, Renu; Kammara, Rajagopal; van Faassen, Henk; Sirard, Jean-Claude; Krishnan, Lakshmi; Sad, Subash

2014-01-01

Ag presentation to CD8+ T cells often commences immediately after infection, which facilitates their rapid expansion and control of infection. Subsequently, the primed cells undergo rapid contraction. We report that this paradigm is not followed during infection with virulent Salmonella enterica, serovar Typhimurium (ST), an intracellular bacterium that replicates within phagosomes of infected cells. Although susceptible mice die rapidly (~7 days), resistant mice (129×1SvJ) harbor a chronic infection lasting ~60–90 days. Using rOVA-expressing ST (ST-OVA), we show that T cell priming is considerably delayed in the resistant mice. CD8+ T cells that are induced during ST-OVA infection undergo delayed expansion, which peaks around day 21, and is followed by protracted contraction. Initially, ST-OVA induces a small population of cycling central phenotype (CD62LhighIL-7RαhighCD44high) CD8+ T cells. However, by day 14–21, majority of the primed CD8+ T cells display an effector phenotype (CD62LlowIL-7RαlowCD44high). Subsequently, a progressive increase in the numbers of effector memory phenotype cells (CD62LlowIL-7RαhighCD44high) occurs. This differentiation program remained unchanged after accelerated removal of the pathogen with antibiotics, as majority of the primed cells displayed an effector memory phenotype even at 6 mo postinfection. Despite the chronic infection, CD8+ T cells induced by ST-OVA were functional as they exhibited killing ability and cytokine production. Importantly, even memory CD8+ T cells failed to undergo rapid expansion in response to ST-OVA infection, suggesting a delay in T cell priming during infection with virulent ST-OVA. Thus, phagosomal lifestyle may allow escape from host CD8+ T cell recognition, conferring a survival advantage to the pathogen. PMID:16849458

Investigation of a bio-inspired lift-enhancing effector on a 2D airfoil.

PubMed

Johnston, Joe; Gopalarathnam, Ashok

2012-09-01

A flap mounted on the upper surface of an airfoil, called a 'lift-enhancing effector', has been shown in wind tunnel tests to have a similar function to a bird's covert feathers, which rise off the wing's surface in response to separated flows. The effector, fabricated from a thin Mylar sheet, is allowed to rotate freely about its leading edge. The tests were performed in the NCSU subsonic wind tunnel at a chord Reynolds number of 4 × 10(5). The maximum lift coefficient with the effector was the same as that for the clean airfoil, but was maintained over an angle-of-attack range from 12° to almost 20°, resulting in a very gentle stall behavior. To better understand the aerodynamics and to estimate the deployment angle of the free-moving effector, fixed-angle effectors fabricated out of stiff wood were also tested. A progressive increase in the stall angle of attack with increasing effector angle was observed, with diminishing returns beyond the effector angle of 60°. Drag tests on both the free-moving and fixed effectors showed a marked improvement in drag at high angles of attack. Oil flow visualization on the airfoil with and without the fixed-angle effectors proved that the effector causes the separation point to move aft on the airfoil, as compared to the clean airfoil. This is thought to be the main mechanism by which an effector improves both lift and drag. A comparison of the fixed-effector results with those from the free-effector tests shows that the free effector's deployment angle is between 30° and 45°. When operating at and beyond the clean airfoil's stall angle, the free effector automatically deploys to progressively higher angles with increasing angles of attack. This slows down the rapid upstream movement of the separation point and avoids the severe reduction in the lift coefficient and an increase in the drag coefficient that are seen on the clean airfoil at the onset of stall. Thus, the effector postpones the stall by 4-8° and makes the stall behavior more gentle. The benefits of using the effector could include care-free operations at high angles of attack during perching and maneuvering flight, especially in gusty conditions.

Cell adhesion and the immune system: a case study using earthworms.

PubMed

Cooper, E L; Cossarizza, A; Kauschke, E; Franceschi, C

1999-02-15

In the earthworm's immune system, cell adhesion, which occurs by putative receptors on leukocytes, is essential after recognition of self vs. non-self. Confrontation with foreign antigens is a normal event in the environment, replete with microbial pathogens that pose a threat to survival. To better understand what happens when an effector cell first recognizes a foreign target followed by its adhesion to it, isolated leukocytes, in sufficient quantities to be subjected to various analyses, have been extremely beneficial. In vitro approaches when accompanied by biochemical, immunological, and molecular technologies, have opened up new vistas concerning the immune response of earthworms and other invertebrates. The most recent discovery includes the preliminary identification of cell differentiation (CD) markers that play vital roles in recognitive and adhesive events. Certain leukocyte effectors show characteristics of natural killer (NK) cells that may act differently depending upon their source, whether autogeneic, allogeneic, xenogeneic, or expressed under normal or varying environmental conditions including exposure to xenobiotics. At the level of earthworm evolution, there is apparently a dissociation of phagocytosis from the process of killing by NK-like effectors. There are at least three future challenges. First, it is essential to determine the precise nature of the CD markers with respect to their molecular structure. Second, once their molecular and biochemical characteristics have been defined, the role of these markers in cellular and humoral mechanisms must be clarified in order to define effector cell products and resulting immune responses. Third, there is a need to differentiate between the several lytic factors that have been found in earthworms with respect to molecular structure, and biochemical and functional characterization.

CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes

PubMed Central

Yao, Shuyu; Huang, Dan; Chen, Crystal Y.; Halliday, Lisa; Wang, Richard C.; Chen, Zheng W.

2014-01-01

The possibility that CD4+ T cells can act as “innate-like” cells to contain very-early M. tuberculosis (Mtb) dissemination and function as master helpers to sustain multiple effector functions of CD8+ T cells and CD3-negative lymphocytes during development of adaptive immunity against primary tuberculosis(TB) has not been demonstrated. We showed that pulmonary Mtb infection of CD4-depleted macaques surprisingly led to very-early extrathoracic Mtb dissemination, whereas CD4 deficiency clearly resulted in rapid TB progression. CD4 depletion during Mtb infection revealed the ability of CD8+ T cells to compensate and rapidly differentiate to Th17-like/Th1-like, and cytotoxic-like effectors, but these effector functions were subsequently unsustainable due to CD4 deficiency. While CD3-negative non-T lymphocytes in presence of CD4+ T cells developed predominant Th22-like and NK-like (perforin production) responses to Mtb infection, CD4 depletion abrogated these Th22-/NK-like effector functions and favored IL-17 production by CD3-negative lymphocytes. CD4-depleted macaques exhibited no or few pulmonary T effector cells constitutively producing IFN-γ, TNFα, IL-17, IL-22, and perforin at the endpoint of more severe TB, but presented pulmonary IL-4+ T effectors. TB granulomas in CD4-depleted macaques contained fewer IL-22+ and perforin+ cells despite presence of IL-17+ and IL-4+ cells. These results implicate previously-unknown “innate-like” ability of CD4+ T cells to contain extrathoracic Mtb dissemination at very early stage. Data also suggest that CD4+ T cells are required to sustain multiple effector functions of CD8+ T cells and CD3-negative lymphocytes and to prevent rapid TB progression during Mtb infection of nonhuman primates. PMID:24489088

Programmed cell death 1 inhibits inflammatory helper T-cell development through controlling the innate immune response

PubMed Central

Rui, Yuxiang; Honjo, Tasuku; Chikuma, Shunsuke

2013-01-01

Programmed cell death 1 (PD-1) is an inhibitory coreceptor on immune cells and is essential for self-tolerance because mice genetically lacking PD-1 (PD-1−/−) develop spontaneous autoimmune diseases. PD-1−/− mice are also susceptible to severe experimental autoimmune encephalomyelitis (EAE), characterized by a massive production of effector/memory T cells against myelin autoantigen, the mechanism of which is not fully understood. We found that an increased primary response of PD-1−/− mice to heat-killed mycobacteria (HKMTB), an adjuvant for EAE, contributed to the enhanced production of T-helper 17 (Th17) cells. Splenocytes from HKMTB-immunized, lymphocyte-deficient PD-1−/− recombination activating gene (RAG)2−/− mice were found to drive antigen-specific Th17 cell differentiation more efficiently than splenocytes from HKMTB-immunized PD-1+/+ RAG2−/− mice. This result suggested PD-1’s involvement in the regulation of innate immune responses. Mice reconstituted with PD-1−/− RAG2−/− bone marrow and PD-1+/+ CD4+ T cells developed more severe EAE compared with the ones reconstituted with PD-1+/+ RAG2−/− bone marrow and PD-1+/+ CD4+ T cells. We found that upon recognition of HKMTB, CD11b+ macrophages from PD-1−/− mice produced very high levels of IL-6, which helped promote naive CD4+ T-cell differentiation into IL-17–producing cells. We propose a model in which PD-1 negatively regulates antimycobacterial responses by suppressing innate immune cells, which in turn prevents autoreactive T-cell priming and differentiation to inflammatory effector T cells. PMID:24043779

The Transcription Factor STAT3 and Type I Interferons Are Mutually Repressive Insulators for Differentiation of Follicular Helper and T Helper_1 Cells

PubMed Central

Ray, John P.; Marshall, Heather D.; Laidlaw, Brian J.; Staron, Matthew M.; Kaech, Susan M.; Craft, Joe

2014-01-01

Summary Follicular helper T (Tfh) cells are required for the establishment of T-dependent B cell memory and high affinity antibody-secreting cells. We have revealed herein opposing roles for signal transducer and activator of transcription 3 (STAT3) and type I interferon (IFN) signaling in the differentiation of Tfh cells following viral infection. STAT3-deficient CD4+ T cells had a profound defect in Tfh cell differentiation, accompanied by decreased germinal center (GC) B cells and antigen-specific antibody production during acute infection with lymphocytic choriomeningitis virus. STAT3-deficient Tfh cells had strikingly increased expression of a number of interferoninducible genes, in addition to enhanced T-bet synthesis, thus adopting a T helper-1 (Th1) cell-like effector phenotype. Conversely, IFNαβ receptor blockade restored Tfh and GC B cell phenotypes in mice containing STAT3-deficient CD4+ T cells. These data suggest mutually repressive roles for STAT3 and type I IFN signaling pathways in the differentiation of Tfh cells following viral infection. PMID:24631156

VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System

PubMed Central

Cianfanelli, Francesca R.; Alcoforado Diniz, Juliana; Guo, Manman; De Cesare, Virginia; Trost, Matthias; Coulthurst, Sarah J.

2016-01-01

The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently (‘specialised’) or non-covalently (‘cargo’ effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a ‘core’ T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery. PMID:27352036

Role of PD-1 during effector CD8 T cell differentiation.

PubMed

Ahn, Eunseon; Araki, Koichi; Hashimoto, Masao; Li, Weiyan; Riley, James L; Cheung, Jeanne; Sharpe, Arlene H; Freeman, Gordon J; Irving, Bryan A; Ahmed, Rafi

2018-05-01

PD-1 (programmed cell death-1) is the central inhibitory receptor regulating CD8 T cell exhaustion during chronic viral infection and cancer. Interestingly, PD-1 is also expressed transiently by activated CD8 T cells during acute viral infection, but the role of PD-1 in modulating T cell effector differentiation and function is not well defined. To address this question, we examined the expression kinetics and role of PD-1 during acute lymphocytic choriomeningitis virus (LCMV) infection of mice. PD-1 was rapidly up-regulated in vivo upon activation of naive virus-specific CD8 T cells within 24 h after LCMV infection and in less than 4 h after peptide injection, well before any cell division had occurred. This rapid PD-1 expression by CD8 T cells was driven predominantly by antigen receptor signaling since infection with a LCMV strain with a mutation in the CD8 T cell epitope did not result in the increase of PD-1 on antigen-specific CD8 T cells. Blockade of the PD-1 pathway using anti-PD-L1 or anti-PD-1 antibodies during the early phase of acute LCMV infection increased mTOR signaling and granzyme B expression in virus-specific CD8 T cells and resulted in faster clearance of the infection. These results show that PD-1 plays an inhibitory role during the naive-to-effector CD8 T cell transition and that the PD-1 pathway can also be modulated at this stage of T cell differentiation. These findings have implications for developing therapeutic vaccination strategies in combination with PD-1 blockade.

CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response

PubMed Central

Peixoto, António; Evaristo, César; Munitic, Ivana; Monteiro, Marta; Charbit, Alain; Rocha, Benedita; Veiga-Fernandes, Henrique

2007-01-01

To study in vivo CD8 T cell differentiation, we quantified the coexpression of multiple genes in single cells throughout immune responses. After in vitro activation, CD8 T cells rapidly express effector molecules and cease their expression when the antigen is removed. Gene behavior after in vivo activation, in contrast, was quite heterogeneous. Different mRNAs were induced at very different time points of the response, were transcribed during different time periods, and could decline or persist independently of the antigen load. Consequently, distinct gene coexpression patterns/different cell types were generated at the various phases of the immune responses. During primary stimulation, inflammatory molecules were induced and down-regulated shortly after activation, generating early cells that only mediated inflammation. Cytotoxic T cells were generated at the peak of the primary response, when individual cells simultaneously expressed multiple killer molecules, whereas memory cells lost killer capacity because they no longer coexpressed killer genes. Surprisingly, during secondary responses gene transcription became permanent. Secondary cells recovered after antigen elimination were more efficient killers than cytotoxic T cells present at the peak of the primary response. Thus, primary responses produced two transient effector types. However, after boosting, CD8 T cells differentiate into long-lived killer cells that persist in vivo in the absence of antigen. PMID:17485515

The IL23R R381Q Gene Variant Protects against Immune-Mediated Diseases by Impairing IL-23-Induced Th17 Effector Response in Humans

PubMed Central

Di Meglio, Paola; Di Cesare, Antonella; Laggner, Ute; Chu, Chung-Ching; Napolitano, Luca; Villanova, Federica; Tosi, Isabella; Capon, Francesca; Trembath, Richard C.; Peris, Ketty; Nestle, Frank O.

2011-01-01

IL-23 and Th17 cells are key players in tissue immunosurveillance and are implicated in human immune-mediated diseases. Genome-wide association studies have shown that the IL23R R381Q gene variant protects against psoriasis, Crohn's disease and ankylosing spondylitis. We investigated the immunological consequences of the protective IL23R R381Q gene variant in healthy donors. The IL23R R381Q gene variant had no major effect on Th17 cell differentiation as the frequency of circulating Th17 cells was similar in carriers of the IL23R protective (A) and common (G) allele. Accordingly, Th17 cells generated from A and G donors produced similar amounts of Th17 cytokines. However, IL-23-mediated Th17 cell effector function was impaired, as Th17 cells from A allele carriers had significantly reduced IL-23-induced IL-17A production and STAT3 phosphorylation compared to G allele carriers. Our functional analysis of a human disease-associated gene variant demonstrates that IL23R R381Q exerts its protective effects through selective attenuation of IL-23-induced Th17 cell effector function without interfering with Th17 differentiation, and highlights its importance in the protection against IL-23-induced tissue pathologies. PMID:21364948

The IL23R R381Q gene variant protects against immune-mediated diseases by impairing IL-23-induced Th17 effector response in humans.

PubMed

Di Meglio, Paola; Di Cesare, Antonella; Laggner, Ute; Chu, Chung-Ching; Napolitano, Luca; Villanova, Federica; Tosi, Isabella; Capon, Francesca; Trembath, Richard C; Peris, Ketty; Nestle, Frank O

2011-02-22

IL-23 and Th17 cells are key players in tissue immunosurveillance and are implicated in human immune-mediated diseases. Genome-wide association studies have shown that the IL23R R381Q gene variant protects against psoriasis, Crohn's disease and ankylosing spondylitis. We investigated the immunological consequences of the protective IL23R R381Q gene variant in healthy donors. The IL23R R381Q gene variant had no major effect on Th17 cell differentiation as the frequency of circulating Th17 cells was similar in carriers of the IL23R protective (A) and common (G) allele. Accordingly, Th17 cells generated from A and G donors produced similar amounts of Th17 cytokines. However, IL-23-mediated Th17 cell effector function was impaired, as Th17 cells from A allele carriers had significantly reduced IL-23-induced IL-17A production and STAT3 phosphorylation compared to G allele carriers. Our functional analysis of a human disease-associated gene variant demonstrates that IL23R R381Q exerts its protective effects through selective attenuation of IL-23-induced Th17 cell effector function without interfering with Th17 differentiation, and highlights its importance in the protection against IL-23-induced tissue pathologies.

Generals die in friendly fire, or modeling immune response to HIV

NASA Astrophysics Data System (ADS)

Rouzine, Igor M.; Murali-Krishna, Kaja; Ahmed, Rafi

2005-12-01

We develop a kinetic model for CD8 T lymphocytes (CTL) whose purpose is to kill cells infected with viruses and intracellular parasites. Using a set of first-order nonlinear differential equations, the model predicts how numbers of different cell types involved in CTL response depend on time. The model postulates that CTL response requires continuous presence of professional antigen-presenting cells (APC) comprised of macrophages and dendritic cells. It assumes that any virus present in excess of a threshold level activates APC that, in turn, activate CTL that expand in number and become armed "effector" cells. In the end, APC are deactivated after virus is cleared. The lack of signal from APC causes effector cells to differentiate, by default, into "transitory cells" that either die, or, in a small part, become long-lived memory cells. Viruses capable of infecting APC will cause premature retirement of effector CTL. If transitory cells encounter virus, which takes place after the premature depletion, CTL become anergic (unresponsive to external stimuli). The model is designed to fit recent experiments on primary CTL response to simian immunodeficiency virus closely related to HIV and lymphocytic choriomeningitis virus. The two viruses are known to infect APC and make them targets for CTL they are supposed to control. Both viruses cause premature depletion and anergy of CTL and persist in the host for life.

Intrinsic disorder in pathogen effectors: protein flexibility as an evolutionary hallmark in a molecular arms race.

PubMed

Marín, Macarena; Uversky, Vladimir N; Ott, Thomas

2013-09-01

Effector proteins represent a refined mechanism of bacterial pathogens to overcome plants' innate immune systems. These modular proteins often manipulate host physiology by directly interfering with immune signaling of plant cells. Even if host cells have developed efficient strategies to perceive the presence of pathogenic microbes and to recognize intracellular effector activity, it remains an open question why only few effectors are recognized directly by plant resistance proteins. Based on in-silico genome-wide surveys and a reevaluation of published structural data, we estimated that bacterial effectors of phytopathogens are highly enriched in long-disordered regions (>50 residues). These structurally flexible segments have no secondary structure under physiological conditions but can fold in a stimulus-dependent manner (e.g., during protein-protein interactions). The high abundance of intrinsic disorder in effectors strongly suggests positive evolutionary selection of this structural feature and highlights the dynamic nature of these proteins. We postulate that such structural flexibility may be essential for (1) effector translocation, (2) evasion of the innate immune system, and (3) host function mimicry. The study of these dynamical regions will greatly complement current structural approaches to understand the molecular mechanisms of these proteins and may help in the prediction of new effectors.

Differential expression of candidate salivary effector proteins in field collections of Hessian fly, Mayetiola destructor

PubMed Central

Johnson, A J; Shukle, R H; Chen, M-S; Srivastava, S; Subramanyam, S; Schemerhorn, B J; Weintraub, P G; Abdel Moniem, H E M; Flanders, K L; Buntin, G D; Williams, C E

2015-01-01

Evidence is emerging that some proteins secreted by gall-forming parasites of plants act as effectors responsible for systemic changes in the host plant, such as galling and nutrient tissue formation. A large number of secreted salivary gland proteins (SSGPs) that are the putative effectors responsible for the physiological changes elicited in susceptible seedling wheat by Hessian fly, Mayetiola destructor (Say), larvae have been documented. However, how the genes encoding these candidate effectors might respond under field conditions is unknown. The goal of this study was to use microarray analysis to investigate variation in SSGP transcript abundance amongst field collections from different geographical regions (southeastern USA, central USA, and the Middle East). Results revealed significant variation in SSGP transcript abundance amongst the field collections studied. The field collections separated into three distinct groups that corresponded to the wheat classes grown in the different geographical regions as well as to recently described Hessian fly populations. These data support previous reports correlating Hessian fly population structure with micropopulation differences owing to agro-ecosystem parameters such as cultivation of regionally adapted wheat varieties, deployment of resistance genes and variation in climatic conditions. PMID:25528896

Epidermal Notch signalling: differentiation, cancer and adhesion.

PubMed

Watt, Fiona M; Estrach, Soline; Ambler, Carrie A

2008-04-01

The Notch pathway plays an important role in regulating epidermal differentiation. Notch ligands, receptors and effectors are expressed in a complex and dynamic pattern in embryonic and adult skin. Genetic ablation or activation of the pathway reveals that Notch signalling promotes differentiation of the hair follicle, sebaceous gland and interfollicular epidermal lineages and that Notch acts as an epidermal tumour suppressor. Notch signalling interacts with a range of other pathways to fulfil these functions and acts via RBP-Jkappa dependent and independent mechanisms. The effects on differentiation can be cell autonomous and non-autonomous, and Notch contributes to stem cell clustering via modulation of cell adhesion.

FAT1 cadherin acts upstream of Hippo signalling through TAZ to regulate neuronal differentiation.

PubMed

Ahmed, Abdulrzag F; de Bock, Charles E; Lincz, Lisa F; Pundavela, Jay; Zouikr, Ihssane; Sontag, Estelle; Hondermarck, Hubert; Thorne, Rick F

2015-12-01

The Hippo pathway is emerging as a critical nexus that balances self-renewal of progenitors against differentiation; however, upstream elements in vertebrate Hippo signalling are poorly understood. High expression of Fat1 cadherin within the developing neuroepithelium and the manifestation of severe neurological phenotypes in Fat1-knockout mice suggest roles in neurogenesis. Using the SH-SY5Y model of neuronal differentiation and employing gene silencing techniques, we show that FAT1 acts to control neurite outgrowth, also driving cells towards terminal differentiation via inhibitory effects on proliferation. FAT1 actions were shown to be mediated through Hippo signalling where it activated core Hippo kinase components and antagonised functions of the Hippo effector TAZ. Suppression of FAT1 promoted the nucleocytoplasmic shuttling of TAZ leading to enhanced transcription of the Hippo target gene CTGF together with accompanying increases in nuclear levels of Smad3. Silencing of TAZ reversed the effects of FAT1 depletion thus connecting inactivation of TAZ-TGFbeta signalling with Hippo signalling mediated through FAT1. These findings establish FAT1 as a new upstream Hippo element regulating early stages of differentiation in neuronal cells.

Systems Modeling of Molecular Mechanisms Controlling Cytokine-driven CD4+ T Cell Differentiation and Phenotype Plasticity

PubMed Central

Carbo, Adria; Hontecillas, Raquel; Kronsteiner, Barbara; Viladomiu, Monica; Pedragosa, Mireia; Lu, Pinyi; Philipson, Casandra W.; Hoops, Stefan; Marathe, Madhav; Eubank, Stephen; Bisset, Keith; Wendelsdorf, Katherine; Jarrah, Abdul; Mei, Yongguo; Bassaganya-Riera, Josep

2013-01-01

Differentiation of CD4+ T cells into effector or regulatory phenotypes is tightly controlled by the cytokine milieu, complex intracellular signaling networks and numerous transcriptional regulators. We combined experimental approaches and computational modeling to investigate the mechanisms controlling differentiation and plasticity of CD4+ T cells in the gut of mice. Our computational model encompasses the major intracellular pathways involved in CD4+ T cell differentiation into T helper 1 (Th1), Th2, Th17 and induced regulatory T cells (iTreg). Our modeling efforts predicted a critical role for peroxisome proliferator-activated receptor gamma (PPARγ) in modulating plasticity between Th17 and iTreg cells. PPARγ regulates differentiation, activation and cytokine production, thereby controlling the induction of effector and regulatory responses, and is a promising therapeutic target for dysregulated immune responses and inflammation. Our modeling efforts predict that following PPARγ activation, Th17 cells undergo phenotype switch and become iTreg cells. This prediction was validated by results of adoptive transfer studies showing an increase of colonic iTreg and a decrease of Th17 cells in the gut mucosa of mice with colitis following pharmacological activation of PPARγ. Deletion of PPARγ in CD4+ T cells impaired mucosal iTreg and enhanced colitogenic Th17 responses in mice with CD4+ T cell-induced colitis. Thus, for the first time we provide novel molecular evidence in vivo demonstrating that PPARγ in addition to regulating CD4+ T cell differentiation also plays a major role controlling Th17 and iTreg plasticity in the gut mucosa. PMID:23592971

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence1[OPEN

PubMed Central

2017-01-01

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M. persicae-host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. PMID:28100451

Cryopreservation has no effect on function of natural killer cells differentiated in vitro from umbilical cord blood CD34(+) cells.

PubMed

Domogala, Anna; Madrigal, J Alejandro; Saudemont, Aurore

2016-06-01

Natural killer (NK) cells offer the potential for a powerful cellular immunotherapy because they can target malignant cells without being direct effectors of graft-versus-host disease. We have previously shown that high numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34(+) cells. To develop a readily available, off-the-shelf cellular product, it is essential that NK cells differentiated in vitro can be frozen and thawed while maintaining the same phenotype and functions. We evaluated the phenotype and function of fresh and frozen NK cells differentiated in vitro. We also assessed whether the concentration of NK cells at the time of freezing had an impact on cell viability. We found that cell concentration of NK cells at the time of freezing did not have an impact on their viability and on cell recovery post-thaw. Moreover, freezing of differentiated NK cells in vitro did not affect their phenotype, cytotoxicity and degranulation capacity toward K562 cells, cytokine production and proliferation. We are therefore able to generate large numbers of functional NK cells from CB CD34(+) cells that maintain the same phenotype and function post-cryopreservation, which will allow for multiple infusions of a highly cytotoxic NK cell product. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation

PubMed Central

Patsoukis, Nikolaos; Bardhan, Kankana; Chatterjee, Pranam; Sari, Duygu; Liu, Bianling; Bell, Lauren N.; Karoly, Edward D.; Freeman, Gordon J.; Petkova, Victoria; Seth, Pankaj; Li, Lequn; Boussiotis, Vassiliki A.

2015-01-01

During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid β-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade. PMID:25809635

Reversion of malignant phenotypes of human glioblastoma cells by β-elemene through β-catenin-mediated regulation of stemness-, differentiation- and epithelial-to-mesenchymal transition-related molecules.

PubMed

Zhu, Tingzhun; Li, Xiaoming; Luo, Lihan; Wang, Xiaogang; Li, Zhiqing; Xie, Peng; Gao, Xu; Song, Zhenquan; Su, Jingyuan; Liang, Guobiao

2015-11-12

Glioblastoma is the most common and lethal type of primary brain tumor. β-Elemene, a natural plant drug extracted from Curcuma wenyujin, has shown strong anti-tumor effects in various tumors with low toxicity. However, the effects of β-elemene on malignant phenotypes of human glioblastoma cells remain to be elucidated. Here we evaluated the effects of β-elemene on cell proliferation, survival, stemness, differentiation and the epithelial-to-mesenchymal transition (EMT) in vitro and in vivo, and investigated the mechanisms underlying these effects. Human primary and U87 glioblastoma cells were treated with β-elemene, cell viability was measured using a cell counting kit-8 assay, and treated cells were evaluated by flow cytometry. Western blot analysis was carried out to determine the expression levels of stemness markers, differentiation-related molecules and EMT-related effectors. Transwell assays were performed to further determine EMT of glioblastoma cells. To evaluate the effect of β-elemene on glioblastoma in vivo, we subcutaneously injected glioblastoma cells into the flank of nude mice and then intraperitoneally injected NaCl or β-elemene. The tumor xenograft volumes were measured every 3 days and the expression of stemness-, differentiation- and EMT-related effectors was determined by Western blot assays in xenografts. β-Elemene inhibited proliferation, promoted apoptosis, impaired invasiveness in glioblastoma cells and suppressed the growth of animal xenografts. The expression levels of the stemness markers CD133 and ATP-binding cassette subfamily G member 2 as well as the mesenchymal markers N-cadherin and β-catenin were significantly downregulated, whereas the expression levels of the differentiation-related effectors glial fibrillary acidic protein, Notch1, and sonic hedgehog as well as the epithelial marker E-cadherin were upregulated by β-elemene in vitro and in vivo. Interestingly, the expression of vimentin was increased by β-elemene in vitro; this result was opposite that for the in vivo procedure. Inhibiting β-catenin enhanced the anti-proliferative, EMT-inhibitory and specific marker expression-regulatory effects of β-elemene. β-Elemene reversed malignant phenotypes of human glioblastoma cells through β-catenin-involved regulation of stemness-, differentiation- and EMT-related molecules. β-Elemene represents a potentially valuable agent for glioblastoma therapy.

CD4 T cell-mediated protection from lethal influenza: perforin and antibody-mediated mechanisms give a one-two punch.

PubMed

Brown, Deborah M; Dilzer, Allison M; Meents, Dana L; Swain, Susan L

2006-09-01

The mechanisms whereby CD4 T cells contribute to the protective response against lethal influenza infection remain poorly characterized. To define the role of CD4 cells in protection against a highly pathogenic strain of influenza, virus-specific TCR transgenic CD4 effectors were generated in vitro and transferred into mice given lethal influenza infection. Primed CD4 effectors conferred protection against lethal infection over a broad range of viral dose. The protection mediated by CD4 effectors did not require IFN-gamma or host T cells, but did result in increased anti-influenza Ab titers compared with untreated controls. Further studies indicated that CD4-mediated protection at high doses of influenza required B cells, and that passive transfer of anti-influenza immune serum was therapeutic in B cell-deficient mice, but only when CD4 effectors were present. Primed CD4 cells also acquired perforin (Pfn)-mediated cytolytic activity during effector generation, suggesting a second mechanism used by CD4 cells to confer protection. Pfn-deficient CD4 effectors were less able to promote survival in intact BALB/c mice and were unable to provide protection in B cell-deficient mice, indicating that Ab-independent protection by CD4 effectors requires Pfn. Therefore, CD4 effectors mediate protection to lethal influenza through at least two mechanisms: Pfn-mediated cytotoxicity early in the response promoted survival independently of Ab production, whereas CD4-driven B cell responses resulted in high titer Abs that neutralized remaining virus.

Dual RNA-Sequencing of Eucalyptus nitens during Phytophthora cinnamomi Challenge Reveals Pathogen and Host Factors Influencing Compatibility

PubMed Central

Meyer, Febé E.; Shuey, Louise S.; Naidoo, Sitha; Mamni, Thandekile; Berger, Dave K.; Myburg, Alexander A.; van den Berg, Noëlani; Naidoo, Sanushka

2016-01-01

Damage caused by Phytophthora cinnamomi Rands remains an important concern on forest tree species. The pathogen causes root and collar rot, stem cankers, and dieback of various economically important Eucalyptus spp. In South Africa, susceptible cold tolerant Eucalyptus plantations have been affected by various Phytophthora spp. with P. cinnamomi considered one of the most virulent. The molecular basis of this compatible interaction is poorly understood. In this study, susceptible Eucalyptus nitens plants were stem inoculated with P. cinnamomi and tissue was harvested five days post inoculation. Dual RNA-sequencing, a technique which allows the concurrent detection of both pathogen and host transcripts during infection, was performed. Approximately 1% of the reads mapped to the draft genome of P. cinnamomi while 78% of the reads mapped to the Eucalyptus grandis genome. The highest expressed P. cinnamomi gene in planta was a putative crinkler effector (CRN1). Phylogenetic analysis indicated the high similarity of this P. cinnamomi CRN1 to that of Phytophthora infestans. Some CRN effectors are known to target host nuclei to suppress defense. In the host, over 1400 genes were significantly differentially expressed in comparison to mock inoculated trees, including suites of pathogenesis related (PR) genes. In particular, a PR-9 peroxidase gene with a high similarity to a Carica papaya PR-9 ortholog previously shown to be suppressed upon infection by Phytophthora palmivora was down-regulated two-fold. This PR-9 gene may represent a cross-species effector target during P. cinnamomi infection. This study identified pathogenicity factors, potential manipulation targets, and attempted host defense mechanisms activated by E. nitens that contributed to the susceptible outcome of the interaction. PMID:26973660

Transcription activation by NtcA and 2-oxoglutarate of three genes involved in heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120.

PubMed

Valladares, Ana; Flores, Enrique; Herrero, Antonia

2008-09-01

In Anabaena sp. strain PCC 7120, differentiation of heterocysts takes place in response to the external cue of combined nitrogen deprivation, allowing the organism to fix atmospheric nitrogen in oxic environments. NtcA, a global transcriptional regulator of cyanobacteria, is required for activation of the expression of multiple genes involved in heterocyst differentiation, including key regulators that are specific to the process. We have set up a fully defined in vitro system, which includes the purified Anabaena RNA polymerase, and have studied the effects of NtcA and its signaling effector 2-oxoglutarate on RNA polymerase binding, open complex formation, and transcript production from promoters of the hetC, nrrA, and devB genes that are activated by NtcA at different stages of heterocyst differentiation. Both RNA polymerase and NtcA could specifically bind to the target DNA in the absence of any effector. 2-Oxoglutarate had a moderate positive effect on NtcA binding, and NtcA had a limited positive effect on RNA polymerase recruitment at the promoters. However, a stringent requirement of both NtcA and 2-oxoglutarate was observed for the detection of open complexes and transcript production at the three investigated promoters. These results support a key role for 2-oxoglutarate in transcription activation in the developing heterocyst.

Trends in Regenerative Medicine: Repigmentation in Vitiligo Through Melanocyte Stem Cell Mobilization.

PubMed

Birlea, Stanca A; Costin, Gertrude-E; Roop, Dennis R; Norris, David A

2017-07-01

Vitiligo is the most frequent human pigmentary disorder, characterized by progressive autoimmune destruction of mature epidermal melanocytes. Of the current treatments offering partial and temporary relief, ultraviolet (UV) light is the most effective, coordinating an intricate network of keratinocyte and melanocyte factors that control numerous cellular and molecular signaling pathways. This UV-activated process is a classic example of regenerative medicine, inducing functional melanocyte stem cell populations in the hair follicle to divide, migrate, and differentiate into mature melanocytes that regenerate the epidermis through a complex process involving melanocytes and other cell lineages in the skin. Using an in-depth correlative analysis of multiple experimental and clinical data sets, we generated a modern molecular research platform that can be used as a working model for further research of vitiligo repigmentation. Our analysis emphasizes the active participation of defined molecular pathways that regulate the balance between stemness and differentiation states of melanocytes and keratinocytes: p53 and its downstream effectors controlling melanogenesis; Wnt/β-catenin with proliferative, migratory, and differentiation roles in different pigmentation systems; integrins, cadherins, tetraspanins, and metalloproteinases, with promigratory effects on melanocytes; TGF-β and its effector PAX3, which control differentiation. Our long-term goal is to design pharmacological compounds that can specifically activate melanocyte precursors in the hair follicle in order to obtain faster, better, and durable repigmentation. © 2016 Wiley Periodicals, Inc.

Trends in Regenerative Medicine: Repigmentation in Vitiligo Through Melanocyte Stem Cell Mobilization

PubMed Central

Birlea, Stanca A.; Costin, Gertrude-E.; Roop, Dennis R.; Norris, David A.

2017-01-01

Vitiligo is the most frequent human pigmentary disorder, characterized by progressive autoimmune destruction of mature epidermal melanocytes. Of the current treatments offering partial and temporary relief, ultraviolet (UV) light is the most effective, coordinating an intricate network of keratinocyte and melanocyte factors that control numerous cellular and molecular signaling pathways. This UV-activated process is a classic example of regenerative medicine, inducing functional melanocyte stem cell populations in the hair follicle to divide, migrate, and differentiate into mature melanocytes that regenerate the epidermis through a complex process involving melanocytes and other cell lineages in the skin. Using an in-depth correlative analysis of multiple experimental and clinical data sets, we generated a modern molecular research platform that can be used as a working model for further research of vitiligo repigmentation. Our analysis emphasizes the active participation of defined molecular pathways that regulate the balance between stemness and differentiation states of melanocytes and keratinocytes: p53 and its downstream effectors controlling melanogenesis; Wnt/β-catenin with proliferative, migratory, and differentiation roles in different pigmentation systems; integrins, cadherins, tetraspanins, and metalloproteinases, with promigratory effects on melanocytes; TGF-β and its effector PAX3, which control differentiation. Our long-term goal is to design pharmacological compounds that can specifically activate melanocyte precursors in the hair follicle in order to obtain faster, better, and durable repigmentation. PMID:28029168

Leptin directly promotes T-cell glycolytic metabolism to drive effector T-cell differentiation in a mouse model of autoimmunity.

PubMed

Gerriets, Valerie A; Danzaki, Keiko; Kishton, Rigel J; Eisner, William; Nichols, Amanda G; Saucillo, Donte C; Shinohara, Mari L; MacIver, Nancie J

2016-08-01

Upon activation, T cells require energy for growth, proliferation, and function. Effector T (Teff) cells, such as Th1 and Th17 cells, utilize high levels of glycolytic metabolism to fuel proliferation and function. In contrast, Treg cells require oxidative metabolism to fuel suppressive function. It remains unknown how Teff/Treg-cell metabolism is altered when nutrients are limited and leptin levels are low. We therefore examined the role of malnutrition and associated hypoleptinemia on Teff versus Treg cells. We found that both malnutrition-associated hypoleptinemia and T cell-specific leptin receptor knockout suppressed Teff-cell number, function, and glucose metabolism, but did not alter Treg-cell metabolism or suppressive function. Using the autoimmune mouse model EAE, we confirmed that fasting-induced hypoleptinemia altered Teff-cell, but not Treg-cell, glucose metabolism, and function in vivo, leading to decreased disease severity. To explore potential mechanisms, we examined HIF-1α, a key regulator of Th17 differentiation and Teff-cell glucose metabolism, and found HIF-1α expression was decreased in T cell-specific leptin receptor knockout Th17 cells, and in Teff cells from fasted EAE mice, but was unchanged in Treg cells. Altogether, these data demonstrate a selective, cell-intrinsic requirement for leptin to upregulate glucose metabolism and maintain function in Teff, but not Treg cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Vitamin C treatment of mouse bone marrow-derived dendritic cells enhanced CD8(+) memory T cell production capacity of these cells in vivo.

PubMed

Jeong, Young-Joo; Kim, Jin-Hee; Hong, Jun-Man; Kang, Jae Seung; Kim, Hang-Rae; Lee, Wang Jae; Hwang, Young-il

2014-07-01

Vitamin C has been found to stimulate dendritic cells (DCs) to secrete more IL-12 and thereby drive naïve CD4(+) T cells to differentiate into Th1 cells. In the present study, we evaluated the effect of these vitamin C-treated DCs on CD8(+) T cell differentiation both in vitro and in vivo. Mouse bone marrow-derived DCs were prepared in the presence of GM-CSF and IL-15. With vitamin C treatment, these DCs, when LPS-stimulated, secreted more IL-12p70 and IL-15 than did untreated DCs. And when co-cultured with T cells, they yielded a higher frequency of IFN-γ(+) CD8(+) T cells. Moreover, we found that administering vitamin C-treated and tumor lysate-loaded DCs into mice yielded a higher frequency of CD44(high) CD62L(low) CD8(+) effector and effector memory T cells, which showed an increased ex vivo killing effect of the tumor cells. These DCs also elicited enhanced protective effects against inoculated tumor cells, most probably by way of the increased cytotoxic T cells, as was revealed by the decreased growth of the inoculated tumor cells in these mice. This ex vivo vitamin C treatment effect on DCs can be considered as a strategy for boosting DC vaccination potency against tumors. Copyright © 2014 Elsevier GmbH. All rights reserved.

DEEP--a tool for differential expression effector prediction.

PubMed

Degenhardt, Jost; Haubrock, Martin; Dönitz, Jürgen; Wingender, Edgar; Crass, Torsten

2007-07-01

High-throughput methods for measuring transcript abundance, like SAGE or microarrays, are widely used for determining differences in gene expression between different tissue types, dignities (normal/malignant) or time points. Further analysis of such data frequently aims at the identification of gene interaction networks that form the causal basis for the observed properties of the systems under examination. To this end, it is usually not sufficient to rely on the measured gene expression levels alone; rather, additional biological knowledge has to be taken into account in order to generate useful hypotheses about the molecular mechanism leading to the realization of a certain phenotype. We present a method that combines gene expression data with biological expert knowledge on molecular interaction networks, as described by the TRANSPATH database on signal transduction, to predict additional--and not necessarily differentially expressed--genes or gene products which might participate in processes specific for either of the examined tissues or conditions. In a first step, significance values for over-expression in tissue/condition A or B are assigned to all genes in the expression data set. Genes with a significance value exceeding a certain threshold are used as starting points for the reconstruction of a graph with signaling components as nodes and signaling events as edges. In a subsequent graph traversal process, again starting from the previously identified differentially expressed genes, all encountered nodes 'inherit' all their starting nodes' significance values. In a final step, the graph is visualized, the nodes being colored according to a weighted average of their inherited significance values. Each node's, or sub-network's, predominant color, ranging from green (significant for tissue/condition A) over yellow (not significant for either tissue/condition) to red (significant for tissue/condition B), thus gives an immediate visual clue on which molecules--differentially expressed or not--may play pivotal roles in the tissues or conditions under examination. The described method has been implemented in Java as a client/server application and a web interface called DEEP (Differential Expression Effector Prediction). The client, which features an easy-to-use graphical interface, can freely be downloaded from the following URL: http://deep.bioinf.med.uni-goettingen.de.

CCR6 and NK1.1 distinguish between IL-17A and IFN-gamma-producing gammadelta effector T cells.

PubMed

Haas, Jan D; González, Frano H Malinarich; Schmitz, Susanne; Chennupati, Vijaykumar; Föhse, Lisa; Kremmer, Elisabeth; Förster, Reinhold; Prinz, Immo

2009-12-01

Gammadelta T cells are a potent source of innate IL-17A and IFN-gamma, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24(low) CD44(high) effector gammadelta T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6+ gammadelta T cells produced IL-17A, while NK1.1+ gammadelta T cells were efficient producers of IFN-gamma but not of IL-17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1+ gammadelta T cells. Accordingly, both gammadelta T-cell subsets were rare in gut-associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL-17A and IFN-gamma in response to TCR-specific and TCR-independent stimuli. IL-12 and IL-18 induced IFN-gamma and IL-23 induced IL-17A production by NK1.1+ or CCR6+ gammadelta T cells, respectively. Importantly, we show that CCR6+ gammadelta T cells are more responsive to TCR stimulation than their NK1.1+ counterparts. In conclusion, our findings support the hypothesis that CCR6+ IL-17A-producing gammadelta T cells derive from less TCR-dependent selection events than IFN-gamma-producing NK1.1+ gammadelta T cells.

GLI1 inhibition promotes epithelial-to-mesenchymal transition in pancreatic cancer cells

PubMed Central

Joost, Simon; Almada, Luciana L.; Rohnalter, Verena; Holz, Philipp S.; Vrabel, Anne M.; Fernandez-Barrena, Maite G.; McWilliams, Robert R.; Krause, Michael; Fernandez-Zapico, Martin E.; Lauth, Matthias

2011-01-01

The Hedgehog (HH) pathway has been identified as an important deregulated signal transduction pathway in pancreatic ductal adenocarcinoma (PDAC), a cancer type characterized by a highly metastatic phenotype. In PDAC, the canonical HH pathway activity is restricted to the stromal compartment while HH signaling in the tumor cells is reduced as a consequence of constitutive KRAS activation. Here we report that in the tumor compartment of PDAC the HH pathway effector transcription factor GLI1 regulates epithelial differentiation. RNAi-mediated knockdown of GLI1 abolished characteristics of epithelial differentiation, increased cell motility and synergized with TGFβ to induce an epithelial-to-mesenchymal transition (EMT). Notably, EMT conversion in PDAC cells occurred in the absence of induction of SNAIL or SLUG, two canonical inducers of EMT in many other settings. Further mechanistic analysis revealed that GLI1 directly regulated the transcription of E-cadherin, a key determinant of epithelial tissue organization. Collectively, our findings identify GLI1 as an important positive regulator of epithelial differentiation, and they offer an explanation for how decreased levels of GLI1 are likely to contribute to the highly metastatic phenotype of PDAC. PMID:22086851

Divide, Conquer, and Sense: CD8+CD28− T Cells in Perspective

PubMed Central

Arosa, Fernando A.; Esgalhado, André J.; Padrão, Carolina A.; Cardoso, Elsa M.

2017-01-01

Understanding the rationale for the generation of a pool of highly differentiated effector memory CD8+ T cells displaying a weakened capacity to scrutinize for peptides complexed with major histocompatibility class I molecules via their T cell receptor, lacking the “signal 2” CD28 receptor, and yet expressing a highly diverse array of innate receptors, from natural killer receptors, interleukin receptors, and damage-associated molecular pattern receptors, among others, is one of the most challenging issues in contemporary human immunology. The prevalence of these differentiated CD8+ T cells, also known as CD8+CD28−, CD8+KIR+, NK-like CD8+ T cells, or innate CD8+ T cells, in non-lymphoid organs and tissues, in peripheral blood of healthy elderly, namely centenarians, but also in stressful and chronic inflammatory conditions suggests that they are not merely end-of-the-line dysfunctional cells. These experienced CD8+ T cells are highly diverse and capable of sensing a variety of TCR-independent signals, which enables them to respond and fine-tune tissue homeostasis. PMID:28096804

Divide, Conquer, and Sense: CD8+CD28- T Cells in Perspective.

PubMed

Arosa, Fernando A; Esgalhado, André J; Padrão, Carolina A; Cardoso, Elsa M

2016-01-01

Understanding the rationale for the generation of a pool of highly differentiated effector memory CD8 + T cells displaying a weakened capacity to scrutinize for peptides complexed with major histocompatibility class I molecules via their T cell receptor, lacking the "signal 2" CD28 receptor, and yet expressing a highly diverse array of innate receptors, from natural killer receptors, interleukin receptors, and damage-associated molecular pattern receptors, among others, is one of the most challenging issues in contemporary human immunology. The prevalence of these differentiated CD8 + T cells, also known as CD8 + CD28 - , CD8 + KIR + , NK-like CD8 + T cells, or innate CD8 + T cells, in non-lymphoid organs and tissues, in peripheral blood of healthy elderly, namely centenarians, but also in stressful and chronic inflammatory conditions suggests that they are not merely end-of-the-line dysfunctional cells. These experienced CD8 + T cells are highly diverse and capable of sensing a variety of TCR-independent signals, which enables them to respond and fine-tune tissue homeostasis.

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence.

PubMed

Rodriguez, Patricia A; Escudero-Martinez, Carmen; Bos, Jorunn I B

2017-03-01

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M persicae -host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. © 2017 American Society of Plant Biologists. All Rights Reserved.

Prevention of tuberculosis in rhesus macaques by a cytomegalovirus-based vaccine.

PubMed

Hansen, Scott G; Zak, Daniel E; Xu, Guangwu; Ford, Julia C; Marshall, Emily E; Malouli, Daniel; Gilbride, Roxanne M; Hughes, Colette M; Ventura, Abigail B; Ainslie, Emily; Randall, Kurt T; Selseth, Andrea N; Rundstrom, Parker; Herlache, Lauren; Lewis, Matthew S; Park, Haesun; Planer, Shannon L; Turner, John M; Fischer, Miranda; Armstrong, Christina; Zweig, Robert C; Valvo, Joseph; Braun, Jackie M; Shankar, Smitha; Lu, Lenette; Sylwester, Andrew W; Legasse, Alfred W; Messerle, Martin; Jarvis, Michael A; Amon, Lynn M; Aderem, Alan; Alter, Galit; Laddy, Dominick J; Stone, Michele; Bonavia, Aurelio; Evans, Thomas G; Axthelm, Michael K; Früh, Klaus; Edlefsen, Paul T; Picker, Louis J

2018-02-01

Despite widespread use of the bacille Calmette-Guérin (BCG) vaccine, tuberculosis (TB) remains a leading cause of global mortality from a single infectious agent (Mycobacterium tuberculosis or Mtb). Here, over two independent Mtb challenge studies, we demonstrate that subcutaneous vaccination of rhesus macaques (RMs) with rhesus cytomegalovirus vectors encoding Mtb antigen inserts (hereafter referred to as RhCMV/TB)-which elicit and maintain highly effector-differentiated, circulating and tissue-resident Mtb-specific CD4 + and CD8 + memory T cell responses-can reduce the overall (pulmonary and extrapulmonary) extent of Mtb infection and disease by 68%, as compared to that in unvaccinated controls, after intrabronchial challenge with the Erdman strain of Mtb at ∼1 year after the first vaccination. Fourteen of 34 RhCMV/TB-vaccinated RMs (41%) across both studies showed no TB disease by computed tomography scans or at necropsy after challenge (as compared to 0 of 17 unvaccinated controls), and ten of these RMs were Mtb-culture-negative for all tissues, an exceptional long-term vaccine effect in the RM challenge model with the Erdman strain of Mtb. These results suggest that complete vaccine-mediated immune control of highly pathogenic Mtb is possible if immune effector responses can intercept Mtb infection at its earliest stages.

Developmental Origin Governs CD8+ T Cell Fate Decisions during Infection.

PubMed

Smith, Norah L; Patel, Ravi K; Reynaldi, Arnold; Grenier, Jennifer K; Wang, Jocelyn; Watson, Neva B; Nzingha, Kito; Yee Mon, Kristel J; Peng, Seth A; Grimson, Andrew; Davenport, Miles P; Rudd, Brian D

2018-06-06

Heterogeneity is a hallmark feature of the adaptive immune system in vertebrates. Following infection, naive T cells differentiate into various subsets of effector and memory T cells, which help to eliminate pathogens and maintain long-term immunity. The current model suggests there is a single lineage of naive T cells that give rise to different populations of effector and memory T cells depending on the type and amounts of stimulation they encounter during infection. Here, we have discovered that multiple sub-populations of cells exist in the naive CD8 + T cell pool that are distinguished by their developmental origin, unique transcriptional profiles, distinct chromatin landscapes, and different kinetics and phenotypes after microbial challenge. These data demonstrate that the naive CD8 + T cell pool is not as homogeneous as previously thought and offers a new framework for explaining the remarkable heterogeneity in the effector and memory T cell subsets that arise after infection. Copyright © 2018 Elsevier Inc. All rights reserved.

Assembly-directed antivirals differentially bind quasiequivalent pockets to modify hepatitis B virus capsid tertiary and quaternary structure.

PubMed

Katen, Sarah P; Tan, Zhenning; Chirapu, Srinivas Reddy; Finn, M G; Zlotnick, Adam

2013-08-06

Hepatitis B virus (HBV) is a major cause of liver disease. Assembly of the HBV capsid is a critical step in virus production and an attractive target for new antiviral therapies. We determined the structure of HBV capsid in complex with AT-130, a member of the phenylpropenamide family of assembly effectors. AT-130 causes tertiary and quaternary structural changes but does not disrupt capsid structure. AT-130 binds a hydrophobic pocket that also accommodates the previously characterized heteroaryldihydropyrimidine compounds but favors a unique quasiequivalent location on the capsid surface. Thus, this pocket is a promiscuous drug-binding site and a likely target for different assembly effectors with a broad range of mechanisms of activity. That AT-130 successfully decreases virus production by increasing capsid assembly rate without disrupting capsid structure delineates a paradigm in antiviral design, that disrupting reaction timing is a viable strategy for assembly effectors of HBV and other viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Transcription Factor Foxo1 Is a Negative Regulator of NK Cell Maturation and Function

PubMed Central

Deng, Youcai; Kerdiles, Yann; Chu, Jianhong; Yuan, Shunzong; Wang, Youwei; Chen, Xilin; Mao, Hsiaoyin; Zhang, Lingling; Zhang, Jianying; Hughes, Tiffany; Deng, Yafei; Zhang, Qi; Wang, Fangjie; Zou, Xianghong; Liu, Chang-Gong; Freud, Aharon G.; Li, Xiaohui; Caligiuri, Michael A; Vivier, Eric; Yu, Jianhua

2015-01-01

SUMMARY Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes through upregulating CD62L expression, and impaired late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21+/− mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions. PMID:25769609

The Rab27a effector exophilin7 promotes fusion of secretory granules that have not been docked to the plasma membrane.

PubMed

Wang, Hao; Ishizaki, Ray; Xu, Jun; Kasai, Kazuo; Kobayashi, Eri; Gomi, Hiroshi; Izumi, Tetsuro

2013-02-01

Granuphilin, an effector of the small GTPase Rab27a, mediates the stable attachment (docking) of insulin granules to the plasma membrane and inhibits subsequent fusion of docked granules, possibly through interaction with a fusion-inhibitory Munc18-1/syntaxin complex. However, phenotypes of insulin exocytosis differ considerably between Rab27a- and granuphilin-deficient pancreatic β cells, suggesting that other Rab27a effectors function in those cells. We found that one of the putative Rab27a effector family proteins, exophilin7/JFC1/Slp1, is expressed in β cells; however, unlike granuphilin, exophilin7 overexpressed in the β-cell line MIN6 failed to show granule-docking or fusion-inhibitory activity. Furthermore, exophilin7 has no affinities to either Munc18-1 or Munc18-1-interacting syntaxin-1a, in contrast to granuphilin. Although β cells of exophilin7-knockout mice show no apparent abnormalities in intracellular distribution or in ordinary glucose-induced exocytosis of insulin granules, they do show impaired fusion in response to some stronger stimuli, specifically from granules that have not been docked to the plasma membrane. Exophilin7 appears to mediate the fusion of undocked granules through the affinity of its C2A domain toward the plasma membrane phospholipids. These findings indicate that the two Rab27a effectors, granuphilin and exophilin7, differentially regulate the exocytosis of either stably or minimally docked granules, respectively.

Immune Cell Metabolism in Systemic Lupus Erythematosus.

PubMed

Choi, Seung-Chul; Titov, Anton A; Sivakumar, Ramya; Li, Wei; Morel, Laurence

2016-11-01

Cellular metabolism represents a newly identified checkpoint of effector functions in the immune system. A solid body of work has characterized the metabolic requirements of normal T cells during activation and differentiation into polarized effector subsets. Similar studies have been initiated to characterize the metabolic requirements for B cells and myeloid cells. Only a few studies though have characterized the metabolism of immune cells in the context of autoimmune diseases. Here, we review what is known on the altered metabolic patterns of CD4 + T cells, B cells, and myeloid cells in lupus patients and lupus-prone mice and how they contribute to lupus pathogenesis. We also discuss how defects in immune metabolism in lupus can be targeted therapeutically.

Akt signaling is critical for memory CD8+ T-cell development and tumor immune surveillance.

PubMed

Rogel, Anne; Willoughby, Jane E; Buchan, Sarah L; Leonard, Henry J; Thirdborough, Stephen M; Al-Shamkhani, Aymen

2017-02-14

Memory CD8 + T cells confer long-term immunity against tumors, and anticancer vaccines therefore should maximize their generation. Multiple memory CD8 + T-cell subsets with distinct functional and homing characteristics exist, but the signaling pathways that regulate their development are ill defined. Here we examined the role of the serine/threonine kinase Akt in the generation of protective immunity by CD8 + T cells. Akt is known to be activated by the T-cell antigen receptor and the cytokine IL-2, but its role in T-cell immunity in vivo has not been explored. Using CD8 + T cells from pdk1 K465E/K465E knockin mice, we found that decreased Akt activity inhibited the survival of T cells during the effector-to-memory cell transition and abolished their differentiation into C-X-C chemokine receptor 3 (CXCR3) lo CD43 lo effector-like memory cells. Consequently, antitumor immunity by CD8 + T cells that display defective Akt signaling was substantially diminished during the memory phase. Reduced memory T-cell survival and altered memory cell differentiation were associated with up-regulation of the proapoptotic protein Bim and the T-box transcription factor eomesodermin, respectively. These findings suggest an important role for effector-like memory CD8 + T cells in tumor immune surveillance and identify Akt as a key signaling node in the development of protective memory CD8 + T-cell responses.

Akt signaling is critical for memory CD8+ T-cell development and tumor immune surveillance

PubMed Central

Rogel, Anne; Willoughby, Jane E.; Buchan, Sarah L.; Leonard, Henry J.; Thirdborough, Stephen M.; Al-Shamkhani, Aymen

2017-01-01

Memory CD8+ T cells confer long-term immunity against tumors, and anticancer vaccines therefore should maximize their generation. Multiple memory CD8+ T-cell subsets with distinct functional and homing characteristics exist, but the signaling pathways that regulate their development are ill defined. Here we examined the role of the serine/threonine kinase Akt in the generation of protective immunity by CD8+ T cells. Akt is known to be activated by the T-cell antigen receptor and the cytokine IL-2, but its role in T-cell immunity in vivo has not been explored. Using CD8+ T cells from pdk1K465E/K465E knockin mice, we found that decreased Akt activity inhibited the survival of T cells during the effector-to-memory cell transition and abolished their differentiation into C-X-C chemokine receptor 3 (CXCR3)loCD43lo effector-like memory cells. Consequently, antitumor immunity by CD8+ T cells that display defective Akt signaling was substantially diminished during the memory phase. Reduced memory T-cell survival and altered memory cell differentiation were associated with up-regulation of the proapoptotic protein Bim and the T-box transcription factor eomesodermin, respectively. These findings suggest an important role for effector-like memory CD8+ T cells in tumor immune surveillance and identify Akt as a key signaling node in the development of protective memory CD8+ T-cell responses. PMID:28137869

Follicular helper T cell in immunity and autoimmunity.

PubMed

Mesquita, D; Cruvinel, W M; Resende, L S; Mesquita, F V; Silva, N P; Câmara, N O S; Andrade, L E C

2016-01-01

The traditional concept that effector T helper (Th) responses are mediated by Th1/Th2 cell subtypes has been broadened by the recent demonstration of two new effector T helper cells, the IL-17 producing cells (Th17) and the follicular helper T cells (Tfh). These new subsets have many features in common, such as the ability to produce IL-21 and to express the IL-23 receptor (IL23R), the inducible co-stimulatory molecule ICOS, and the transcription factor c-Maf, all of them essential for expansion and establishment of the final pool of both subsets. Tfh cells differ from Th17 by their ability to home to B cell areas in secondary lymphoid tissue through interactions mediated by the chemokine receptor CXCR5 and its ligand CXCL13. These CXCR5+ CD4+ T cells are considered an effector T cell type specialized in B cell help, with a transcriptional profile distinct from Th1 and Th2 cells. The role of Tfh cells and its primary product, IL-21, on B-cell activation and differentiation is essential for humoral immunity against infectious agents. However, when deregulated, Tfh cells could represent an important mechanism contributing to exacerbated humoral response and autoantibody production in autoimmune diseases. This review highlights the importance of Tfh cells by focusing on their biology and differentiation processes in the context of normal immune response to infectious microorganisms and their role in the pathogenesis of autoimmune diseases.

mTOR Complex 1 Signaling Regulates the Generation and Function of Central and Effector Foxp3+ Regulatory T Cells.

PubMed

Sun, Im-Hong; Oh, Min-Hee; Zhao, Liang; Patel, Chirag H; Arwood, Matthew L; Xu, Wei; Tam, Ada J; Blosser, Richard L; Wen, Jiayu; Powell, Jonathan D

2018-06-08

The mechanistic/mammalian target of rapamycin (mTOR) has emerged as a critical integrator of signals from the immune microenvironment capable of regulating T cell activation, differentiation, and function. The precise role of mTOR in the control of regulatory T cell (Treg) differentiation and function is complex. Pharmacologic inhibition and genetic deletion of mTOR promotes the generation of Tregs even under conditions that would normally promote generation of effector T cells. Alternatively, mTOR activity has been observed to be increased in Tregs, and the genetic deletion of the mTOR complex 1 (mTORC1)-scaffold protein Raptor inhibits Treg function. In this study, by employing both pharmacologic inhibitors and genetically altered T cells, we seek to clarify the role of mTOR in Tregs. Our studies demonstrate that inhibition of mTOR during T cell activation promotes the generation of long-lived central Tregs with a memory-like phenotype in mice. Metabolically, these central memory Tregs possess enhanced spare respiratory capacity, similar to CD8 + memory cells. Alternatively, the generation of effector Tregs (eTregs) requires mTOR function. Indeed, genetic deletion of Rptor leads to the decreased expression of ICOS and PD-1 on the eTregs. Overall, our studies define a subset of mTORC1 hi eTregs and mTORC1 lo central Tregs. Copyright © 2018 by The American Association of Immunologists, Inc.

High immunosuppressive burden in advanced hepatocellular carcinoma patients: Can effector functions be restored?

PubMed

Lugade, Amit A; Kalathil, Suresh; Miller, Austin; Iyer, Renuka; Thanavala, Yasmin

2013-07-01

The accumulation of immunosuppressive cells and exhausted effector T cells highlight an important immune dysfunction in advanced stage hepatocellular carcinoma (HCC) patients. These cells significantly hamper the efficacy immunotherapies and facilitate HCC progression. We have recently demonstrated that the multipronged depletion of immunosuppressive cells potentially restores effector T-cell function in HCC.

BET bromodomain inhibition enhances T cell persistence and function in adoptive immunotherapy models.

PubMed

Kagoya, Yuki; Nakatsugawa, Munehide; Yamashita, Yuki; Ochi, Toshiki; Guo, Tingxi; Anczurowski, Mark; Saso, Kayoko; Butler, Marcus O; Arrowsmith, Cheryl H; Hirano, Naoto

2016-09-01

Adoptive immunotherapy is a potentially curative therapeutic approach for patients with advanced cancer. However, the in vitro expansion of antitumor T cells prior to infusion inevitably incurs differentiation towards effector T cells and impairs persistence following adoptive transfer. Epigenetic profiles regulate gene expression of key transcription factors over the course of immune cell differentiation, proliferation, and function. Using comprehensive screening of chemical probes with defined epigenetic targets, we found that JQ1, an inhibitor of bromodomain and extra-terminal motif (BET) proteins, maintained CD8+ T cells with functional properties of stem cell-like and central memory T cells. Mechanistically, the BET protein BRD4 directly regulated expression of the transcription factor BATF in CD8+ T cells, which was associated with differentiation of T cells into an effector memory phenotype. JQ1-treated T cells showed enhanced persistence and antitumor effects in murine T cell receptor and chimeric antigen receptor gene therapy models. Furthermore, we found that histone acetyltransferase p300 supported the recruitment of BRD4 to the BATF promoter region, and p300 inhibition similarly augmented antitumor effects of the adoptively transferred T cells. These results demonstrate that targeting the BRD4-p300 signaling cascade supports the generation of superior antitumor T cell grafts for adoptive immunotherapy.

Evaluation of Late Effects of Heavy-Ion Radiation on Mesenchymal Stem Cells

NASA Technical Reports Server (NTRS)

Gonda, S.R.; Behravesh, E.; Huff, J.L.; Johnson, F.

2005-01-01

The overall objective of this recently funded study is to utilize well-characterized model test systems to assess the impact of pluripotent stem cell differentiation on biological effects associated with high-energy charged particle radiation. These stem cells, specifically mesenchymal stem cells (MSCs), have the potential for differentiation into bone, cartilage, fat, tendons, and other tissue types. The characterization of the regulation mechanisms of MSC differentiation to the osteoblastic lineage by transcription factors, such as Runx2/Cbfa1 and Osterix, and osteoinductive proteins such as members of the bone morphogenic protein family are well established. More importantly, for late biological effects, MSCs have been shown to contribute to tissue restructuring and repair after tissue injury. The complex regulation of and interactions between inflammation and repair determine the eventual outcome of the responses to tissue injury, for which MSCs play a crucial role. Additionally, MSCs have been shown to respond to reactive oxygen species, a secondary effector of radiation, by differentiating. With this, we hypothesized that differentiation of MSCs can alter or exacerbate the damage initiated by radiation, which can ultimately lead to late biological effects of misrepair/fibrosis which may ultimately lead to carcinogenesis. Currently, studies are underway to examine high-energy X-ray radiation at low and high doses, approximately 20 and 200 Rad, respectively, on cytogenetic damage and gene modulation of isolated MSCs. These cells, positive for MSC surface markers, were obtained from three persons. In vitro cell samples were harvested during cellular proliferation and after both cellular recovery and differentiation. Future work will use established in vitro models of increasing complexity to examine the value of traditional 2D tissue-culture techniques, and utilize 3D in vitro tissue culture techniques that can better assess late effects associated with radiation.

The Genome Biology of Effector Gene Evolution in Filamentous Plant Pathogens.

PubMed

Sánchez-Vallet, Andrea; Fouché, Simone; Fudal, Isabelle; Hartmann, Fanny E; Soyer, Jessica L; Tellier, Aurélien; Croll, Daniel

2018-05-16

Filamentous pathogens, including fungi and oomycetes, pose major threats to global food security. Crop pathogens cause damage by secreting effectors that manipulate the host to the pathogen's advantage. Genes encoding such effectors are among the most rapidly evolving genes in pathogen genomes. Here, we review how the major characteristics of the emergence, function, and regulation of effector genes are tightly linked to the genomic compartments where these genes are located in pathogen genomes. The presence of repetitive elements in these compartments is associated with elevated rates of point mutations and sequence rearrangements with a major impact on effector diversification. The expression of many effectors converges on an epigenetic control mediated by the presence of repetitive elements. Population genomics analyses showed that rapidly evolving pathogens show high rates of turnover at effector loci and display a mosaic in effector presence-absence polymorphism among strains. We conclude that effective pathogen containment strategies require a thorough understanding of the effector genome biology and the pathogen's potential for rapid adaptation. Expected final online publication date for the Annual Review of Phytopathology Volume 56 is August 25, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Differential association of GABAB receptors with their effector ion channels in Purkinje cells.

PubMed

Luján, Rafael; Aguado, Carolina; Ciruela, Francisco; Cózar, Javier; Kleindienst, David; de la Ossa, Luis; Bettler, Bernhard; Wickman, Kevin; Watanabe, Masahiko; Shigemoto, Ryuichi; Fukazawa, Yugo

2018-04-01

Metabotropic GABA B receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABA B receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABA B1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABA B receptors with two key effector ion channels, the G protein-gated inwardly rectifying K + (GIRK/Kir3) channel and the voltage-dependent Ca 2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABA B receptors co-assembled with GIRK and Ca V 2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABA B1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABA B1 and Ca V 2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABA B1 and GIRK2 or Ca V 2.1 channels was detected, inter-cluster distance for GABA B1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABA B1 and Ca V 2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABA B receptors are associated with GIRK and Ca V 2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABA B receptors and their effector ion channels in the cerebellar network.

Comparative genomics and prediction of conditionally dispensable sequences in legume-infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors.

PubMed

Williams, Angela H; Sharma, Mamta; Thatcher, Louise F; Azam, Sarwar; Hane, James K; Sperschneider, Jana; Kidd, Brendan N; Anderson, Jonathan P; Ghosh, Raju; Garg, Gagan; Lichtenzveig, Judith; Kistler, H Corby; Shea, Terrance; Young, Sarah; Buck, Sally-Anne G; Kamphuis, Lars G; Saxena, Rachit; Pande, Suresh; Ma, Li-Jun; Varshney, Rajeev K; Singh, Karam B

2016-03-05

Soil-borne fungi of the Fusarium oxysporum species complex cause devastating wilt disease on many crops including legumes that supply human dietary protein needs across many parts of the globe. We present and compare draft genome assemblies for three legume-infecting formae speciales (ff. spp.): F. oxysporum f. sp. ciceris (Foc-38-1) and f. sp. pisi (Fop-37622), significant pathogens of chickpea and pea respectively, the world's second and third most important grain legumes, and lastly f. sp. medicaginis (Fom-5190a) for which we developed a model legume pathosystem utilising Medicago truncatula. Focusing on the identification of pathogenicity gene content, we leveraged the reference genomes of Fusarium pathogens F. oxysporum f. sp. lycopersici (tomato-infecting) and F. solani (pea-infecting) and their well-characterised core and dispensable chromosomes to predict genomic organisation in the newly sequenced legume-infecting isolates. Dispensable chromosomes are not essential for growth and in Fusarium species are known to be enriched in host-specificity and pathogenicity-associated genes. Comparative genomics of the publicly available Fusarium species revealed differential patterns of sequence conservation across F. oxysporum formae speciales, with legume-pathogenic formae speciales not exhibiting greater sequence conservation between them relative to non-legume-infecting formae speciales, possibly indicating the lack of a common ancestral source for legume pathogenicity. Combining predicted dispensable gene content with in planta expression in the model legume-infecting isolate, we identified small conserved regions and candidate effectors, four of which shared greatest similarity to proteins from another legume-infecting ff. spp. We demonstrate that distinction of core and potential dispensable genomic regions of novel F. oxysporum genomes is an effective tool to facilitate effector discovery and the identification of gene content possibly linked to host specificity. While the legume-infecting isolates didn't share large genomic regions of pathogenicity-related content, smaller regions and candidate effector proteins were highly conserved, suggesting that they may play specific roles in inducing disease on legume hosts.

The metabolism of malate by cultured rat brain astrocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

McKenna, M.C.; Tildon, J.T.; Couto, R.

1990-12-01

Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-(U-14C)malate in rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasicmore » kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain.« less

A transcriptome-based model of central memory CD4 T cell death in HIV infection.

PubMed

Olvera-García, Gustavo; Aguilar-García, Tania; Gutiérrez-Jasso, Fany; Imaz-Rosshandler, Iván; Rangel-Escareño, Claudia; Orozco, Lorena; Aguilar-Delfín, Irma; Vázquez-Pérez, Joel A; Zúñiga, Joaquín; Pérez-Patrigeon, Santiago; Espinosa, Enrique

2016-11-22

Human central memory CD4 T cells are characterized by their capacity of proliferation and differentiation into effector memory CD4 T cells. Homeostasis of central memory CD4 T cells is considered a key factor sustaining the asymptomatic stage of Human Immunodeficiency Virus type 1 (HIV-1) infection, while progression to acquired immunodeficiency syndrome is imputed to central memory CD4 T cells homeostatic failure. We investigated if central memory CD4 T cells from patients with HIV-1 infection have a gene expression profile impeding proliferation and survival, despite their activated state. Using gene expression microarrays, we analyzed mRNA expression patterns in naive, central memory, and effector memory CD4 T cells from healthy controls, and naive and central memory CD4 T cells from patients with HIV-1 infection. Differentially expressed genes, defined by Log 2 Fold Change (FC) ≥ |0.5| and Log (odds) > 0, were used in pathway enrichment analyses. Central memory CD4 T cells from patients and controls showed comparable expression of differentiation-related genes, ruling out an effector-like differentiation of central memory CD4 T cells in HIV infection. However, 210 genes were differentially expressed in central memory CD4 T cells from patients compared with those from controls. Expression of 75 of these genes was validated by semi quantitative RT-PCR, and independently reproduced enrichment results from this gene expression signature. The results of functional enrichment analysis indicated movement to cell cycle phases G1 and S (increased CCNE1, MKI67, IL12RB2, ADAM9, decreased FGF9, etc.), but also arrest in G2/M (increased CHK1, RBBP8, KIF11, etc.). Unexpectedly, the results also suggested decreased apoptosis (increased CSTA, NFKBIA, decreased RNASEL, etc.). Results also suggested increased IL-1β, IFN-γ, TNF, and RANTES (CCR5) activity upstream of the central memory CD4 T cells signature, consistent with the demonstrated milieu in HIV infection. Our findings support a model where progressive loss of central memory CD4 T cells in chronic HIV-1 infection is driven by increased cell cycle entry followed by mitotic arrest, leading to a non-apoptotic death pathway without actual proliferation, possibly contributing to increased turnover.

Low expression of CD39+/CD45RA+ on regulatory T cells (Treg) cells in type 1 diabetic children in contrast to high expression of CD101+/CD129+ on Treg cells in children with coeliac disease

PubMed Central

Åkesson, K; Tompa, A; Rydén, A; Faresjö, M

2015-01-01

Type 1 diabetes (T1D) and coeliac disease are both characterized by an autoimmune feature. As T1D and coeliac disease share the same risk genes, patients risk subsequently developing the other disease. This study aimed to investigate the expression of T helper (Th), T cytotoxic (Tc) and regulatory T cells (Treg) in T1D and/or coeliac disease children in comparison to healthy children. Subgroups of T cells (Th : CD4+ or Tc : CD8+); naive (CD27+CD28+CD45RA+CCR7+), central memory (CD27+CD28+CD45RA−CCR7+), effector memory (early differentiated; CD27+CD28+CD45RA−CCR7− and late differentiated; CD27−CD28−CD45RA−CCR7−), terminally differentiated effector cells (TEMRA; CD27−CD28−CD45RA+CCR7−) and Treg (CD4+CD25+FOXP3+CD127−) cells, and their expression of CD39, CD45RA, CD101 and CD129, were studied by flow cytometry in T1D and/or coeliac disease children or without any of these diseases (reference group). Children diagnosed with both T1D and coeliac disease showed a higher percentage of TEMRA CD4+ cells (P < 0·05), but lower percentages of both early and late effector memory CD8+ cells (P < 0·05) compared to references. Children with exclusively T1D had lower median fluorescence intensity (MFI) of forkhead box protein 3 (FoxP3) (P < 0·05) and also a lower percentage of CD39+ and CD45RA+ within the Treg population (CD4+CD25+FOXP3+CD127−) (P < 0·05). Children with exclusively coeliac disease had a higher MFI of CD101 (P < 0·01), as well as a higher percentage of CD129+ (P < 0·05), in the CD4+CD25hi lymphocyte population, compared to references. In conclusion, children with combined T1D and coeliac disease have a higher percentage of differentiated CD4+ cells compared to CD8+ cells. T1D children show signs of low CD39+/CD45RA+ Treg cells that may indicate loss of suppressive function. Conversely, children with coeliac disease show signs of CD101+/CD129+ Treg cells that may indicate suppressor activity. PMID:25421756

Wind Tunnel Test of an RPV with Shape-Change Control Effector and Sensor Arrays

NASA Technical Reports Server (NTRS)

Raney, David L.; Cabell, Randolph H.; Sloan, Adam R.; Barnwell, William G.; Lion, S. Todd; Hautamaki, Bret A.

2004-01-01

A variety of novel control effector concepts have recently emerged that may enable new approaches to flight control. In particular, the potential exists to shift the composition of the typical aircraft control effector suite from a small number of high authority, specialized devices (rudder, aileron, elevator, flaps), toward larger numbers of smaller, less specialized, distributed device arrays. The concept envisions effector and sensor networks composed of relatively small high-bandwidth devices able to simultaneously perform a variety of control functions using feedback from disparate data sources. To investigate this concept, a remotely piloted flight vehicle has been equipped with an array of 24 trailing edge shape-change effectors and associated pressure measurements. The vehicle, called the Multifunctional Effector and Sensor Array (MESA) testbed, was recently tested in NASA Langley's 12-ft Low Speed wind tunnel to characterize its stability properties, control authorities, and distributed pressure sensitivities for use in a dynamic simulation prior to flight testing. Another objective was to implement and evaluate a scheme for actively controlling the spanwise pressure distribution using the shape-change array. This report describes the MESA testbed, design of the pressure distribution controller, and results of the wind tunnel test.

The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaynes, J.B.; Johnson, J.E.; Buskin, J.N.

1988-01-01

Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. The authors examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer.more » This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.« less

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins.

PubMed

Pombo, Marina A; Zheng, Yi; Fernandez-Pozo, Noe; Dunham, Diane M; Fei, Zhangjun; Martin, Gregory B

2014-01-01

Plants have two related immune systems to defend themselves against pathogen attack. Initially,pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses. We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling. Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

The making of a lymphocyte: the choice among disparate cell fates and the IKAROS enigma.

PubMed

Georgopoulos, Katia

2017-03-01

Lymphocyte differentiation is set to produce myriad immune effector cells with the ability to respond to multitudinous foreign substances. The uniqueness of this developmental system lies in not only the great diversity of cellular functions that it can generate but also the ability of its differentiation intermediates and mature effector cells to expand upon demand, thereby providing lifelong immunity. Surprisingly, the goals of this developmental system are met by a relatively small group of DNA-binding transcription factors that work in concert to control the timing and magnitude of gene expression and fulfill the demands for cellular specialization, expansion, and maintenance. The cellular and molecular mechanisms through which these lineage-promoting transcription factors operate have been a focus of basic research in immunology. The mechanisms of development discerned in this effort are guiding clinical research on disorders with an immune cell base. Here, I focus on IKAROS, one of the earliest regulators of lymphoid lineage identity and a guardian of lymphocyte homeostasis. © 2017 Georgopoulos; Published by Cold Spring Harbor Laboratory Press.

MicroRNA-202 maintains spermatogonial stem cells by inhibiting cell cycle regulators and RNA binding proteins

PubMed Central

Chen, Jian; Cai, Tanxi; Zheng, Chunwei; Lin, Xiwen; Wang, Guojun; Liao, Shangying; Wang, Xiuxia; Gan, Haiyun; Zhang, Daoqin; Hu, Xiangjing; Wang, Si; Li, Zhen; Feng, Yanmin

2017-01-01

Abstract miRNAs play important roles during mammalian spermatogenesis. However, the function of most miRNAs in spermatogenesis and the underlying mechanisms remain unknown. Here, we report that miR-202 is highly expressed in mouse spermatogonial stem cells (SSCs), and is oppositely regulated by Glial cell-Derived Neurotrophic Factor (GDNF) and retinoic acid (RA), two key factors for SSC self-renewal and differentiation. We used inducible CRISPR-Cas9 to knockout miR-202 in cultured SSCs, and found that the knockout SSCs initiated premature differentiation accompanied by reduced stem cell activity and increased mitosis and apoptosis. Target genes were identified with iTRAQ-based proteomic analysis and RNA sequencing, and are enriched with cell cycle regulators and RNA-binding proteins. Rbfox2 and Cpeb1 were found to be direct targets of miR-202 and Rbfox2 but not Cpeb1, is essential for the differentiation of SSCs into meiotic cells. Accordingly, an SSC fate-regulatory network composed of signaling molecules of GDNF and RA, miR-202 and diverse downstream effectors has been identified. PMID:27998933

The effector candidate repertoire of the arbuscular mycorrhizal fungus Rhizophagus clarus.

PubMed

Sędzielewska Toro, Kinga; Brachmann, Andreas

2016-02-09

Arbuscular mycorrhizal fungi (AMF) form an ecologically important symbiosis with more than two thirds of studied land plants. Recent studies of plant-pathogen interactions showed that effector proteins play a key role in host colonization by controlling the plant immune system. We hypothesise that also for symbiotic-plant interactions the secreted effectome of the fungus is a major component of communication and the conservation level of effector proteins between AMF species may be indicative whether they play a fundamental role. In this study, we used a bioinformatics pipeline to predict and compare the effector candidate repertoire of the two AMF species, Rhizophagus irregularis and Rhizophagus clarus. Our in silico pipeline revealed a list of 220 R. irregularis candidate effector genes that create a valuable information source to elucidate the mechanism of plant infection and colonization by fungi during AMF symbiotic interaction. While most of the candidate effectors show no homologies to known domains or proteins, the candidates with homologies point to potential roles in signal transduction, cell wall modification or transcription regulation. A remarkable aspect of our work is presence of a large portion of the effector proteins involved in symbiosis, which are not unique to each fungi or plant species, but shared along the Glomeromycota phylum. For 95% of R. irregularis candidates we found homologs in a R. clarus genome draft generated by Illumina high-throughput sequencing. Interestingly, 9% of the predicted effectors are at least as conserved between the two Rhizophagus species as proteins with housekeeping functions (similarity > 90%). Therefore, we state that this group of highly conserved effector proteins between AMF species may play a fundamental role during fungus-plant interaction. We hypothesise that in symbiotic interactions the secreted effectome of the fungus might be an important component of communication. Identification and functional characterization of the primary AMF effectors that regulate symbiotic development will help in understanding the mechanisms of fungus-plant interaction.

Functionally Redundant RXLR Effectors from Phytophthora infestans Act at Different Steps to Suppress Early flg22-Triggered Immunity

PubMed Central

Fraiture, Malou; Liu, Xiaoyu; Boevink, Petra C.; Gilroy, Eleanor M.; Chen, Ying; Kandel, Kabindra; Sessa, Guido; Birch, Paul R. J.; Brunner, Frédéric

2014-01-01

Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular mechanisms underlying the manipulation of host MAMP-triggered immunity (MTI) by P. infestans and to understand the basis of host versus non-host resistance in plants towards P. infestans. PMID:24763622

Pak3 promotes cell cycle exit and differentiation of β-cells in the embryonic pancreas and is necessary to maintain glucose homeostasis in adult mice.

PubMed

Piccand, Julie; Meunier, Aline; Merle, Carole; Jia, Zhengping; Barnier, Jean-Vianney; Gradwohl, Gérard

2014-01-01

The transcription factor neurogenin3 (Ngn3) triggers islet cell differentiation in the developing pancreas. However, little is known about the molecular mechanisms coupling cell cycle exit and differentiation in Ngn3(+) islet progenitors. We identified a novel effector of Ngn3 endocrinogenic function, the p21 protein-activated kinase Pak3, known to control neuronal differentiation and implicated in X-linked intellectual disability in humans. We show that Pak3 expression is initiated in Ngn3(+) endocrine progenitor cells and next maintained in maturing hormone-expressing cells during pancreas development as well as in adult islet cells. In Pak3-deficient embryos, the proliferation of Ngn3(+) progenitors and β-cells is transiently increased concomitantly with an upregulation of Ccnd1. β-Cell differentiation is impaired at E15.5 but resumes at later stages. Pak3-deficient mice do not develop overt diabetes but are glucose intolerant under high-fat diet (HFD). In the intestine, Pak3 is expressed in enteroendocrine cells but is not necessary for their differentiation. Our results indicate that Pak3 is a novel regulator of β-cell differentiation and function. Pak3 acts downstream of Ngn3 to promote cell cycle exit and differentiation in the embryo by a mechanism that might involve repression of Ccnd1. In the adult, Pak3 is required for the proper control of glucose homeostasis under challenging HFD.

Transcriptional Regulation of Th17 Cell Differentiation

PubMed Central

Ivanov, Ivaylo I.; Zhou, Liang; Littman, Dan R.

2009-01-01

The paradigm of effector T helper cell differentiation into either Th1 or Th2 lineages has been profoundly shaken by the discovery of T cells that secrete IL-17 and other inflammatory cytokines. This subset, referred to as Th17, is centrally involved in autoimmune disease and is important in host defense at mucosal surfaces. In mouse, a series of cytokines, including IL-6, IL-21, IL-23, and TGF-β, function sequentially or synergistically to induce the Th17 lineage. Other cytokines, including IL-2, IL-4, IFNγ, and IL-27, inhibit differentiation of this lineage. Here we review how the nuclear orphan receptor RORγt functions to coordinate the diverse cytokine-induced signals and thus control Th17 cell differentiation. PMID:18053739

Gene targeting technologies in rats: zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats.

PubMed

Mashimo, Tomoji

2014-01-01

The laboratory rat has been widely used as an animal model in biomedical science for more than 150 years. Applying zinc-finger nucleases or transcription activator-like effector nucleases to rat embryos via microinjection is an efficient genome editing tool for generating targeted knockout rats. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonucleases have been used as an effective tool for precise and multiplex genome editing in mice and rats. In this review, the advantages and disadvantages of these site-specific nuclease technologies for genetic analysis and manipulation in rats are discussed. © 2013 The Author Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Regulation of Tissue Growth by the Mammalian Hippo Signaling Pathway

PubMed Central

Watt, Kevin I.; Harvey, Kieran F.; Gregorevic, Paul

2017-01-01

The integrative control of diverse biological processes such as proliferation, differentiation, apoptosis and metabolism is essential to maintain cellular and tissue homeostasis. Disruption of these underlie the development of many disease states including cancer and diabetes, as well as many of the complications that arise as a consequence of aging. These biological outputs are governed by many cellular signaling networks that function independently, and in concert, to convert changes in hormonal, mechanical and metabolic stimuli into alterations in gene expression. First identified in Drosophila melanogaster as a powerful mediator of cell division and apoptosis, the Hippo signaling pathway is a highly conserved regulator of mammalian organ size and functional capacity in both healthy and diseased tissues. Recent studies have implicated the pathway as an effector of diverse physiological cues demonstrating an essential role for the Hippo pathway as an integrative component of cellular homeostasis. In this review, we will: (a) outline the critical signaling elements that constitute the mammalian Hippo pathway, and how they function to regulate Hippo pathway-dependent gene expression and tissue growth, (b) discuss evidence that shows this pathway functions as an effector of diverse physiological stimuli and (c) highlight key questions in this developing field. PMID:29225579

Comparative phenotypic and functional analysis of migratory dendritic cell subsets from human oral mucosa and skin

PubMed Central

van de Ven, Rieneke; Thon, Maria; Gibbs, Susan; de Gruijl, Tanja D.

2017-01-01

Antigen exposure to oral mucosa is generally thought to lead to immune tolerance induction. However, very little is known about the subset composition and function of dendritic cells (DC) migrating from human oral mucosa. Here we show that migratory DC from healthy human gingival explants consist of the same phenotypic subsets in the same frequency distribution as DC migrating from human skin. The gingival CD1a+ Langerhans cell and interstitial DC subsets lacked CXCR4 expression in contrast to their cutaneous counterparts, pointing to different migration mechanisms, consistent with previous observations in constructed skin and gingival equivalents. Remarkably, without any exogenous conditioning, gingival explants released higher levels of inflammatory cytokines than human skin explants, resulting in higher DC migration rates and a superior ability of migrated DC to prime allogeneic T cells and to induce type-1 effector T cell differentiation. From these observations we conclude that rather than an intrinsic ability to induce T cell tolerance, DC migrating from oral mucosa may have a propensity to induce effector T cell immunity and maintain a high state of alert against possible pathogenic intruders in the steady state. These findings may have implications for oral immunization strategies. PMID:28704477

Effector Gene Suites in Some Soil Isolates of Fusarium oxysporum Are Not Sufficient Predictors of Vascular Wilt in Tomato.

PubMed

Jelinski, Nicolas A; Broz, Karen; Jonkers, Wilfried; Ma, Li-Jun; Kistler, H Corby

2017-07-01

Seventy-four Fusarium oxysporum soil isolates were assayed for known effector genes present in an F. oxysporum f. sp. lycopersici race 3 tomato wilt strain (FOL MN-25) obtained from the same fields in Manatee County, Florida. Based on the presence or absence of these genes, four haplotypes were defined, two of which represented 96% of the surveyed isolates. These two most common effector haplotypes contained either all or none of the assayed race 3 effector genes. We hypothesized that soil isolates with all surveyed effector genes, similar to FOL MN-25, would be pathogenic toward tomato, whereas isolates lacking all effectors would be nonpathogenic. However, inoculation experiments revealed that presence of the effector genes alone was not sufficient to ensure pathogenicity on tomato. Interestingly, a nonpathogenic isolate containing the full suite of unmutated effector genes (FOS 4-4) appears to have undergone a chromosomal rearrangement yet remains vegetatively compatible with FOL MN-25. These observations confirm the highly dynamic nature of the F. oxysporum genome and support the conclusion that pathogenesis among free-living populations of F. oxysporum is a complex process. Therefore, the presence of effector genes alone may not be an accurate predictor of pathogenicity among soil isolates of F. oxysporum.

YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity.

PubMed

Ma, Ka-Wai; Ma, Wenbo

2016-12-01

Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted "effector" proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Experimental study of trajectory planning and control of a high precision robot manipulator

NASA Technical Reports Server (NTRS)

Nguyen, Charles C.; Antrazi, Sami S.

1991-01-01

The kinematic and trajectory planning is presented for a 6 DOF end-effector whose design was based on the Stewart Platform mechanism. The end-effector was used as a testbed for studying robotic assembly of NASA hardware with passive compliance. Vector analysis was employed to derive a closed-form solution for the end-effector inverse kinematic transformation. A computationally efficient numerical solution was obtained for the end-effector forward kinematic transformation using Newton-Raphson method. Three trajectory planning schemes, two for fine motion and one for gross motion, were developed for the end-effector. Experiments conducted to evaluate the performance of the trajectory planning schemes showed excellent tracking quality with minimal errors. Current activities focus on implementing the developed trajectory planning schemes on mating and demating space-rated connectors and using the compliant platform to acquire forces/torques applied on the end-effector during the assembly task.

Five Xanthomonas type III effectors suppress cell death induced by components of immunity-associated MAP kinase cascades

PubMed Central

Teper, Doron; Sunitha, Sukumaran; Martin, Gregory B; Sessa, Guido

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in signaling of plant immunity and mediate elicitation of cell death. Xanthomonas spp. manipulate plant signaling by using a type III secretion system to deliver effector proteins into host cells. We examined the ability of 33 Xanthomonas effectors to inhibit cell death induced by overexpression of components of MAPK cascades in Nicotiana benthamiana plants. Five effectors inhibited cell death induced by overexpression of MAPKKKα and MEK2, but not of MAP3Kϵ. In addition, expression of AvrBs1 in yeast suppressed activation of the high osmolarity glycerol MAPK pathway, suggesting that the target of this effector is conserved in eukaryotic organisms. These results indicate that Xanthomonas employs several type III effectors to suppress immunity-associated cell death mediated by MAPK cascades. PMID:26237448

Flow cytometry analysis of T-cell subsets in cerebrospinal fluid of narcolepsy type 1 patients with long-lasting disease.

PubMed

Moresco, Monica; Lecciso, Mariangela; Ocadlikova, Darina; Filardi, Marco; Melzi, Silvia; Kornum, Birgitte Rahbek; Antelmi, Elena; Pizza, Fabio; Mignot, Emmanuel; Curti, Antonio; Plazzi, Giuseppe

2018-04-01

Type 1 narcolepsy (NT1) is a central hypersomnia linked to the destruction of hypocretin-producing neurons. A great body of genetic and epidemiological data points to likely autoimmune disease aetiology. Recent reports have characterized peripheral blood T-cell subsets in NT1, whereas data regarding the cerebrospinal fluid (CSF) immune cell composition are lacking. The current study aimed to characterize the T-cell and natural killer (NK) cell subsets in NT1 patients with long disease course. Immune cell subsets from CSF and peripheral blood mononuclear cell (PBMC) samples were analysed by flow cytometry in two age-balanced and sex-balanced groups of 14 NT1 patients versus 14 healthy controls. The frequency of CSF cell groups was compared with PBMCs. Non-parametric tests were used for statistical analyses. The NT1 patients did not show significant differences of CSF immune cell subsets compared to controls, despite a trend towards higher CD4 + terminally differentiated effector memory T cells. T cells preferentially displayed a memory phenotype in the CSF compared to PBMCs. Furthermore, a reduced frequency of CD4 + terminally differentiated effector memory T cells and an increased frequency of NK CD56 bright cells was observed in PBMCs from patients compared to controls. Finally, the ratio between CSF and peripheral CD4 + terminally differentiated effector memory T cells was two-fold increased in NT1 patients versus controls. Significant differences in PBMCs and in CSF/PBMC ratios of immune cell profile were found in NT1 patients compared to healthy controls. These differences might have arisen from the different HLA status, or be primary or secondary to hypocretin deficiency. Further functional studies in patients close to disease onset are required to understand NT1 pathophysiology. Copyright © 2017 Elsevier B.V. All rights reserved.

Prostaglandin E2 inhibits Tr1 cell differentiation through suppression of c-Maf

PubMed Central

Hooper, Kirsten Mary; Kong, Weimin

2017-01-01

Prostaglandin E2 (PGE2), a major lipid mediator abundant at inflammatory sites, acts as a proinflammatory agent in models of inflammatory/autoimmune diseases by promoting CD4 Th1/Th17 differentiation. Regulatory T cells, including the IL-10 producing Tr1 cells counterbalance the proinflammatory activity of effector Th1/Th17 cells. Tr1 cell differentiation and function are induced by IL-27, and depend primarily on sustained expression of c-Maf in addition to AhR and Blimp-1. In agreement with the in vivo proinflammatory role of PGE2, here we report for the first time that PGE2 inhibits IL-27-induced differentiation and IL-10 production of murine CD4+CD49b+LAG-3+Foxp3- Tr1 cells. The inhibitory effect of PGE2 was mediated through EP4 receptors and induction of cAMP, leading to a significant reduction in c-Maf expression. Although PGE2 reduced IL-21 production in differentiating Tr1 cells, its inhibitory effect on Tr1 differentiation and c-Maf expression also occurred independent of IL-21 signaling. PGE2 did not affect STAT1/3 activation, AhR expression and only marginally reduced Egr-2/Blimp-1 expression. The effect of PGE2 on CD4+CD49b+LAG-3+ Tr1 differentiation was not associated with either induction of Foxp3 or IL-17 production, suggesting a lack of transdifferentiation into Foxp3+ Treg or effector Th17 cells. We recently reported that PGE2 inhibits the expression and production of IL-27 from activated conventional dendritic cells (cDC) in vivo and in vitro. The present study indicates that PGE2 also reduces murine Tr1 differentiation and function directly by acting on IL-27-differentiating Tr1 cells. Together, the ability of PGE2 to inhibit IL-27 production by cDC, and the direct inhibitory effect on Tr1 differentiation mediated through reduction in c-Maf expression, represent a new mechanistic perspective for the proinflammatory activity of PGE2. PMID:28604806

YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity

PubMed Central

2016-01-01

SUMMARY Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted “effector” proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. PMID:27784797

Multi-color flow cytometry for evaluating age-related changes in memory lymphocyte subsets in dogs.

PubMed

Withers, Sita S; Moore, Peter F; Chang, Hong; Choi, Jin W; McSorley, Stephen J; Kent, Michael S; Monjazeb, Arta M; Canter, Robert J; Murphy, William J; Sparger, Ellen E; Rebhun, Robert B

2018-05-31

While dogs are increasingly being utilized as large-animal models of disease, important features of age-related immunosenescence in the dog have yet to be evaluated due to the lack of defined naïve vs. memory T lymphocyte phenotypes. We therefore performed multi-color flow cytometry on peripheral blood mononuclear cells from young and aged beagles, and determined the differential cytokine production by proposed memory subsets. CD4+ and CD8+ T lymphocytes in aged dogs displayed increased cytokine production, and decreased proliferative capacity. Antibodies targeting CD45RA and CD62L, but less so CD28 or CD44, defined canine cells that consistently exhibited properties of naïve-, central memory-, effector memory-, and terminal effector-like CD4+ and CD8+ T lymphocyte subsets. Older dogs demonstrated decreased frequencies of naïve-like CD4+ and CD8+ T lymphocytes, and an increased frequency of terminal effector-like CD8+ T lymphocytes. Overall findings revealed that aged dogs displayed features of immunosenescence similar to those reported in other species. Copyright © 2018 Elsevier Ltd. All rights reserved.

¹H, ¹³C and ¹⁵N resonance assignment for the human K-Ras at physiological pH.

PubMed

Vo, Uybach; Embrey, Kevin J; Breeze, Alexander L; Golovanov, Alexander P

2013-10-01

K-Ras, a member of the Ras family of small GTPases, is involved in cell growth, proliferation, differentiation and apoptosis and is frequently mutated in cancer. The activity of Ras is mediated by the inter-conversion between GTP- and GDP- bound states. This conversion is regulated by binding of effector proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Previously, NMR signals from these effector-binding regions of Ras often remained unassigned and largely unobservable due to conformational exchange and polysterism inherent to this protein. In this paper, we report the complete backbone and C(β), as well as partial H(α), H(β) and C(γ), NMR assignment for human K-Ras (residues 1-166) in the GDP-bound form at a physiological pH of 7.4. These data thereby make possible detailed monitoring of the functional cycle of Ras and its interactions with nucleotides and effector proteins through the observation of fingerprint signals from all the functionally important regions of the protein.

Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

PubMed Central

Shames, Stephanie R.; Liu, Luying; Havey, James C.; Schofield, Whitman B.; Goodman, Andrew L.; Roy, Craig R.

2017-01-01

Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis. PMID:29133401

HMBPP-deficient Listeria mutant immunization alters pulmonary/systemic responses, effector functions, and memory polarization of Vγ2Vδ2 T cells

PubMed Central

Frencher, James T.; Shen, Hongbo; Yan, Lin; Wilson, Jessica O.; Freitag, Nancy E.; Rizzo, Alicia N.; Chen, Crystal Y.; Chen, Zheng W.

2014-01-01

Whereas infection or immunization of humans/primates with microbes coproducing HMBPP/IPP can remarkably activate Vγ2Vδ2 T cells, in vivo studies have not been done to dissect HMBPP- and IPP-driven expansion, pulmonary trafficking, effector functions, and memory polarization of Vγ2Vδ2 T cells. We define these phosphoantigen-host interplays by comparative immunizations of macaques with the HMBPP/IPP-coproducing Listeria ΔactA prfA* and HMBPP-deficient Listeria ΔactAΔgcpE prfA* mutant. The HMBPP-deficient ΔgcpE mutant shows lower ability to expand Vγ2Vδ2 T cells in vitro than the parental HMBPP-producing strain but displays comparably attenuated infectivity or immunogenicity. Respiratory immunization of macaques with the HMBPP-deficient mutant elicits lower pulmonary and systemic responses of Vγ2Vδ2 T cells compared with the HMBPP-producing vaccine strain. Interestingly, HMBPP-deficient mutant reimmunization or boosting elicits enhanced responses of Vγ2Vδ2 T cells, but the magnitude is lower than that by HMBPP-producing listeria. HMBPP-deficient listeria differentiated fewer Vγ2Vδ2 T effector cells capable of coproducing IFN-γ and TNF-α and inhibiting intracellular listeria than HMBPP-producing listeria. Furthermore, HMBPP deficiency in listerial immunization influences memory polarization of Vγ2Vδ2 T cells. Thus, both HMBPP and IPP production in listerial immunization or infection elicit systemic/pulmonary responses and differentiation of Vγ2Vδ2 T cells, but a role for HMBPP is more dominant. Findings may help devise immune intervention. PMID:25114162

MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects.

PubMed

Naor, Adit; Panas, Michael W; Marino, Nicole; Coffey, Michael J; Tonkin, Christopher J; Boothroyd, John C

2018-04-03

The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma , we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔ myr1 , and RHΔ asp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, "hidden" responses arising in RHΔ myr1 - but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite's ability to co-opt host cell functions. IMPORTANCE Toxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1-dependent effectors reveals previously unknown activities that are masked or hidden by the action of these proteins. Copyright © 2018 Naor et al.

Disease implications of the Hippo/YAP pathway

PubMed Central

Plouffe, Steven W; Hong, Audrey W; Guan, Kun-Liang

2015-01-01

The Hippo signaling pathway is important for controlling organ size and tissue homeostasis. Originally identified in Drosophila melanogaster, the core components of the Hippo pathway are highly conserved in mammals. The Hippo pathway can be modulated by a wide range of stimuli, including G protein coupled receptor (GPCR) signaling, changes in the actin cytoskeleton, cell-cell contact, and cell polarity. When activated, the Hippo pathway functions as a tumor suppressor to limit cell growth. However, dysregulation by genetic inactivation of core pathway components, or amplification or gene fusion of its downstream effectors, results in increased cell proliferation and decreased apoptosis and differentiation. Not surprisingly, this can lead to tissue overgrowth, tumorigenesis, and many other diseases. PMID:25702974

Evidence of the immunomodulatory role of dual PI3K/mTOR inhibitors in transplantation: an experimental study in mice.

PubMed

Vilchez, Valery; Turcios, Lilia; Butterfield, David A; Mitov, Mihail I; Coquillard, Cristin L; Brandon, Ja Anthony; Cornea, Virgilius; Gedaly, Roberto; Marti, Francesc

2017-10-01

The PI3K/mTOR signaling cascade is fundamental in T-cell activation and fate decisions. We showed the distinct regulation of PI3K/mTOR in regulatory and effector T-cells and proposed the potential therapeutic benefit of targeting this pathway to control the balance between effector and regulatory T-cell activities. Substantial adverse effects in long-term clinical usage of rapamycin suggest the use of alternative treatments in restraining effector T-cell function in transplant patients. We hypothesize that dual PI3K/mTOR inhibitors may represent an immunosuppressant alternative. Here we show that dual PI3K/mTOR PI-103 and PKI-587 inhibitors interfered IL-2-dependent responses in T-cells. However, in contrast to the inhibitory effects in non-Treg T-cell proliferation and effector functions, dual inhibitors increased the differentiation, preferential expansion, and suppressor activity of iTregs. Rapamycin, PI-103, and PKI-587 targeted different signaling events and induced different metabolic patterns in primary T-cells. Similar to rapamycin, in vivo administration of PI-103 and PKI-587 controlled effectively the immunological response against allogeneic skin graft. These results characterize specific regulatory mechanisms of dual PI3K/mTOR inhibitors in T-cells and support their potential as a novel therapeutic option in transplantation. © 2017 Steunstichting ESOT.

Epigenetic control of CD8+ T cell differentiation.

PubMed

Henning, Amanda N; Roychoudhuri, Rahul; Restifo, Nicholas P

2018-05-01

Upon stimulation, small numbers of naive CD8 + T cells proliferate and differentiate into a variety of memory and effector cell types. CD8 + T cells can persist for years and kill tumour cells and virally infected cells. The functional and phenotypic changes that occur during CD8 + T cell differentiation are well characterized, but the epigenetic states that underlie these changes are incompletely understood. Here, we review the epigenetic processes that direct CD8 + T cell differentiation and function. We focus on epigenetic modification of DNA and associated histones at genes and their regulatory elements. We also describe structural changes in chromatin organization that affect gene expression. Finally, we examine the translational potential of epigenetic interventions to improve CD8 + T cell function in individuals with chronic infections and cancer.

Differentiation of Effector CD4 T Cell Populations*

PubMed Central

Zhu, Jinfang; Yamane, Hidehiro; Paul, William E.

2012-01-01

CD4 T cells play critical roles in mediating adaptive immunity to a variety of pathogens. They are also involved in autoimmunity, asthma, and allergic responses as well as in tumor immunity. During TCR activation in a particular cytokine milieu, naive CD4 T cells may differentiate into one of several lineages of T helper (Th) cells, including Th1, Th2, Th17, and iTreg, as defined by their pattern of cytokine production and function. In this review, we summarize the discovery, functions, and relationships among Th cells; the cytokine and signaling requirements for their development; the networks of transcription factors involved in their differentiation; the epigenetic regulation of their key cytokines and transcription factors; and human diseases involving defective CD4 T cell differentiation. PMID:20192806

PKCλ/ι regulates Th17 differentiation and house dust mite-induced allergic airway inflammation.

PubMed

Yang, Yingying; Dong, Panpan; Zhao, Jing; Zhou, Wei; Zhou, Yonghua; Xu, Yongliang; Mei, Congjin; Guo, Fukun; Zheng, Yi; Yang, Jun-Qi

2018-03-01

Asthma is a chronic airway inflammation in which Th2 and Th17 cells play critical roles in its pathogenesis. We have reported that atypical protein kinase (PKC) λ/ι is a new regulator for Th2 differentiation and function. However, the role of PKCλ/ι for Th17 cells remains elusive. In this study, we explored the effect of PKCλ/ι on Th17 cells in the context of ex vivo cell culture systems and an in vivo murine model of allergic airway inflammation with the use of activated T cell-specific conditional PKCλ/ι-deficient mice. Our findings indicate that PKCλ/ι regulates Th17 cells. The secretion of Th17 effector cytokines, including IL-17, IL-21 and IL-22, were inhibited from PKCλ/ι-deficient T cells under non-skewing or Th17-skewing culture conditions. Moreover, the impaired Th17 differentiation and function by the PKCλ/ι-deficiency was associated with the downregulation of Stat3 and Rorγt, key Th17 transcription factors. We developed a model of Th17 and neutrophil-involved allergic airway inflammation by intratracheal inoculation of house dust mites. PKCλ/ι-deficiency significantly inhibited airway inflammations. The infiltrating cells in the lungs and bronchoalveolar lavage fluids were significantly reduced in conditional PKCλ/ι-deficient mice. Th17 effector cytokines were reduced in the bronchoalveolar lavage fluids and lungs at protein and mRNA levels. Thus, PKCλ/ι emerges as a critical regulator of Th17 differentiation and allergic airway hyperresponsiveness. Copyright © 2018 Elsevier B.V. All rights reserved.

Control of Innate and Adaptive Lymphocytes by the RAR-Retinoic Acid Axis.

PubMed

Kim, Chang H

2018-02-01

Lymphocytes, such as T cells, B cells, and innate lymphoid cells (ILCs), play central roles in regulating immune responses. Retinoic acids (RAs) are vitamin A metabolites, produced and metabolized by certain tissue cells and myeloid cells in a tissue-specific manner. It has been established that RAs induce gut-homing receptors on T cells, B cells, and ILCs. A mounting body of evidence indicates that RAs exert far-reaching effects on functional differentiation and fate of these lymphocytes. For example, RAs promote effector T cell maintenance, generation of induced gut-homing regulatory and effector T cell subsets, antibody production by B cells, and functional maturation of ILCs. Key functions of RAs in regulating major groups of innate and adaptive lymphocytes are highlighted in this article.

Differential Kinematics Of Contemporary Industrial Robots

NASA Astrophysics Data System (ADS)

Szkodny, T.

2014-08-01

The paper presents a simple method of avoiding singular configurations of contemporary industrial robot manipulators of such renowned companies as ABB, Fanuc, Mitsubishi, Adept, Kawasaki, COMAU and KUKA. To determine the singular configurations of these manipulators a global form of description of the end-effector kinematics was prepared, relative to the other links. On the basis of this description , the formula for the Jacobian was defined in the end-effector coordinates. Next, a closed form of the determinant of the Jacobian was derived. From the formula, singular configurations, where the determinant's value equals zero, were determined. Additionally, geometric interpretations of these configurations were given and they were illustrated. For the exemplary manipulator, small corrections of joint variables preventing the reduction of the Jacobian order were suggested. An analysis of positional errors, caused by these corrections, was presented

Human-like characteristics for high degree of freedom robotic door-opening end-effector

NASA Astrophysics Data System (ADS)

Gray, Jeremy P.; Campagna, Frank

2011-05-01

In the field of military Unmanned Ground Vehicles (UGV's), military units are forced to sweep largely populated cities and towns in search of hostile enemies. These urban types of operations are referred to as MOUT (Military Operations on Urban Terrain). During urban operations, these UGV's encounter difficulties when opening doors. Current manipulator end effectors have these difficulties, because they are not designed to mimic human hand operations. This paper explains the mechanical nature of the Modular Universal Door Opening End-effector (MUDOE). MUDOE is a result of our development research to improve robotic manipulators ability to negotiate closed doors. The presented solution has the ability to mimic human hand characteristics when opening doors. The end-effector possesses an ability to maintain a high Degree of Freedom (DoF), and grasp the doorknob by applying equally distributed forces to all points of contact.

Effectors from Wheat Rust Fungi Suppress Multiple Plant Defense Responses.

PubMed

Ramachandran, Sowmya R; Yin, Chuntao; Kud, Joanna; Tanaka, Kiwamu; Mahoney, Aaron K; Xiao, Fangming; Hulbert, Scot H

2017-01-01

Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection, the fungus secretes a number of effector proteins. Although a large repository of putative effectors has been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study, we mined the available transcriptomes of Puccinia graminis and P. striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (<300 amino acids), secreted proteins, with no predicted functions were selected for the HR suppression assay using Nicotiana benthamiana, in which each of the proteins were transiently expressed and evaluated for their ability to suppress HR caused by four cytotoxic effector-R gene combinations (Cp/Rx, ATR13/RPP13, Rpt2/RPS-2, and GPA/RBP-1) and one mutated R gene-Pto(Y207D). Nine out of twenty proteins, designated Shr1 to Shr9 (suppressors of hypersensitive response), were found to suppress HR in N. benthamiana. These effectors varied in the effector-R gene defenses they suppressed, indicating these pathogens can interfere with a variety of host defense pathways. In addition to HR suppression, effector Shr7 also suppressed PAMP-triggered immune response triggered by flg22. Finally, delivery of Shr7 through Pseudomonas fluorescens EtHAn suppressed nonspecific HR induced by Pseudomonas syringae DC3000 in wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.

New insights into the mechanisms of acetic acid resistance in Acetobacter pasteurianus using iTRAQ-dependent quantitative proteomic analysis.

PubMed

Xia, Kai; Zang, Ning; Zhang, Junmei; Zhang, Hong; Li, Yudong; Liu, Ye; Feng, Wei; Liang, Xinle

2016-12-05

Acetobacter pasteurianus is the main starter in rice vinegar manufacturing due to its remarkable abilities to resist and produce acetic acid. Although several mechanisms of acetic acid resistance have been proposed and only a few effector proteins have been identified, a comprehensive depiction of the biological processes involved in acetic acid resistance is needed. In this study, iTRAQ-based quantitative proteomic analysis was adopted to investigate the whole proteome of different acidic titers (3.6, 7.1 and 9.3%, w/v) of Acetobacter pasteurianus Ab3 during the vinegar fermentation process. Consequently, 1386 proteins, including 318 differentially expressed proteins (p<0.05), were identified. Compared to that in the low titer circumstance, cells conducted distinct biological processes under high acetic acid stress, where >150 proteins were differentially expressed. Specifically, proteins involved in amino acid metabolic processes and fatty acid biosynthesis were differentially expressed, which may contribute to the acetic acid resistance of Acetobacter. Transcription factors, two component systems and toxin-antitoxin systems were implicated in the modulatory network at multiple levels. In addition, the identification of proteins involved in redox homeostasis, protein metabolism, and the cell envelope suggested that the whole cellular system is mobilized in response to acid stress. These findings provide a differential proteomic profile of acetic acid resistance in Acetobacter pasteurianus and have potential application to highly acidic rice vinegar manufacturing. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid.

PubMed

Asai, Shuta; Rallapalli, Ghanasyam; Piquerez, Sophie J M; Caillaud, Marie-Cécile; Furzer, Oliver J; Ishaque, Naveed; Wirthmueller, Lennart; Fabro, Georgina; Shirasu, Ken; Jones, Jonathan D G

2014-10-01

Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.

Force Control and Its Relation to Timing. Cognitive Science Program, Technical Report No. 86-4.

ERIC Educational Resources Information Center

Keele, Steven W.; And Others

Timing and speed are suggested to be the two general factors of coordination that differentiate people across a variety of motor movements. This study provides evidence for a third general factor of coordination, that of force control. Subjects that exhibit low variability in reproducing a target force with one effector, such as the finger, show…

Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000[OPEN

PubMed Central

Lewis, Laura A.; Polanski, Krzysztof; de Torres-Zabala, Marta; Bowden, Laura; Jenkins, Dafyd J.; Hill, Claire; Baxter, Laura; Truman, William; Prusinska, Justyna; Hickman, Richard; Wild, David L.; Ott, Sascha; Buchanan-Wollaston, Vicky; Beynon, Jim

2015-01-01

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae. PMID:26566919

Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000.

PubMed

Lewis, Laura A; Polanski, Krzysztof; de Torres-Zabala, Marta; Jayaraman, Siddharth; Bowden, Laura; Moore, Jonathan; Penfold, Christopher A; Jenkins, Dafyd J; Hill, Claire; Baxter, Laura; Kulasekaran, Satish; Truman, William; Littlejohn, George; Prusinska, Justyna; Mead, Andrew; Steinbrenner, Jens; Hickman, Richard; Rand, David; Wild, David L; Ott, Sascha; Buchanan-Wollaston, Vicky; Smirnoff, Nick; Beynon, Jim; Denby, Katherine; Grant, Murray

2015-11-01

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae. © 2015 American Society of Plant Biologists. All rights reserved.

Architectural and Mechanical Cues Direct Mesenchymal Stem Cell Interactions with Crosslinked Gelatin Scaffolds

PubMed Central

McAndrews, Kathleen M.; Kim, Min Jeong; Lam, Tuyet Y.; McGrail, Daniel J.

2014-01-01

Naturally derived biomaterials have emerged as modulators of cell function and tissue substitutes. Here, we developed crosslinked glutaraldehyde (GTA) scaffolds for the expansion and differentiation of mesenchymal stem cells (MSCs). The mechanical and architectural properties of the scaffolds were altered by varying the concentration of gelatin and GTA. Higher GTA concentrations were associated with an increase in more confined pores and osteogenic differentiation. In addition, myogenic potential varied with crosslinking degree, although bulk mechanical properties were unaltered. Correlation analysis revealed that ALP activity of differentiated MSCs on higher gelatin concentration scaffolds was dependent on traditional effectors, including environment elasticity and spread area. In contrast, the differentiation capacity of cells cultured on lower gelatin concentration scaffolds did not correlate with these factors, instead it was dependent on the hydrated pore structure. These results suggest that scaffold composition can determine what factors direct differentiation and may have critical implications for biomaterial design. PMID:24873687

Specific lymphocyte subsets predict response to adoptive cell therapy using expanded autologous tumor-infiltrating lymphocytes in metastatic melanoma patients

PubMed Central

Radvanyi, Laszlo G.; Bernatchez, Chantale; Zhang, Minying; Fox, Patricia S.; Miller, Priscilla; Chacon, Jessica; Wu, Richard; Lizee, Gregory; Mahoney, Sandy; Alvarado, Gladys; Glass, Michelle; Johnson, Valen E.; McMannis, John D.; Shpall, Elizabeth; Prieto, Victor; Papadopoulos, Nicholas; Kim, Kevin; Homsi, Jade; Bedikian, Agop; Hwu, Wen-Jen; Patel, Sapna; Ross, Merrick I.; Lee, Jeffrey E.; Gershenwald, Jeffrey E.; Lucci, Anthony; Royal, Richard; Cormier, Janice N.; Davies, Michael A.; Mansaray, Rahmatu; Fulbright, Orenthial J.; Toth, Christopher; Ramachandran, Renjith; Wardell, Seth; Gonzalez, Audrey; Hwu, Patrick

2012-01-01

Purpose Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) is a promising treatment for metastatic melanoma unresponsive to conventional therapies. We report here on the results of an ongoing Phase II clinical trial testing the efficacy of ACT using TIL in metastatic melanoma patients and the association of specific patient clinical characteristics and the phenotypic attributes of the infused TIL with clinical response. Experimental Design Altogether, 31 transiently lymphodepleted patients were treated with their expanded TIL followed by two cycles of high-dose (HD) IL-2 therapy. The effects of patient clinical features and the phenotypes of the T-cells infused on clinical response were determined. Results Overall, 15/31 (48.4%) patients had an objective clinical response using immune-related response criteria (irRC), with two patients (6.5%) having a complete response. Progression-free survival of >12 months was observed for 9/15 (60%) of the responding patients. Factors significantly associated with objective tumor regression included a higher number of TIL infused, a higher proportion of CD8+ T-cells in the infusion product, a more differentiated effector phenotype of the CD8+ population and a higher frequency of CD8+ T-cells co-expressing the negative costimulation molecule “B- and T-lymphocyte attenuator” (BTLA). No significant difference in telomere lengths of TIL between responders and non-responders was identified. Conclusion These results indicate that immunotherapy with expanded autologous TIL is capable of achieving durable clinical responses in metastatic melanoma patients and that CD8+ T-cells in the infused TIL, particularly differentiated effectors cells and cells expressing BTLA, are associated with tumor regression. PMID:23032743

Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34.

PubMed

Marlin, Jerry W; Chang, Yu-Wen E; Ober, Margaret; Handy, Amy; Xu, Wenhao; Jakobi, Rolf

2011-06-01

p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.

Novel Control Effectors for Truss Braced Wing

NASA Technical Reports Server (NTRS)

White, Edward V.; Kapania, Rakesh K.; Joshi, Shiv

2015-01-01

At cruise flight conditions very high aspect ratio/low sweep truss braced wings (TBW) may be subject to design requirements that distinguish them from more highly swept cantilevered wings. High aspect ratio, short chord length and relative thinness of the airfoil sections all contribute to relatively low wing torsional stiffness. This may lead to aeroelastic issues such as aileron reversal and low flutter margins. In order to counteract these issues, high aspect ratio/low sweep wings may need to carry additional high speed control effectors to operate when outboard ailerons are in reversal and/or must carry additional structural weight to enhance torsional stiffness. The novel control effector evaluated in this study is a variable sweep raked wing tip with an aileron control surface. Forward sweep of the tip allows the aileron to align closely with the torsional axis of the wing and operate in a conventional fashion. Aft sweep of the tip creates a large moment arm from the aileron to the wing torsional axis greatly enhancing aileron reversal. The novelty comes from using this enhanced and controllable aileron reversal effect to provide roll control authority by acting as a servo tab and providing roll control through intentional twist of the wing. In this case the reduced torsional stiffness of the wing becomes an advantage to be exploited. The study results show that the novel control effector concept does provide roll control as described, but only for a restricted class of TBW aircraft configurations. For the configuration studied (long range, dual aisle, Mach 0.85 cruise) the novel control effector provides significant benefits including up to 12% reduction in fuel burn.

Magnaporthe oryzae Effector AVR-Pii Helps to Establish Compatibility by Inhibition of the Rice NADP-Malic Enzyme Resulting in Disruption of Oxidative Burst and Host Innate Immunity

PubMed Central

Singh, Raksha; Dangol, Sarmina; Chen, Yafei; Choi, Jihyun; Cho, Yoon-Seong; Lee, Jea-Eun; Choi, Mi-Ok; Jwa, Nam-Soo

2016-01-01

Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant immune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVR-Pii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen. PMID:27126515

Ras and relatives--job sharing and networking keep an old family together.

PubMed

Ehrhardt, Annette; Ehrhardt, Götz R A; Guo, Xuecui; Schrader, John W

2002-10-01

Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

Activation and propagation of tumor infiltrating lymphocytes on clinical-grade designer artificial antigen presenting cells for adoptive immunotherapy of melanoma

PubMed Central

Forget, Marie-Andrée; Malu, Shruti; Liu, Hui; Toth, Christopher; Maiti, Sourindra; Kale, Charuta; Haymaker, Cara; Bernatchez, Chantale; Huls, Helen; Wang, Ena; Marincola, Francesco M.; Hwu, Patrick; Cooper, Laurence J. N.; Radvanyi, Laszlo G.

2014-01-01

PURPOSE Adoptive cell therapy (ACT) with autologous tumor infiltrating lymphocytes (TIL) is a therapy for metastatic melanoma with response rates up to 50%. However, the generation of the TIL transfer product is challenging, requiring pooled allogeneic normal donor peripheral blood mononuclear cells (PBMC) used in vitro as “feeders” to support a rapid expansion protocol (REP). Here, we optimized a platform to propagate TIL to a clinical scale using K562-cells genetically modified to express costimulatory molecules such as CD86, CD137-ligand and membrane-bound IL-15 to function as artificial antigen-presenting cell (aAPC) as an alternative to using PBMC feeders. EXPERIMENTAL DESIGN We used aAPC or γ-irradiated PBMC feeders to propagate TIL and measured rates of expansion. The activation and differentiation state was evaluated by flow cytometry and differential gene expression analyses. Clonal diversity was assessed based on pattern of T-cell receptor (TCR) usage. T-cell effector function was measured by evaluation of cytotoxic granule content and killing of target cells. RESULTS The aAPC propagated TIL at numbers equivalent to that found with PBMC feeders, while increasing the frequency of CD8+ T-cell expansion with a comparable effector-memory phenotype. mRNA profiling revealed an up-regulation of genes in the Wnt and stem-cell pathways with the aAPC. The aAPC platform did not skew clonal diversity and CD8+ T cells showed comparable anti-tumor function as those expanded with PBMC feeders. CONCLUSIONS TIL can be rapidly expanded with aAPC to clinical scale generating T cells with similar phenotypic and effector profiles as with PBMC feeders. These data support the clinical-application of aAPC to manufacture TIL for the treatment of melanoma. PMID:25304728

Characterization of Human CD8 T Cell Responses in Dengue Virus-Infected Patients from India

PubMed Central

Chandele, Anmol; Sewatanon, Jaturong; Gunisetty, Sivaram; Singla, Mohit; Onlamoon, Nattawat; Akondy, Rama S.; Kissick, Haydn Thomas; Nayak, Kaustuv; Reddy, Elluri Seetharami; Kalam, Haroon; Kumar, Dhiraj; Verma, Anil; Panda, HareKrushna; Wang, Siyu; Angkasekwinai, Nasikarn; Pattanapanyasat, Kovit; Chokephaibulkit, Kulkanya; Lodha, Rakesh; Kabra, Sushil; Ahmed, Rafi

2016-01-01

ABSTRACT Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR− CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue. IMPORTANCE Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro. Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects. PMID:27707928

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

PubMed

Verma, Anju; Lee, Chris; Morriss, Stephanie; Odu, Fiona; Kenning, Charlotte; Rizzo, Nancy; Spollen, William G; Lin, Marriam; McRae, Amanda G; Givan, Scott A; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Baum, Thomas J; Mitchum, Melissa G

2018-05-04

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Memory T cells and vaccines.

PubMed

Esser, Mark T; Marchese, Rocio D; Kierstead, Lisa S; Tussey, Lynda G; Wang, Fubao; Chirmule, Narendra; Washabaugh, Michael W

2003-01-17

T lymphocytes play a central role in the generation of a protective immune response in many microbial infections. After immunization, dendritic cells take up microbial antigens and traffic to draining lymph nodes where they present processed antigens to naïve T cells. These naïve T cells are stimulated to proliferate and differentiate into effector and memory T cells. Activated, effector and memory T cells provide B cell help in the lymph nodes and traffic to sites of infection where they secrete anti-microbial cytokines and kill infected cells. At least two types of memory cells have been defined in humans based on their functional and migratory properties. T central-memory (T(CM)) cells are found predominantly in lymphoid organs and can not be immediately activated, whereas T effector-memory (T(EM)) cells are found predominantly in peripheral tissue and sites of inflammation and exhibit rapid effector function. Most currently licensed vaccines induce antibody responses capable of mediating long-term protection against lytic viruses such as influenza and small pox. In contrast, vaccines against chronic pathogens that require cell-mediated immune responses to control, such as malaria, Mycobacterium tuberculosis (TB), human immunodeficiency virus (HIV) and hepatitis C virus (HCV), are currently not available or are ineffective. Understanding the mechanisms by which long-lived cellular immune responses are generated following vaccination should facilitate the development of safe and effective vaccines against these emerging diseases. Here, we review the current literature with respect to memory T cells and their implications to vaccine development.

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.

Many pathogenic bacteria of the familyEnterobacteriaceaeuse type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from theEnterobacteriaceaeintracellular pathogensSalmonella entericaserovar Typhimurium andCitrobacter rodentium. We identified 54 high-confidence host interactors for theSalmonellaeffectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for theCitrobactereffectors EspT,more » NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfHSalmonellaprotein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCEDuring infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets ofSalmonellaandCitrobactereffectors, which will help elucidate their mechanisms of action.« less

The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the basis of parasitism and virulence.

PubMed

Eves-van den Akker, Sebastian; Laetsch, Dominik R; Thorpe, Peter; Lilley, Catherine J; Danchin, Etienne G J; Da Rocha, Martine; Rancurel, Corinne; Holroyd, Nancy E; Cotton, James A; Szitenberg, Amir; Grenier, Eric; Montarry, Josselin; Mimee, Benjamin; Duceppe, Marc-Olivier; Boyes, Ian; Marvin, Jessica M C; Jones, Laura M; Yusup, Hazijah B; Lafond-Lapalme, Joël; Esquibet, Magali; Sabeh, Michael; Rott, Michael; Overmars, Hein; Finkers-Tomczak, Anna; Smant, Geert; Koutsovoulos, Georgios; Blok, Vivian; Mantelin, Sophie; Cock, Peter J A; Phillips, Wendy; Henrissat, Bernard; Urwin, Peter E; Blaxter, Mark; Jones, John T

2016-06-10

The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes. We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative 'effector islands' in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking. These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.

High energy laser demonstrators for defense applications

NASA Astrophysics Data System (ADS)

Jung, M.; Riesbeck, Th.; Schmitz, J.; Baumgärtel, Th.; Ludewigt, K.; Graf, A.

2017-01-01

Rheinmetall Waffe Munition has worked since 30 years in the area of High Energy Laser (HEL) for defence applications, starting from pulsed CO2 to pulsed glass rods lasers. In the last decade Rheinmetall Waffe Munition changed to diode pumped solid state laser (DPSSL) technology and has successfully developed, realised and tested a variety of versatile HEL weapon demonstrators for air- and ground defence scenarios like countering rocket, artillery, mortar, missile (RAMM), unmanned aerial systems (UAS) and unexploded ordnances clearing. By employing beam superimposing technology and a modular laser weapon concept, the total optical power has been successively increased. Stationary weapon platforms, military vehicles and naval platforms have been equipped with high energy laser effectors. The contribution gives a summary of the most recent development stages of Rheinmetalls HEL weapon program. In addition to the stationary 30 kW laser weapon demonstrator, we present vehicle based HEL demonstrators: the 5 kW class Mobile HEL Effector Track V, the 20 kW class Mobile HEL Effector Wheel XX and the 50 kW class Mobile HEL Effector Container L and the latest 10 kW HEL effector integrated in the naval weapon platform MLG 27. We describe the capabilities of these demonstrators against different potential targets. Furthermore, we will show the capability of the 30 kW stationary Laser Weapon Demonstrator integrated into an existing ground based air defence system to defeat saturated attacks of RAMM and UAS targets.

Assembly of Customized TAL Effectors Through Advanced ULtiMATE System.

PubMed

Yang, Junjiao; Guo, Shengjie; Yuan, Pengfei; Wei, Wensheng

2016-01-01

Transcription activator-like effectors (TALEs) have been widely applied in gene targeting. Here we describe an advanced ULtiMATE (USER-based Ligation-Mediated Assembly of TAL Effector) system that utilizes USER fusion technique and archive of 512 tetramer templates to achieve highly efficient construction of TALEs, which takes only half a day to accomplish the assembly of any given TALE construct. This system is also suitable for large-scale assembly of TALENs and any other TALE-based constructions.

Quiescence of Memory CD8(+) T Cells Is Mediated by Regulatory T Cells through Inhibitory Receptor CTLA-4.

PubMed

Kalia, Vandana; Penny, Laura Anne; Yuzefpolskiy, Yevgeniy; Baumann, Florian Martin; Sarkar, Surojit

2015-06-16

Immune memory cells are poised to rapidly expand and elaborate effector functions upon reinfection yet exist in a functionally quiescent state. The paradigm is that memory T cells remain inactive due to lack of T cell receptor (TCR) stimuli. Here, we report that regulatory T (Treg) cells orchestrate memory T cell quiescence by suppressing effector and proliferation programs through inhibitory receptor, cytotoxic-T-lymphocyte-associated protein-4 (CTLA-4). Loss of Treg cells resulted in activation of genome-wide transcriptional programs characteristic of effector T cells and drove transitioning as well as established memory CD8(+) T cells toward terminally differentiated KLRG-1(hi)IL-7Rα(lo)GzmB(hi) phenotype, with compromised metabolic fitness, longevity, polyfunctionality, and protective efficacy. CTLA-4 functionally replaced Treg cells in trans to rescue memory T cell defects and restore homeostasis. These studies present the CTLA-4-CD28-CD80/CD86 axis as a potential target to accelerate vaccine-induced immunity and improve T cell memory quality in current cancer immunotherapies proposing transient Treg cell ablation. Copyright © 2015 Elsevier Inc. All rights reserved.

Functions of Calcium-Dependent Protein Kinases in Plant Innate Immunity

PubMed Central

Gao, Xiquan; Cox, Kevin L.; He, Ping

2014-01-01

An increase of cytosolic Ca2+ is generated by diverse physiological stimuli and stresses, including pathogen attack. Plants have evolved two branches of the immune system to defend against pathogen infections. The primary innate immune response is triggered by the detection of evolutionarily conserved pathogen-associated molecular pattern (PAMP), which is called PAMP-triggered immunity (PTI). The second branch of plant innate immunity is triggered by the recognition of specific pathogen effector proteins and known as effector-triggered immunity (ETI). Calcium (Ca2+) signaling is essential in both plant PTI and ETI responses. Calcium-dependent protein kinases (CDPKs) have emerged as important Ca2+ sensor proteins in transducing differential Ca2+ signatures, triggered by PAMPs or effectors and activating complex downstream responses. CDPKs directly transmit calcium signals by calcium binding to the elongation factor (EF)-hand domain at the C-terminus and substrate phosphorylation by the catalytic kinase domain at the N-terminus. Emerging evidence suggests that specific and overlapping CDPKs phosphorylate distinct substrates in PTI and ETI to regulate diverse plant immune responses, including production of reactive oxygen species, transcriptional reprogramming of immune genes, and the hypersensitive response. PMID:27135498

Development of SH2 probes and pull-down assays to detect pathogen-induced, site-specific tyrosine phosphorylation of the TLR adaptor SCIMP.

PubMed

Luo, Lin; Tong, Samuel J; Wall, Adam A; Khromykh, Tatiana; Sweet, Matthew J; Stow, Jennifer L

2017-07-01

Protein tyrosine phosphorylation guides many molecular interactions for cellular functions. SCIMP is a transmembrane adaptor protein (TRAP) family member that mediates selective proinflammatory cytokine responses generated by pathogen-activated Toll-like receptor (TLR) pathways in macrophages. TLR activation triggers SCIMP phosphorylation and selective phosphorylation of distinct tyrosine residues on this adaptor offers the potential for regulating or biasing inflammatory responses. To analyze site-specific phosphorylation events, we developed three probes based on the SH2 domains of known SCIMP effectors, and used them for pull-downs from macrophage extracts. CRISPR-mediated SCIMP-deficient RAW264.7 macrophage-like cells were reconstituted with various phosphorylation-deficient (Y58F, Y96F, Y120F) SCIMPs, and used to demonstrate the specificity of LPS/TLR4-induced, site-specific phosphorylation of SCIMP for the temporal recruitment of the effectors Grb2, Csk and SLP65. Our findings reveal potential for differential SCIMP phosphorylation and specific effectors to influence TLR signaling and inflammatory programs. Furthermore, the use of Csk-SH2 pull-downs to identify additional known and new Csk targets in LPS-activated macrophages reveals the wider utility of our SH2 probes.

Expression changes and novel interaction partners of talin 1 in effector cells of autoimmune uveitis.

PubMed

Degroote, Roxane L; Hauck, Stefanie M; Treutlein, Gudrun; Amann, Barbara; Fröhlich, Kristina J H; Kremmer, Elisabeth; Merl, Juliane; Stangassinger, Manfred; Ueffing, Marius; Deeg, Cornelia A

2013-12-06

Autoimmune uveitis is characterized by crossing of blood-retinal barrier (BRB) by autoaggressive immune cells. Equine recurrent uveitis (ERU) is a valuable spontaneous model for autoimmune uveitis and analyses of differentially expressed proteins in ERU unraveled changed protein clusters in target tissues and immune system. Healthy eyes are devoid of leukocytes. In ERU, however, leukocytes enter the inner eye and subsequently destroy it. Molecular mechanisms enabling cell migration through BRB still remain elusive. Previously, we detected decreased talin 1 expression in blood-derived granulocytes of ERU cases, linking the innate immune system to ERU. Because changes in leukocyte protein expression pattern may play a role in pathological abnormalities leading to migration ability, we aimed at identifying interactors of talin 1 in leukocytes with immunoprecipitation, followed by LC-MS/MS for candidate identification. This enabled us to identify CD90 (Thy1) as novel interactor of talin 1 besides several other interactors. In blood-derived granulocytes from healthy individuals, CD90 was highly abundant and significantly reduced in ERU, especially in effector cells. Connection between talin 1 and CD90 and their expression differences in inflammation is an interesting novel finding allowing deeper insight into immune response of innate immune system and granulocyte migration ability in this organ-specific autoimmune disease.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srikannathasan, Velupillai; English, Grant; Bui, Nhat Khai

Crystal structures of type VI secretion system-associated immunity proteins, a peptidoglycan endopeptidase and a complex of the endopeptidase and its cognate immunity protein are reported together with assays of endopeptidase activity and functional assessment. Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-d-glutamic acid and l-meso-diaminopimelic acid with differentmore » specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure–activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1–Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2–Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector–immunity protein interactions.« less

Caveolin-mediated endocytosis of the Chlamydia M278 outer membrane peptide encapsulated in poly(lactic acid)-Poly(ethylene glycol) nanoparticles by mouse primary dendritic cells enhances specific immune effectors mediated by MHC class II and CD4+ T cells.

PubMed

Dixit, Saurabh; Sahu, Rajnish; Verma, Richa; Duncan, Skyla; Giambartolomei, Guillermo H; Singh, Shree R; Dennis, Vida A

2018-03-01

We previously developed a Chlamydia trachomatis nanovaccine (PPM) by encapsulating a chlamydial M278 peptide within poly(lactic acid)-poly(ethylene glycol) biodegradable nanoparticles that immunopotentiated Chlamydia-specific immune effector responses in mice. Herein, we investigated the mechanistic interactions of PPM with mouse bone marrow-derived dendritic cells (DCs) for its uptake, trafficking, and T cell activation. Our results reveal that PPM triggered enhanced expression of effector cytokines and chemokines, surface activation markers (Cd1d2, Fcgr1), pathogen-sensing receptors (TLR2, Nod1), co-stimulatory (CD40, CD80, CD86) and MHC class I and II molecules. Co-culturing of PPM-primed DCs with T cells from C. muridarum vaccinated mice yielded an increase in Chlamydia-specific immune effector responses including CD3 + lymphoproliferation, CD3 + CD4 + IFN-γ-secreting cells along with CD3 + CD4 + memory (CD44 high and CD62L high ) and effector (CD44 high and CD62L low ) phenotypes. Intracellular trafficking analyses revealed an intense expression and colocalization of PPM predominantly in endosomes. PPM also upregulated the transcriptional and protein expression of the endocytic mediator, caveolin-1 in DCs. More importantly, the specific inhibition of caveolin-1 led to decreased expression of PPM-induced cytokines and co-stimulatory molecules. Our investigation shows that PPM provided enhancement of uptake, probably by exploiting the caveolin-mediated endocytosis pathway, endosomal processing, and MHC II presentation to immunopotentiate Chlamydia-specific immune effector responses mediated by CD4 + T cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

CXCL13-producing TFH cells link immune suppression and adaptive memory in human breast cancer

PubMed Central

Gu-Trantien, Chunyan; Migliori, Edoardo; de Wind, Alexandre; Brohée, Sylvain; Garaud, Soizic; Noël, Grégory; Dang Chi, Vu Luan; Lodewyckx, Jean-Nicolas; Naveaux, Céline; Duvillier, Hugues; Larsimont, Denis

2017-01-01

T follicular helper cells (TFH cells) are important regulators of antigen-specific B cell responses. The B cell chemoattractant CXCL13 has recently been linked with TFH cell infiltration and improved survival in human cancer. Although human TFH cells can produce CXCL13, their immune functions are currently unknown. This study presents data from human breast cancer, advocating a role for tumor-infiltrating CXCL13-producing (CXCR5–) TFH cells, here named TFHX13 cells, in promoting local memory B cell differentiation. TFHX13 cells potentially trigger tertiary lymphoid structure formation and thereby generate germinal center B cell responses at the tumor site. Follicular DCs are not potent CXCL13 producers in breast tumor tissues. We used the TFH cell markers PD-1 and ICOS to identify distinct effector and regulatory CD4+ T cell subpopulations in breast tumors. TFHX13 cells are an important component of the PD-1hiICOSint effector subpopulation and coexpanded with PD-1intICOShiFOXP3hi Tregs. IL2 deprivation induces CXCL13 expression in vitro with a synergistic effect from TGFβ1, providing insight into TFHX13 cell differentiation in response to Treg accumulation, similar to conventional TFH cell responses. Our data suggest that human TFHX13 cell differentiation may be a key factor in converting Treg-mediated immune suppression to de novo activation of adaptive antitumor humoral responses in the chronic inflammatory breast cancer microenvironment. PMID:28570278

MicroRNA profiling of the murine hematopoietic system

PubMed Central

Monticelli, Silvia; Ansel, K Mark; Xiao, Changchun; Socci, Nicholas D; Krichevsky, Anna M; Thai, To-Ha; Rajewsky, Nikolaus; Marks, Debora S; Sander, Chris; Rajewsky, Klaus; Rao, Anjana; Kosik, Kenneth S

2005-01-01

Background MicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene expression during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete. Results We used oligonucleotide arrays to analyze miRNA expression in the murine hematopoietic system. Complementary oligonucleotides capable of hybridizing to 181 miRNAs were immobilized on a membrane and probed with radiolabeled RNA derived from low molecular weight fractions of total RNA from several different hematopoietic and neuronal cells. This method allowed us to analyze cell type-specific patterns of miRNA expression and to identify miRNAs that might be important for cell lineage specification and/or cell effector functions. Conclusion This is the first report of systematic miRNA gene profiling in cells of the hematopoietic system. As expected, miRNA expression patterns were very different between hematopoietic and non-hematopoietic cells, with further subtle differences observed within the hematopoietic group. Interestingly, the most pronounced similarities were observed among fully differentiated effector cells (Th1 and Th2 lymphocytes and mast cells) and precursors at comparable stages of differentiation (double negative thymocytes and pro-B cells), suggesting that in addition to regulating the process of commitment to particular cellular lineages, miRNAs might have an important general role in the mechanism of cell differentiation and maintenance of cell identity. PMID:16086853

Interconnected subsets of memory follicular helper T cells have different effector functions.

PubMed

Asrir, Assia; Aloulou, Meryem; Gador, Mylène; Pérals, Corine; Fazilleau, Nicolas

2017-10-10

Follicular helper T cells regulate high-affinity antibody production. Memory follicular helper T cells can be local in draining lymphoid organs and circulate in the blood, but the underlying mechanisms of this subdivision are unresolved. Here we show that both memory follicular helper T subsets sustain B-cell responses after reactivation. Local cells promote more plasma cell differentiation, whereas circulating cells promote more secondary germinal centers. In parallel, local memory B cells are homogeneous and programmed to become plasma cells, whereas circulating memory B cells are able to rediversify. Local memory follicular helper T cells have higher affinity T-cell receptors, which correlates with expression of peptide MHC-II at the surface of local memory B cells only. Blocking T-cell receptor-peptide MHC-II interactions induces the release of local memory follicular helper T cells in the circulating compartment. Our studies show that memory follicular helper T localization is highly intertwined with memory B cells, a finding that has important implications for vaccine design.Tfh cells can differentiate into memory cells. Here the authors describe distinct functional and phenotypic profiles of these memory Tfh cells dependent on their anatomical localization to the lymphoid organs or to the circulation.

Bach2 Promotes Regulatory T-cell Development by Limiting Effector Differentiation | Center for Cancer Research

Cancer.gov

A number of allergic and autoimmune disorders, such as Crohn’s disease, asthma, type I diabetes and multiple sclerosis, are associated with polymorphisms in a gene encoding the transcription factor, BACH2. Despite this, the mechanism Bach2 uses to prevent immune-mediated diseases was not known. To function appropriately, the immune system relies on a delicate balance between

Robotics, a Kennedy Educate to Innovate (KETI) PowerPoint Presentation

NASA Technical Reports Server (NTRS)

Davila, Dina

2010-01-01

This presentation is a series of lecture notes for a lecture on Robotics. It describes the concept of robots and differentiates between robotic devices and "true robots". It also reviews the reasons for why we use robots, generally, and specificaly.why NASA uses robots. It also explains what an end effector is and explores some of the careers available in the field of robotics.

Specific Eph receptor-cytoplasmic effector signaling mediated by SAM-SAM domain interactions.

PubMed

Wang, Yue; Shang, Yuan; Li, Jianchao; Chen, Weidi; Li, Gang; Wan, Jun; Liu, Wei; Zhang, Mingjie

2018-05-11

The Eph receptor tyrosine kinase (RTK) family is the largest subfamily of RTKs playing critical roles in many developmental processes such as tissue patterning, neurogenesis and neuronal circuit formation, angiogenesis, etc. How the 14 Eph proteins, via their highly similar cytoplasmic domains, can transmit diverse and sometimes opposite cellular signals upon engaging ephrins is a major unresolved question. Here we systematically investigated the bindings of each SAM domain of Eph receptors to the SAM domains from SHIP2 and Odin, and uncover a highly specific SAM-SAM interaction-mediated cytoplasmic Eph-effector binding pattern. Comparative X-ray crystallographic studies of several SAM-SAM heterodimer complexes, together with biochemical and cell biology experiments, not only revealed the exquisite specificity code governing Eph/effector interactions but also allowed us to identify SAMD5 as a new Eph binding partner. Finally, these Eph/effector SAM heterodimer structures can explain many Eph SAM mutations identified in patients suffering from cancers and other diseases. © 2018, Wang et al.

Global study of holistic morphological effectors in the budding yeast Saccharomyces cerevisiae.

PubMed

Suzuki, Godai; Wang, Yang; Kubo, Karen; Hirata, Eri; Ohnuki, Shinsuke; Ohya, Yoshikazu

2018-02-20

The size of the phenotypic effect of a gene has been thoroughly investigated in terms of fitness and specific morphological traits in the budding yeast Saccharomyces cerevisiae, but little is known about gross morphological abnormalities. We identified 1126 holistic morphological effectors that cause severe gross morphological abnormality when deleted, and 2241 specific morphological effectors with weak holistic effects but distinctive effects on yeast morphology. Holistic effectors fell into many gene function categories and acted as network hubs, affecting a large number of morphological traits, interacting with a large number of genes, and facilitating high protein expression. Holistic morphological abnormality was useful for estimating the importance of a gene to morphology. The contribution of gene importance to fitness and morphology could be used to efficiently classify genes into functional groups. Holistic morphological abnormality can be used as a reproducible and reliable gene feature for high-dimensional morphological phenotyping. It can be used in many functional genomic applications.

ZNF750 is a p63 Target Gene that Induces KLF4 to Drive Terminal Epidermal Differentiation

PubMed Central

Sen, George L.; Boxer, Lisa D.; Webster, Dan E.; Bussat, Rose T.; Qu, Kun; Zarnegar, Brian J.; Johnston, Danielle; Siprashvili, Zurab; Khavari, Paul A.

2012-01-01

SUMMARY Disrupted epidermal differentiation characterizes numerous diseases that impact >25% of the population. In a search for dominant mediators of differentiation, we defined a requirement for ZNF750 in terminal epidermal differentiation. ZNF750 controlled genes mutated in numerous human skin diseases, including FLG, LOR, LCE3B, ALOXE3, and SPINK5. ZNF750 induced progenitor differentiation via an evolutionarily conserved C2H2 zinc finger motif. The epidermal master regulator, p63, bound the ZNF750 promoter and was necessary for its induction. ZNF750 restored differentiation to p63-deficient tissue, suggesting it acts downstream of p63. A search for functionally important ZNF750 targets via analysis of ZNF750-regulated genes identified KLF4, a transcription factor that activates late epidermal differentiation. ZNF750 binds to KLF4 at multiple sites flanking the transcriptional start site and controls its expression. ZNF750 thus directly links a tissue-specifying factor, p63, to an effector of terminal differentiation, KLF4, and represents a potential future target for disorders of this process. PMID:22364861

Silicon protects soybean plants against Phytophthora sojae by interfering with effector-receptor expression.

PubMed

Rasoolizadeh, Aliyeh; Labbé, Caroline; Sonah, Humira; Deshmukh, Rupesh K; Belzile, François; Menzies, James G; Bélanger, Richard R

2018-05-30

Silicon (Si) is known to protect against biotrophic and hemibiotrophic plant pathogens; however, the mechanisms by which it exerts its prophylactic role remain unknown. In an attempt to obtain unique insights into the mode of action of Si, we conducted a full comparative transcriptomic analysis of soybean (Glycine max) plants and Phytophthora sojae, a hemibiotroph that relies heavily on effectors for its virulence. Supplying Si to inoculated plants provided a strong protection against P. sojae over the course of the experiment (21 day). Our results showed that the response of Si-free (Si - ) plants to inoculation was characterized early (4 dpi) by a high expression of defense-related genes, including plant receptors, which receded over time as the pathogen progressed into the roots. The infection was synchronized with a high expression of effectors by P. sojae, the nature of which changed over time. By contrast, the transcriptomic response of Si-fed (Si + ) plants was remarkably unaffected by the presence of P. sojae, and the expression of effector-coding genes by the pathogen was significantly reduced. Given that the apoplast is a key site of interaction between effectors and plant defenses and receptors in the soybean-P. sojae complex, as well as the site of amorphous-Si accumulation, our results indicate that Si likely interferes with the signaling network between P. sojae and the plant, preventing or decreasing the release of effectors reaching plant receptors, thus creating a form of incompatible interaction.

Insights into the role of Bcl6 in follicular Th cells using a new conditional mutant mouse model.

PubMed

Hollister, Kristin; Kusam, Saritha; Wu, Hao; Clegg, Ninah; Mondal, Arpita; Sawant, Deepali V; Dent, Alexander L

2013-10-01

The transcriptional repressor Bcl6 controls development of the follicular Th cell (T(FH)) lineage, but the precise mechanisms by which Bcl6 regulates this process are unclear. A model has been proposed whereby Bcl6 represses the differentiation of T cells into alternative effector lineages, thus favoring T(FH) cell differentiation. Analysis of T cell differentiation using Bcl6-deficient mice has been complicated by the strong proinflammatory phenotype of Bcl6-deficient myeloid cells. In this study, we report data from a novel mouse model where Bcl6 is conditionally deleted in T cells (Bcl6(fl/fl)Cre(CD4) mice). After immunization, programmed death -1 (PD-1)(high) T(FH) cells in Bcl6(fl/fl)Cre(CD4) mice are decreased >90% compared with control mice, and Ag-specific IgG is sharply reduced. Residual PD-1(high)CXCR5(+) T(FH) cells in Bcl6(fl/fl)Cre(CD4) mice show a significantly higher rate of apoptosis than do PD-1(high)CXCR5(+) T(FH) cells in control mice. Immunization of Bcl6(fl/fl)Cre(CD4) mice did not reveal enhanced differentiation into Th1, Th2, or Th17 lineages, although IL-10 expression by CD4 T cells was markedly elevated. Thus, T cell-extrinsic factors appear to promote the increased Th1, Th2, and Th17 responses in germline Bcl6-deficient mice. Furthermore, IL-10 may be a key target gene for Bcl6 in CD4 T cells, which enables Bcl6 to promote the T(FH) cell phenotype. Finally, our data reveal a novel mechanism for the role of Bcl6 in promoting T(FH) cell survival.

A transcription activator-like effector from Xanthomonas oryzae pv. oryzicola elicits dose-dependent resistance in rice.

PubMed

Hummel, Aaron W; Wilkins, Katherine E; Wang, Li; Cernadas, R Andres; Bogdanove, Adam J

2017-01-01

Xanthomonas spp. reduce crop yields and quality worldwide. During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter. TAL effectors may contribute to disease by activating the expression of susceptibility genes or trigger resistance associated with the hypersensitive reaction (HR) by activating an executor resistance (R) gene. The rice bacterial leaf streak pathogen X. oryzae pv. oryzicola (Xoc) is known to suppress host resistance, and no host R gene has been identified against it, despite considerable effort. To further investigate Xoc suppression of host resistance, we conducted a screen of effectors from BLS256 and identified Tal2a as an HR elicitor in rice when delivered heterologously by a strain of the closely related rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) or by the soybean pathogen X. axonopodis pv. glycines. The HR required the Tal2a activation domain, suggesting an executor R gene. Tal2a activity was differentially distributed among geographically diverse Xoc isolates, being largely conserved among Asian isolates. We identified four genes induced by Tal2a in next-generation RNA sequencing experiments and confirmed them using quantitative real-time reverse transcription-polymerase chain reaction (qPCR). However, neither individual nor collective activation of these genes by designer TAL effectors resulted in HR. A tal2a knockout mutant of BLS256 showed virulence comparable with the wild-type, but plasmid-based overexpression of tal2a at different levels in the wild-type reduced virulence in a directly corresponding way. Overall, the results reveal that host resistance suppression by Xoc plays a critical role in pathogenesis. Further, the dose-dependent avirulence activity of Tal2a and the apparent lack of a single canonical target that accounts for HR point to a novel, activation domain-dependent mode of action, which might involve, for example, a non-coding gene or a specific pattern of activation across multiple targets. © 2016 BSPP and John Wiley & Sons Ltd.

Immunoregulation by Mesenchymal Stem Cells: Biological Aspects and Clinical Applications

PubMed Central

Castro-Manrreza, Marta E.; Montesinos, Juan J.

2015-01-01

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into mesenchymal lineages and that can be isolated from various tissues and easily cultivated in vitro. Currently, MSCs are of considerable interest because of the biological characteristics that confer high potential applicability in the clinical treatment of many diseases. Specifically, because of their high immunoregulatory capacity, MSCs are used as tools in cellular therapies for clinical protocols involving immune system alterations. In this review, we discuss the current knowledge about the capacity of MSCs for the immunoregulation of immunocompetent cells and emphasize the effects of MSCs on T cells, principal effectors of the immune response, and the immunosuppressive effects mediated by the secretion of soluble factors and membrane molecules. We also describe the mechanisms of MSC immunoregulatory modulation and the participation of MSCs as immune response regulators in several autoimmune diseases, and we emphasize the clinical application in graft versus host disease (GVHD). PMID:25961059

Regulation of 5'-adenosine monophosphate deaminase in the freeze tolerant wood frog, Rana sylvatica.

PubMed

Dieni, Christopher A; Storey, Kenneth B

2008-04-22

The wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65-70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD) plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle. Wood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to approximately 1.0) and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg.ATP and Mg.ADP and inhibited by Mg.GTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated) enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5 degrees C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing. Maintenance of long term viability under the ischemic conditions in frozen muscle requires attention to the control of cellular energetics. Differential regulatory controls on AMPD by mechanisms including binding to muscle proteins, actions allosteric effectors, glucose and temperature effects and reversible phosphorylation adjust enzyme function for an optimal role in controlling cellular adenylate levels in ischemic frozen muscle. Stable modification of AMPD properties via freeze-responsive phosphorylation may contribute both to AMPD control and to coordinating AMPD function with other enzymes of energy metabolism in cold ischemic muscle.

Rapamycin resistant murine th9 cells have a stable in vivo phenotype and inhibit graft-versus-host reactivity.

PubMed

Mangus, Courtney W; Massey, Paul R; Fowler, Daniel H; Amarnath, Shoba

2013-01-01

The cytokine micro-environment can direct murine CD4(+) T cells towards various differentiation lineages such as Th1, Th2 and Tregs even in the presence of rapamycin, which results in T cells that mediate increased in vivo effects. Recently, a new lineage of T cells known as Th9 cells that secrete increased IL-9 have been described. However, it is not known whether Th9 differentiation occurs in the presence of rapamycin or whether adoptively transferred donor Th9 cells would augment or restrict alloreactivity after experimental bone marrow transplantation. We found that CD4(+) T cells that were co-stimulated and polarized with TGF-β and IL-4 in the presence or absence of rapamycin each yielded effector cells of Th9 phenotype that secreted increased IL-9 and expressed a transcription factor profile characteristic of both Th9 and Th2 cells (high GATA-3/low T-bet). Augmentation of T cell replete allografts with manufactured rapamycin resistant Th9 cells markedly reduced both CD4(+) and CD8(+) T cell engraftment and strongly inhibited allo-specific T cell secretion of IFN-γ. The potency of Th9 cell inhibition of alloreactivity was similar to that of rapamycin resistant Th2 cells. Importantly, rapamycin resistant Th9 cells persisted and maintained their cytokine phenotype, thereby indicating limited differentiation plasticity of the Th9 subset. As such, Th9 differentiation proceeds in the presence of rapamycin to generate a cell therapy product that maintains high IL-9 expression in vivo while inhibiting IFN-γ driven alloreactivity.

Mobile and stationary laser weapon demonstrators of Rheinmetall Waffe Munition

NASA Astrophysics Data System (ADS)

Ludewigt, K.; Riesbeck, Th.; Baumgärtel, Th.; Schmitz, J.; Graf, A.; Jung, M.

2014-10-01

For some years Rheinmetall Waffe Munition has successfully developed, realised and tested a variety of versatile high energy laser (HEL) weapon systems for air- and ground-defence scenarios like C-RAM, UXO clearing. By employing beam superimposition technology and a modular laser weapon concept, the total optical power has been successively increased. Stationary weapon platforms and now military mobile vehicles were equipped with high energy laser effectors. Our contribution summarises the most recent development stages of Rheinmetalls high energy laser weapon program. We present three different vehicle based HEL demonstrators: the 5 kW class Mobile HEL Effector Track V integrated in an M113 tank, the 20 kW class Mobile HEL Effector Wheel XX integrated in a multirole armoured vehicle GTK Boxer 8x8 and the 50 kW class Mobile HEL Effector Container L integrated in a reinforced container carried by an 8x8 truck. As a highlight, a stationary 30 kW Laser Weapon Demonstrator shows the capability to defeat saturated attacks of RAM targets and unmanned aerial vehicles. 2013 all HEL demonstrators were tested in a firing campaign at the Rheinmetall testing centre in Switzerland. Major results of these tests are presented.

Antimicrobial peptide hLF1-11 directs granulocyte-macrophage colony-stimulating factor-driven monocyte differentiation toward macrophages with enhanced recognition and clearance of pathogens.

PubMed

van der Does, Anne M; Bogaards, Sylvia J P; Ravensbergen, Bep; Beekhuizen, Henry; van Dissel, Jaap T; Nibbering, Peter H

2010-02-01

The human lactoferrin-derived peptide hLF1-11 displays antimicrobial activities in vitro and is effective against infections with antibiotic-resistant bacteria and fluconazole-resistant Candida albicans in animals. However, the mechanisms underlying these activities remain largely unclear. Since hLF1-11 is ineffective in vitro at physiological salt concentrations, we suggested modulation of the immune system as an additional mechanism of action of the peptide. We investigated whether hLF1-11 affects human monocyte-macrophage differentiation and determined the antimicrobial activities of the resulting macrophages. Monocytes were cultured for 7 days with GM-CSF in the presence of hLF1-11, control peptide, or saline for various intervals. At day 6, the cells were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or heat-killed C. albicans for 24 h. Thereafter, the levels of cytokines in the culture supernatants, the expression of pathogen recognition receptors, and the antimicrobial activities of these macrophages were determined. The results showed that a short exposure of monocytes to hLF1-11 during GM-CSF-driven differentiation is sufficient to direct differentiation of monocytes toward a macrophage subset characterized by both pro- and anti-inflammatory cytokine production and increased responsiveness to microbial structures. Moreover, these macrophages are highly effective against C. albicans and Staphylococcus aureus. In conclusion, hLF1-11 directs GM-CSF-driven differentiation of monocytes toward macrophages with enhanced effector functions.

A surgeons' guide to renal transplant immunopathology, immunology, and immunosuppression.

PubMed

Gaber, Lillian W; Knight, Richard J; Patel, Samir J

2013-12-01

The response to allografting involves adaptive and innate immune mechanisms. In the adaptive system, activated T cells differentiate to cytotoxic effectors that attack the graft and trigger B cells to differentiation to plasma cells that produce anti-HLA antibodies. The innate immune system recognizes antigens in a non-specific manner and recruits immune cells to the graft through the productions of chemotactic factors, and activation of cytokines and the complement cascade. In the kidney the tubules and the endothelium are the targets of the rejection response. Immune suppression is effective in modulating the adaptive immune system effect on graft histology. Copyright © 2013 Elsevier Inc. All rights reserved.

Multilevel regulation of gene expression by microRNAs.

PubMed

Makeyev, Eugene V; Maniatis, Tom

2008-03-28

MicroRNAs (miRNAs) are approximately 22-nucleotide-long noncoding RNAs that normally function by suppressing translation and destabilizing messenger RNAs bearing complementary target sequences. Some miRNAs are expressed in a cell- or tissue-specific manner and may contribute to the establishment and/or maintenance of cellular identity. Recent studies indicate that tissue-specific miRNAs may function at multiple hierarchical levels of gene regulatory networks, from targeting hundreds of effector genes incompatible with the differentiated state to controlling the levels of global regulators of transcription and alternative pre-mRNA splicing. This multilevel regulation may allow individual miRNAs to profoundly affect the gene expression program of differentiated cells.

Connections Between Metabolism and Epigenetics in Programming Cellular Differentiation.

PubMed

Chisolm, Danielle A; Weinmann, Amy S

2018-04-26

Researchers are intensifying efforts to understand the mechanisms by which changes in metabolic states influence differentiation programs. An emerging objective is to define how fluctuations in metabolites influence the epigenetic states that contribute to differentiation programs. This is because metabolites such as S-adenosylmethionine, acetyl-CoA, α-ketoglutarate, 2-hydroxyglutarate, and butyrate are donors, substrates, cofactors, and antagonists for the activities of epigenetic-modifying complexes and for epigenetic modifications. We discuss this topic from the perspective of specialized CD4 + T cells as well as effector and memory T cell differentiation programs. We also highlight findings from embryonic stem cells that give mechanistic insight into how nutrients processed through pathways such as glycolysis, glutaminolysis, and one-carbon metabolism regulate metabolite levels to influence epigenetic events and discuss similar mechanistic principles in T cells. Finally, we highlight how dysregulated environments, such as the tumor microenvironment, might alter programming events.

Effector analogues detect varied allosteric roles for conserved protein-effector interactions in pyruvate kinase isozymes†

PubMed Central

Alontaga, Aileen Y.; Fenton, Aron W.

2011-01-01

The binding site for allosteric inhibitor (amino acid) is highly conserved between human liver pyruvate kinase (hL-PYK) and the rabbit muscle isozyme (rM1-PYK). To detail similarities/differences in the allosteric function of these two homologs, we quantified the binding of 45 amino acid analogues to hL-PYK and their allosteric impact on affinity for the substrate, phosphoenolpyruvate (PEP). This complements a similar study previously completed for rM1-PYK. In hL-PYK, the minimum chemical requirements for effector binding are the same as those identified for rM1-PYK (i.e. the L-2-aminopropanaldehyde substructure of the effector is primarily responsible for binding). However different regions of the effector determine the magnitude of the allosteric response in hL-PYK vs. rM1-PYK. This finding is inconsistent with the idea that allosteric pathways are conserved between homologs of a protein family. PMID:21261284

Contact detection for nanomanipulation in a scanning electron microscope.

PubMed

Ru, Changhai; To, Steve

2012-07-01

Nanomanipulation systems require accurate knowledge of the end-effector position in all three spatial coordinates, XYZ, for reliable manipulation of nanostructures. Although the images acquired by a scanning electron microscope (SEM) provide high resolution XY information, the lack of depth information in the Z-direction makes 3D nanomanipulation time-consuming. Existing approaches for contact detection of end-effectors inside SEM typically utilize fragile touch sensors that are difficult to integrate into a nanomanipulation system. This paper presents a method for determining the contact between an end-effector and a target surface during nanomanipulation inside SEM, purely based on the processing of SEM images. A depth-from-focus method is used in the fast approach of the end-effector to the substrate, followed by fine contact detection. Experimental results demonstrate that the contact detection approach is capable of achieving an accuracy of 21.5 nm at 50,000× magnification while inducing little end-effector damage. Copyright © 2012 Elsevier B.V. All rights reserved.

Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA-seq

PubMed Central

Kakaradov, Boyko; Arsenio, Janilyn; Widjaja, Christella E.; He, Zhaoren; Aigner, Stefan; Metz, Patrick J.; Yu, Bingfei; Wehrens, Ellen J.; Lopez, Justine; Kim, Stephanie H.; Zuniga, Elina I.; Goldrath, Ananda W.; Chang, John T.; Yeo, Gene W.

2017-01-01

SUMMARY During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants controlling CD8+ T lymphocyte fate specification. These findings suggest a model of terminal effector cell differentiation initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, highlighting the power and necessity of single-cell approaches. PMID:28218746

p21WAF1 immunohistochemical expression in breast carcinoma: correlations with clinicopathological data, oestrogen receptor status, MIB1 expression, p53 gene and protein alterations and relapse-free survival.

PubMed Central

Barbareschi, M.; Caffo, O.; Doglioni, C.; Fina, P.; Marchetti, A.; Buttitta, F.; Leek, R.; Morelli, L.; Leonardi, E.; Bevilacqua, G.; Dalla Palma, P.; Harris, A. L.

1996-01-01

p21 protein (p21) inhibitor of cyclin-dependent kinases is a critical downstream effector in the p53-specific pathway of growth control. p21 can also be induced by p53-independent pathways in relation to terminal differentiation. We investigated p21 immunoreactivity in normal breast and in 91 breast carcinomas [three in situ ductal carcinomas (DCIS) with microinfiltration and 88 infiltrating carcinomas, 17 of which with an associated DCIS; 57 node negative and 34 node positive] with long-term follow-up (median = 58 months). Seven additional breast carcinomas with known p53 gene mutations were investigated. In normal breast p21 expression was seen in the nuclei of rare luminal cells of acinar structures, and in occasional myoepithelial cells. Poorly differentiated DCIS showed high p21 expression, whereas well-differentiated DCIS tumours showed few p21-reactive cells. p21 was seen in 82 (90%) infiltrating tumours; staining was heterogeneous; the percentage of reactive nuclei ranged from 1% to 35%. High p21 expression (more than 10% of reactive cells) was seen in 24 (26%) cases, and was associated with high tumour grade (P = 0.032); no associations were seen with tumour size, metastases, oestrogen receptor status, MIB1 expression and p53 expression. p21 expression in cases with p53 gene mutations was low in six cases and high in one. High p21 expression was associated with short relapse-free survival (P = 0.003). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8688323

Shigella IpaH Family Effectors as a Versatile Model for Studying Pathogenic Bacteria.

PubMed

Ashida, Hiroshi; Sasakawa, Chihiro

2015-01-01

Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis). Via the type III secretion system (T3SS), Shigella deliver a subset of virulence proteins (effectors) that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC). Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections.

Shigella IpaH Family Effectors as a Versatile Model for Studying Pathogenic Bacteria

PubMed Central

Ashida, Hiroshi; Sasakawa, Chihiro

2016-01-01

Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis). Via the type III secretion system (T3SS), Shigella deliver a subset of virulence proteins (effectors) that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC). Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections. PMID:26779450

Impaired Subset Progression and Polyfunctionality of T Cells in Mice Exposed to Methamphetamine during Chronic LCMV Infection

PubMed Central

Sriram, Uma; Hill, Beth L.; Cenna, Jonathan M.; Gofman, Larisa; Fernandes, Nicole C.; Haldar, Bijayesh; Potula, Raghava

2016-01-01

Methamphetamine (METH) is a widely used psychostimulant that severely impacts the host’s innate and adaptive immune systems and has profound immunological implications. T cells play a critical role in orchestrating immune responses. We have shown recently how chronic exposure to METH affects T cell activation using a murine model of lymphocytic choriomeningitis virus (LCMV) infection. Using the TriCOM (trinary state combinations) feature of GemStone™ to study the polyfunctionality of T cells, we have analyzed how METH affected the cytokine production pattern over the course of chronic LCMV infection. Furthermore, we have studied in detail the effects of METH on splenic T cell functions, such as cytokine production and degranulation, and how they regulate each other. We used the Probability State Modeling (PSM) program to visualize the differentiation of effector/memory T cell subsets during LCMV infection and analyze the effects of METH on T cell subset progression. We recently demonstrated that METH increased PD-1 expression on T cells during viral infection. In this study, we further analyzed the impact of PD-1 expression on T cell functional markers as well as its expression in the effector/memory subsets. Overall, our study indicates that analyzing polyfunctionality of T cells can provide additional insight into T cell effector functions. Analysis of T cell heterogeneity is important to highlight changes in the evolution of memory/effector functions during chronic viral infections. Our study also highlights the impact of METH on PD-1 expression and its consequences on T cell responses. PMID:27760221

The quartet theory of human emotions: An integrative and neurofunctional model

NASA Astrophysics Data System (ADS)

Koelsch, Stefan; Jacobs, Arthur M.; Menninghaus, Winfried; Liebal, Katja; Klann-Delius, Gisela; von Scheve, Christian; Gebauer, Gunter

2015-06-01

Despite an explosion of research in the affective sciences during the last few decades, interdisciplinary theories of human emotions are lacking. Here we present a neurobiological theory of emotions that includes emotions which are uniquely human (such as complex moral emotions), considers the role of language for emotions, advances the understanding of neural correlates of attachment-related emotions, and integrates emotion theories from different disciplines. We propose that four classes of emotions originate from four neuroanatomically distinct cerebral systems. These emotional core systems constitute a quartet of affect systems: the brainstem-, diencephalon-, hippocampus-, and orbitofrontal-centred affect systems. The affect systems were increasingly differentiated during the course of evolution, and each of these systems generates a specific class of affects (e.g., ascending activation, pain/pleasure, attachment-related affects, and moral affects). The affect systems interact with each other, and activity of the affect systems has effects on - and interacts with - biological systems denoted here as emotional effector systems. These effector systems include motor systems (which produce actions, action tendencies, and motoric expression of emotion), peripheral physiological arousal, as well as attentional and memory systems. Activity of affect systems and effector systems is synthesized into an emotion percept (pre-verbal subjective feeling), which can be transformed (or reconfigured) into a symbolic code such as language. Moreover, conscious cognitive appraisal (involving rational thought, logic, and usually language) can regulate, modulate, and partly initiate, activity of affect systems and effector systems. Our emotion theory integrates psychological, neurobiological, sociological, anthropological, and psycholinguistic perspectives on emotions in an interdisciplinary manner, aiming to advance the understanding of human emotions and their neural correlates.

Impaired Subset Progression and Polyfunctionality of T Cells in Mice Exposed to Methamphetamine during Chronic LCMV Infection.

PubMed

Sriram, Uma; Hill, Beth L; Cenna, Jonathan M; Gofman, Larisa; Fernandes, Nicole C; Haldar, Bijayesh; Potula, Raghava

2016-01-01

Methamphetamine (METH) is a widely used psychostimulant that severely impacts the host's innate and adaptive immune systems and has profound immunological implications. T cells play a critical role in orchestrating immune responses. We have shown recently how chronic exposure to METH affects T cell activation using a murine model of lymphocytic choriomeningitis virus (LCMV) infection. Using the TriCOM (trinary state combinations) feature of GemStone™ to study the polyfunctionality of T cells, we have analyzed how METH affected the cytokine production pattern over the course of chronic LCMV infection. Furthermore, we have studied in detail the effects of METH on splenic T cell functions, such as cytokine production and degranulation, and how they regulate each other. We used the Probability State Modeling (PSM) program to visualize the differentiation of effector/memory T cell subsets during LCMV infection and analyze the effects of METH on T cell subset progression. We recently demonstrated that METH increased PD-1 expression on T cells during viral infection. In this study, we further analyzed the impact of PD-1 expression on T cell functional markers as well as its expression in the effector/memory subsets. Overall, our study indicates that analyzing polyfunctionality of T cells can provide additional insight into T cell effector functions. Analysis of T cell heterogeneity is important to highlight changes in the evolution of memory/effector functions during chronic viral infections. Our study also highlights the impact of METH on PD-1 expression and its consequences on T cell responses.

Global impact of Salmonella type III secretion effector SteA on host cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cardenal-Muñoz, Elena, E-mail: e_cardenal@us.es; Gutiérrez, Gabriel, E-mail: ggpozo@us.es; Ramos-Morales, Francisco, E-mail: framos@us.es

Highlights: • We analyzed HeLa cells transcriptome in response to Salmonella SteA. • Significant differential expression was detected for 58 human genes. • They are involved in ECM organization and regulation of some signaling pathways. • Cell death, cell adhesion and cell migration were decreased in SteA-expressing cells. • These results contribute to understand the role of SteA during infections. - Abstract: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. Thesemore » systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration.« less

Blocking Glycolytic Metabolism Increases Memory T Cells and Antitumor Function | Center for Cancer Research

Cancer.gov

CD8+ T cells are a major component of the cellular immune response, which is necessary to control a variety of bacterial and viral infections. CD8+ T cells also play a major role in the cell-mediated antitumor immune response. After encountering antigen, naïve CD8+ T cells undergo an extensive period of proliferation and expansion, and differentiate into effector cells and

The role of hemocytes in A. gambiae antiplasmodial immunity

PubMed Central

Ramirez, Jose Luis; Garver, Lindsey S.; Brayner, Fábio André; Alves, Luiz Carlos; Rodrigues, Janneth; Molina-Cruz, Alvaro; Barillas-Mury, Carolina

2013-01-01

Hemocytes synthesize key components of the mosquito complement-like system, but their role in the activation of antiplasmodial responses has not been established. The effect of activating Toll signaling in hemocytes on Plasmodium survival was investigated by transferring hemocytes or cell-free hemolymph from donor mosquitoes in which the suppressor cactus was silenced. These transfers greatly enhanced antiplasmodial immunity, indicating that hemocytes are active players in the activation of the complement-like system, through an effector(s) regulated by the Toll pathway. A comparative analysis of hemocyte populations between susceptible (S) G3 and the refractory (R) L3-5 A. gambiae mosquito strains did not reveal significant differences under basal conditions or in response to Plasmodium berghei infection. The response of S mosquitoes to different Plasmodium species revealed similar kinetics following infection with P. berghei, P. yoelii or P. falciparum, but the strength of the priming response was stronger in less compatible mosquito-parasite pairs. The Toll, Imd, STAT or JNK signaling cascades were not essential for the production of hemocyte differentiation factor (HDF) in response to P. berghei infection, but disruption of Toll, STAT or JNK abolished hemocyte differentiation in response to HDF. We conclude that hemocytes are key mediators of A. gambiae antiplasmodial responses. PMID:23886925

PI3K: A Crucial Piece in the RAS Signaling Puzzle.

PubMed

Krygowska, Agata Adelajda; Castellano, Esther

2018-06-01

RAS proteins are key signaling switches essential for control of proliferation, differentiation, and survival of eukaryotic cells. RAS proteins are mutated in 30% of human cancers. In addition, mutations in upstream or downstream signaling components also contribute to oncogenic activation of the pathway. RAS proteins exert their functions through activation of several signaling pathways and dissecting the contributions of these effectors in normal cells and in cancer is an ongoing challenge. In this review, we summarize our current knowledge about how RAS regulates type I phosphatidylinositol 3-kinase (PI3K), one of the main RAS effectors. RAS signaling through PI3K is necessary for normal lymphatic vasculature development and for RAS-induced transformation in vitro and in vivo, especially in lung cancer, where it is essential for tumor initiation and necessary for tumor maintenance. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

PubMed

Altman, Amnon; Kong, Kok-Fai

2016-05-20

The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

Multipronged CD4 T cell effector and memory responses cooperate to provide potent immunity against respiratory virus

PubMed Central

Strutt, Tara M.; McKinstry, K. Kai; Marshall, Nikki B.; Vong, Allen M.; Dutton, Richard W.; Swain, Susan L.

2014-01-01

Summary Over the last decade, the known spectrum of CD4 T cell effect or subsets has become much broader and it has become clear that there are multiple dimensions by which subsets with a particular cytokine commitment can be further defined, including their stage of differentiation, their location and most importantly, their ability to carryout discrete functions. Here we focus on our studies that highlight the synergy among discrete subsets, especially those defined by helper and cytotoxic function, in mediating viral protection and on distinctions between CD4 T cell effectors located in spleen, draining lymph node, and in tissue sites of infection. What emerges is a surprising multiplicity of CD4 T cell functions that indicate a large arsenal of mechanisms by which CD4 T cells act to combat viruses. PMID:23947353

Complement-Mediated Regulation of Metabolism and Basic Cellular Processes.

PubMed

Hess, Christoph; Kemper, Claudia

2016-08-16

Complement is well appreciated as a critical arm of innate immunity. It is required for the removal of invading pathogens and works by directly destroying them through the activation of innate and adaptive immune cells. However, complement activation and function is not confined to the extracellular space but also occurs within cells. Recent work indicates that complement activation regulates key metabolic pathways and thus can impact fundamental cellular processes, such as survival, proliferation, and autophagy. Newly identified functions of complement include a key role in shaping metabolic reprogramming, which underlies T cell effector differentiation, and a role as a nexus for interactions with other effector systems, in particular the inflammasome and Notch transcription-factor networks. This review focuses on the contributions of complement to basic processes of the cell, in particular the integration of complement with cellular metabolism and the potential implications in infection and other disease settings. Copyright © 2016 Elsevier Inc. All rights reserved.

Ral signaling pathway in health and cancer.

PubMed

Moghadam, Adel Rezaei; Patrad, Elham; Tafsiri, Elham; Peng, Warner; Fangman, Benjamin; Pluard, Timothy J; Accurso, Anthony; Salacz, Michael; Shah, Kushal; Ricke, Brandon; Bi, Danse; Kimura, Kyle; Graves, Leland; Najad, Marzieh Khajoie; Dolatkhah, Roya; Sanaat, Zohreh; Yazdi, Mina; Tavakolinia, Naeimeh; Mazani, Mohammad; Amani, Mojtaba; Ghavami, Saeid; Gartell, Robyn; Reilly, Colleen; Naima, Zaid; Esfandyari, Tuba; Farassati, Faris

2017-12-01

The Ral (Ras-Like) signaling pathway plays an important role in the biology of cells. A plethora of effects is regulated by this signaling pathway and its prooncogenic effectors. Our team has demonstrated the overactivation of the RalA signaling pathway in a number of human malignancies including cancers of the liver, ovary, lung, brain, and malignant peripheral nerve sheath tumors. Additionally, we have shown that the activation of RalA in cancer stem cells is higher in comparison with differentiated cancer cells. In this article, we review the role of Ral signaling in health and disease with a focus on the role of this multifunctional protein in the generation of therapies for cancer. An improved understanding of this pathway can lead to development of a novel class of anticancer therapies that functions on the basis of intervention with RalA or its downstream effectors. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

In sílico identification and characterization of putative Dot/Icm secreted virulence effectors in the fish pathogen Piscirickettsia salmonis.

PubMed

Labra, Álvaro; Arredondo-Zelada, Oscar; Flores-Herrera, Patricio; Marshall, Sergio H; Gómez, Fernando A

2016-03-01

Piscirickettsia salmonis seriously affects the Chilean salmon industry. The bacterium is phylogenetically related to Legionella pneumophila and Coxiella burnetii, sharing a Dot/Icm secretion system with them. Although it is well documented that L. pneumophila and C. burnetii secrete different virulence effectors via this Dot/Icm system in order to attenuate host cell responses, to date there have been no reported virulence effectors secreted by the Dot/Icm system of P. salmonis. Using several annotations of P. salmonis genome, here we report an in silico analyses of 4 putative Dot/Icm effectors. Three of them contain ankyrin repeat domains and the typical conserved 3D structures of this protein family. The fourth one is highly similar to one of the Dot/Icm-dependent effectors of L. pneumophila. Additionally, all the potential P. salmonis effectors contain a classical Dot/Icm secretion signal in their C-terminus, consisting of: an E-Block, a hydrophobic residue in -3 or -4 and an electronegative charge. Finally, qPCR analysis demonstrated that these proteins are overexpressed early in infection, perhaps contributing to the generation of a replicative vacuole, a key step in the neutralizing strategy proposed for the Dot/Icm system. In summary, this report identifies four Dot/Icm-dependent effectors in P. salmonis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Y4lO of Rhizobium sp. Strain NGR234 Is a Symbiotic Determinant Required for Symbiosome Differentiation▿

PubMed Central

Yang, Feng-Juan; Cheng, Li-Li; Zhang, Ling; Dai, Wei-Jun; Liu, Zhe; Yao, Nan; Xie, Zhi-Ping; Staehelin, Christian

2009-01-01

Type 3 (T3) effector proteins, secreted by nitrogen-fixing rhizobia with a bacterial T3 secretion system, affect the nodulation of certain host legumes. The open reading frame y4lO of Rhizobium sp. strain NGR234 encodes a protein with sequence similarities to T3 effectors from pathogenic bacteria (the YopJ effector family). Transcription studies showed that the promoter activity of y4lO depended on the transcriptional activator TtsI. Recombinant Y4lO protein expressed in Escherichia coli did not acetylate two representative mitogen-activated protein kinase kinases (human MKK6 and MKK1 from Medicago truncatula), indicating that YopJ-like proteins differ with respect to their substrate specificities. The y4lO gene was mutated in NGR234 (strain NGRΩy4lO) and in NGRΩnopL, a mutant that does not produce the T3 effector NopL (strain NGRΩnopLΩy4lO). When used as inoculants, the symbiotic properties of the mutants differed. Tephrosia vogelii, Phaseolus vulgaris cv. Yudou No. 1, and Vigna unguiculata cv. Sui Qing Dou Jiao formed pink effective nodules with NGR234 and NGRΩnopLΩy4lO. Nodules induced by NGRΩy4lO were first pink but rapidly turned greenish (ineffective nodules), indicating premature senescence. An ultrastructural analysis of the nodules induced by NGRΩy4lO revealed abnormal formation of enlarged infection droplets in ineffective nodules, whereas symbiosomes harboring a single bacteroid were frequently observed in effective nodules induced by NGR234 or NGRΩnopLΩy4lO. It is concluded that Y4lO is a symbiotic determinant involved in the differentiation of symbiosomes. Y4lO mitigated senescence-inducing effects caused by the T3 effector NopL, suggesting synergistic effects for Y4lO and NopL in nitrogen-fixing nodules. PMID:19060155

Genome and Transcriptome Analyses Provide Insight into the Euryhaline Adaptation Mechanism of Crassostrea gigas

PubMed Central

Zhang, Linlin; Li, Chunyan; Li, Li; She, Zhicai; Huang, Baoyu; Zhang, Guofan

2013-01-01

Background The Pacific oyster, Crassostrea gigas, has developed special mechanisms to regulate its osmotic balance to adapt to fluctuations of salinities in coastal zones. To understand the oyster’s euryhaline adaptation, we analyzed salt stress effectors metabolism pathways under different salinities (salt 5, 10, 15, 20, 25, 30 and 40 for 7 days) using transcriptome data, physiology experiment and quantitative real-time PCR. Results Transcriptome data uncovered 189, 480, 207 and 80 marker genes for monitoring physiology status of oysters and the environment conditions. Three known salt stress effectors (involving ion channels, aquaporins and free amino acids) were examined. The analysis of ion channels and aquaporins indicated that 7 days long-term salt stress inhibited voltage-gated Na+/K+ channel and aquaporin but increased calcium-activated K+ channel and Ca2+ channel. As the most important category of osmotic stress effector, we analyzed the oyster FAAs metabolism pathways (including taurine, glycine, alanine, beta-alanine, proline and arginine) and explained FAAs functional mechanism for oyster low salinity adaptation. FAAs metabolism key enzyme genes displayed expression differentiation in low salinity adapted individuals comparing with control which further indicated that FAAs played important roles for oyster salinity adaptation. A global metabolic pathway analysis (iPath) of oyster expanded genes displayed a co-expansion of FAAs metabolism in C. gigas compared with seven other species, suggesting oyster’s powerful ability regarding FAAs metabolism, allowing it to adapt to fluctuating salinities, which may be one important mechanism underlying euryhaline adaption in oyster. Additionally, using transcriptome data analysis, we uncovered salt stress transduction networks in C. gigas. Conclusions Our results represented oyster salt stress effectors functional mechanisms under salt stress conditions and explained the expansion of FAAs metabolism pathways as the most important effectors for oyster euryhaline adaptation. This study was the first to explain oyster euryhaline adaptation at a genome-wide scale in C. gigas. PMID:23554902

Pluripotent Stem Cells in Research and Treatment of Hemoglobinopathies

PubMed Central

Arora, Natasha; Daley, George Q.

2012-01-01

Pluripotent stem cells (PSCs) hold great promise for research and treatment of hemoglobinopathies. In principle, patient-specific induced pluripotent stem cells could be derived from a blood sample, genetically corrected to repair the disease-causing mutation, differentiated into hematopoietic stem cells (HSCs), and returned to the patient to provide a cure through autologous gene and cell therapy. However, there are many challenges at each step of this complex treatment paradigm. Gene repair is currently inefficient in stem cells, but use of zinc finger nucleases and transcription activator-like effector nucleases appear to be a major advance. To date, no successful protocol exists for differentiating PSCs into definitive HSCs. PSCs can be directly differentiated into primitive red blood cells, but not yet in sufficient numbers to enable treating patients, and the cost of clinical scale differentiation is prohibitively expensive with current differentiation methods and efficiencies. Here we review the progress, promise, and remaining hurdles in realizing the potential of PSCs for cell therapy. PMID:22474618

Transcription factor interplay in T helper cell differentiation.

PubMed

Evans, Catherine M; Jenner, Richard G

2013-11-01

The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity.

Receptor Tyrosine Kinases in Drosophila Development

PubMed Central

Sopko, Richelle; Perrimon, Norbert

2013-01-01

Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

Reversal of human allergen-specific CRTH2+ T(H)2 cells by IL-12 or the PS-DSP30 oligodeoxynucleotide.

PubMed

Annunziato, F; Cosmi, L; Manetti, R; Brugnolo, F; Parronchi, P; Maggi, E; Nagata, K; Romagnani, S

2001-11-01

The chemoattractant receptor homologous molecule expressed on T(H)2 cells (CRTH2) is a receptor for prostaglandin D(2), which among human T cells is selectively expressed by T(H)2 and type 2 cytotoxic effectors. Our purpose was to assess whether the cytokine production profile of T(H)2 effectors could be reversed by exploiting their selective expression of CRTH2. CRTH2(+) T cells were purified from the blood of allergic subjects, stimulated with the specific allergen in the absence or presence of IL-12, and assessed by flow cytometry at the single-cell level for their ability to produce IL-4 and/or IFN-gamma after antigen or polyclonal stimulation. Both IL-12 and the PS-DSP30 oligodeoxynucleotide enabled CRTH2(+) allergen-stimulated T(H)2 cells to produce IFN-gamma. This change in the profile of cytokine production by T(H)2 cells from allergic subjects was related to the upregulation of IL-12 receptor beta2 chain and was associated with the loss of CRTH2. These data demonstrate that the cytokine production pattern of fully differentiated T(H)2 effectors can be changed to a less polarized profile, thus providing the physiologic basis for new immunotherapeutic strategies in allergic disorders.

Identification of new ligands for the methionine biosynthesis transcriptional regulator (MetJ) by FAC-MS.

PubMed

Martí-Arbona, Ricardo; Teshima, Munehiro; Anderson, Penelope S; Nowak-Lovato, Kristy L; Hong-Geller, Elizabeth; Unkefer, Clifford J; Unkefer, Pat J

2012-01-01

We have developed a high-throughput approach using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) for the identification and characterization of the small molecules that modulate transcriptional regulator (TR) binding to TR targets. We tested this approach using the methionine biosynthesis regulator (MetJ). We used effector mixtures containing S-adenosyl-L-methionine (SAM) and S-adenosyl derivatives as potential ligands for MetJ binding. The differences in the elution time of different compounds allowed us to rank the binding affinity of each compound. Consistent with previous results, FAC-MS showed that SAM binds to MetJ with the highest affinity. In addition, adenine and 5'-deoxy-5'-(methylthio)adenosine bind to the effector binding site on MetJ. Our experiments with MetJ demonstrate that FAC-MS is capable of screening complex mixtures of molecules and identifying high-affinity binders to TRs. In addition, FAC-MS experiments can be used to discriminate between specific and nonspecific binding of the effectors as well as to estimate the dissociation constant (K(d)) for effector-TR binding. Copyright © 2012 S. Karger AG, Basel.

Effect of human bone marrow mesenchymal stromal cells on cytokine production by peripheral blood naive, memory, and effector T cells.

PubMed

Laranjeira, Paula; Pedrosa, Monia; Pedreiro, Susana; Gomes, Joana; Martinho, Antonio; Antunes, Brigida; Ribeiro, Tania; Santos, Francisco; Trindade, Helder; Paiva, Artur

2015-01-05

The different distribution of T cells among activation/differentiation stages in immune disorders may condition the outcome of mesenchymal stromal cell (MSC)-based therapies. Indeed, the effect of MSCs in the different functional compartments of T cells is not completely elucidated. We investigated the effect of human bone marrow MSCs on naturally occurring peripheral blood functional compartments of CD4(+) and CD8(+) T cells: naive, central memory, effector memory, and effector compartments. For that, mononuclear cells (MNCs) stimulated with phorbol myristate acetate (PMA) plus ionomycin were cultured in the absence/presence of MSCs. The percentage of cells expressing tumor necrosis factor-alpha (TNF-α), interferon gamma (IFNγ), and interleukin-2 (IL-2), IL-17, IL-9, and IL-6 and the amount of cytokine produced were assessed by flow cytometry. mRNA levels of IL-4, IL-10, transforming growth factor-beta (TGF-β), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) in purified CD4(+) and CD8(+) T cells, and phenotypic and mRNA expression changes induced by PMA + ionomycin stimulation in MSCs, were also evaluated. MSCs induced the reduction of the percentage of CD4(+) and CD8(+) T cells producing TNF-α, IFNγ, and IL-2 in all functional compartments, except for naive IFNγ(+)CD4(+) T cells. This inhibitory effect differentially affected CD4(+) and CD8(+) T cells as well as the T-cell functional compartments; remarkably, different cytokines showed distinct patterns of inhibition regarding both the percentage of producing cells and the amount of cytokine produced. Likewise, the percentages of IL-17(+), IL-17(+)TNF-α(+), and IL-9(+) within CD4(+) and CD8(+) T cells and of IL-6(+)CD4(+) T cells were decreased in MNC-MSC co-cultures. MSCs decreased IL-10 and increased IL-4 mRNA expression in stimulated CD4(+) and CD8(+) T cells, whereas TGF-β was reduced in CD8(+) and augmented in CD4(+) T cells, with no changes for CTLA4. Finally, PMA + ionomycin stimulation did not induce significant alterations on MSCs phenotype but did increase indoleamine-2,3-dioxygenase (IDO), inducible costimulatory ligand (ICOSL), IL-1β, IL-8, and TNF-α mRNA expression. Overall, our study showed that MSCs differentially regulate the functional compartments of CD4(+) and CD8(+) T cells, which may differentially impact their therapeutic effect in immune disorders. Furthermore, the influence of MSCs on IL-9 expression can open new possibilities for MSC-based therapy in allergic diseases.

CMV-Specific CD8 T Cell Differentiation and Localization: Implications for Adoptive Therapies.

PubMed

Smith, Corinne J; Quinn, Michael; Snyder, Christopher M

2016-01-01

Human cytomegalovirus (HCMV) is a ubiquitous virus that causes chronic infection and, thus, is one of the most common infectious complications of immune suppression. Adoptive transfer of HCMV-specific T cells has emerged as an effective method to reduce the risk for HCMV infection and/or reactivation by restoring immunity in transplant recipients. However, the CMV-specific CD8 + T cell response is comprised of a heterogenous mixture of subsets with distinct functions and localization, and it is not clear if current adoptive immunotherapy protocols can reconstitute the full spectrum of CD8 + T cell immunity. The aim of this review is to briefly summarize the role of these T cell subsets in CMV immunity and to describe how current adoptive immunotherapy practices might affect their reconstitution in patients. The bulk of the CMV-specific CD8 + T cell population is made up of terminally differentiated effector T cells with immediate effector function and a short life span. Self-renewing memory T cells within the CMV-specific population retain the capacity to expand and differentiate upon challenge and are important for the long-term persistence of the CD8 + T cell response. Finally, mucosal organs, which are frequent sites of CMV reactivation, are primarily inhabited by tissue-resident memory T cells, which do not recirculate. Future work on adoptive transfer strategies may need to focus on striking a balance between the formation of these subsets to ensure the development of long lasting and protective immune responses that can access the organs affected by CMV disease.

Human liver infiltrating γδ T cells are composed of clonally expanded circulating and tissue-resident populations.

PubMed

Hunter, Stuart; Willcox, Carrie R; Davey, Martin S; Kasatskaya, Sofya A; Jeffery, Hannah C; Chudakov, Dmitriy M; Oo, Ye H; Willcox, Benjamin E

2018-05-18

γδ T-cells comprise a substantial proportion of tissue-associated lymphocytes. However, our current understanding of human γδ T-cells is primarily based on peripheral blood subsets, while the immunobiology of tissue-associated subsets remains largely unclear. To address this, we characterised the TCR diversity, immunophenotype and function of human liver infiltrating γδ T-cells, focussing on the predominant tissue-associated Vδ2 neg γδ subset, which is implicated in liver immunopathology. Intrahepatic Vδ2 neg γδ T-cells were highly clonally focussed, with single expanded clonotypes featuring complex, private TCR rearrangements frequently dominating the compartment. Such T-cells were predominantly CD27 lo/neg effector lymphocytes, whereas naïve CD27 hi , TCR diverse populations present in matched blood were generally absent in the liver. Furthermore, while a CD45RA hi Vδ2 neg γδ effector subset present in both liver and peripheral blood contained overlapping TCR clonotypes, the liver Vδ2 neg γδ T-cell pool also included a phenotypically distinct CD45RA lo effector compartment that was enriched for expression of the tissue tropism marker CD69, the hepatic homing chemokine receptors CXCR3 and CXCR6, and liver-restricted TCR clonotypes, suggestive of intrahepatic tissue residency. Liver infiltrating Vδ2 neg γδ cells were capable of polyfunctional cytokine secretion, and unlike peripheral blood subsets, were responsive to both TCR and innate stimuli. These findings suggest the ability of Vδ2 neg γδ T-cells to undergo clonotypic expansion and differentiation is crucial in permitting access to solid tissues such as the liver, and can result in functionally distinct peripheral and liver-resident memory γδ T-cell subsets. They highlight the inherent functional plasticity within the Vδ2 neg γδ T-cell compartment, and may inform design of cellular therapies involving intrahepatic trafficking of γδ T-cells to suppress liver inflammation or combat liver cancer. γδ T cells are frequently enriched in many solid tissues, however the immunobiology of such tissue-associated subsets in humans has remained unclear. We show that intrahepatic γδ T cells are enriched for clonally expanded effector T cells, whereas naïve γδ T cells are largely excluded; moreover, whereas a distinct proportion of circulating T cell clonotypes was present in both the liver tissue and peripheral blood, a functionally and clonotypically distinct population of liver-resident γδ T cells was also evident. Our findings suggest that factors triggering γδ T cell clonal selection and differentiation, such as infection, can drive enrichment of γδ T cells into liver tissue, allowing the development of functionally distinct tissue-restricted memory populations specialised in local hepatic immunosurveillance. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes

PubMed Central

van der Does, H. Charlotte; Schmidt, Sarah M.; Langereis, Léon; Hughes, Timothy R.

2016-01-01

Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called ‘effectors’. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the ‘pathogenicity’ chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol pathogenicity chromosome may be partially transcriptionally autonomous, but there are also extensive transcriptional connections between core and accessory chromosomes. PMID:27855160

Gene expression profiling during asexual development of the late blight pathogen Phytophthora infestans reveals a highly dynamic transcriptome.

PubMed

Judelson, Howard S; Ah-Fong, Audrey M V; Aux, George; Avrova, Anna O; Bruce, Catherine; Cakir, Cahid; da Cunha, Luis; Grenville-Briggs, Laura; Latijnhouwers, Maita; Ligterink, Wilco; Meijer, Harold J G; Roberts, Samuel; Thurber, Carrie S; Whisson, Stephen C; Birch, Paul R J; Govers, Francine; Kamoun, Sophien; van West, Pieter; Windass, John

2008-04-01

Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle. Approximately half of P. infestans genes were found to exhibit significant differential expression between developmental transitions, with approximately (1)/(10) being stage-specific and most changes occurring during zoosporogenesis. Quantitative reverse-transcription polymerase chain reaction assays confirmed the robustness of the array results and showed that similar patterns of differential expression were obtained regardless of whether hyphae were from laboratory media or infected tomato. Differentially expressed genes encode potential cellular regulators, especially protein kinases; metabolic enzymes such as those involved in glycolysis, gluconeogenesis, or the biosynthesis of amino acids or lipids; regulators of DNA synthesis; structural proteins, including predicted flagellar proteins; and pathogenicity factors, including cell-wall-degrading enzymes, RXLR effector proteins, and enzymes protecting against plant defense responses. Curiously, some stage-specific transcripts do not appear to encode functional proteins. These findings reveal many new aspects of oomycete biology, as well as potential targets for crop protection chemicals.

ClpP-deletion impairs the virulence of Legionella pneumophila and the optimal translocation of effector proteins.

PubMed

Zhao, Bei-Bei; Li, Xiang-Hui; Zeng, Yong-Lun; Lu, Yong-Jun

2016-08-02

The opportunistic bacterial pathogen Legionella pneumophila uses substrate effectors of Dot/Icm type IVB secretion system (T4BSS) to accomplish survival and replication in amoebae cells and mammalian alveolar macrophages. During the conversion between its highly resistant, infectious dormant form and vigorously growing, uninfectious replicative form, L. pneumophila utilizes a complicated regulatory network in which proteolysis may play a significant role. As a highly conserved core protease, ClpP is involved in various cellular processes as well as virulence in bacteria, and has been proved to be required for the expression of transmission traits and cell division of L. pneumophila. The clpP-deficient L. pneumophila strain failed to replicate and was digested in the first 3 h post-infection in mammalian cells J774A.1. Further investigation demonstrates that the clpP deficient mutant strain was unable to escape the endosome-lysosomal pathway in host cells. We also found that the clpP deficient mutant strain still expresses T4BSS components, induces contact-dependent cytotoxicity and translocate effector proteins RalF and LegK2, indicating that its T4BSS was overall functional. Interestingly, we further found that the translocation of several effector proteins is significantly reduced without ClpP. The data indicate that ClpP plays an important role in regulating the virulence and effector translocation of Legionella pneumophila.

Rap1b Is an Effector of Axin2 Regulating Crosstalk of Signaling Pathways During Skeletal Development.

PubMed

Maruyama, Takamitsu; Jiang, Ming; Abbott, Alycia; Yu, H-M Ivy; Huang, Qirong; Chrzanowska-Wodnicka, Magdalena; Chen, Emily I; Hsu, Wei

2017-09-01

Recent identification and isolation of suture stem cells capable of long-term self-renewal, clonal expanding, and differentiating demonstrate their essential role in calvarial bone development, homeostasis, and injury repair. These bona fide stem cells express a high level of Axin2 and are able to mediate bone regeneration and repair in a cell autonomous fashion. The importance of Axin2 is further demonstrated by its genetic inactivation in mice causing skeletal deformities resembling craniosynostosis in humans. The fate determination and subsequent differentiation of Axin2+ stem cells are highly orchestrated by a variety of evolutionary conserved signaling pathways including Wnt, FGF, and BMP. These signals are often antagonistic of each other and possess differential effects on osteogenic and chondrogenic cell types. However, the mechanisms underlying the interplay of these signaling transductions remain largely elusive. Here we identify Rap1b acting downstream of Axin2 as a signaling interrogator for FGF and BMP. Genetic analysis reveals that Rap1b is essential for development of craniofacial and body skeletons. Axin2 regulates Rap1b through modulation of canonical BMP signaling. The BMP-mediated activation of Rap1b promotes chondrogenic fate and chondrogenesis. Furthermore, by inhibiting MAPK signaling, Rap1b mediates the antagonizing effect of BMP on FGF to repress osteoblast differentiation. Disruption of Rap1b in mice not only enhances osteoblast differentiation but also impairs chondrocyte differentiation during intramembranous and endochondral ossifications, respectively, leading to severe defects in craniofacial and body skeletons. Our findings reveal a dual role of Rap1b in development of the skeletogenic cell types. Rap1b is critical for balancing the signaling effects of BMP and FGF during skeletal development and disease. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

The Greenland shark Somniosus microcephalus-Hemoglobins and ligand-binding properties.

PubMed

Russo, Roberta; Giordano, Daniela; Paredi, Gianluca; Marchesani, Francesco; Milazzo, Lisa; Altomonte, Giovanna; Del Canale, Pietro; Abbruzzetti, Stefania; Ascenzi, Paolo; di Prisco, Guido; Viappiani, Cristiano; Fago, Angela; Bruno, Stefano; Smulevich, Giulietta; Verde, Cinzia

2017-01-01

A large amount of data is currently available on the adaptive mechanisms of polar bony fish hemoglobins, but structural information on those of cartilaginous species is scarce. This study presents the first characterisation of the hemoglobin system of one of the longest-living vertebrate species (392 ± 120 years), the Arctic shark Somniosus microcephalus. Three major hemoglobins are found in its red blood cells and are made of two copies of the same α globin combined with two copies of three very similar β subunits. The three hemoglobins show very similar oxygenation and carbonylation properties, which are unaffected by urea, a very important compound in marine elasmobranch physiology. They display identical electronic absorption and resonance Raman spectra, indicating that their heme-pocket structures are identical or highly similar. The quaternary transition equilibrium between the relaxed (R) and the tense (T) states is more dependent on physiological allosteric effectors than in human hemoglobin, as also demonstrated in polar teleost hemoglobins. Similar to other cartilaginous fishes, we found no evidence for functional differentiation among the three isoforms. The very similar ligand-binding properties suggest that regulatory control of O2 transport may be at the cellular level and that it may involve changes in the cellular concentrations of allosteric effectors and/or variations of other systemic factors. The hemoglobins of this polar shark have evolved adaptive decreases in O2 affinity in comparison to temperate sharks.

The Greenland shark Somniosus microcephalus—Hemoglobins and ligand-binding properties

PubMed Central

Paredi, Gianluca; Marchesani, Francesco; Milazzo, Lisa; Altomonte, Giovanna; Del Canale, Pietro; Abbruzzetti, Stefania; Ascenzi, Paolo; di Prisco, Guido; Viappiani, Cristiano; Fago, Angela; Bruno, Stefano; Smulevich, Giulietta

2017-01-01

A large amount of data is currently available on the adaptive mechanisms of polar bony fish hemoglobins, but structural information on those of cartilaginous species is scarce. This study presents the first characterisation of the hemoglobin system of one of the longest-living vertebrate species (392 ± 120 years), the Arctic shark Somniosus microcephalus. Three major hemoglobins are found in its red blood cells and are made of two copies of the same α globin combined with two copies of three very similar β subunits. The three hemoglobins show very similar oxygenation and carbonylation properties, which are unaffected by urea, a very important compound in marine elasmobranch physiology. They display identical electronic absorption and resonance Raman spectra, indicating that their heme-pocket structures are identical or highly similar. The quaternary transition equilibrium between the relaxed (R) and the tense (T) states is more dependent on physiological allosteric effectors than in human hemoglobin, as also demonstrated in polar teleost hemoglobins. Similar to other cartilaginous fishes, we found no evidence for functional differentiation among the three isoforms. The very similar ligand-binding properties suggest that regulatory control of O2 transport may be at the cellular level and that it may involve changes in the cellular concentrations of allosteric effectors and/or variations of other systemic factors. The hemoglobins of this polar shark have evolved adaptive decreases in O2 affinity in comparison to temperate sharks. PMID:29023598

Systems analysis of BCL2 protein family interactions establishes a model to predict responses to chemotherapy.

PubMed

Lindner, Andreas U; Concannon, Caoimhín G; Boukes, Gerhardt J; Cannon, Mary D; Llambi, Fabien; Ryan, Deborah; Boland, Karen; Kehoe, Joan; McNamara, Deborah A; Murray, Frank; Kay, Elaine W; Hector, Suzanne; Green, Douglas R; Huber, Heinrich J; Prehn, Jochen H M

2013-01-15

Apoptotic desensitization is a hallmark of cancer cells, but present knowledge of molecular systems controlling apoptosis has yet to provide significant prognostic insights. Here, we report findings from a systems study of the intrinsic pathway of apoptosis by BCL2 family proteins and clinical translation of its findings into a model with applications in colorectal cancer (CRC). By determining absolute protein quantifications in CRC cells and patient tumor samples, we found that BAK and BAX were expressed more highly than their antiapoptotic inhibitors. This counterintuitive finding suggested that sole inhibition of effector BAX and BAK could not be sufficient for systems stability in nonstressed cells. Assuming a model of direct effector activation by BH3-only proteins, we calculated that the amount of stress-induced BH3-only proteins required to activate mitochondrial apoptosis could predict individual death responses of CRC cells to 5-fluorouracil/oxaliplatin. Applying this model predictor to protein profiles in tumor and matched normal tissue samples from 26 patients with CRCs, we found that differences in protein quantities were sufficient to model the increased tumor sensitivity to chemotherapy compared with normal tissue. In addition, these differences were sufficient to differentiate clinical responders from nonresponders with high confidence. Applications of our model, termed DR_MOMP, were used to assess the impact of apoptosis-sensitizing drugs in lowering the necessary dose of state-of-the-art chemotherapy in individual patients. Together, our findings offer a ready clinical tool with the potential to tailor chemotherapy to individual patients.

Noncanonical Roles of the Immune System in Eliciting Oncogene Addiction

PubMed Central

Casey, Stephanie C.; Bellovin, David I.; Felsher, Dean W.

2013-01-01

Summary Cancer is highly complex. The magnitude of this complexity makes it highly surprising that even the brief suppression of an oncogene can sometimes result in rapid and sustained tumor regression illustrating that cancers can be “oncogene addicted” [1-10]. The essential implication is that oncogenes may not only fuel the initiation of tumorigenesis, but in some cases necessarily their surfeit of activation is paramaount to maintain a neoplastic state [11]. Oncogene suppression acutely restores normal physiological programs that effectively overrides secondary genetic events and a cancer collapses [12,13]. Oncogene addiction is mediated both through both tumor intrinsic cell-autonomous mechanisms including proliferative arrest, apoptosis, differentiation and cellular senescence [1,2,4,12] but also host-dependent mechanisms that interact with these tumor intrinsic programs [14,15]. Notably, oncogene inactivation elicits a host immune response that involves specific immune effectors and cytokines that facilitate a remodeling of the tumor microenvironment including the shut down of angiogenesis and the induction of cellular senescence of tumor cells [16]. Hence, immune effectors are critically involved in tumor initiation and prevention [17-19] and progression [20], but also appear to be essential to tumor regression upon oncogene inactivation [21-23]. The understanding how the inactivation of an oncogene elicits a systemic signal in the host that prompts a deconstruction of a tumor could have important implications. The combination of oncogene-targeted therapy together with immunomodulatory therapy may be ideal for the development of both a robust tumor intrinsic as well as immunological effectively leading to sustained tumor regression. PMID:23571026

Engineered Human Antibody Constant Domains with Increased Stability*S⃞

PubMed Central

Gong, Rui; Vu, Bang K.; Feng, Yang; Prieto, DaRue A.; Dyba, Marzena A.; Walsh, Joseph D.; Prabakaran, Ponraj; Veenstra, Timothy D.; Tarasov, Sergey G.; Ishima, Rieko; Dimitrov, Dimiter S.

2009-01-01

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability γ1 CH2 was cloned and a panel of cysteine mutants was constructed. Human γ1 CH2 unfolded at a higher temperature (Tm = 54.1 °C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 °C). One mutant (m01) was remarkably stable (Tm = 73.8 °C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases. PMID:19307178

Antigen-Specific Induction of Osteopontin Contributes to the Chronification of Allergic Contact Dermatitis

PubMed Central

Seier, Anne M.; Renkl, Andreas C.; Schulz, Guido; Uebele, Tanja; Sindrilaru, Anca; Iben, Sebastian; Liaw, Lucy; Kon, Shigeyuki; Uede, Toshimitsu; Weiss, Johannes M.

2010-01-01

Allergic contact dermatitis is a T cell-mediated immune response, which in its relapsing chronic form is of high socioeconomic impact. The phosphoglycoprotein osteopontin (OPN) has chemotactic and Th1 cytokine functions and in various models is essential for robust T cell-mediated immunity. Here we demonstrate that OPN is abundantly expressed by both effector T cells and keratinocytes in allergic contact dermatitis lesions. T cells from nickel-allergic donors secrete high levels of OPN following antigen-specific stimulation. OPN may substitute for missing IFN-γ secretion in T effector cells because low IFN-γ-producing T cell clones secrete high levels of OPN, and OPN down-modulates their interleukin-4 expression. Furthermore, interferon-γ from T effector cells augments OPN in allergic contact dermatitis by inducing OPN in keratinocytes, which in turn polarizes dendritic cells and attracts inflammatory cells. In the murine contact hypersensitivity (CHS) model for allergic contact dermatitis, OPN is strongly induced in antigen-specific proliferating T cells, and OPN null mice display a reduced chronic CHS inflammatory response due to a decreased influx of effector T cells. Importantly, because of its function for chronic allergic contact dermatitis, OPN may well be a therapeutic target, because anti-OPN antibody treatment in part suppresses established chronic CHS. PMID:20008129

Neem leaf glycoprotein promotes dual generation of central and effector memory CD8(+) T cells against sarcoma antigen vaccine to induce protective anti-tumor immunity.

PubMed

Ghosh, Sarbari; Sarkar, Madhurima; Ghosh, Tithi; Guha, Ipsita; Bhuniya, Avishek; Saha, Akata; Dasgupta, Shayani; Barik, Subhasis; Bose, Anamika; Baral, Rathindranath

2016-03-01

We have previously shown that Neem Leaf Glycoprotein (NLGP) mediates sustained tumor protection by activating host immune response. Now we report that adjuvant help from NLGP predominantly generates CD44(+)CD62L(high)CCR7(high) central memory (TCM; in lymph node) and CD44(+)CD62L(low)CCR7(low) effector memory (TEM; in spleen) CD8(+) T cells of Swiss mice after vaccination with sarcoma antigen (SarAg). Generated TCM and TEM participated either to replenish memory cell pool for sustained disease free states or in rapid tumor eradication respectively. TCM generated after SarAg+NLGP vaccination underwent significant proliferation and IL-2 secretion following SarAg re-stimulation. Furthermore, SarAg+NLGP vaccination helps in greater survival of the memory precursor effector cells at the peak of the effector response and their maintenance as mature memory cells, in comparison to single modality treatment. Such response is corroborated with the reduced phosphorylation of FOXO in the cytosol and increased KLF2 in the nucleus associated with enhanced CD62L, CCR7 expression of lymph node-resident CD8(+) T cells. However, spleen-resident CD8(+) T memory cells show superior efficacy for immediate memory-to-effector cell conversion. The data support in all aspects that SarAg+NLGP demonstrate superiority than SarAg vaccination alone that benefits the host by rapid effector functions whenever required, whereas, central-memory cells are thought to replenish the memory cell pool for ultimate sustained disease free survival till 60 days following post-vaccination tumor inoculation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Active Flow Effectors for Noise and Separation Control

NASA Technical Reports Server (NTRS)

Turner, Travis L.

2011-01-01

New flow effector technology for separation control and enhanced mixing is based upon shape memory alloy hybrid composite (SMAHC) technology. The technology allows for variable shape control of aircraft structures through actively deformable surfaces. The flow effectors are made by embedding shape memory alloy actuator material in a composite structure. When thermally actuated, the flow effector def1ects into or out of the flow in a prescribed manner to enhance mixing or induce separation for a variety of applications, including aeroacoustic noise reduction, drag reduction, and f1ight control. The active flow effectors were developed for noise reduction as an alternative to fixed-configuration effectors, such as static chevrons, that cannot be optimized for airframe installation effects or variable operating conditions and cannot be retracted for off-design or fail-safe conditions. Benefits include: Increased vehicle control, overall efficiency, and reduced noise throughout all f1ight regimes, Reduced flow noise, Reduced drag, Simplicity of design and fabrication, Simplicity of control through direct current stimulation, autonomous re sponse to environmental heating, fast re sponse, and a high degree of geometric stability. The concept involves embedding prestrained SMA actuators on one side of the chevron neutral axis in order to generate a thermal moment and def1ect the structure out of plane when heated. The force developed in the host structure during def1ection and the aerodynamic load is used for returning the structure to the retracted position. The chevron design is highly scalable and versatile, and easily affords active and/or autonomous (environmental) control. The technology offers wide-ranging market applications, including aerospace, automotive, and any application that requires flow separation or noise control.

Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells.

PubMed

Hamuro, Junji; Toda, Munetoyo; Asada, Kazuko; Hiraga, Asako; Schlötzer-Schrehardt, Ursula; Montoya, Monty; Sotozono, Chie; Ueno, Morio; Kinoshita, Shigeru

2016-09-01

To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy. Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na+/K+ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. Flow cytometry analysis identified the effector cell expressing CD166+CD105-CD44-∼+/-CD26-CD24-, but CD200-, and the presence of other SPs with CD166+ CD105-CD44+++ (CD26 and CD24, either + or -) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Na+/K+ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200∼220 μm2, and the density of cHCECs exceeded 2500 cells/mm2. A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.

Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization

PubMed Central

Kombrink, Anja; Hansen, Guido; Valkenburg, Dirk-Jan

2013-01-01

While host immune receptors detect pathogen-associated molecular patterns to activate immunity, pathogens attempt to deregulate host immunity through secreted effectors. Fungi employ LysM effectors to prevent recognition of cell wall-derived chitin by host immune receptors, although the mechanism to compete for chitin binding remained unclear. Structural analysis of the LysM effector Ecp6 of the fungal tomato pathogen Cladosporium fulvum reveals a novel mechanism for chitin binding, mediated by intrachain LysM dimerization, leading to a chitin-binding groove that is deeply buried in the effector protein. This composite binding site involves two of the three LysMs of Ecp6 and mediates chitin binding with ultra-high (pM) affinity. Intriguingly, the remaining singular LysM domain of Ecp6 binds chitin with low micromolar affinity but can nevertheless still perturb chitin-triggered immunity. Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex. DOI: http://dx.doi.org/10.7554/eLife.00790.001 PMID:23840930

Effects of altered auditory feedback across effector systems: production of melodies by keyboard and singing.

PubMed

Pfordresher, Peter Q; Mantell, James T

2012-01-01

We report an experiment that tested whether effects of altered auditory feedback (AAF) during piano performance differ from its effects during singing. These effector systems differ with respect to the mapping between motor gestures and pitch content of auditory feedback. Whereas this action-effect mapping is highly reliable during phonation in any vocal motor task (singing or speaking), mapping between finger movements and pitch occurs only in limited situations, such as piano playing. Effects of AAF in both tasks replicated results previously found for keyboard performance (Pfordresher, 2003), in that asynchronous (delayed) feedback slowed timing whereas alterations to feedback pitch increased error rates, and the effect of asynchronous feedback was similar in magnitude across tasks. However, manipulations of feedback pitch had larger effects on singing than on keyboard production, suggesting effector-specific differences in sensitivity to action-effect mapping with respect to feedback content. These results support the view that disruption from AAF is based on abstract, effector independent, response-effect associations but that the strength of associations differs across effector systems. Copyright © 2011. Published by Elsevier B.V.

Role of Th17 Cell in Tubercle Bacillus Infection

NASA Astrophysics Data System (ADS)

Zhang, Dandan

2018-01-01

Tuberculosis is mainly a kind of lung disease. Normal immune cell expression can inhibit proliferation of tubercle bacillus in the lungs, but this may also lead to chronic inflammation and pathological lesion. Th17 cell is a newly discovered CD4 + effector T cell subsets, whose differentiation and roles are influenced by various cytokines in the surrounding environment. Th17 cell plays an important role in resisting tubercle bacillus infection, but also it may cause pathological damage through the inflammatory response. Therefore, to balance two kinds of roles of Th17 cells in tubercle bacillus infection can effectively protect the body. This paper intends to do a summary on differentiation, regulation, and biological functions of Th17 cell.

Functional differences between PD-1+ and PD-1- CD4+ effector T cells in healthy donors and patients with glioblastoma multiforme

PubMed Central

Lucca, Liliana E.; Lerner, Benjamin A.; Gunel, Murat; Raddassi, Khadir; Coric, Vlad; Hafler, David A.; Love, J. Christopher

2017-01-01

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) have been highly successful in the treatment of cancer. While PD-1 expression has been widely investigated, its role in CD4+ effector T cells in the setting of health and cancer remains unclear, particularly in the setting of glioblastoma multiforme (GBM), the most aggressive and common form of brain cancer. We examined the functional and molecular features of PD-1+CD4+CD25—CD127+Foxp3—effector cells in healthy subjects and in patients with GBM. In healthy subjects, we found that PD-1+CD4+ effector cells are dysfunctional: they do not proliferate but can secrete large quantities of IFNγ. Strikingly, blocking antibodies against PD-1 did not rescue proliferation. RNA-sequencing revealed features of exhaustion in PD-1+ CD4 effectors. In the context of GBM, tumors were enriched in PD-1+ CD4+ effectors that were similarly dysfunctional and unable to proliferate. Furthermore, we found enrichment of PD-1+TIM-3+ CD4+ effectors in tumors, suggesting that co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial. RNA-sequencing of blood and tumors from GBM patients revealed distinct differences between CD4+ effectors from both compartments with enrichment in multiple gene sets from tumor infiltrating PD-1—CD4+ effectors cells. Enrichment of these gene sets in tumor suggests a more metabolically active cell state with signaling through other co-receptors. PD-1 expression on CD4 cells identifies a dysfunctional subset refractory to rescue with PD-1 blocking antibodies, suggesting that the influence of immune checkpoint inhibitors may involve recovery of function in the PD-1—CD4+ T cell compartment. Additionally, co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial. PMID:28880903

Nanorobotic end-effectors: Design, fabrication, and in situ characterization

NASA Astrophysics Data System (ADS)

Fan, Zheng

Nano-robotic end-effectors have promising applications for nano-fabrication, nano-manufacturing, nano-optics, nano-medical, and nano-sensing; however, low performances of the conventional end-effectors have prevented the widespread utilization of them in various fields. There are two major difficulties in developing the end-effectors: their nano-fabrication and their advanced characterization in the nanoscale. Here we introduce six types of end-effectors: the nanotube fountain pen (NFP), the super-fine nanoprobe, the metal-filled carbon nanotube (m CNT)-based sphere-on-pillar (SOP) nanoantennas, the tunneling nanosensor, and the nanowire-based memristor. The investigations on the NFP are focused on nano-fluidics and nano-fabrications. The NFP could direct write metallic "inks" and fabricating complex metal nanostructures from 0D to 3D with a position servo control, which is critically important to future large-scale, high-throughput nanodevice production. With the help of NFP, we could fabricate the end-effectors such as super-fine nanoprobe and m CNT-based SOP nanoantennas. Those end-effectors are able to detect local flaws or characterize the electrical/mechanical properties of the nanostructure. Moreover, using electron-energy-loss-spectroscopy (EELS) technique during the operation of the SOP optical antenna opens a new basis for the application of nano-robotic end-effectors. The technique allows advanced characterization of the physical changes, such as carrier diffusion, that are directly responsible for the device's properties. As the device was coupled with characterization techniques of scanning-trasmission-electron-microscopy (STEM), the development of tunneling nanosensor advances this field of science into quantum world. Furthermore, the combined STEM-EELS technique plays an important role in our understanding of the memristive switching performance in the nanowire-based memristor. The developments of those nano-robotic end-effectors expend the study abilities in investigating the in situ nanotechnology, providing efficient ways in in situ nanostructure fabrication and the advanced characterization of the nanomaterials.

The cathelicidin protein CRAMP is a potential atherosclerosis self-antigen in ApoE(-/-) mice

PubMed Central

Mihailovic, Peter M.; Lio, Wai Man; Yano, Juliana; Zhao, Xiaoning; Zhou, Jianchang; Chyu, Kuang-Yuh; Shah, Prediman K.; Cercek, Bojan

2017-01-01

Auto-immunity is believed to contribute to inflammation in atherosclerosis. The antimicrobial peptide LL-37, a fragment of the cathelicidin protein precursor hCAP18, was previously identified as an autoantigen in psoriasis. Given the reported link between psoriasis and coronary artery disease, the biological relevance of the autoantigen to atherosclerosis was tested in vitro using a truncated (t) form of the mouse homolog of hCAP18, CRAMP, on splenocytes from athero-prone ApoE(-/-) mice. Stimulation with tCRAMP resulted in increased CD8+ T cells with Central Memory and Effector Memory phenotypes in ApoE(-/-) mice, differentially activated by feeding with normal chow or high fat diet. Immunization of ApoE(-/-) with different doses of the shortened peptide (Cramp) resulted in differential outcomes with a lower dose reducing atherosclerosis whereas a higher dose exacerbating the disease with increased neutrophil infiltration of the atherosclerotic plaques. Low dose Cramp immunization also resulted in increased splenic CD8+ T cell degranulation and reduced CD11b+CD11c+ conventional dendritic cells (cDCs), whereas high dose increased CD11b+CD11c+ cDCs. Our results identified CRAMP, the mouse homolog of hCAP-18, as a potential self-antigen involved in the immune response to atherosclerosis in the ApoE(-/-) mouse model. PMID:29091929

The quartet theory of human emotions: An integrative and neurofunctional model.

PubMed

Koelsch, Stefan; Jacobs, Arthur M; Menninghaus, Winfried; Liebal, Katja; Klann-Delius, Gisela; von Scheve, Christian; Gebauer, Gunter

2015-06-01

Despite an explosion of research in the affective sciences during the last few decades, interdisciplinary theories of human emotions are lacking. Here we present a neurobiological theory of emotions that includes emotions which are uniquely human (such as complex moral emotions), considers the role of language for emotions, advances the understanding of neural correlates of attachment-related emotions, and integrates emotion theories from different disciplines. We propose that four classes of emotions originate from four neuroanatomically distinct cerebral systems. These emotional core systems constitute a quartet of affect systems: the brainstem-, diencephalon-, hippocampus-, and orbitofrontal-centred affect systems. The affect systems were increasingly differentiated during the course of evolution, and each of these systems generates a specific class of affects (e.g., ascending activation, pain/pleasure, attachment-related affects, and moral affects). The affect systems interact with each other, and activity of the affect systems has effects on - and interacts with - biological systems denoted here as emotional effector systems. These effector systems include motor systems (which produce actions, action tendencies, and motoric expression of emotion), peripheral physiological arousal, as well as attentional and memory systems. Activity of affect systems and effector systems is synthesized into an emotion percept (pre-verbal subjective feeling), which can be transformed (or reconfigured) into a symbolic code such as language. Moreover, conscious cognitive appraisal (involving rational thought, logic, and usually language) can regulate, modulate, and partly initiate, activity of affect systems and effector systems. Our emotion theory integrates psychological, neurobiological, sociological, anthropological, and psycholinguistic perspectives on emotions in an interdisciplinary manner, aiming to advance the understanding of human emotions and their neural correlates. Copyright © 2015 Elsevier B.V. All rights reserved.

Polyfunctional response by ImmTAC (IMCgp100) redirected CD8+ and CD4+ T cells.

PubMed

Boudousquie, Caroline; Bossi, Giovanna; Hurst, Jacob M; Rygiel, Karolina A; Jakobsen, Bent K; Hassan, Namir J

2017-11-01

The success of immune system-based cancer therapies depends on a broad immune response engaging a range of effector cells and mechanisms. Immune mobilizing monoclonal T cell receptors (TCRs) against cancer (ImmTAC™ molecules: fusion proteins consisting of a soluble, affinity enhanced TCR and an anti-CD3 scFv antibody) were previously shown to redirect CD8 + and CD4 + T cells against tumours. Here we present evidence that IMCgp100 (ImmTAC recognizing a peptide derived from the melanoma-specific protein, gp100, presented by HLA-A*0201) efficiently redirects and activates effector and memory cells from both CD8 + and CD4 + repertoires. Using isolated subpopulations of T cells, we find that both terminally differentiated and effector memory CD8 + T cells redirected by IMCgp100 are potent killers of melanoma cells. Furthermore, CD4 + effector memory T cells elicit potent cytotoxic activity leading to melanoma cell killing upon redirection by IMCgp100. The majority of T cell subsets belonging to both the CD8 + and CD4 + repertoires secrete key pro-inflammatory cytokines (tumour necrosis factor-α, interferon-γ, interleukin-6) and chemokines (macrophage inflammatory protein-1α-β, interferon-γ-inducible protein-10, monocyte chemoattractant protein-1). At an individual cell level, IMCgp100-redirected T cells display a polyfunctional phenotype, which is a hallmark of a potent anti-cancer response. This study demonstrates that IMCgp100 induces broad immune responses that extend beyond the induction of CD8 + T cell-mediated cytotoxicity. These findings are of particular importance because IMCgp100 is currently undergoing clinical trials as a single agent or in combination with check point inhibitors for patients with malignant melanoma. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

PubMed Central

Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2015-01-01

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

Deregulation of Rab and Rab Effector Genes in Bladder Cancer

PubMed Central

Ho, Joel R.; Chapeaublanc, Elodie; Kirkwood, Lisa; Nicolle, Remy; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Southgate, Jennifer; Radvanyi, François; Goud, Bruno

2012-01-01

Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer. PMID:22724020

Predictive Computational Modeling of the Mucosal Immune Responses during Helicobacter pylori Infection

PubMed Central

Carbo, Adria; Bassaganya-Riera, Josep; Pedragosa, Mireia; Viladomiu, Monica; Marathe, Madhav; Eubank, Stephen; Wendelsdorf, Katherine; Bisset, Keith; Hoops, Stefan; Deng, Xinwei; Alam, Maksudul; Kronsteiner, Barbara; Mei, Yongguo; Hontecillas, Raquel

2013-01-01

T helper (Th) cells play a major role in the immune response and pathology at the gastric mucosa during Helicobacter pylori infection. There is a limited mechanistic understanding regarding the contributions of CD4+ T cell subsets to gastritis development during H. pylori colonization. We used two computational approaches: ordinary differential equation (ODE)-based and agent-based modeling (ABM) to study the mechanisms underlying cellular immune responses to H. pylori and how CD4+ T cell subsets influenced initiation, progression and outcome of disease. To calibrate the model, in vivo experimentation was performed by infecting C57BL/6 mice intragastrically with H. pylori and assaying immune cell subsets in the stomach and gastric lymph nodes (GLN) on days 0, 7, 14, 30 and 60 post-infection. Our computational model reproduced the dynamics of effector and regulatory pathways in the gastric lamina propria (LP) in silico. Simulation results show the induction of a Th17 response and a dominant Th1 response, together with a regulatory response characterized by high levels of mucosal Treg) cells. We also investigated the potential role of peroxisome proliferator-activated receptor γ (PPARγ) activation on the modulation of host responses to H. pylori by using loss-of-function approaches. Specifically, in silico results showed a predominance of Th1 and Th17 cells in the stomach of the cell-specific PPARγ knockout system when compared to the wild-type simulation. Spatio-temporal, object-oriented ABM approaches suggested similar dynamics in induction of host responses showing analogous T cell distributions to ODE modeling and facilitated tracking lesion formation. In addition, sensitivity analysis predicted a crucial contribution of Th1 and Th17 effector responses as mediators of histopathological changes in the gastric mucosa during chronic stages of infection, which were experimentally validated in mice. These integrated immunoinformatics approaches characterized the induction of mucosal effector and regulatory pathways controlled by PPARγ during H. pylori infection affecting disease outcomes. PMID:24039925

CD31+ Cells From Peripheral Blood Facilitate Bone Regeneration in Biologically Impaired Conditions Through Combined Effects on Immunomodulation and Angiogenesis.

PubMed

Sass, F Andrea; Schmidt-Bleek, Katharina; Ellinghaus, Agnes; Filter, Sebastian; Rose, Alexander; Preininger, Bernd; Reinke, Simon; Geissler, Sven; Volk, Hans-Dieter; Duda, Georg N; Dienelt, Anke

2017-05-01

Controlled revascularization and inflammation are key elements regulating endogenous regeneration after (bone) tissue trauma. Peripheral blood-derived cell subsets, such as regulatory T-helper cells and circulating (endothelial) progenitor cells, respectively, can support endogenous tissue healing, whereas effector T cells that are associated with an aged immune system can hinder bone regeneration. CD31 is expressed by diverse leukocytes and is well recognized as a marker of circulating endothelial (precursor) cells; however, CD31 is absent from the surface of differentiated effector T cells. Thus, we hypothesized that by separating the inhibitory fractions from the supportive fractions of circulating cells within the peripheral blood (PB) using the CD31 marker, bone regeneration in biologically compromised conditions, such as those observed in aged patients, could be improved. In support of our hypothesis, we detected an inverse correlation between CD31+ cells and effector T cells in the hematomas of human fracture patients, dependent on the age of the patient. Furthermore, we demonstrated the regenerative capacity of human PB-CD31+ cells in vitro. These findings were translated to a clinically relevant rat model of impaired bone healing. The transplantation of rat PB-CD31+ cells advanced bone tissue restoration in vivo and was associated with an early anti-inflammatory response, the stimulation of (re)vascularization, and reduced fibrosis. Interestingly, the depletion or enrichment of the highly abundant CD31+/14+ monocytes from the mixed CD31+ cell population diminished tissue regeneration at different levels, suggesting combined effects within the PB-CD31+ subsets. In summary, an intraoperative enrichment of PB-CD31+ cells might be a novel option to facilitate endogenous regeneration under biologically impaired situations by supporting immunomodulation and vascularization. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion.

PubMed

Kaartinen, Tanja; Luostarinen, Annu; Maliniemi, Pilvi; Keto, Joni; Arvas, Mikko; Belt, Heini; Koponen, Jonna; Loskog, Angelica; Mustjoki, Satu; Porkka, Kimmo; Ylä-Herttuala, Seppo; Korhonen, Matti

2017-06-01

Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (T SCM , CD95 + CD45RO - CD45RA + CD27 + ) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

The basis of distinctive IL-2- and IL-15-dependent signaling: weak CD122-dependent signaling favors CD8+ T central-memory cell survival but not T effector-memory cell development.

PubMed

Castro, Iris; Yu, Aixin; Dee, Michael J; Malek, Thomas R

2011-11-15

Recent work suggests that IL-2 and IL-15 induce distinctive levels of signaling through common receptor subunits and that such varied signaling directs the fate of Ag-activated CD8(+) T cells. In this study, we directly examined proximal signaling by IL-2 and IL-15 and CD8(+) T cell primary and memory responses as a consequence of varied CD122-dependent signaling. Initially, IL-2 and IL-15 induced similar p-STAT5 and p-S6 activation, but these activities were only sustained by IL-2. Transient IL-15-dependent signaling is due to limited expression of IL-15Rα. To investigate the outcome of varied CD122 signaling for CD8(+) T cell responses in vivo, OT-I T cells were used from mouse models where CD122 signals were attenuated by mutations within the cytoplasmic tail of CD122 or intrinsic survival function was provided in the absence of CD122 expression by transgenic Bcl-2. In the absence of CD122 signaling, generally normal primary response occurred, but the primed CD8(+) T cells were not maintained. In marked contrast, weak CD122 signaling supported development and survival of T central-memory (T(CM)) but not T effector-memory (T(EM)) cells. Transgenic expression of Bcl-2 in CD122(-/-) CD8(+) T cells also supported the survival and persistence of T(CM) cells but did not rescue T(EM) development. These data indicate that weak CD122 signals readily support T(CM) development largely through providing survival signals. However, stronger signals, independent of Bcl-2, are required for T(EM) development. Our findings are consistent with a model whereby low, intermediate, and high CD122 signaling support T(CM) memory survival, T(EM) programming, and terminal T effector cell differentiation, respectively.

Plant-pathogen interactions: toward development of next-generation disease-resistant plants.

PubMed

Nejat, Naghmeh; Rookes, James; Mantri, Nitin L; Cahill, David M

2017-03-01

Briskly evolving phytopathogens are dire threats to our food supplies and threaten global food security. From the recent advances made toward high-throughput sequencing technologies, understanding of pathogenesis and effector biology, and plant innate immunity, translation of these means into new control tools is being introduced to develop durable disease resistance. Effectoromics as a powerful genetic tool for uncovering effector-target genes, both susceptibility genes and executor resistance genes in effector-assisted breeding, open up new avenues to improve resistance. TALENs (Transcription Activator-Like Effector Nucleases), engineered nucleases and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems are breakthrough and powerful techniques for genome editing, providing efficient mechanisms for targeted crop protection strategies in disease resistance programs. In this review, major advances in plant disease management to confer durable disease resistance and novel strategies for boosting plant innate immunity are highlighted.

Development of kinematic equations and determination of workspace of a 6 DOF end-effector with closed-kinematic chain mechanism

NASA Technical Reports Server (NTRS)

Nguyen, Charles C.; Pooran, Farhad J.

1989-01-01

This report presents results from the research grant entitled Active Control of Robot Manipulators, funded by the Goddard Space Flight Center, under Grant NAG5-780, for the period July 1, 1988 to January 1, 1989. An analysis is presented of a 6 degree-of-freedom robot end-effector built to study telerobotic assembly of NASA hardware in space. Since the end-effector is required to perform high precision motion in a limited workspace, closed-kinematic mechanisms are chosen for its design. A closed-form solution is obtained for the inverse kinematic problem and an iterative procedure employing Newton-Raphson method is proposed to solve the forward kinematic problem. A study of the end-effector workspace results in a general procedure for the workspace determination based on link constraints. Computer simulation results are presented.

Innate immunity in rice

PubMed Central

Chen, Xuewei; Ronald, Pamela C.

2011-01-01

Advances in studies of rice innate immunity have led to the identification and characterization of host sensors encoding receptor kinases that perceive conserved microbial signatures. The non-RD domain, a newly recognized hallmark of these receptor kinases is highly expanded in rice (Oryza sativa) compared with Arabidopsis (Arabidopsis thaliana). Researchers have also identified a diverse array of microbial effectors from bacterial and fungal pathogens that triggers immune responses upon perception. These include both, effectors that indirectly target host Nucleotide binding site/Leucine rice repeat (NBS-LRR) proteins and transcription activator-like (TAL) effectors that directly bind promoters of host genes. Here we review the recognition and signaling events that govern rice innate immunity. PMID:21602092

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment

PubMed Central

Bagley, Elena E.

2014-01-01

Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector. PMID:25009497

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

PubMed

Bagley, Elena E

2014-01-01

Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than E k . Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector.

Systems analysis of effector caspase activation and its control by X-linked inhibitor of apoptosis protein

PubMed Central

Rehm, Markus; Huber, Heinrich J; Dussmann, Heiko; Prehn, Jochen H M

2006-01-01

Activation of effector caspases is a final step during apoptosis. Single-cell imaging studies have demonstrated that this process may occur as a rapid, all-or-none response, triggering a complete substrate cleavage within 15 min. Based on biochemical data from HeLa cells, we have developed a computational model of apoptosome-dependent caspase activation that was sufficient to remodel the rapid kinetics of effector caspase activation observed in vivo. Sensitivity analyses predicted a critical role for caspase-3-dependent feedback signalling and the X-linked-inhibitor-of-apoptosis-protein (XIAP), but a less prominent role for the XIAP antagonist Smac. Single-cell experiments employing a caspase fluorescence resonance energy transfer substrate verified these model predictions qualitatively and quantitatively. XIAP was predicted to control this all-or-none response, with concentrations as high as 0.15 μM enabling, but concentrations >0.30 μM significantly blocking substrate cleavage. Overexpression of XIAP within these threshold concentrations produced cells showing slow effector caspase activation and submaximal substrate cleavage. Our study supports the hypothesis that high levels of XIAP control caspase activation and substrate cleavage, and may promote apoptosis resistance and sublethal caspase activation in vivo. PMID:16932741

Robust generation and expansion of skeletal muscle progenitors and myocytes from human pluripotent stem cells.

PubMed

Shelton, Michael; Kocharyan, Avetik; Liu, Jun; Skerjanc, Ilona S; Stanford, William L

2016-05-15

Human pluripotent stem cells provide a developmental model to study early embryonic and tissue development, tease apart human disease processes, perform drug screens to identify potential molecular effectors of in situ regeneration, and provide a source for cell and tissue based transplantation. Highly efficient differentiation protocols have been established for many cell types and tissues; however, until very recently robust differentiation into skeletal muscle cells had not been possible unless driven by transgenic expression of master regulators of myogenesis. Nevertheless, several breakthrough protocols have been published in the past two years that efficiently generate cells of the skeletal muscle lineage from pluripotent stem cells. Here, we present an updated version of our recently described 50-day protocol in detail, whereby chemically defined media are used to drive and support muscle lineage development from initial CHIR99021-induced mesoderm through to PAX7-expressing skeletal muscle progenitors and mature skeletal myocytes. Furthermore, we report an optional method to passage and expand differentiating skeletal muscle progenitors approximately 3-fold every 2weeks using Collagenase IV and continued FGF2 supplementation. Both protocols have been optimized using a variety of human pluripotent stem cell lines including patient-derived induced pluripotent stem cells. Taken together, our differentiation and expansion protocols provide sufficient quantities of skeletal muscle progenitors and myocytes that could be used for a variety of studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Circulating precursor CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure.

PubMed

He, Jing; Tsai, Louis M; Leong, Yew Ann; Hu, Xin; Ma, Cindy S; Chevalier, Nina; Sun, Xiaolin; Vandenberg, Kirsten; Rockman, Steve; Ding, Yan; Zhu, Lei; Wei, Wei; Wang, Changqi; Karnowski, Alexander; Belz, Gabrielle T; Ghali, Joanna R; Cook, Matthew C; Riminton, D Sean; Veillette, André; Schwartzberg, Pamela L; Mackay, Fabienne; Brink, Robert; Tangye, Stuart G; Vinuesa, Carola G; Mackay, Charles R; Li, Zhanguo; Yu, Di

2013-10-17

Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory. Copyright © 2013 Elsevier Inc. All rights reserved.

Population-expression models of immune response

NASA Astrophysics Data System (ADS)

Stromberg, Sean P.; Antia, Rustom; Nemenman, Ilya

2013-06-01

The immune response to a pathogen has two basic features. The first is the expansion of a few pathogen-specific cells to form a population large enough to control the pathogen. The second is the process of differentiation of cells from an initial naive phenotype to an effector phenotype which controls the pathogen, and subsequently to a memory phenotype that is maintained and responsible for long-term protection. The expansion and the differentiation have been considered largely independently. Changes in cell populations are typically described using ecologically based ordinary differential equation models. In contrast, differentiation of single cells is studied within systems biology and is frequently modeled by considering changes in gene and protein expression in individual cells. Recent advances in experimental systems biology make available for the first time data to allow the coupling of population and high dimensional expression data of immune cells during infections. Here we describe and develop population-expression models which integrate these two processes into systems biology on the multicellular level. When translated into mathematical equations, these models result in non-conservative, non-local advection-diffusion equations. We describe situations where the population-expression approach can make correct inference from data while previous modeling approaches based on common simplifying assumptions would fail. We also explore how model reduction techniques can be used to build population-expression models, minimizing the complexity of the model while keeping the essential features of the system. While we consider problems in immunology in this paper, we expect population-expression models to be more broadly applicable.

Plakophilin-1, a Novel Wnt Signaling Regulator, Is Critical for Tooth Development and Ameloblast Differentiation

PubMed Central

Arai, Chieko; Yamada, Aya; Saito, Kan; Ishikawa, Masaki; Xue, Han; Funada, Keita; Haruyama, Naoto; Yamada, Yoshihiko; Fukumoto, Satoshi; Takahashi, Ichiro

2016-01-01

Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme, and the Wnt signaling pathway is involved in this process. We found that Plakophilin (PKP)1, which is associated with diseases such as ectodermal dysplasia/skin fragility syndrome, was highly expressed in teeth and skin, and was upregulated during tooth development. We hypothesized that PKP1 regulates Wnt signaling via its armadillo repeat domain in a manner similar to β-catenin. To determine its role in tooth development, we performed Pkp1 knockdown experiments using ex vivo organ cultures and cell cultures. Loss of Pkp1 reduced the size of tooth germs and inhibited dental epithelial cell proliferation, which was stimulated by Wnt3a. Furthermore, transfected PKP1-emerald green fluorescent protein was translocated from the plasma membrane to the nucleus upon stimulation with Wnt3a and LiCl, which required the PKP1 N terminus (amino acids 161 to 270). Localization of PKP1, which is known as an adhesion-related desmosome component, shifted to the plasma membrane during ameloblast differentiation. In addition, Pkp1 knockdown disrupted the localization of Zona occludens 1 in tight junctions and inhibited ameloblast differentiation; the two proteins were shown to directly interact by immunoprecipitation. These results implicate the participation of PKP1 in early tooth morphogenesis as an effector of canonical Wnt signaling that controls ameloblast differentiation via regulation of the cell adhesion complex. PMID:27015268

Human reinforcement learning subdivides structured action spaces by learning effector-specific values

PubMed Central

Gershman, Samuel J.; Pesaran, Bijan; Daw, Nathaniel D.

2009-01-01

Humans and animals are endowed with a large number of effectors. Although this enables great behavioral flexibility, it presents an equally formidable reinforcement learning problem of discovering which actions are most valuable, due to the high dimensionality of the action space. An unresolved question is how neural systems for reinforcement learning – such as prediction error signals for action valuation associated with dopamine and the striatum – can cope with this “curse of dimensionality.” We propose a reinforcement learning framework that allows for learned action valuations to be decomposed into effector-specific components when appropriate to a task, and test it by studying to what extent human behavior and BOLD activity can exploit such a decomposition in a multieffector choice task. Subjects made simultaneous decisions with their left and right hands and received separate reward feedback for each hand movement. We found that choice behavior was better described by a learning model that decomposed the values of bimanual movements into separate values for each effector, rather than a traditional model that treated the bimanual actions as unitary with a single value. A decomposition of value into effector-specific components was also observed in value-related BOLD signaling, in the form of lateralized biases in striatal correlates of prediction error and anticipatory value correlates in the intraparietal sulcus. These results suggest that the human brain can use decomposed value representations to “divide and conquer” reinforcement learning over high-dimensional action spaces. PMID:19864565

Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease.

PubMed

de Lange, Orlando; Schreiber, Tom; Schandry, Niklas; Radeck, Jara; Braun, Karl Heinz; Koszinowski, Julia; Heuer, Holger; Strauß, Annett; Lahaye, Thomas

2013-08-01

Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5' terminal guanine, contrasting with the TALE preference for a 5' thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Human reinforcement learning subdivides structured action spaces by learning effector-specific values.

PubMed

Gershman, Samuel J; Pesaran, Bijan; Daw, Nathaniel D

2009-10-28

Humans and animals are endowed with a large number of effectors. Although this enables great behavioral flexibility, it presents an equally formidable reinforcement learning problem of discovering which actions are most valuable because of the high dimensionality of the action space. An unresolved question is how neural systems for reinforcement learning-such as prediction error signals for action valuation associated with dopamine and the striatum-can cope with this "curse of dimensionality." We propose a reinforcement learning framework that allows for learned action valuations to be decomposed into effector-specific components when appropriate to a task, and test it by studying to what extent human behavior and blood oxygen level-dependent (BOLD) activity can exploit such a decomposition in a multieffector choice task. Subjects made simultaneous decisions with their left and right hands and received separate reward feedback for each hand movement. We found that choice behavior was better described by a learning model that decomposed the values of bimanual movements into separate values for each effector, rather than a traditional model that treated the bimanual actions as unitary with a single value. A decomposition of value into effector-specific components was also observed in value-related BOLD signaling, in the form of lateralized biases in striatal correlates of prediction error and anticipatory value correlates in the intraparietal sulcus. These results suggest that the human brain can use decomposed value representations to "divide and conquer" reinforcement learning over high-dimensional action spaces.

Vitamin D Pathway Status and the Identification of Target Genes in the Mouse Mammary Gland

DTIC Science & Technology

2013-01-01

12 Palmer HG et al. The vitamin D receptor is a Wnt effector that controls hair follicle differentiation and specifies tumor type in adult epidermis...AD_________________ Award Number: W81XWH-11-1-0152 TITLE: Vitamin D pathway status and the...December 2012 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-11-1-0152 Vitamin D pathway status and the identification of target genes in the

The lupus susceptibility gene Pbx1 regulates the balance between follicular helper T cell and regulatory T cell differentiation

PubMed Central

Choi, Seung-Chul; Hutchinson, Tarun E.; Titov, Anton A.; Seay, Howard R.; Li, Shiwu; Brusko, Todd M.; Croker, Byron P.; Salek-Ardakani, Shahram; Morel, Laurence

2016-01-01

Pbx1 controls chromatin accessibility to a large number of genes and is entirely conserved between mice and humans. The Pbx1-d dominant negative isoform is more frequent in the CD4+ T cells from lupus patients than from healthy controls. Pbx1-d is associated with the production of autoreactive T cells in mice carrying the Sle1a1 lupus susceptibility locus. Transgenic expression of Pbx1-d in CD4+ T cells reproduced the phenotypes of Sle1a1 mice, with increased inflammatory functions of CD4+ T cells and impaired regulatory T cell homeostasis. Pbx1-d Tg also expanded the number of follicular helper T cells in a cell-intrinsic and antigen-specific manner that was enhanced in recall responses, and resulted in TH1-biased antibodies. Moreover, Pbx1-d Tg CD4+ T cells upregulated the expression of miR-10a, miR-21 and miR-155, which have been implicated in Treg and TFH cell homeostasis. Our results suggest that Pbx1-d impacts lupus development by regulating effector T cell differentiation and promoting TFH cells at the expense of Treg cells. In addition, our results identify Pbx1 as a novel regulator of CD4+ T cell effector function. PMID:27296664

Killing of targets by effector CD8 T cells in the mouse spleen follows the law of mass action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganusov, Vitaly V

2009-01-01

In contrast with antibody-based vaccines, it has been difficult to measure the efficacy of T cell-based vaccines and to correlate the efficacy of CD8 T cell responses with protection again viral infections. In part, this difficulty is due to poor understanding of the in vivo efficacy of CD8 T cells produced by vaccination. Using a: recently developed experimental method of in vivo cytotoxicity we have investigated quantitative aspects of killing of peptide-pulsed targets by effector and memory CD8 T cells, specific to three epitopes of lymphocytic choriomeningitis virus (LCMV), in the mouse spleen. By analyzing data on killing of targetsmore » with varying number of epitope-specific effector and memory CD8 T cells, we find that killing of targets by effectors follows the law of mass-action, that is the death rate of peptide-pulsed targets is proportional to the frequency of CTLs in the spleen. In contrast, killing of targets by memory CD8 T cells does not follow the mass action law because the death rate of targets saturates at high frequencies of memory CD8 T cells. For both effector and memory cells, we also find little support for the killing term that includes the decrease of the death rate of targets with target cell density. Interestingly, our analysis suggests that at low CD8 T cell frequencies, memory CD8 T cells on the per capita basis are more efficient at killing peptide-pulsed targets than effectors, but at high frequencies, effectors are more efficient killers than memory T cells. Comparison of the estimated killing efficacy of effector T cells with the value that is predicted from theoretical physics and based on motility of T cells in lymphoid tissues, suggests that limiting step in the killing of peptide-pulsed targets is delivering the lethal hit and not finding the target. Our results thus form a basis for quantitative understanding of the process of killing of virus-infected cells by T cell responses in tissues and can be used to correlate the phenotype of vaccine-induced memory CD8 T cells with their killing efficacy in vivo.« less

Uncovering the Legionella genus effector repertoire - strength in diversity and numbers

PubMed Central

Burstein, David; Amaro, Francisco; Zusman, Tal; Lifshitz, Ziv; Cohen, Ofir; Gilbert, Jack A; Pupko, Tal; Shuman, Howard A; Segal, Gil

2016-01-01

Infection by the human pathogen Legionella pneumophila relies on the translocation of ~300 virulence proteins, termed effectors, which manipulate host-cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species, and predicted their effector repertoire using a previously validated machine-learning approach. This analysis revealed a treasure trove of 5,885 predicted effectors. The effector repertoire of different Legionella species was found to be largely non-overlapping, and only seven core-effectors were shared among all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from their natural protozoan hosts. Furthermore, we detected numerous novel conserved effector domains, and discovered new domain combinations, which allowed inferring yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution. PMID:26752266

Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.

PubMed

Tanaka, Shigeyuki; Djamei, Armin; Presti, Libera Lo; Schipper, Kerstin; Winterberg, Sarah; Amati, Simone; Becker, Dirk; Büchner, Heike; Kumlehn, Jochen; Reissmann, Stefanie; Kahmann, Regine

2015-01-01

The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion. Effectors can remain in the extracellular space, i.e. the plant apoplast (apoplastic effectors), or can cross the plant plasma membrane and exert their function inside the host cell (cytoplasmic effectors). The U. maydis effectors lack conserved motifs in their primary sequences that could allow a classification of the effectome into apoplastic/cytoplasmic effectors. This represents a significant obstacle in functional effector characterization. Here we describe our attempts to establish a system for effector classification into apoplastic and cytoplasmic members, using U. maydis for effector delivery. Copyright © 2015 Elsevier GmbH. All rights reserved.

Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora.

PubMed

Sharma, Rahul; Xia, Xiaojuan; Cano, Liliana M; Evangelisti, Edouard; Kemen, Eric; Judelson, Howard; Oome, Stan; Sambles, Christine; van den Hoogen, D Johan; Kitner, Miloslav; Klein, Joël; Meijer, Harold J G; Spring, Otmar; Win, Joe; Zipper, Reinhard; Bode, Helge B; Govers, Francine; Kamoun, Sophien; Schornack, Sebastian; Studholme, David J; Van den Ackerveken, Guido; Thines, Marco

2015-10-05

Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily. Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic. The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.

Progressive CD4+ central memory T cell decline results in CD4+ effector memory insufficiency and overt disease in chronic SIV infection.

PubMed

Okoye, Afam; Meier-Schellersheim, Martin; Brenchley, Jason M; Hagen, Shoko I; Walker, Joshua M; Rohankhedkar, Mukta; Lum, Richard; Edgar, John B; Planer, Shannon L; Legasse, Alfred; Sylwester, Andrew W; Piatak, Michael; Lifson, Jeffrey D; Maino, Vernon C; Sodora, Donald L; Douek, Daniel C; Axthelm, Michael K; Grossman, Zvi; Picker, Louis J

2007-09-03

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4(+) CCR5(+) effector-memory T (T(EM)) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4(+) memory T cell proliferation appears to prevent collapse of effector site CD4(+) T(EM) cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4(+) T(EM) cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4(+) T(EM) cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4(+) T(EM) cells from central-memory T (T(CM)) cell precursors. The instability of effector site CD4(+) T(EM) cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5(-) CD4(+) T(CM) cells. These data suggest that although CD4(+) T(EM) cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4(+) T(CM) cells.

High pressure stability of protein complexes studied by static and dynamic light scattering

NASA Astrophysics Data System (ADS)

Gebhardt, Ronald; Kulozik, Ulrich

2011-03-01

The high pressure dissociation of hemocyanin prepared from the lobster Homarus americanus and casein micelles from cow milk were observed by in situ light scattering. The hemocyanin dodecamer dissociated via a hexamer into monomers in a two-step three-species reaction. The influence of ligands and the effector l-lactate on the dissociation behavior was investigated. While no effect by carbon monoxide after exchanging the ligand oxygen was observed, the addition of the effector l-lactate led to a decrease in the pressure stability. Due to a trimer intermediate which was found to be stabilized by l-lactate, the dissociation reaction in the presence of the effector was analyzed by a three-step four-species reaction. In the case of casein micelles, a two-step dissociation mechanism was found. The stabilizing interactions of casein micelles were identified and separated.

Expansion of PD-1-positive effector CD4 T cells in an experimental model of SLE: contribution to the self-organized criticality theory.

PubMed

Miyazaki, Yumi; Tsumiyama, Ken; Yamane, Takashi; Ito, Mitsuhiro; Shiozawa, Shunichi

2013-04-18

We have developed a systems biology concept to explain the origin of systemic autoimmunity. From our studies of systemic lupus erythematosus (SLE) we have concluded that this disease is the inevitable consequence of over-stimulating the host's immune system by repeated exposure to antigen to levels that surpass a critical threshold, which we term the system's "self-organized criticality". We observed that overstimulation of CD4 T cells in mice led to the development of autoantibody-inducing CD4 T cells (aiCD4 T) capable of generating various autoantibodies and pathological lesions identical to those observed in SLE. We show here that this is accompanied by the significant expansion of a novel population of effector T cells characterized by expression of programmed death-1 (PD-1)-positive, CD27(low), CD127(low), CCR7(low) and CD44(high)CD62L(low) markers, as well as increased production of IL-2 and IL-6. In addition, repeated immunization caused the expansion of CD8 T cells into fully-matured cytotoxic T lymphocytes (CTL) that express Ly6C(high)CD122(high) effector and memory markers. Thus, overstimulation with antigen leads to the expansion of a novel effector CD4 T cell population that expresses an unusual memory marker, PD-1, and that may contribute to the pathogenesis of SLE.

mTOR Complex Signaling through the SEMA4A-Plexin B2 Axis Is Required for Optimal Activation and Differentiation of CD8+ T Cells.

PubMed

Ito, Daisuke; Nojima, Satoshi; Nishide, Masayuki; Okuno, Tatsusada; Takamatsu, Hyota; Kang, Sujin; Kimura, Tetsuya; Yoshida, Yuji; Morimoto, Keiko; Maeda, Yohei; Hosokawa, Takashi; Toyofuku, Toshihiko; Ohshima, Jun; Kamimura, Daisuke; Yamamoto, Masahiro; Murakami, Masaaki; Morii, Eiichi; Rakugi, Hiromi; Isaka, Yoshitaka; Kumanogoh, Atsushi

2015-08-01

Mammalian target of rapamycin (mTOR) plays crucial roles in activation and differentiation of diverse types of immune cells. Although several lines of evidence have demonstrated the importance of mTOR-mediated signals in CD4(+) T cell responses, the involvement of mTOR in CD8(+) T cell responses is not fully understood. In this study, we show that a class IV semaphorin, SEMA4A, regulates CD8(+) T cell activation and differentiation through activation of mTOR complex (mTORC) 1. SEMA4A(-/-) CD8(+) T cells exhibited impairments in production of IFN-γ and TNF-α and induction of the effector molecules granzyme B, perforin, and FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8(+) T cell responses were significantly impaired in SEMA4A(-/-) mice. Furthermore, SEMA4A(-/-) CD8(+) T cells exhibited reduced mTORC1 activity and elevated mTORC2 activity, suggesting that SEMA4A is required for optimal activation of mTORC1 in CD8(+) T cells. IFN-γ production and mTORC1 activity in SEMA4A(-/-) CD8(+) T cells were restored by administration of recombinant Sema4A protein. In addition, we show that plexin B2 is a functional receptor of SEMA4A in CD8(+) T cells. Collectively, these results not only demonstrate the role of SEMA4A in CD8(+) T cells, but also reveal a novel link between a semaphorin and mTOR signaling. Copyright © 2015 by The American Association of Immunologists, Inc.

Induction of neurite extension and survival in pheochromocytoma cells by the Rit GTPase.

PubMed

Spencer, Michael L; Shao, Haipeng; Andres, Douglas A

2002-06-07

The Rit, Rin, and Ric proteins comprise a distinct and evolutionarily conserved subfamily of the Ras-like small G-proteins. Although these proteins share the majority of core effector domain residues with Ras, recent studies suggest that Rit uses novel effector pathways to regulate NIH3T3 cell proliferation and transformation, while the functions of Rin and Ric remain largely unknown. Since we demonstrate that Rit is expressed in neurons, we investigated the role of Rit signaling in promoting the differentiation and survival of pheochromocytoma cells. In this study, we show that expression of constitutively active Rit (RitL79) in PC6 cells results in neuronal differentiation, characterized by the elaboration of an extensive network of neurite-like processes that are morphologically distinct from those mediated by the expression of oncogenic Ras. Although activated Rit fails to stimulate mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways in COS cells, RitL79 induced the phosphorylation of ERK1/2 in PC6 cells. We also find that Rit-mediated effects on neurite outgrowth can be blocked by co-expression of dominant-negative mutants of C-Raf1 or mitogen-activated protein kinase kinase 1 (MEK1). Moreover, expression of dominant-negative Rit is sufficient to inhibit NGF-induced neurite outgrowth. Expression of active Rit inhibits growth factor-withdrawal mediated apoptosis of PC6 cells, but does not induce phosphorylation of Akt/protein kinase B, suggesting that survival does not utilize the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Instead, pharmacological inhibitors of MEK block Rit-stimulated cell survival. Taken together, these studies suggest that Rit represents a distinct regulatory protein, capable of mediating differentiation and cell survival in PC6 cells using a MEK-dependent signaling pathway to achieve its effects.

Effector and regulatory dendritic cells display distinct patterns of miRNA expression.

PubMed

Lombardi, Vincent; Luce, Sonia; Moussu, Hélène; Morizur, Lise; Gueguen, Claire; Neukirch, Catherine; Chollet-Martin, Sylvie; Mascarell, Laurent; Aubier, Michel; Baron-Bodo, Véronique; Moingeon, Philippe

2017-09-01

MicroRNAs (miRNAs) contribute to the regulation of dendritic cell (DC) polarization, thereby influencing the balance of adaptive immune responses. Herein, we studied the expression of miRNAs in polarized DCs and analyzed whether expression of these miRNAs could be associated with allergic rhinitis and allergen immunotherapy (AIT) outcome. Using specific culture conditions, we differentiated immature human monocyte-derived DCs into DC1, DC2, and DCreg subsets (supporting the differentiation of T H 1, T H 2 or regulatory T cells, respectively). Profiling of miRNA expression was performed in these DC subpopulations using microarrays. Levels of miRNAs specific for polarized DCs were then evaluated in a cohort of 58 patients with allergic rhinitis and 25 non-allergic controls, as well as in samples from 30 subjects treated with sublingual grass pollen tablets or placebo for four months. We successfully identified 16 miRNAs differentially regulated between immature DCs, DC1, DC2, and DCreg cells. In allergic rhinoconjunctivitis patients, the expression of two of those miRNAs (miR-132 and miR-155), was down-regulated compared to non-allergic individuals. However, the levels of these miRNAs were not significantly modified following four months of grass pollen immunotherapy. Studying polarized DCs and clinical samples from subjects with or without allergic rhinoconjunctivitis, we demonstrated that the expression of two miRNAs linked to effector DCs (i.e., DC1 and/or DC2 cells), was reduced in the blood of patients with allergic rhinoconjunctivitis. Nevertheless, these miRNAs did not represent relevant biomarkers to predict or follow-up AIT efficacy. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

Effector and regulatory dendritic cells display distinct patterns of miRNA expression

PubMed Central

Luce, Sonia; Moussu, Hélène; Morizur, Lise; Gueguen, Claire; Neukirch, Catherine; Chollet‐Martin, Sylvie; Mascarell, Laurent; Aubier, Michel; Baron‐Bodo, Véronique; Moingeon, Philippe

2017-01-01

Abstract Introduction MicroRNAs (miRNAs) contribute to the regulation of dendritic cell (DC) polarization, thereby influencing the balance of adaptive immune responses. Herein, we studied the expression of miRNAs in polarized DCs and analyzed whether expression of these miRNAs could be associated with allergic rhinitis and allergen immunotherapy (AIT) outcome. Method Using specific culture conditions, we differentiated immature human monocyte‐derived DCs into DC1, DC2, and DCreg subsets (supporting the differentiation of TH1, TH2 or regulatory T cells, respectively). Profiling of miRNA expression was performed in these DC subpopulations using microarrays. Levels of miRNAs specific for polarized DCs were then evaluated in a cohort of 58 patients with allergic rhinitis and 25 non‐allergic controls, as well as in samples from 30 subjects treated with sublingual grass pollen tablets or placebo for four months. Results We successfully identified 16 miRNAs differentially regulated between immature DCs, DC1, DC2, and DCreg cells. In allergic rhinoconjunctivitis patients, the expression of two of those miRNAs (miR‐132 and miR‐155), was down‐regulated compared to non‐allergic individuals. However, the levels of these miRNAs were not significantly modified following four months of grass pollen immunotherapy. Conclusions Studying polarized DCs and clinical samples from subjects with or without allergic rhinoconjunctivitis, we demonstrated that the expression of two miRNAs linked to effector DCs (i.e., DC1 and/or DC2 cells), was reduced in the blood of patients with allergic rhinoconjunctivitis. Nevertheless, these miRNAs did not represent relevant biomarkers to predict or follow‐up AIT efficacy. PMID:28497578

T cell-intrinsic requirement for NF-kappa B induction in postdifferentiation IFN-gamma production and clonal expansion in a Th1 response.

PubMed

Corn, Radiah A; Aronica, Mark A; Zhang, Fuping; Tong, Yingkai; Stanley, Sarah A; Kim, Se Ryoung Agnes; Stephenson, Linda; Enerson, Ben; McCarthy, Susan; Mora, Ana; Boothby, Mark

2003-08-15

NF-kappaB/Rel transcription factors are linked to innate immune responses and APC activation. Whether and how the induction of NF-kappaB signaling in normal CD4(+) T cells regulates effector function are not well-understood. The liberation of NF-kappaB dimers from inhibitors of kappaB (IkappaBs) constitutes a central checkpoint for physiologic regulation of most forms of NF-kappaB. To investigate the role of NF-kappaB induction in effector T cell responses, we targeted inhibition of the NF-kappaB/Rel pathway specifically to T cells. The Th1 response in vivo is dramatically weakened when T cells defective in their NF-kappaB induction (referred to as IkappaBalpha(DeltaN) transgenic cells) are activated by a normal APC population. Analyses in vivo, and IL-12-supplemented T cell cultures in vitro, reveal that the mechanism underlying this T cell-intrinsic requirement for NF-kappaB involves activation of the IFN-gamma gene in addition to clonal expansion efficiency. The role of NF-kappaB in IFN-gamma gene expression includes a modest decrease in Stat4 activation, T box expressed in T cell levels, and differentiation efficiency along with a more prominent postdifferentiation step. Further, induced expression of Bcl-3, a trans-activating IkappaB-like protein, is decreased in T cells as a consequence of NF-kappaB inhibition. Together, these findings indicate that NF-kappaB induction in T cells regulates efficient clonal expansion, Th1 differentiation, and IFN-gamma production by Th1 lymphocytes at a control point downstream from differentiation.

Activation of the Hedgehog Signaling Pathway in the Developing Lens Stimulates Ectopic FoxE3 Expression and Disruption in Fiber Cell Differentiation

PubMed Central

Kerr, Christine L.; Huang, Jian; Williams, Trevor; West-Mays, Judith A.

2012-01-01

Purpose. The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens. Methods. We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm. Results. Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death. Conclusions. Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3. PMID:22491411

Modulation of CD4(+) T cell-dependent specific cytotoxic CD8(+) T cells differentiation and proliferation by the timing of increase in the pathogen load.

PubMed

Tzelepis, Fanny; Persechini, Pedro M; Rodrigues, Mauricio M

2007-04-25

Following infection with viruses, bacteria or protozoan parasites, naïve antigen-specific CD8(+) T cells undergo a process of differentiation and proliferation to generate effector cells. Recent evidences suggest that the timing of generation of specific effector CD8(+) T cells varies widely according to different pathogens. We hypothesized that the timing of increase in the pathogen load could be a critical parameter governing this process. Using increasing doses of the protozoan parasite Trypanosoma cruzi to infect C57BL/6 mice, we observed a significant acceleration in the timing of parasitemia without an increase in mouse susceptibility. In contrast, in CD8 deficient mice, we observed an inverse relationship between the parasite inoculum and the timing of death. These results suggest that in normal mice CD8(+) T cells became protective earlier, following the accelerated development of parasitemia. The evaluation of specific cytotoxic responses in vivo to three distinct epitopes revealed that increasing the parasite inoculum hastened the expansion of specific CD8(+) cytotoxic T cells following infection. The differentiation and expansion of T. cruzi-specific CD8(+) cytotoxic T cells is in fact dependent on parasite multiplication, as radiation-attenuated parasites were unable to activate these cells. We also observed that, in contrast to most pathogens, the activation process of T. cruzi-specific CD8(+) cytotoxic T cells was dependent on MHC class II restricted CD4(+) T cells. Our results are compatible with our initial hypothesis that the timing of increase in the pathogen load can be a critical parameter governing the kinetics of CD4(+) T cell-dependent expansion of pathogen-specific CD8(+) cytotoxic T cells.

Differential effector responses by circulating/blood and tissue/peritoneal neutrophils following burn combined with Enterococcus faecalis infection.

PubMed

Fazal, Nadeem; Shelip, Alla; Siddiqui, Erum; Ali, Ashraf; Azim, Anser C; Al-Ghoul, Walid M

2012-03-01

Recently we found that superimposition of Enterococcus faecalis infection on burn injury caused an eruption of host mortality not seen with either individual challenge. We hypothesized that the Enterococcus bacteria, and/or factors related to these organisms, aggravate burn-induced modulations in host defense by neutrophils. Our study focuses on alterations in neutrophils' oxidative, proteolytic, and adhesive functions and transendothelial migration of neutrophils in burn rats inoculated with E. faecalis. Rats were subjected to burn (30% total body surface area) and then intra-abdominally inoculated with E. faecalis (10(4)CFU kg(-1) b.w). Polymorphonuclear neutrophils (PMNs) were harvested from circulating/blood and tissue/peritoneal cavity at day-2 post injury. Extracellular release of O(-)(2) anion production was determined by luminometry, and intracellular production of reactive oxygen species was measured by digital imaging technique. Fluoroscan analysis and confocal microscopy determined intracellular elastase production. The expression of adhesion molecule CD11b/CD18 was performed by flow cytometry. Calcein AM-labeled PMNs were co-cultured with TNF-α-stimulated rat lung microvascular endothelial cells, and their ability to adhere was assessed by fluorometry and digital imaging and finally, chemotaxis was measured by neutrophil transmigration assays. The results showed differential effector responses by circulatory and/or tissue PMNs. Tissue/peritoneal PMNs produced more O(-)(2), less intracellular elastase, and increased expression of CD11b/CD18 accompanied with increased adhesivity of MIP-2-stimulated PMNs to endothelial cells as compared to circulatory/blood PMNs. This differential effect was more pronounced following burn plus E. faecalis infection, indicating that the combined injury changed neutrophil functions. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Distributed delays in a hybrid model of tumor-immune system interplay.

PubMed

Caravagna, Giulio; Graudenzi, Alex; d'Onofrio, Alberto

2013-02-01

A tumor is kinetically characterized by the presence of multiple spatio-temporal scales in which its cells interplay with, for instance, endothelial cells or Immune system effectors, exchanging various chemical signals. By its nature, tumor growth is an ideal object of hybrid modeling where discrete stochastic processes model low-numbers entities, and mean-field equations model abundant chemical signals. Thus, we follow this approach to model tumor cells, effector cells and Interleukin-2, in order to capture the Immune surveillance effect. We here present a hybrid model with a generic delay kernel accounting that, due to many complex phenomena such as chemical transportation and cellular differentiation, the tumor-induced recruitment of effectors exhibits a lag period. This model is a Stochastic Hybrid Automata and its semantics is a Piecewise Deterministic Markov process where a two-dimensional stochastic process is interlinked to a multi-dimensional mean-field system. We instantiate the model with two well-known weak and strong delay kernels and perform simulations by using an algorithm to generate trajectories of this process. Via simulations and parametric sensitivity analysis techniques we (i) relate tumor mass growth with the two kernels, we (ii) measure the strength of the Immune surveillance in terms of probability distribution of the eradication times, and (iii) we prove, in the oscillatory regime, the existence of a stochastic bifurcation resulting in delay-induced tumor eradication.

Rab15 Effector Protein: A Novel Protein for Receptor Recycling from the Endocytic Recycling CompartmentD⃞

PubMed Central

Strick, David J.; Elferink, Lisa A.

2005-01-01

Sorting endosomes and the endocytic recycling compartment are critical intracellular stores for the rapid recycling of internalized membrane receptors to the cell surface in multiple cell types. However, the molecular mechanisms distinguishing fast receptor recycling from sorting endosomes and slow receptor recycling from the endocytic recycling compartment remain poorly understood. We previously reported that Rab15 differentially regulates transferrin receptor trafficking through sorting endosomes and the endocytic recycling compartment, suggesting a role for distinct Rab15-effector interactions at these endocytic compartments. In this study, we identified the novel protein Rab15 effector protein (REP15) as a binding partner for Rab15-GTP. REP15 is compartment specific, colocalizing with Rab15 and Rab11 on the endocytic recycling compartment but not with Rab15, Rab4, or early endosome antigen 1 on sorting endosomes. REP15 interacts directly with Rab15-GTP but not with Rab5 or Rab11. Consistent with its localization, REP15 overexpression and small interfering RNA-mediated depletion inhibited transferrin receptor recycling from the endocytic recycling compartment, without affecting receptor entry into or recycling from sorting endosomes. Our data identify REP15 as a compartment-specific protein for receptor recycling from the endocytic recycling compartment, highlighting that the rapid and slow modes of transferrin receptor recycling are mechanistically distinct pathways. PMID:16195351

Integration of T Cell Receptor, Notch and Cytokine Signals Programs in Mouse γδ T Cell Effector Differentiation.

PubMed

Zarin, Payam; In, Tracy S H; Chen, Edward L Y; Singh, Jastaranpreet; Wong, Gladys W; Mohtashami, Mahmood; Wiest, David L; Anderson, Michele K; Zúñiga-Pflücker, Juan Carlos

2018-05-13

γδ T-cells perform a wide range of tissue and disease specific functions that are dependent on the effector cytokines produced by these cells. However, the aggregate signals required for the development of interferon-γ (IFNγ) and interleukin-17 (IL-17) producing γδ T-cells remain unknown. Here, we define the cues involved in the functional programming of γδ T-cells, by examining the roles of T-cell receptor (TCR), Notch, and cytokine-receptor signaling. KN6 γδTCR-transduced Rag2 -/- T-cell progenitors were cultured on stromal cells variably expressing TCR and Notch ligands, supplemented with different cytokines. We found that distinct combinations of these signals are required to program IFNγ versus IL-17 producing γδ T cell subsets, with Notch and weak TCR ligands optimally enabling development of γδ17 cells in the presence of IL-1β, IL-21 and IL-23. Notably, these cytokines were also shown to be required for the intrathymic development of γδ17 cells. Together, this work provides a framework of how signals downstream of TCR, Notch and cytokine receptors integrate to program the effector function of IFNγ and IL-17 producing γδ T-cell subsets. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Dendritic cell immunization route determines CD8+ T cell trafficking to inflamed skin: role for tissue microenvironment and dendritic cells in establishment of T cell-homing subsets.

PubMed

Dudda, Jan C; Simon, Jan C; Martin, Stefan

2004-01-15

The effector/memory T cell pool branches in homing subsets selectively trafficking to organs such as gut or skin. Little is known about the critical factors in the generation of skin-homing CD8+ T cells, although they are crucial effectors in skin-restricted immune responses such as contact hypersensitivity and melanoma defense. In this study, we show that intracutaneous, but not i.v. injection of bone marrow-derived dendritic cells induced skin-homing CD8+ T cells with up-regulated E-selectin ligand expression and effector function in contact hypersensitivity. The skin-homing potential and E-selectin ligand expression remained stable in memory phase without further Ag contact. In contrast, i.p. injection induced T cells expressing the gut-homing integrin alpha(4)beta(7). Although differential expression of these adhesion molecules was strictly associated with the immunization route, the postulated skin-homing marker CCR4 was transiently up-regulated in all conditions. Interestingly, dendritic cells from different tissues effectively induced the corresponding homing markers on T cells in vitro. Our results suggest a crucial role for the tissue microenvironment and dendritic cells in the instruction of T cells for tissue-selective homing and demonstrate that Langerhans cells are specialized to target T cells to inflamed skin.

Transcription factor interplay in T helper cell differentiation

PubMed Central

Evans, Catherine M.

2013-01-01

The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity. PMID:23878131

Dynamic regulation of nuclear architecture and mechanics—a rheostatic role for the nucleus in tailoring cellular mechanosensitivity

PubMed Central

Lee, David A.

2017-01-01

ABSTRACT Nuclear architecture, a function of both chromatin and nucleoskeleton structure, is known to change with stem cell differentiation and differs between various somatic cell types. These changes in nuclear architecture are associated with the regulation of gene expression and genome function in a cell-type specific manner. Biophysical stimuli are known effectors of differentiation and also elicit stimuli-specific changes in nuclear architecture. This occurs via the process of mechanotransduction whereby extracellular mechanical forces activate several well characterized signaling cascades of cytoplasmic origin, and potentially some recently elucidated signaling cascades originating in the nucleus. Recent work has demonstrated changes in nuclear mechanics both with pluripotency state in embryonic stem cells, and with differentiation progression in adult mesenchymal stem cells. This review explores the interplay between cytoplasmic and nuclear mechanosensitivity, highlighting a role for the nucleus as a rheostat in tuning the cellular mechano-response. PMID:28152338

Dynamic regulation of nuclear architecture and mechanics-a rheostatic role for the nucleus in tailoring cellular mechanosensitivity.

PubMed

Thorpe, Stephen D; Lee, David A

2017-05-04

Nuclear architecture, a function of both chromatin and nucleoskeleton structure, is known to change with stem cell differentiation and differs between various somatic cell types. These changes in nuclear architecture are associated with the regulation of gene expression and genome function in a cell-type specific manner. Biophysical stimuli are known effectors of differentiation and also elicit stimuli-specific changes in nuclear architecture. This occurs via the process of mechanotransduction whereby extracellular mechanical forces activate several well characterized signaling cascades of cytoplasmic origin, and potentially some recently elucidated signaling cascades originating in the nucleus. Recent work has demonstrated changes in nuclear mechanics both with pluripotency state in embryonic stem cells, and with differentiation progression in adult mesenchymal stem cells. This review explores the interplay between cytoplasmic and nuclear mechanosensitivity, highlighting a role for the nucleus as a rheostat in tuning the cellular mechano-response.

Exposure of Human CD8+ T Cells to Type-2 Cytokines Impairs Division and Differentiation and Induces Limited Polarization.

PubMed

Fox, Annette; Harland, Kim L; Kedzierska, Katherine; Kelso, Anne

2018-01-01

Effector CD8 + T cells generally produce type-1 cytokines and mediators of the perforin/granzyme cytolytic pathway, yet type-2-polarized CD8 + cells (Tc2) are detected in type-2 (T2) cytokine-driven diseases such as asthma. It is unclear whether T2 cytokine exposure during activation is sufficient to polarize human CD8 + T cells. To address this question, a protocol was developed for high-efficiency activation of human CD8 + T cells in which purified single cells or populations were stimulated with plate-bound anti-CD3 and anti-CD11a mAb for up to 8 days in T2 polarizing or neutral conditions, before functional analysis. Activation of CD8 + naïve T cells (T N ) in T2 compared with neutral conditions decreased the size of single-cell clones, although early division kinetics were equivalent, indicating an effect on overall division number. Activation of T N in T2 conditions followed by brief anti-CD3 mAb restimulation favored expression of T2 cytokines, GATA3 and Eomes , and lowered expression of type-1 cytokines, Prf1 , Gzmb, T-BET, and Prdm1 . However, IL-4 was only weakly expressed, and PMA and ionomycin restimulation favored IFN-γ over IL-4 expression. Activation of T N in T2 compared with neutral conditions prevented downregulation of costimulatory (CD27, CD28) and lymph-node homing receptors (CCR7) and CD95 acquisition, which typically occur during differentiation into effector phenotypes. CD3 was rapidly and substantially induced after activation in neutral, but not T2 conditions, potentially contributing to greater division and differentiation in neutral conditions. CD8 + central memory T cells (T CM ) were less able to enter division upon reactivation in T2 compared with neutral conditions, and were more refractory to modulating IFN-γ and IL-4 production than CD8 + T N. In summary, while activation of T N in T2 conditions can generate T2 cytokine-biased cells, IL-4 expression is weak, T2 bias is lost upon strong restimulation, differentiation, and division are arrested, and reactivation of T CM is reduced in T2 conditions. Taken together, this suggests that exposure to T2 cytokines during activation may not be sufficient to generate and retain human Tc2 cells.

What makes Ras an efficient molecular switch: a computational, biophysical, and structural study of Ras-GDP interactions with mutants of Raf.

PubMed

Filchtinski, Daniel; Sharabi, Oz; Rüppel, Alma; Vetter, Ingrid R; Herrmann, Christian; Shifman, Julia M

2010-06-11

Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (Ras(GTP)) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras(GDP)) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with Ras(GDP). Most of our designed mutations narrow the gap between the affinity of Raf for Ras(GTP) and Ras(GDP), producing the desired shift in binding specificity towards Ras(GDP). A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras(GDP). The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras(GDP) bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras(GDP).Raf mutant complex is found in a conformation similar to that of Ras(GTP) and not Ras(GDP). Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras(GTP) is likely to explain the natural low affinity of Raf and other Ras effectors to Ras(GDP). Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch. Copyright 2010 Elsevier Ltd. All rights reserved.

Mitochondrial Ca2+ and membrane potential, an alternative pathway for Interleukin 6 to regulate CD4 cell effector function

PubMed Central

Yang, Rui; Lirussi, Dario; Thornton, Tina M; Jelley-Gibbs, Dawn M; Diehl, Sean A; Case, Laure K; Madesh, Muniswamy; Taatjes, Douglas J; Teuscher, Cory; Haynes, Laura; Rincón, Mercedes

2015-01-01

IL-6 plays an important role in determining the fate of effector CD4 cells and the cytokines that these cells produce. Here we identify a novel molecular mechanism by which IL-6 regulates CD4 cell effector function. We show that IL-6-dependent signal facilitates the formation of mitochondrial respiratory chain supercomplexes to sustain high mitochondrial membrane potential late during activation of CD4 cells. Mitochondrial hyperpolarization caused by IL-6 is uncoupled from the production of ATP by oxidative phosphorylation. However, it is a mechanism to raise the levels of mitochondrial Ca2+ late during activation of CD4 cells. Increased levels of mitochondrial Ca2+ in the presence of IL-6 are used to prolong Il4 and Il21 expression in effector CD4 cells. Thus, the effect of IL-6 on mitochondrial membrane potential and mitochondrial Ca2+ is an alternative pathway by which IL-6 regulates effector function of CD4 cells and it could contribute to the pathogenesis of inflammatory diseases. DOI: http://dx.doi.org/10.7554/eLife.06376.001 PMID:25974216

Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake.

PubMed

Yildiz, Ozkan; Kalthoff, Christoph; Raunser, Stefan; Kühlbrandt, Werner

2007-01-24

A binary complex of the ammonia channel Amt1 from Methanococcus jannaschii and its cognate P(II) signalling protein GlnK1 has been produced and characterized. Complex formation is prevented specifically by the effector molecules Mg-ATP and 2-ketoglutarate. Single-particle electron microscopy of the complex shows that GlnK1 binds on the cytoplasmic side of Amt1. Three high-resolution X-ray structures of GlnK1 indicate that the functionally important T-loop has an extended, flexible conformation in the absence of Mg-ATP, but assumes a compact, tightly folded conformation upon Mg-ATP binding, which in turn creates a 2-ketoglutarate-binding site. We propose a regulatory mechanism by which nitrogen uptake is controlled by the binding of both effector molecules to GlnK1. At normal effector levels, a 2-ketoglutarate molecule binding at the apex of the compact T-loop would prevent complex formation, ensuring uninhibited ammonia uptake. At low levels of Mg-ATP, the extended loops would seal the ammonia channels in the complex. Binding of both effector molecules to P(II) signalling proteins may thus represent an effective feedback mechanism for regulating ammonium uptake through the membrane.

Role of the glucocorticoid-induced TNFR-related protein (GITR)-GITR ligand pathway in innate and adaptive immunity.

PubMed

Azuma, Miyuki

2010-01-01

Glucocorticoid-induced TNF receptor-related protein (GITR) is expressed in regulatory T cells at high levels, but is also inducible in conventional effector T cells after activation. Initial studies using an agonistic anti- GITR mAb mislead this line of research with respect to the contribution of GITR stimulation on the function of regulatory T cells. In fact, GITR acts as a costimulatory receptor for both effector and regulatory T cells by enhancing effector and regulatory functions, respectively. Unlike other costimulatory ligands, GITR ligand (GITRL) expression on mature myeloid dendritic cells (DCs) is extremely limited and the GITR-GITRL pathway does not contribute markedly to direct interactions with T cells and antigen-presenting cells in the secondary lymphoid tissues. Rather, GITRL is constitutively expressed on parenchymal tissue cells and interacts with GITR expressed on tissue-infiltrating macrophages and DCs, or effector and regulatory T cells. Interactions with GITR and GITRL at local inflammatory sites induce site-specific production of cytokines and chemokines, resulting in control activation of tissue-infiltrating effector or regulatory cells and their migration. This review summarizes recent reports on the GITR-GITRL pathway, which controls both innate and adaptive immune responses.

Effector proteins of rust fungi.

PubMed

Petre, Benjamin; Joly, David L; Duplessis, Sébastien

2014-01-01

Rust fungi include many species that are devastating crop pathogens. To develop resistant plants, a better understanding of rust virulence factors, or effector proteins, is needed. Thus far, only six rust effector proteins have been described: AvrP123, AvrP4, AvrL567, AvrM, RTP1, and PGTAUSPE-10-1. Although some are well established model proteins used to investigate mechanisms of immune receptor activation (avirulence activities) or entry into plant cells, how they work inside host tissues to promote fungal growth remains unknown. The genome sequences of four rust fungi (two Melampsoraceae and two Pucciniaceae) have been analyzed so far. Genome-wide analyses of these species, as well as transcriptomics performed on a broader range of rust fungi, revealed hundreds of small secreted proteins considered as rust candidate secreted effector proteins (CSEPs). The rust community now needs high-throughput approaches (effectoromics) to accelerate effector discovery/characterization and to better understand how they function in planta. However, this task is challenging due to the non-amenability of rust pathosystems (obligate biotrophs infecting crop plants) to traditional molecular genetic approaches mainly due to difficulties in culturing these species in vitro. The use of heterologous approaches should be promoted in the future.

Autonomous TNF is critical for in vivo monocyte survival in steady state and inflammation

PubMed Central

Wolf, Yochai; Shemer, Anat; Polonsky, Michal; Gross, Mor; Mildner, Alexander; David, Eyal; Amit, Ido; Heikenwalder, Mathias; Nedospasov, Sergei; Prinz, Marco; Friedman, Nir

2017-01-01

Monocytes are circulating mononuclear phagocytes, poised to extravasate to sites of inflammation and differentiate into monocyte-derived macrophages and dendritic cells. Tumor necrosis factor (TNF) and its receptors are up-regulated during monopoiesis and expressed by circulating monocytes, as well as effector monocytes infiltrating certain sites of inflammation, such as the spinal cord, during experimental autoimmune encephalomyelitis (EAE). In this study, using competitive in vitro and in vivo assays, we show that monocytes deficient for TNF or TNF receptors are outcompeted by their wild-type counterpart. Moreover, monocyte-autonomous TNF is critical for the function of these cells, as TNF ablation in monocytes/macrophages, but not in microglia, delayed the onset of EAE in challenged animals and was associated with reduced acute spinal cord infiltration of Ly6Chi effector monocytes. Collectively, our data reveal a previously unappreciated critical cell-autonomous role of TNF on monocytes for their survival, maintenance, and function. PMID:28330904

Rho GTPases and their downstream effectors in megakaryocyte biology.

PubMed

Pleines, Irina; Cherpokova, Deya; Bender, Markus

2018-06-18

Megakaryocytes differentiate from hematopoietic stem cells in the bone marrow. The transition of megakaryocytes to platelets is a complex process. Thereby, megakaryocytes extend proplatelets into sinusoidal blood vessels, where the proplatelets undergo fission to release platelets. Defects in platelet production can lead to a low platelet count (thrombocytopenia) with increased bleeding risk. Rho GTPases comprise a family of small signaling G proteins that have been shown to be master regulators of the cytoskeleton controlling many aspects of intracellular processes. The generation of Pf4-Cre transgenic mice was a major breakthrough that enabled studies in megakaryocyte-/platelet-specific knockout mouse lines and provided new insights into the central regulatory role of Rho GTPases in megakaryocyte maturation and platelet production. In this review, we will summarize major findings on the role of Rho GTPases in megakaryocyte biology with a focus on mouse lines in which knockout strategies have been applied to study the function of the best-characterized members Rac1, Cdc42 and RhoA and their downstream effector proteins.

Crossed-uncrossed difference (CUD) in a new light: anatomy of the negative CUD in Poffenberger's paradigm.

PubMed

Derakhshan, I

2006-03-01

Crossed Uncrossed Differentials (CUDs) have long been used as surrogate for the interhemispheric transfer time (IHTT). Evidence is presented that macular vision is the province of the major hemisphere, wherein all commands are initiated regardless of the laterality of the effectors of such commands. Using clinical and time-resolved data it is shown also that the above arrangement (i.e. neural handedness) corresponds to the subject's behavioral avowed (avowed, self-declared) handedness only in a statistical sense; with a substantial minority of humanity displaying a disparity of neural and behavioral handedness. Evidence is provided that the negative CUD in previously reported studies was a reflection of such incongruity in those subjects studied. Thus, to lateralize the command center it is sufficient to determine the reaction time of two symmetrically located effectors on the body. The side with longer reaction time is ipsilateral to the major hemisphere, with the difference of the two sides commensurate to transcallosal IHTT.

Regulation of Asymmetric Division and CD8+ T Lymphocyte Fate Specification by PKCζ and PKCλ/ι

PubMed Central

Metz, Patrick J.; Arsenio, Janilyn; Kakaradov, Boyko; Kim, Stephanie H.; Remedios, Kelly A.; Oakley, Katherine; Akimoto, Kazunori; Ohno, Shigeo; Yeo, Gene W.; Chang, John T.

2015-01-01

During an immune response against a microbial pathogen, activated naïve T lymphocytes give rise to effector cells that provide acute host defense and memory cells that provide long-lived immunity. It has been shown that T lymphocytes can undergo asymmetric division, enabling the daughter cells to inherit unequal amounts of fate-determining proteins and thereby acquire distinct fates from their inception. Here, we show that the absence of the atypical protein kinase C (aPKC) isoforms, PKCζ and PKCλ/ι, disrupts asymmetric CD8+ T lymphocyte division. These alterations were associated with aberrant acquisition of a ‘pre-effector’ transcriptional program, detected by single-cell gene expression analyses, in lymphocytes that had undergone their first division in vivo and enhanced differentiation toward effector fates at the expense of memory fates. Together, these results demonstrate a role for aPKC in regulating asymmetric division and the specification of divergent CD8+ T lymphocyte fates early during an immune response. PMID:25617472

A Near-Complete Haplotype-Phased Genome of the Dikaryotic Wheat Stripe Rust Fungus Puccinia striiformis f. sp. tritici Reveals High Interhaplotype Diversity

PubMed Central

Sperschneider, Jana; Garnica, Diana P.; Miller, Marisa E.; Taylor, Jennifer M.; Dodds, Peter N.; Park, Robert F.

2018-01-01

ABSTRACT A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high-quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here, we present a diploid-aware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long reads using the FALCON-Unzip assembler. Transcriptome sequencing data sets were used to infer high-quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 of 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high interhaplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When we investigated genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet, candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales. In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant-pathogenic fungus previously hidden in collapsed and fragmented genome assemblies. PMID:29463659

Mucins in Gastric Cancer – An Update

PubMed Central

Boltin, Doron; Niv, Yaron

2013-01-01

Mucins are high-molecular-weight glycoproteins expressed throughout the gastrointestinal tract, with a key role in mucosal protection and function. In gastric cancer expression of MUC5AC and MUC1 is reduced and denovo expression of MUC2 occurs. With progressive loss of tumor differentiation and increased tumor stage, expression of MUC5AC and MUC1 is further reduced, and MUC2 decreases. Isolated MUC2 expression (the intestinal phenotype) correlates with metastatic spread and poor survival. There is emerging evidence that MUC1 acts as an oncoprotein when overexpressed. The cytoplasmic tail of MUC1 interacts with the H. pylori virulence factor cagA and is a major effector of the wnt-β catenin intracellular signalling cascade. Polymorphism in the MUC1 gene has been identified in gastric cancer patients and may have a prospective role in the stratification of high-risk subjects. The MUC1 gene also mediates resistance to the recombinant HER2/neu antibody trastuzumab. Future research efforts will examine targeting MUC1 for therapeutic purposes. PMID:24077811

An animated landscape representation of CD4+ T-cell differentiation, variability, and plasticity: Insights into the behavior of populations versus cells

PubMed Central

Rebhahn, Jonathan A; Deng, Nan; Sharma, Gaurav; Livingstone, Alexandra M; Huang, Sui; Mosmann, Tim R

2014-01-01

Recent advances in understanding CD4+ T-cell differentiation suggest that previous models of a few distinct, stable effector phenotypes were too simplistic. Although several well-characterized phenotypes are still recognized, some states display plasticity, and intermediate phenotypes exist. As a framework for reexamining these concepts, we use Waddington's landscape paradigm, augmented with explicit consideration of stochastic variations. Our animation program “LAVA” visualizes T-cell differentiation as cells moving across a landscape of hills and valleys, leading to attractor basins representing stable or semistable differentiation states. The model illustrates several principles, including: (i) cell populations may behave more predictably than individual cells; (ii) analogous to reticulate evolution, differentiation may proceed through a network of interconnected states, rather than a single well-defined pathway; (iii) relatively minor changes in the barriers between attractor basins can change the stability or plasticity of a population; (iv) intrapopulation variability of gene expression may be an important regulator of differentiation, rather than inconsequential noise; (v) the behavior of some populations may be defined mainly by the behavior of outlier cells. While not a quantitative representation of actual differentiation, our model is intended to provoke discussion of T-cell differentiation pathways, particularly highlighting a probabilistic view of transitions between states. PMID:24945794

Adoptive T-cell Therapy Using Autologous Tumor-infiltrating Lymphocytes for Metastatic Melanoma: Current Status and Future Outlook

PubMed Central

Wu, Richard; Forget, Marie-Andree; Chacon, Jessica; Bernatchez, Chantale; Haymaker, Cara; Chen, Jie Qing; Hwu, Patrick; Radvanyi, Laszlo

2012-01-01

Immunotherapy using autologous T-cells has emerged to be a powerful treatment option for patients with metastatic melanoma. These include the adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL), T-cells transduced with high-affinity T-cell receptors (TCR) against major melanosomal tumor antigens, and T cells transduced with chimeric antigen receptors (CAR) composed of hybrid immunoglobulin light chains with endo-domains of T-cell signaling molecules. Among these and other options for T-cell therapy, TIL together with high-dose IL-2 has had the longest clinical history with multiple clinical trials in centers across the world consistently demonstrating durable clinical response rates near 50% or more. A distinct advantage of TIL therapy making it still the T-cell therapy of choice is the broad nature of the T-cell recognition against both defined as well as un-defined tumors antigens against all possible MHC, rather than the single specificity and limited MHC coverage of the newer TCR and CAR transduction technologies. In the past decade, significant inroads have been made in defining the phenotypes of T cells in TIL mediating tumor regression. CD8+ T cells are emerging to be critical, although the exact subset of CD8+ T cells exhibiting the highest clinical activity in terms of memory and effector markers is still controversial. We present a model in which both effector-memory and more differentiated effector T cells ultimately may need to cooperate to mediate long-term tumor control in responding patients. Although TIL therapy has shown great potential to treat metastatic melanoma, a number of issues have emerged that need to be addressed to bring it more into the mainstream of melanoma care. First, we have a reached the point where a pivotal phase II or phase III trials are needed in an attempt to gain regulatory approval of TIL as standard-of-care. Second, improvements in how we expand TIL for therapy are needed, that minimize the time the T-cells are in culture and improve the memory and effector characteristics of the T cells for longer persistence and enhanced anti-tumor activity in vivo. Third, there is a critical need to identify surrogate and predictive biomarkers in order to better select suitable patients for TIL therapy in order to improve response rate and duration. Overall, the outlook for TIL therapy for melanoma is very bright. We predict that TIL will indeed emerge to become an approved treatment in the upcoming years through pivotal clinical trials. Moreover, new approaches combining TIL with targeted signaling pathway drugs, such as mutant B-RAF inhibitors, and synergistic immunomodulatory interventions enhancing T-cell costimulation and preventing negative regulation, should further increase therapeutic efficacy and durable complete response rates. PMID:22453018

Transfer of Motor Learning Is More Pronounced in Proximal Compared to Distal Effectors in Upper Extremities

PubMed Central

Aune, Tore K.; Aune, Morten A.; Ingvaldsen, Rolf P.; Vereijken, Beatrix

2017-01-01

The current experiment investigated generalizability of motor learning in proximal versus distal effectors in upper extremities. Twenty-eight participants were divided into three groups: training proximal effectors, training distal effectors, and no training control group (CG). Performance was tested pre- and post-training for specific learning and three learning transfer conditions: (1) bilateral learning transfer between homologous effectors, (2) lateral learning transfer between non-homologous effectors, and (3) bilateral learning transfer between non-homologous effectors. With respect to specific learning, both training groups showed significant, similar improvement for the trained proximal and distal effectors, respectively. In addition, there was significant learning transfer to all three transfer conditions, except for bilateral learning transfer between non-homologous effectors for the distal training group. Interestingly, the proximal training group showed significantly larger learning transfer to other effectors compared to the distal training group. The CG did not show significant improvements from pre- to post-test. These results show that learning is partly effector independent and generalizable to different effectors, even though transfer is suboptimal compared to specific learning. Furthermore, there is a proximal-distal gradient in generalizability, in that learning transfer from trained proximal effectors is larger than from trained distal effectors, which is consistent with neuroanatomical differences in activation of proximal and distal muscles. PMID:28943857

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

PubMed Central

Zihni, Ceniz; Munro, Peter M.G.; Elbediwy, Ahmed; Keep, Nicholas H.; Terry, Stephen J.; Harris, John

2014-01-01

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation. PMID:24379416

In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire

PubMed Central

Liu, Yunxiao; Lan, Xia; Song, Shiren; Yin, Ling; Dry, Ian B.; Qu, Junjie; Xiang, Jiang; Lu, Jiang

2018-01-01

Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs) were cloned from the P. viticola isolate “JL-7-2” genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83) could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria. PMID:29706971

Colonization and effector functions of innate lymphoid cells in mucosal tissues

PubMed Central

Kim, Myunghoo; Kim, Chang H.

2016-01-01

Innate lymphoid cells (ILCs) protect mucosal barrier tissues to fight infection and maintain tissue integrity. ILCs and their progenitors are developmentally programmed to migrate, differentiate and populate various mucosal tissues and associated lymphoid tissues. Functionally mature ILC subsets respond to diverse pathogens such as bacteria, viruses, fungi and parasites in subset-specific manners. In this review, we will discuss how ILCs populate mucosal tissues and regulate immune responses to distinct pathogens to protect the host and maintain tissue integrity. PMID:27365193

Affinity of antigen encounter and other early B-cell signals determine B-cell fate

PubMed Central

Benson, Micah J; Erickson, Loren D; Gleeson, Michael W; Noelle, Randolph J

2010-01-01

Three possible effector fates await the naïve follicular B cell following antigen stimulation in thymus-dependent reactions. Short-lived plasma cells produce an initial burst of germline-encoded protective antibodies, and long-lived plasma cells and memory B cells arise from the germinal center and function to enhance and sustain the humoral immune response. The inherent B-cell receptor affinity of naïve follicular B cells and the contribution of other early B-cell signals pre-determines the pattern of transcription factor expression and the differentiation path taken by these cells. High initial B-cell receptor affinity shunts naïve follicular B-cell clones towards the short-lived plasma cell fate, whereas modest-affinity clones are skewed towards a plasma cell fate and low-affinity clones are recruited into the germinal center and are selected for both long-lived plasma cells and memory B cell pathways. In the germinal center reaction, increased levels of the transcription factor interferon regulatory factor-4 drive the molecular program that dictates differentiation into the long-lived plasma cell phenotype but has no impact on the memory B cell compartment. We hypothesize that graded interferon regulatory factor-4 levels driven by signals to B cells, including B-cell receptor signal strength, are responsible for this branch point in the B-cell terminal differentiation pathway. PMID:17433651

CMV drives the expansion of highly functional memory T cells expressing NK-cell receptors in renal transplant recipients.

PubMed

Makwana, Nandini; Foley, Bree; Fernandez, Sonia; Lee, Silvia; Irish, Ashley; Pircher, Hanspeter; Price, Patricia

2017-08-01

Cytomegalovirus (CMV) is a common opportunistic infection encountered in renal transplant recipients (RTRs) and may be reactivated without symptoms at any time post-transplant. We describe how active and latent CMV affect T-cell subsets in RTRs who are stable on maintenance therapy. T-cell responses to CMV were assessed in RTRs (n = 54) >2 years post-transplant, and healthy controls (n = 38). Seven RTRs had CMV DNA detectable in plasma. CMV antibody and DNA aligned with increased proportions of CD8 + T cells and reduced CD4/CD8 ratios. This paralleled an expansion of effector memory T-cell (T EM ), terminally differentiated T-cell (T EMRA ) and CD57 + T EMRA cell populations. Expression of NK-cell receptors, LIR-1 and KLRG1 on CD4 + and CD8 + CD57 + T EM and T EMRA cells correlated with elevated interferon-γ and cytotoxic responses to anti-CD3 and increased cytotoxic responses to CMV phosphoprotein (pp) 65 in RTRs who carried CMV DNA. CD8 + T cells from all CMV seropositive RTRs responded efficiently to CMV immediate early (IE) -1 peptides. The data show that latent and active CMV infection can alter T-cell subsets in RTRs many years after transplantation, and up-regulate T-cell expression of NK-cell receptors. This may enhance effector responses of CD4 + and CD8 + T cells against CMV. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative immunophenotypic analysis of antigen-presenting cells involved in ectromelia virus antigen presentation in BALB/c and C57BL/6 mice.

PubMed

Szulc-Dąbrowska, Lidia; Gieryńska, Małgorzata; Boratyńska-Jasińska, Anna; Martyniszyn, Lech; Winnicka, Anna; Niemiałtowski, Marek G

2013-08-01

During mousepox in resistant (C57BL/6) or susceptible (BALB/c) strains of mice, stimulation of Th1 or Th2 cytokine immune response, respectively, is observed. Because mechanisms of different polarization of T cells remain elusive, in this study, we quantitatively assessed the phenotype of antigen-presenting cells (APCs) involved in ectromelia virus (ECTV) antigen presentation and cluster formation with effector cells in secondary lymphoid organs of BALB/c and C57BL/6 mice. We showed that both strains of mice display similar dynamics and kinetics of viral antigen presentation by CD11c(+) , CD11b(+) , and CD19(+) cells. CD11c(+) and CD11b(+) cells highly participated in viral antigen presentation during all stages of mousepox, whereas CD19(+) cells presented viral peptides later in infection. The main population of dendritic cells (DCs) engaged in ECTV antigen presentation and cell junction formation with effector cells was a population of myeloid CD11b(+) DCs (mDCs). We suggest that, on the one hand, ECTV may differentially affect the functions of APCs depending on the strain of mice. On the other hand, we suggest that some types of APCs, such as mDCs or other DCs subsets, have different abilities to direct the shape of immune response depending on the host resistance to mousepox. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptors.

PubMed

Hintersteiner, Beate; Lingg, Nico; Zhang, Peiqing; Woen, Susanto; Hoi, Kong Meng; Stranner, Stefan; Wiederkum, Susanne; Mutschlechner, Oliver; Schuster, Manfred; Loibner, Hans; Jungbauer, Alois

We identified active isoforms of the chimeric anti-GD2 antibody, ch14.18, a recombinant antibody produced in Chinese hamster ovary cells, which is already used in clinical trials. 1,2,3 We separated the antibody by high resolution ion-exchange chromatography with linear pH gradient elution into acidic, main and basic charge variants on a preparative scale yielding enough material for an in-depth study of the sources and the effects of microheterogeneity. The binding affinity of the charge variants toward the antigen and various cell surface receptors was studied by Biacore. Effector functions were evaluated using cellular assays for antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. Basic charge variants showed increased binding to cell surface receptor FcγRIIIa, which plays a major role in regulating effector functions. Furthermore, increased binding of the basic fractions to the neonatal receptor was observed. As this receptor mediates the prolonged half-life of IgG in human serum, this data may well hint at an increased serum half-life of these basic variants compared to their more acidic counterparts. Different glycoform patterns, C-terminal lysine clipping and N-terminal pyroglutamate formation were identified as the main structural sources for the observed isoform pattern. Potential differences in structural stability between individual charge variant fractions by nano differential scanning calorimetry could not been detected. Our in-vitro data suggests that the connection between microheterogeneity and the biological activity of recombinant antibody therapeutics deserves more attention than commonly accepted.

G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

PubMed Central

El Far, Oussama; Betz, Heinrich

2002-01-01

G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity. PMID:12006104

Isoenzymes of protein kinase C in rat mammary tissue: changes in properties and relative amounts during pregnancy and lactation.

PubMed

Connor, K; Clegg, R A

1993-05-01

Protein kinase isoenzymes belonging to the protein kinase C (PK-C) family present in rat mammary tissue have been resolved from one another by chromatography on hydroxyapatite, and characterized. PK-C alpha is the predominant isoenzyme and is present at a constant level of activity throughout mammary-gland development and differentiation. In contrast, marked changes in the relative abundance of other mammary PK-C isoenzymes accompany the transition from pregnancy to lactation. The sensitivity of mammary PK-C alpha to Ca2+ is greater in tissue from pregnant than from lactating rats. This isoenzyme has other atypical properties consistent with its being more highly phosphorylated than PK-C alpha in rat brain and spleen. One of the protein kinase isoenzymes resolved from mammary tissue recognizes the peptide substrate used to assay AMP-activated kinase and may thus interfere in the determination of this activity. Another is fully active in the absence of Ca2+ and is more than 80% active in the absence of added lipid effectors. A 'housekeeping' role is proposed for PK-C alpha in mammary tissue, whereas the less abundant PK-C isoenzymes may be involved in mammary cell proliferation and differentiation.

Black rice (Oryza sativa L.) extracts induce osteoblast differentiation and protect against bone loss in ovariectomized rats.

PubMed

Jang, Woo-Seok; Seo, Cho-Rong; Jang, Hwan Hee; Song, No-Joon; Kim, Jong-Keun; Ahn, Jee-Yin; Han, Jaejoon; Seo, Woo Duck; Lee, Young Min; Park, Kye Won

2015-01-01

Osteoporosis, an age associated skeletal disease, exhibits increased adipogenesis at the expense of osteogenesis from common osteoporotic bone marrow cells. In this study, black rice (Oryza sativa L.) extracts (BRE) were identified as osteogenic inducers. BRE stimulated the alkaline phosphatase (ALP) activity in both C3H10T1/2 and primary bone marrow cells. Similarly, BRE increased mRNA expression of ALP and osterix. Oral administration of BRE in OVX rats prevented decreases in bone density and strength. By contrast, BRE inhibited adipocyte differentiation of mesenchymal C3H10T1/2 cells and prevented increases in body weight and fat mass in high fat diet fed obese mice, further suggesting the dual effects of BRE on anti-adipogenesis and pro-osteogenesis. UPLC analysis identified cyanidin-3-O-glucoside and peonidin-3-O-glucoside as main anti-adipogenic effectors but not for pro-osteogenic induction. In mechanism studies, BRE selectively stimulated Wnt-driven luciferase activities. BRE treatment also induced Wnt-specific target genes such as Axin2, WISP2, and Cyclin D1. Taken together, these data suggest that BRE is a potentially useful ingredient to protect against age related osteoporosis and diet induced obesity.

Cannabidiol in medical marijuana: Research vistas and potential opportunities.

PubMed

Rong, Carola; Lee, Yena; Carmona, Nicole E; Cha, Danielle S; Ragguett, Renee-Marie; Rosenblat, Joshua D; Mansur, Rodrigo B; Ho, Roger C; McIntyre, Roger S

2017-07-01

The high and increasing prevalence of medical marijuana consumption in the general population invites the need for quality evidence regarding its safety and efficacy. Herein, we synthesize extant literature pertaining to the phytocannabinoid cannabidiol (CBD) and its brain effects. The principle phytocannabinoid Δ 9 -tetrahydrocannabinol (Δ 9 -THC) and CBD are the major pharmacologically active cannabinoids. The effect of CBD on brain systems as well as on phenomenological measures (e.g. cognitive function) are distinct and in many cases opposite to that of Δ 9 -THC. Cannabidiol is without euphoriant properties, and exerts antipsychotic, anxiolytic, anti-seizure, as well as anti-inflammatory properties. It is essential to parcellate phytocannabinoids into their constituent moieties as the most abundant cannabinoid have differential effects on physiologic systems in psychopathology measures. Disparate findings and reports related to effects of cannabis consumption reflect differential relative concentration of Δ 9 -THC and CBD. Existing literature, notwithstanding its deficiencies, provides empirical support for the hypothesis that CBD may exert beneficial effects on brain effector systems/substrates subserving domain-based phenomenology. Interventional studies with purified CBD are warranted with a call to target-engagement proof-of-principle studies using the research domain criteria (RDoC) framework. Copyright © 2017 Elsevier Ltd. All rights reserved.

Populus trichocarpa encodes small, effector-like secreted proteins that are highly induced during mutualistic symbiosis

DOE PAGES

Plett, Jonathan M.; Yin, Hengfu; Mewalal, Ritesh; ...

2017-03-23

During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as ‘effectors’, i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa andmore » 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. Finally, we conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.« less

Populus trichocarpa encodes small, effector-like secreted proteins that are highly induced during mutualistic symbiosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plett, Jonathan M.; Yin, Hengfu; Mewalal, Ritesh

During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as ‘effectors’, i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa andmore » 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. Finally, we conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.« less

Anionic lipids and the cytoskeletal proteins MreB and RodZ define the spatio-temporal distribution and function of membrane stress controller PspA in Escherichia coli.

PubMed

Jovanovic, Goran; Mehta, Parul; Ying, Liming; Buck, Martin

2014-11-01

All cell types must maintain the integrity of their membranes. The conserved bacterial membrane-associated protein PspA is a major effector acting upon extracytoplasmic stress and is implicated in protection of the inner membrane of pathogens, formation of biofilms and multi-drug-resistant persister cells. PspA and its homologues in Gram-positive bacteria and archaea protect the cell envelope whilst also supporting thylakoid biogenesis in cyanobacteria and higher plants. In enterobacteria, PspA is a dual function protein negatively regulating the Psp system in the absence of stress and acting as an effector of membrane integrity upon stress. We show that in Escherichia coli the low-order oligomeric PspA regulatory complex associates with cardiolipin-rich, curved polar inner membrane regions. There, cardiolipin and the flotillin 1 homologue YqiK support the PspBC sensors in transducing a membrane stress signal to the PspA-PspF inhibitory complex. After stress perception, PspA high-order oligomeric effector complexes initially assemble in polar membrane regions. Subsequently, the discrete spatial distribution and dynamics of PspA effector(s) in lateral membrane regions depend on the actin homologue MreB and the peptidoglycan machinery protein RodZ. The consequences of loss of cytoplasmic membrane anionic lipids, MreB, RodZ and/or YqiK suggest that the mode of action of the PspA effector is closely associated with cell envelope organization. © 2014 The Authors.

PSTha5a23, a candidate effector from the obligate biotrophic pathogen Puccinia striiformis f. sp. tritici, is involved in plant defense suppression and rust pathogenicity.

PubMed

Cheng, Yulin; Wu, Kuan; Yao, Juanni; Li, Shumin; Wang, Xiaojie; Huang, Lili; Kang, Zhensheng

2017-05-01

During the infection of host plants, pathogens can deliver virulence-associated 'effector' proteins to promote plant susceptibility. However, little is known about effector function in the obligate biotrophic pathogen Puccinia striiformis f. sp. tritici (Pst) that is an important fungal pathogen in wheat production worldwide. Here, they report their findings on an in planta highly induced candidate effector from Pst, PSTha5a23. The PSTha5a23 gene is unique to Pst and shows a low level of intra-species polymorphism. It has a functional N-terminal signal peptide and is translocated to the host cytoplasm after infection. Overexpression of PSTha5a23 in Nicotiana benthamiana was found to suppress the programmed cell death triggered by BAX, PAMP-INF1 and two resistance-related mitogen-activated protein kinases (MKK1 and NPK1). Overexpression of PSTha5a23 in wheat also suppressed pattern-triggered immunity (PTI)-associated callose deposition. In addition, silencing of PSTha5a23 did not change Pst virulence phenotypes; however, overexpression of PSTha5a23 significantly enhanced Pst virulence in wheat. These results indicate that the Pst candidate effector PSTha5a23 plays an important role in plant defense suppression and rust pathogenicity, and also highlight the utility of gene overexpression in plants as a tool for studying effectors from obligate biotrophic pathogens. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida.

PubMed

Thorpe, Peter; Mantelin, Sophie; Cock, Peter Ja; Blok, Vivian C; Coke, Mirela C; Eves-van den Akker, Sebastian; Guzeeva, Elena; Lilley, Catherine J; Smant, Geert; Reid, Adam J; Wright, Kathryn M; Urwin, Peter E; Jones, John T

2014-10-23

The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure - the syncytium - which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium. The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure. This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors.

PubMed

Wei, Hai-Lei; Collmer, Alan

2017-12-25

Pseudomonas syringae pv. tomato DC3000 and its derivatives cause disease in tomato, Arabidopsis and Nicotiana benthamiana. The primary virulence factors include a repertoire of 29 effector proteins injected into plant cells by the type III secretion system and the phytotoxin coronatine. The complete repertoire of effector genes and key coronatine biosynthesis genes have been progressively deleted and minimally reassembled to reconstitute basic pathogenic ability in N. benthamiana, and in Arabidopsis plants that have mutations in target genes that mimic effector actions. This approach and molecular studies of effector activities and plant immune system targets have highlighted a small subset of effectors that contribute to essential processes in pathogenesis. Most notably, HopM1 and AvrE1 redundantly promote an aqueous apoplastic environment, and AvrPtoB and AvrPto redundantly block early immune responses, two conditions that are sufficient for substantial bacterial growth in planta. In addition, disarmed DC3000 polymutants have been used to identify the individual effectors responsible for specific activities of the complete repertoire and to more effectively study effector domains, effector interplay and effector actions on host targets. Such work has revealed that AvrPtoB suppresses cell death elicitation in N. benthamiana that is triggered by another effector in the DC3000 repertoire, highlighting an important aspect of effector interplay in native repertoires. Disarmed DC3000 polymutants support the natural delivery of test effectors and infection readouts that more accurately reveal effector functions in key pathogenesis processes, and enable the identification of effectors with similar activities from a broad range of other pathogens that also defeat plants with cytoplasmic effectors. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Direct and Indirect Visualization of Bacterial Effector Delivery into Diverse Plant Cell Types during Infection[OPEN

PubMed Central

Henry, Elizabeth; Jauneau, Alain; Deslandes, Laurent

2017-01-01

To cause disease, diverse pathogens deliver effector proteins into host cells. Pathogen effectors can inhibit defense responses, alter host physiology, and represent important cellular probes to investigate plant biology. However, effector function and localization have primarily been investigated after overexpression in planta. Visualizing effector delivery during infection is challenging due to the plant cell wall, autofluorescence, and low effector abundance. Here, we used a GFP strand system to directly visualize bacterial effectors delivered into plant cells through the type III secretion system. GFP is a beta barrel that can be divided into 11 strands. We generated transgenic Arabidopsis thaliana plants expressing GFP1-10 (strands 1 to 10). Multiple bacterial effectors tagged with the complementary strand 11 epitope retained their biological function in Arabidopsis and tomato (Solanum lycopersicum). Infection of plants expressing GFP1-10 with bacteria delivering GFP11-tagged effectors enabled direct effector detection in planta. We investigated the temporal and spatial delivery of GFP11-tagged effectors during infection with the foliar pathogen Pseudomonas syringae and the vascular pathogen Ralstonia solanacearum. Thus, the GFP strand system can be broadly used to investigate effector biology in planta. PMID:28600390

Epstein-Barr virus-encoded EBNA-5 binds to Epstein-Barr virus-induced Fte1/S3a protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kashuba, Elena; Yurchenko, Mariya; Szirak, Krisztina

Epstein-Barr virus (EBV) transforms resting human B cells into immortalized immunoblasts. EBV-encoded nuclear antigens EBNA-5 (also called EBNA-LP) is one of the earliest viral proteins expressed in freshly infected B cells. We have recently shown that EBNA-5 binds p14ARF, a nucleolar protein that regulates the p53 pathway. Here, we report the identification of another protein with partially nucleolar localization, the v-fos transformation effector Fte-1 (Fte-1/S3a), as an EBNA-5 binding partner. In transfected cells, Fte-1/S3a and EBNA-5 proteins showed high levels of colocalization in extranucleolar inclusions. Fte-1/S3a has multiple biological functions. It enhances v-fos-mediated cellular transformation and is part of themore » small ribosomal subunit. It also interacts with the transcriptional factor CHOP and apoptosis regulator poly(ADP-ribose) polymerase (PARP). Fte-1/S3a is regularly expressed at high levels in both tumors and cancer cell lines. Its high expression favors the maintenance of malignant phenotype and undifferentiated state, whereas its down-regulation is associated with cellular differentiation and growth arrest. Here, we show that EBV-induced B cell transformation leads to the up-regulation of Fte-1/S3a. We suggest that EBNA-5 through binding may influence the growth promoting, differentiation inhibiting, or apoptosis regulating functions of Fte-1/S3a.« less

Controlled-rate freezer cryopreservation of highly concentrated peripheral blood mononuclear cells results in higher cell yields and superior autologous T-cell stimulation for dendritic cell-based immunotherapy.

PubMed

Buhl, Timo; Legler, Tobias J; Rosenberger, Albert; Schardt, Anke; Schön, Michael P; Haenssle, Holger A

2012-11-01

Availability of large quantities of functionally effective dendritic cells (DC) represents one of the major challenges for immunotherapeutic trials against infectious or malignant diseases. Low numbers or insufficient T-cell activation of DC may result in premature termination of treatment and unsatisfying immune responses in clinical trials. Based on the notion that cryopreservation of monocytes is superior to cryopreservation of immature or mature DC in terms of resulting DC quantity and immuno-stimulatory capacity, we aimed to establish an optimized protocol for the cryopreservation of highly concentrated peripheral blood mononuclear cells (PBMC) for DC-based immunotherapy. Cryopreserved cell preparations were analyzed regarding quantitative recovery, viability, phenotype, and functional properties. In contrast to standard isopropyl alcohol (IPA) freezing, PBMC cryopreservation in an automated controlled-rate freezer (CRF) with subsequent thawing and differentiation resulted in significantly higher cell yields of immature and mature DC. Immature DC yields and total protein content after using CRF were comparable with results obtained with freshly prepared PBMC and exceeded results of standard IPA freezing by approximately 50 %. While differentiation markers, allogeneic T-cell stimulation, viability, and cytokine profiles were similar to DC from standard freezing procedures, DC generated from CRF-cryopreserved PBMC induced a significantly higher antigen-specific IFN-γ release from autologous effector T cells. In summary, automated controlled-rate freezing of highly concentrated PBMC represents an improved method for increasing DC yields and autologous T-cell stimulation.

Visualization of Notch signaling oscillation in cells and tissues.

PubMed

Shimojo, Hiromi; Harima, Yukiko; Kageyama, Ryoichiro

2014-01-01

The Notch signaling effectors Hes1 and Hes7 exhibit oscillatory expression with a period of about 2-3 h during embryogenesis. Hes1 oscillation is important for proliferation and differentiation of neural stem cells, whereas Hes7 oscillation regulates periodic formation of somites. Continuous expression of Hes1 and Hes7 inhibits these developmental processes. Thus, expression dynamics are very important for gene functions, but it is difficult to distinguish between oscillatory and persistent expression by conventional methods such as in situ hybridization and immunostaining. Here, we describe time-lapse imaging methods using destabilized luciferase reporters and a highly sensitive cooled charge-coupled device camera, which can monitor dynamic gene expression. Furthermore, the expression of two genes can be examined simultaneously by a dual reporter system using two-color luciferase reporters. Time-lapse imaging analyses reveal how dynamically gene expression changes in many biological events.

Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana

PubMed Central

Chen, Changlong; Chen, Yongpan; Jian, Heng; Yang, Dan; Dai, Yiran; Pan, Lingling; Shi, Fengwei; Yang, Shanshan; Liu, Qian

2018-01-01

Heterodera avenae is one of the most important plant pathogens and causes vast losses in cereal crops. As a sedentary endoparasitic nematode, H. avenae secretes effectors that modify plant defenses and promote its biotrophic infection of its hosts. However, the number of effectors involved in the interaction between H. avenae and host defenses remains unclear. Here, we report the identification of putative effectors in H. avenae that regulate plant defenses on a large scale. Our results showed that 78 of the 95 putative effectors suppressed programmed cell death (PCD) triggered by BAX and that 7 of the putative effectors themselves caused cell death in Nicotiana benthamiana. Among the cell-death-inducing effectors, three were found to be dependent on their specific domains to trigger cell death and to be expressed in esophageal gland cells by in situ hybridization. Ten candidate effectors that suppressed BAX-triggered PCD also suppressed PCD triggered by the elicitor PsojNIP and at least one R-protein/cognate effector pair, suggesting that they are active in suppressing both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Notably, with the exception of isotig16060, these putative effectors could also suppress PCD triggered by cell-death-inducing effectors from H. avenae, indicating that those effectors may cooperate to promote nematode parasitism. Collectively, our results indicate that the majority of the tested effectors of H. avenae may play important roles in suppressing cell death induced by different elicitors in N. benthamiana. PMID:29379510

Repeat-containing protein effectors of plant-associated organisms

PubMed Central

Mesarich, Carl H.; Bowen, Joanna K.; Hamiaux, Cyril; Templeton, Matthew D.

2015-01-01

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms. PMID:26557126

Repeat-containing protein effectors of plant-associated organisms.

PubMed

Mesarich, Carl H; Bowen, Joanna K; Hamiaux, Cyril; Templeton, Matthew D

2015-01-01

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

Comparative secretome analysis of Colletotrichum falcatum identifies a cerato-platanin protein (EPL1) as a potential pathogen-associated molecular pattern (PAMP) inducing systemic resistance in sugarcane.

PubMed

Ashwin, N M R; Barnabas, Leonard; Ramesh Sundar, Amalraj; Malathi, Palaniyandi; Viswanathan, Rasappa; Masi, Antonio; Agrawal, Ganesh Kumar; Rakwal, Randeep

2017-10-03

Colletotrichum falcatum, an intriguing hemibiotrophic fungal pathogen causes red rot, a devastating disease of sugarcane. Repeated in vitro subculturing of C. falcatum under dark condition alters morphology and reduces virulence of the culture. Hitherto, no information is available on this phenomenon at molecular level. In this study, the in vitro secretome of C. falcatum cultured under light and dark conditions was analyzed using 2-DE coupled with MALDI TOF/TOF MS. Comparative analysis identified nine differentially abundant proteins. Among them, seven proteins were less abundant in the dark-cultured C. falcatum, wherein only two protein species of a cerato-platanin protein called EPL1 (eliciting plant response-like protein) were found to be highly abundant. Transcriptional expression of candidate high abundant proteins was profiled during host-pathogen interaction using qRT-PCR. Comprehensively, this comparative secretome analysis identified five putative effectors, two pathogenicity-related proteins and one pathogen-associated molecular pattern (PAMP) of C. falcatum. Functional characterization of three distinct domains of the PAMP (EPL1) showed that the major cerato-platanin domain (EPL1∆N1-92) is exclusively essential for inducing defense and hypersensitive response (HR) in sugarcane and tobacco, respectively. Further, priming with EPL1∆N1-92 protein induced systemic resistance and significantly suppressed the red rot severity in sugarcane. Being the first secretomic investigation of C. falcatum, this study has identified five potential effectors, two pathogenicity-related proteins and a PAMP. Although many reports have highlighted the influence of light on pathogenicity, this study has established a direct link between light and expression of effectors, for the first time. This study has presented the influence of a novel N-terminal domain of EPL1 in physical and biological properties and established the functional role of major cerato-platanin domain of EPL1 as a potential elicitor inducing systemic resistance in sugarcane. Comprehensively, the study has identified proteins that putatively contribute to virulence of C. falcatum and for the first time, demonstrated the potential role of EPL1 in inducing PAMP-triggered immunity (PTI) in sugarcane. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of legionella effectors using bioinformatic approaches.

PubMed

Segal, Gil

2013-01-01

Legionella pneumophila the causative agent of Legionnaires' disease, actively manipulates host cell processes to establish a replication niche inside host cells. The establishment of its replication niche requires a functional Icm/Dot type IV secretion system which translocates about 300 effector proteins into host cells during infection. Many of these effectors were first identified as effector candidates by several bioinformatic approaches, and these predicted effectors were later examined experimentally for translocation and a large number of which were validated as effector proteins. Here, I summarized the bioinformatic approaches that were used to identify these effectors.

Intelligent nanomedicine integrating diagnosis and therapy

NASA Technical Reports Server (NTRS)

Li, Na (Inventor); Tan, Winny (Inventor)

2012-01-01

A method of controlling the activity of a biologically active compound. The method concerns an oligonucleotide-based compound, comprising a hairpin-forming oligonucleotide, an effector moiety physically associated with the oligonucleotide, where the effector moiety possesses a biological activity, and a regulating moiety physically associated with the oligonucleotide, where the regulating moiety controls the biological activity of the effector moiety by interacting with the effector moiety. The oligonucleotide can assume a hairpin configuration, where the effector and regulating moieties interact, or an open configuration, where the effector and regulating moieties fail to interact. Depending on the nature of the effector and regulating moieties, either configuration can result in the expression of the biological activity of the effector moiety.

Bacterial virulence effectors and their activities.

PubMed

Hann, Dagmar R; Gimenez-Ibanez, Selena; Rathjen, John P

2010-08-01

The major virulence strategy for plant pathogenic bacteria is deployment of effector molecules within the host cytoplasm. Each bacterial strain possesses a set of 20-30 effectors which have overlapping activities, are functionally interchangeable, and diverge in composition between strains. Effectors target host molecules to suppress immunity. Two main strategies are apparent. Effectors that target host proteins seem to attack conserved structural domains but otherwise lack specificity. On the other hand, those that influence host gene transcription directly do so with extreme specificity. In both cases, examples are known where the host has exploited effector-target affinities to establish immune recognition of effectors. The molecular activity of each effector links virulence and immune outcomes. Copyright 2010 Elsevier Ltd. All rights reserved.

Functional characterization of mouse spinal cord infiltrating CD8+ lymphocytes

PubMed Central

Deb, Chandra; Howe, Charles L

2011-01-01

Understanding the immunopathogenesis of neuroimmunological diseases of the CNS requires a robust method for isolating and characterizing the immune effector cells that infiltrate the spinal cord in animal models. We have developed a simple and rapid isolation method that produces high yields of spinal cord infiltrating leukocytes from a single demyelinated spinal cord and which maintains high surface expression of key immunophenotyping antigens. Using this method and the Theiler’s virus model of chronic demyelination, we report the presence of spinal cord infiltrating acute effector CD8+ lymphocytes that are CD45hiCD44loCD62L− and a population of spinal cord infiltrating target effector memory CD8+ lymphocytes that are CD45hiCD44hiCD62L−. These cells respond robustly to ex vivo stimulation by producing interferon γ but do not exhibit specificity for Theiler’s virus in a cytotoxicity assay. We conclude that target-derived lymphocytes in a mouse model of chronic spinal cord demyelination may have unique functional specificities. PMID:19596449

LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell

PubMed Central

Sperschneider, Jana; Catanzariti, Ann-Maree; DeBoer, Kathleen; Petre, Benjamin; Gardiner, Donald M.; Singh, Karam B.; Dodds, Peter N.; Taylor, Jennifer M.

2017-01-01

Pathogens secrete effector proteins and many operate inside plant cells to enable infection. Some effectors have been found to enter subcellular compartments by mimicking host targeting sequences. Although many computational methods exist to predict plant protein subcellular localization, they perform poorly for effectors. We introduce LOCALIZER for predicting plant and effector protein localization to chloroplasts, mitochondria, and nuclei. LOCALIZER shows greater prediction accuracy for chloroplast and mitochondrial targeting compared to other methods for 652 plant proteins. For 107 eukaryotic effectors, LOCALIZER outperforms other methods and predicts a previously unrecognized chloroplast transit peptide for the ToxA effector, which we show translocates into tobacco chloroplasts. Secretome-wide predictions and confocal microscopy reveal that rust fungi might have evolved multiple effectors that target chloroplasts or nuclei. LOCALIZER is the first method for predicting effector localisation in plants and is a valuable tool for prioritizing effector candidates for functional investigations. LOCALIZER is available at http://localizer.csiro.au/. PMID:28300209

Selective CD28 blockade attenuates CTLA-4–dependent CD8+ memory T cell effector function and prolongs graft survival

PubMed Central

Liu, Danya; Badell, I. Raul; Ford, Mandy L.

2018-01-01

Memory T cells pose a significant problem to successful therapeutic control of unwanted immune responses during autoimmunity and transplantation, as they are differentially controlled by cosignaling receptors such as CD28 and CTLA-4. Treatment with abatacept and belatacept impede CD28 signaling by binding to CD80 and CD86, but they also have the unintended consequence of blocking the ligands for CTLA-4, a process that may inadvertently boost effector responses. Here, we show that a potentially novel anti-CD28 domain antibody (dAb) that selectively blocks CD28 but preserves CTLA-4 coinhibition confers improved allograft survival in sensitized recipients as compared with CTLA-4 Ig. However, both CTLA-4 Ig and anti-CD28 dAb similarly and significantly reduced the accumulation of donor-reactive CD8+ memory T cells, demonstrating that regulation of the expansion of CD8+ memory T cell populations is controlled in part by CD28 signals and is not significantly impacted by CTLA-4. In contrast, selective CD28 blockade was superior to CTLA-4 Ig in inhibiting IFN-γ, TNF, and IL-2 production by CD8+ memory T cells, which in turn resulted in reduced recruitment of innate CD11b+ monocytes into allografts. Importantly, this superiority was CTLA-4 dependent, demonstrating that effector function of CD8+ memory T cells is regulated by the balance of CD28 and CTLA-4 signaling. PMID:29321374

Advances in Therapeutic Fc Engineering – Modulation of IgG-Associated Effector Functions and Serum Half-life

PubMed Central

Saxena, Abhishek; Wu, Donghui

2016-01-01

Today, monoclonal immunoglobulin gamma (IgG) antibodies have become a major option in cancer therapy especially for the patients with advanced or metastatic cancers. Efficacy of monoclonal antibodies (mAbs) is achieved through both its antigen-binding fragment (Fab) and crystallizable fragment (Fc). Fab can specifically recognize tumor-associated antigen (TAA) and thus modulate TAA-linked downstream signaling pathways that may lead to the inhibition of tumor growth, induction of tumor apoptosis, and differentiation. The Fc region can further improve mAbs’ efficacy by mediating effector functions such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cell-mediated phagocytosis. Moreover, Fc is the region interacting with the neonatal Fc receptor in a pH-dependent manner that can slow down IgG’s degradation and extend its serum half-life. Loss of the antibody Fc region dramatically shortens its serum half-life and weakens its anticancer effects. Given the essential roles that the Fc region plays in the modulation of the efficacy of mAb in cancer treatment, Fc engineering has been extensively studied in the past years. This review focuses on the recent advances in therapeutic Fc engineering that modulates its related effector functions and serum half-life. We also discuss the progress made in aglycosylated mAb development that may substantially reduce the cost of manufacture but maintain similar efficacies as conventional glycosylated mAb. Finally, we highlight several Fc engineering-based mAbs under clinical trials. PMID:28018347

CFTR modulates RPS27 gene expression using chloride anion as signaling effector.

PubMed

Valdivieso, Ángel G; Mori, Consuelo; Clauzure, Mariángeles; Massip-Copiz, Macarena; Santa-Coloma, Tomás A

2017-11-01

In Cystic Fibrosis (CF), the impairment of the CFTR channel activity leads to a variety of alterations, including differential gene expression. However, the CFTR signaling mechanisms remain unclear. Recently, culturing IB3-1 CF cells under different intracellular Cl - concentrations ([Cl - ] i ), we observed several Cl - -dependent genes and further characterized one of them as RPS27. Thus, we hypothesized that Cl - might act as a signaling effector for CFTR signaling. Here, to test this idea, we study RPS27 expression in T84 cells modulating the CFTR activity by using CFTR inhibitors. First, we observed that incubation of T84 cells with increasing concentrations of the CFTR inhibitors CFTR(inh)-172 or GlyH-101 determined a progressive increase in the relative [Cl - ] i (using the Cl - fluorescent probe SPQ). The [Cl - ] i rise was concomitant with a dose-dependent down-regulation of RPS27. These results imply that CFTR inhibition produce Cl - accumulation and that RPS27 expression can be modulated by CFTR inhibition. Therefore, Cl - behaves as a signaling effector for CFTR in the modulation of RPS27 expression. In addition, the IL-1β receptor antagonist IL1RN or the JNK inhibitor SP600125, both restored the down-regulation of RPS27 induced by CFTRinh-172, implying a role of autocrine IL-1β and JNK signaling downstream of Cl - in RPS27 modulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Three-dimensional structure of the NLRP7 pyrin domain: insight into pyrin-pyrin-mediated effector domain signaling in innate immunity.

PubMed

Pinheiro, Anderson S; Proell, Martina; Eibl, Clarissa; Page, Rebecca; Schwarzenbacher, Robert; Peti, Wolfgang

2010-08-27

The innate immune system provides an initial line of defense against infection. Nucleotide-binding domain- and leucine-rich repeat-containing protein (NLR or (NOD-like)) receptors play a critical role in the innate immune response by surveying the cytoplasm for traces of intracellular invaders and endogenous stress signals. NLRs themselves are multi-domain proteins. Their N-terminal effector domains (typically a pyrin or caspase activation and recruitment domain) are responsible for driving downstream signaling and initiating the formation of inflammasomes, multi-component complexes necessary for cytokine activation. However, the currently available structures of NLR effector domains have not yet revealed the mechanism of their differential modes of interaction. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP7 (NLRP7 PYD) obtained by NMR spectroscopy. The NLRP7 PYD adopts a six-alpha-helix bundle death domain fold. A comparison of conformational and dynamics features of the NLRP7 PYD with other PYDs showed distinct differences for helix alpha3 and loop alpha2-alpha3, which, in NLRP7, is stabilized by a strong hydrophobic cluster. Moreover, the NLRP7 and NLRP1 PYDs have different electrostatic surfaces. This is significant, because death domain signaling is driven by electrostatic contacts and stabilized by hydrophobic interactions. Thus, these results provide new insights into NLRP signaling and provide a first molecular understanding of inflammasome formation.

The CD4+ T cell regulatory network mediates inflammatory responses during acute hyperinsulinemia: a simulation study.

PubMed

Martinez-Sanchez, Mariana E; Hiriart, Marcia; Alvarez-Buylla, Elena R

2017-06-26

Obesity is frequently linked to insulin resistance, high insulin levels, chronic inflammation, and alterations in the behaviour of CD4+ T cells. Despite the biomedical importance of this condition, the system-level mechanisms that alter CD4+ T cell differentiation and plasticity are not well understood. We model how hyperinsulinemia alters the dynamics of the CD4+ T regulatory network, and this, in turn, modulates cell differentiation and plasticity. Different polarizing microenvironments are simulated under basal and high levels of insulin to assess impacts on cell-fate attainment and robustness in response to transient perturbations. In the presence of high levels of insulin Th1 and Th17 become more stable to transient perturbations, and their basin sizes are augmented, Tr1 cells become less stable or disappear, while TGFβ producing cells remain unaltered. Hence, the model provides a dynamic system-level framework and explanation to further understand the documented and apparently paradoxical role of TGFβ in both inflammation and regulation of immune responses, as well as the emergence of the adipose Treg phenotype. Furthermore, our simulations provide new predictions on the impact of the microenvironment in the coexistence of the different cell types, suggesting that in pro-Th1, pro-Th2 and pro-Th17 environments effector and regulatory cells can coexist, but that high levels of insulin severely diminish regulatory cells, especially in a pro-Th17 environment. This work provides a first step towards a system-level formal and dynamic framework to integrate further experimental data in the study of complex inflammatory diseases.

Dynamics of Tissue-Specific CD8+ T Cell Responses during West Nile Virus Infection.

PubMed

Aguilar-Valenzuela, Renan; Netland, Jason; Seo, Young-Jin; Bevan, Michael J; Grakoui, Arash; Suthar, Mehul S

2018-05-15

The mouse model of West Nile virus (WNV), which is a leading cause of mosquito-borne encephalitis worldwide, has provided fundamental insights into the host and viral factors that regulate viral pathogenesis and infection outcome. In particular, CD8 + T cells are critical for controlling WNV replication and promoting protection against infection. Here, we present the characterization of a T cell receptor (TCR)-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein (here referred to as transgenic WNV-I mice). Using an adoptive-transfer model, we found that WNV-I CD8 + T cells behave similarly to endogenous CD8 + T cell responses, with an expansion phase in the periphery beginning around day 7 postinfection (p.i.) followed by a contraction phase through day 15 p.i. Through the use of in vivo intravascular immune cell staining, we determined the kinetics, expansion, and differentiation into effector and memory subsets of WNV-I CD8 + T cells within the spleen and brain. We found that red-pulp WNV-I CD8 + T cells were more effector-like than white-pulp WNV-I CD8 + T cells, which displayed increased differentiation into memory precursor cells. Within the central nervous system (CNS), we found that WNV-I CD8 + T cells were polyfunctional (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), displayed tissue-resident characteristics (CD69 + and CD103 + ), persisted in the brain through day 15 p.i., and reduced the viral burden within the brain. The use of these TCR-transgenic WNV-I mice provides a new resource to dissect the immunological mechanisms of CD8 + T cell-mediated protection during WNV infection. IMPORTANCE West Nile Virus (WNV) is the leading cause of mosquito-borne encephalitis worldwide. There are currently no approved therapeutics or vaccines for use in humans to treat or prevent WNV infection. CD8 + T cells are critical for controlling WNV replication and protecting against infection. Here, we present a comprehensive characterization of a novel TCR-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein. In this study, we determine the kinetics, proliferation, differentiation into effector and memory subsets, homing, and clearance of WNV in the CNS. Our findings provide a new resource to dissect the immunological mechanisms of CD8 + T cell-mediated protection during WNV infection. Copyright © 2018 American Society for Microbiology.

The rise of the undead:Pseudokinases as mediators of effector-triggered immunity

USDA-ARS?s Scientific Manuscript database

Pathogens use effector proteins to suppress host immunity and promote infection. However, plants can recognize specific effectors and mount an effector-triggered immune response that suppresses pathogen growth. The YopJ/HopZ family of type III secreted effector proteins is broadly distributed in bac...

Integrated roles of BclA and DD-carboxypeptidase 1 in Bradyrhizobium differentiation within NCR-producing and NCR-lacking root nodules.

PubMed

Barrière, Quentin; Guefrachi, Ibtissem; Gully, Djamel; Lamouche, Florian; Pierre, Olivier; Fardoux, Joël; Chaintreuil, Clémence; Alunni, Benoît; Timchenko, Tatiana; Giraud, Eric; Mergaert, Peter

2017-08-22

Legumes harbor in their symbiotic nodule organs nitrogen fixing rhizobium bacteria called bacteroids. Some legumes produce Nodule-specific Cysteine-Rich (NCR) peptides in the nodule cells to control the intracellular bacterial population. NCR peptides have antimicrobial activity and drive bacteroids toward terminal differentiation. Other legumes do not produce NCR peptides and their bacteroids are not differentiated. Bradyrhizobia, infecting NCR-producing Aeschynomene plants, require the peptide uptake transporter BclA to cope with the NCR peptides as well as a specific peptidoglycan-modifying DD-carboxypeptidase, DD-CPase1. We show that Bradyrhizobium diazoefficiens strain USDA110 forms undifferentiated bacteroids in NCR-lacking soybean nodules. Unexpectedly, in Aeschynomene afraspera nodules the nitrogen fixing USDA110 bacteroids are hardly differentiated despite the fact that this host produces NCR peptides, suggesting that USDA110 is insensitive to the host peptide effectors and that nitrogen fixation can be uncoupled from differentiation. In agreement with the absence of bacteroid differentiation, USDA110 does not require its bclA gene for nitrogen fixing symbiosis with these two host plants. Furthermore, we show that the BclA and DD-CPase1 act independently in the NCR-induced morphological differentiation of bacteroids. Our results suggest that BclA is required to protect the rhizobia against the NCR stress but not to induce the terminal differentiation pathway.

Phosphoinositide 3-kinase/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.

PubMed

Lim, Jeong A; Woo, Joo Hong; Kim, Hye Sun

2008-09-01

In this study, it was found that undifferentiated myoblasts were more vulnerable to menadione-induced oxidative stress than differentiated myotubes. Cell death occurred with a relatively low concentration of menadione in myoblasts compared to myotubes. With the same concentration of menadione, the Bcl-2/Bax ratio decreased and nuclei containing condensed chromatin were observed in myoblasts to a greater extent than in myotubes. However, myotubes became increasingly susceptible to menadione when phosphoinositide 3-kinase (PI3-K) was blocked by pre-incubation with LY294002, a PI3-K inhibitor. Actually, PI3-K activity was reduced by menadione in myoblasts but not in myotubes. In addition, the phosphorylation of Akt, a downstream effector of PI3-K, was inhibited in myoblasts by menadione but increased in myotubes. Both LY294002 and API-2, an Akt inhibitor, decreased the Bcl-2/Bax ratio in menadione-exposed myotubes. These results suggest that the differential activity of PI3-K/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.

Identification of transcription coactivator OCA-B-dependent genes involved in antigen-dependent B cell differentiation by cDNA array analyses.

PubMed

Kim, Unkyu; Siegel, Rachael; Ren, Xiaodi; Gunther, Cary S; Gaasterland, Terry; Roeder, Robert G

2003-07-22

The tissue-specific transcriptional coactivator OCA-B is required for antigen-dependent B cell differentiation events, including germinal center formation. However, the identity of OCA-B target genes involved in this process is unknown. This study has used large-scale cDNA arrays to monitor changes in gene expression patterns that accompany mature B cell differentiation. B cell receptor ligation alone induces many genes involved in B cell expansion, whereas B cell receptor and helper T cell costimulation induce genes associated with B cell effector function. OCA-B expression is induced by both B cell receptor ligation alone and helper T cell costimulation, suggesting that OCA-B is involved in B cell expansion as well as B cell function. Accordingly, several genes involved in cell proliferation and signaling, such as Lck, Kcnn4, Cdc37, cyclin D3, B4galt1, and Ms4a11, have been identified as OCA-B-dependent genes. Further studies on the roles played by these genes in B cells will contribute to an understanding of B cell differentiation.

Costimulation through the CD137/4-1BB pathway protects human melanoma tumor-infiltrating lymphocytes from activation-induced cell death and enhances antitumor effector function.

PubMed

Hernandez-Chacon, Jessica Ann; Li, Yufeng; Wu, Richard C; Bernatchez, Chantale; Wang, Yijun; Weber, Jeffrey S; Hwu, Patrick; Radvanyi, Laszlo G

2011-04-01

Adoptive T-cell therapy (ACT) using expanded tumor-infiltrating lymphocytes (TIL) with high-dose interleukin-2 is a promising form of immunotherapy for stage IV melanoma having clinical response rates of 50% or more. One of the major problems preventing further success of this therapy is that the current protocols used to highly expand TIL for infusion drive CD8(+) T cells to differentiate into effector cells losing key costimulatory molecules such as CD28 and CD27. This has been associated with a lack of persistence in vivo for reasons not entirely clear. In this study, we demonstrate that while human melanoma CD8(+) TIL lost CD27 and CD28 expression during the rapid expansion for ACT, they gained expression of the alternative costimulatory molecule CD137/4-1BB, and to a lesser extent CD134/OX40. Postrapid expansion protocol (REP) TIL were found to be highly sensitive to activation-induced cell death when reactivated through the T-cell receptor with low levels of OKT3 antibody. However, coligation of 4-1BB using 2 different agonistic anti-4-1BB antibodies potently prevented activation-induced cell death of post-REP CD8(+) TIL, including those specific for melanoma antigen recognized by T cells, and facilitated even further cell expansion. This was correlated with increased levels of bcl-2 and bcl-xL together with decreased bim expression. 4-1BB costimulated post-REP TIL also expressed increased levels of the cytolytic granule proteins and exhibited enhanced cytotoxic T-cell activity against melanoma cells. Lastly, post-REP CD8(+) TIL were protected from cell death by anti-4-1BB ligation when exposed to human leukocyte antigen-matched melanoma cells. Our results indicate that 4-1BB costimulation may significantly improve TIL survival during melanoma ACT and boost antitumor cytolytic activity.

Cytomegalovirus persistence and T-cell immunosenescence in people aged fifty and older: A systematic review.

PubMed

Weltevrede, Marlies; Eilers, Renske; de Melker, Hester E; van Baarle, Debbie

2016-05-01

Immunosenescence is the age-related deterioration of immunocompetence which is reflected in a poorer response to new antigens. This process is characterized by decreases in naïve T cells and increases in memory T cells. The highly prevalent β-herpesvirus cytomegalovirus (CMV) is thought to enhance T-cell immunosenescence. The aim of this study was to perform a systematic review on the current evidence regarding the relation between CMV-infection and immunosenescence in Western people aged fifty years and older. Studies that investigated the relation between CMV infection and immune parameters in Western people aged 50 years and older were eligible for inclusion. No restrictions were placed on study type. This article focuses on the relation between CMV infections as measured by serology and T cell subsets. A narrative approach to data synthesis was applied. In the majority of included studies higher levels of Effector Memory (EM) and TEMRA (Effector Memory T cells re-expressing CD45RA) cells were found in the CD4+ and the CD8+ T cell pools in CMV-seropositive elderly compared to CMV-seronegative elderly. No clear evidence was found for lower levels of naïve T cells in CMV-seropositive elderly compared to CMV-seronegative elderly. The total CD8+ T cell pool appeared to be larger in CMV-seropositive elderly in three out of four studies, while the total CD4+ T cell pool appeared to be smaller in CMV-seropositive elderly in two out of four studies. CMV seems to enhance immunosenescence based on the high levels of the highly differentiated EM and TEMRA cells in the CD8+ and CD4+ T cell pools. The relation of the shifts within the T cell compartments in CMV-seropositive elderly in relation to susceptibility to infectious diseases remains to be investigated. Copyright © 2016 Elsevier Inc. All rights reserved.

Co-stimulation through the CD137/4-1BB pathway protects human melanoma tumor-infiltrating lymphocytes from activation-induced cell death and enhances anti-tumor effector function

PubMed Central

Hernandez-Chacon, Jessica Ann; Li, Yufeng; Wu, Richard C.; Bernatchez, Chantale; Wang, Yijun; Weber, Jeffrey; Hwu, Patrick; Radvanyi, Laszlo

2011-01-01

Adoptive T-cell therapy (ACT) using expanded tumor-infiltrating lymphocytes (TIL) with high-dose IL-2 is a promising form of immunotherapy for Stage IV melanoma having clinical response rates of 50% or more. One of the major problems preventing further success of this therapy is that the current protocols used to highly expand TIL for infusion drive CD8+ T cells to differentiate into effector cells losing key co-stimulatory molecules such as CD28 and CD27. This has been associated with a lack of persistence in vivo for reasons not entirely clear. In this study, we demonstrate that while human melanoma CD8+ TIL lost CD27 and CD28 expression during the rapid expansion for ACT, they gained expression of the alternative co-stimulatory molecule CD137/4-1BB, and to a lesser extent CD134/OX40. Post-REP TIL were found to be highly sensitive to activation-induced cell death (AICD) when re-activated through the TCR with low levels of OKT3 antibody. However, co-ligation of 4-1BB using two different agonistic anti-4-1BB antibodies potently prevented AICD of post-REP CD8+ TIL, including those specific for MART-1, and facilitated even further cell expansion. This was correlated with increased levels of bcl-2 and bcl-xL together with decreased bim expression. 4-1BB-co-stimulated post-REP TIL also expressed increased levels of the cytolytic granule proteins and exhibited enhanced CTL activity against melanoma cells. Lastly, post-REP CD8+ TIL were protected from cell death by anti-4-1BB ligation when exposed to HLA-matched melanoma cells. Our results indicate that 4-1BB co-stimulation may significantly improve TIL survival during melanoma ACT and boost anti-tumor cytolytic activity. PMID:21389874

Metabolic pathways in T cell activation and lineage differentiation.

PubMed

Almeida, Luís; Lochner, Matthias; Berod, Luciana; Sparwasser, Tim

2016-10-01

Recent advances in the field of immunometabolism support the concept that fundamental processes in T cell biology, such as TCR-mediated activation and T helper lineage differentiation, are closely linked to changes in the cellular metabolic programs. Although the major task of the intermediate metabolism is to provide the cell with a constant supply of energy and molecular precursors for the production of biomolecules, the dynamic regulation of metabolic pathways also plays an active role in shaping T cell responses. Key metabolic processes such as glycolysis, fatty acid and mitochondrial metabolism are now recognized as crucial players in T cell activation and differentiation, and their modulation can differentially affect the development of T helper cell lineages. In this review, we describe the diverse metabolic processes that T cells engage during their life cycle from naïve towards effector and memory T cells. We consider in particular how the cellular metabolism may actively support the function of T cells in their different states. Moreover, we discuss how molecular regulators such as mTOR or AMPK link environmental changes to adaptations in the cellular metabolism and elucidate the consequences on T cell differentiation and function. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

The different effector function capabilities of the seven equine IgG subclasses have implications for vaccine strategies

PubMed Central

Lewis, Melanie J.; Wagner, Bettina; Woof, Jenny M.

2008-01-01

Recombinant versions of the seven equine IgG subclasses were expressed in CHO cells. All assembled into intact immunoglobulins stabilised by disulphide bridges, although, reminiscent of human IgG4, a small proportion of equine IgG4 and IgG7 were held together by non-covalent bonds alone. All seven IgGs were N-glycosylated. In addition IgG3 appeared to be O-glycosylated and could bind the lectin jacalin. Staphylococcal protein A displayed weak binding for the equine IgGs in the order: IgG1 > IgG3 > IgG4 > IgG7 > IgG2 = IgG5 > IgG6. Streptococcal protein G bound strongly to IgG1, IgG4 and IgG7, moderately to IgG3, weakly to IgG2 and IgG6, and not at all to IgG5. Analysis of antibody effector functions revealed that IgG1, IgG3, IgG4, IgG5 and IgG7, but not IgG2 and IgG6, were able to elicit a strong respiratory burst from equine peripheral blood leukocytes, predicting that the former five IgG subclasses are able to interact with Fc receptors on effector cells. IgG1, IgG3, IgG4 and IgG7, but not IgG2, IgG5 and IgG6, were able to bind complement C1q and activate complement via the classical pathway. The differential effector function capabilities of the subclasses suggest that, for maximum efficacy, equine vaccine strategies should seek to elicit antibody responses of the IgG1, IgG3, IgG4, and IgG7 subclasses. PMID:17669496

Piloted evaluation of an integrated propulsion and flight control simulator

NASA Technical Reports Server (NTRS)

Bright, Michelle M.; Simon, Donald L.

1992-01-01

A piloted evaluation of the integrated flight and propulsion control simulator for advanced integrated propulsion and airframe control design is described. The evaluation will cover control effector gains and deadbands, control effectiveness and control authority, and heads up display functionality. For this evaluation the flight simulator is configured for transition flight using an advanced Short Take-Off and Vertical Landing fighter aircraft model, a simplified high-bypass turbofan engine model, fighter cockpit displays, and pilot effectors. The piloted tasks used for rating displays and control effector gains are described. Pilot comments and simulation results confirm that the display symbology and control gains are very adequate for the transition flight task. Additionally, it is demonstrated that this small-scale, fixed base flight simulator facility can adequately perform a real time, piloted control evaluation.

Killing of intrafamilial leukocytes by earthworm effector cells.

PubMed

Suzuki, M M; Cooper, E L

1995-01-01

When Lumbricus and Eisenia coelomocytes are cultured together in intrafamilial xenogeneic combinations, significant cytotoxicity occurs at 24 h but not at 5 nor 72 h, as shown by trypan blue assay. In a 4.5-h assay, measuring 51Cr release, using an effector/target ratio of 25:1, unpooled cells from a single Lumbricus killed Eisenia cells at levels of 6% and 14%. However, Eisenia coelomocyte survival was high and identical in either cell-free xenogeneic (Lumbricus) coelomic fluid or in artificial medium. In this 1-way assay, earthworm (Lumbricus) coelomocytes act as effector cells that kill non-self target cells, even those of other earthworms. Comparisons with previous results reveal greater reliability and consistently repeatable results when the 51Cr release assay is used to measure cytotoxicity regardless of the targets.

Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering

PubMed Central

Blaževitš, Olga; Mideksa, Yonatan G.; Šolman, Maja; Ligabue, Alessio; Ariotti, Nicholas; Nakhaeizadeh, Hossein; Fansa, Eyad K.; Papageorgiou, Anastassios C.; Wittinghofer, Alfred; Ahmadian, Mohammad R.; Abankwa, Daniel

2016-01-01

Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling. PMID:27087647

Expression of Terminal Effector Genes in Mammalian Neurons Is Maintained by a Dynamic Relay of Transient Enhancers.

PubMed

Rhee, Ho Sung; Closser, Michael; Guo, Yuchun; Bashkirova, Elizaveta V; Tan, G Christopher; Gifford, David K; Wichterle, Hynek

2016-12-21

Generic spinal motor neuron identity is established by cooperative binding of programming transcription factors (TFs), Isl1 and Lhx3, to motor-neuron-specific enhancers. How expression of effector genes is maintained following downregulation of programming TFs in maturing neurons remains unknown. High-resolution exonuclease (ChIP-exo) mapping revealed that the majority of enhancers established by programming TFs are rapidly deactivated following Lhx3 downregulation in stem-cell-derived hypaxial motor neurons. Isl1 is released from nascent motor neuron enhancers and recruited to new enhancers bound by clusters of Onecut1 in maturing neurons. Synthetic enhancer reporter assays revealed that Isl1 operates as an integrator factor, translating the density of Lhx3 or Onecut1 binding sites into transient enhancer activity. Importantly, independent Isl1/Lhx3- and Isl1/Onecut1-bound enhancers contribute to sustained expression of motor neuron effector genes, demonstrating that outwardly stable expression of terminal effector genes in postmitotic neurons is controlled by a dynamic relay of stage-specific enhancers. Copyright © 2016 Elsevier Inc. All rights reserved.

Real-Time Adaptive Control Allocation Applied to a High Performance Aircraft

NASA Technical Reports Server (NTRS)

Davidson, John B.; Lallman, Frederick J.; Bundick, W. Thomas

2001-01-01

Abstract This paper presents the development and application of one approach to the control of aircraft with large numbers of control effectors. This approach, referred to as real-time adaptive control allocation, combines a nonlinear method for control allocation with actuator failure detection and isolation. The control allocator maps moment (or angular acceleration) commands into physical control effector commands as functions of individual control effectiveness and availability. The actuator failure detection and isolation algorithm is a model-based approach that uses models of the actuators to predict actuator behavior and an adaptive decision threshold to achieve acceptable false alarm/missed detection rates. This integrated approach provides control reconfiguration when an aircraft is subjected to actuator failure, thereby improving maneuverability and survivability of the degraded aircraft. This method is demonstrated on a next generation military aircraft Lockheed-Martin Innovative Control Effector) simulation that has been modified to include a novel nonlinear fluid flow control control effector based on passive porosity. Desktop and real-time piloted simulation results demonstrate the performance of this integrated adaptive control allocation approach.

The Ustilago maydis repetitive effector Rsp3 blocks the antifungal activity of mannose-binding maize proteins.

PubMed

Ma, Lay-Sun; Wang, Lei; Trippel, Christine; Mendoza-Mendoza, Artemio; Ullmann, Steffen; Moretti, Marino; Carsten, Alexander; Kahnt, Jörg; Reissmann, Stefanie; Zechmann, Bernd; Bange, Gert; Kahmann, Regine

2018-04-27

To cause disease in maize, the biotrophic fungus Ustilago maydis secretes a large arsenal of effector proteins. Here, we functionally characterize the repetitive effector Rsp3 (repetitive secreted protein 3), which shows length polymorphisms in field isolates and is highly expressed during biotrophic stages. Rsp3 is required for virulence and anthocyanin accumulation. During biotrophic growth, Rsp3 decorates the hyphal surface and interacts with at least two secreted maize DUF26-domain family proteins (designated AFP1 and AFP2). AFP1 binds mannose and displays antifungal activity against the rsp3 mutant but not against a strain constitutively expressing rsp3. Maize plants silenced for AFP1 and AFP2 partially rescue the virulence defect of rsp3 mutants, suggesting that blocking the antifungal activity of AFP1 and AFP2 by the Rsp3 effector is an important virulence function. Rsp3 orthologs are present in all sequenced smut fungi, and the ortholog from Sporisorium reilianum can complement the rsp3 mutant of U. maydis, suggesting a novel widespread fungal protection mechanism.

Oomycetes, effectors, and all that jazz.

PubMed

Bozkurt, Tolga O; Schornack, Sebastian; Banfield, Mark J; Kamoun, Sophien

2012-08-01

Plant pathogenic oomycetes secrete a diverse repertoire of effector proteins that modulate host innate immunity and enable parasitic infection. Understanding how effectors evolve, translocate and traffic inside host cells, and perturb host processes are major themes in the study of oomycete-plant interactions. The last year has seen important progress in the study of oomycete effectors with, notably, the elucidation of the 3D structures of five RXLR effectors, and novel insights into how cytoplasmic effectors subvert host cells. In this review, we discuss these and other recent advances and highlight the most important open questions in oomycete effector biology. Copyright © 2012 Elsevier Ltd. All rights reserved.

Existence and stability of periodic solutions of an impulsive differential equation and application to CD8 T-cell differentiation.

PubMed

Girel, Simon; Crauste, Fabien

2018-06-01

Unequal partitioning of the molecular content at cell division has been shown to be a source of heterogeneity in a cell population. We propose to model this phenomenon with the help of a scalar, nonlinear impulsive differential equation (IDE). To study the effect of molecular partitioning at cell division on the effector/memory cell-fate decision in a CD8 T-cell lineage, we study an IDE describing the concentration of the protein Tbet in a CD8 T-cell, where impulses are associated to cell division. We discuss how the degree of asymmetry of molecular partitioning can affect the process of cell differentiation and the phenotypical heterogeneity of a cell population. We show that a moderate degree of asymmetry is necessary and sufficient to observe irreversible differentiation. We consider, in a second part, a general autonomous IDE with fixed times of impulse and a specific form of impulse function. We establish properties of the solutions of that equation, most of them obtained under the hypothesis that impulses occur periodically. In particular, we show how to investigate the existence of periodic solutions and their stability by studying the flow of an autonomous differential equation. Then we apply those properties to prove the results presented in the first part.

Notch1 engagement by Delta-like-1 promotes differentiation of B lymphocytes to antibody-secreting cells

PubMed Central

Santos, Margarida Almeida; Sarmento, Leonor Morais; Rebelo, Manuel; Doce, Ana Agua; Maillard, Ivan; Dumortier, Alexis; Neves, Helia; Radtke, Freddy; Pear, Warren S.; Parreira, Leonor; Demengeot, Jocelyne

2007-01-01

Notch signaling regulates B and T lymphocyte development and T cell effector class decision. In this work, we tested whether Notch activity affects mature B cell activation and differentiation to antibody-secreting cells (ASC). We show increased frequency of ASC in cultures of splenic B cells activated with LPS or anti-CD40 when provided exogenous Notch ligand Delta-like-1 (Dll1). Our results indicate that Notch–Dll1 interaction releases a default pathway that otherwise inhibits Ig secretion upon B cell activation. Thus, Dll1 enhanced spontaneous Ig secretion by naturally activated marginal zone B and B1 cells and reversed the inhibition of ASC differentiation mediated by B cell receptor crosslinking during LPS. Moreover, suppression of Notch signaling in B cell expression of either a dominant-negative mutant form of Mastermind-like 1 or a null mutation of Notch1 not only prevented Dll1-mediated enhancement of ASC differentiation but also reduced dramatically LPS-induced Ig secretion. Finally, we show that Dll1 and Jagged-1 are differentially expressed in discrete areas of the spleen, and that the effect of Notch engagement on Ig secretion is ligand-specific. These results indicate that Notch ligands participate in the definition of the mature B cell microenvironment that influences their terminal differentiation. PMID:17878313

Fine regulation of RhoA and Rock is required for skeletal muscle differentiation.

PubMed

Castellani, Loriana; Salvati, Erica; Alemà, Stefano; Falcone, Germana

2006-06-02

The RhoA GTPase controls a variety of cell functions such as cell motility, cell growth, and gene expression. Previous studies suggested that RhoA mediates signaling inputs that promote skeletal myogenic differentiation. We show here that levels and activity of RhoA protein are down-regulated in both primary avian myoblasts and mouse satellite cells undergoing differentiation, suggesting that a fine regulation of this GTPase is required. In addition, ectopic expression of activated RhoA in primary quail myocytes, but not in mouse myocytes, inhibits accumulation of muscle-specific proteins and cell fusion. By disrupting RhoA signaling with specific inhibitors, we have shown that this GTPase, although required for cell identity in proliferating myoblasts, is not essential for commitment to terminal differentiation and muscle gene expression. Ectopic expression of an activated form of its downstream effector, Rock, impairs differentiation of both avian and mouse myoblasts. Conversely, Rock inhibition with specific inhibitors and small interfering RNA-mediated gene silencing leads to accelerated progression in the lineage and enhanced cell fusion, underscoring a negative regulatory function of Rock in myogenesis. Finally, we have reported that Rock acts independently from RhoA in preventing myoblast exit from the cell cycle and commitment to differentiation and may receive signaling inputs from Raf-1 kinase.

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila.

PubMed

Urbanus, Malene L; Quaile, Andrew T; Stogios, Peter J; Morar, Mariya; Rao, Chitong; Di Leo, Rosa; Evdokimova, Elena; Lam, Mandy; Oatway, Christina; Cuff, Marianne E; Osipiuk, Jerzy; Michalska, Karolina; Nocek, Boguslaw P; Taipale, Mikko; Savchenko, Alexei; Ensminger, Alexander W

2016-12-16

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector-effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector-effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila-translocated substrates. While capturing all known examples of effector-effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct-a hallmark of an emerging class of proteins called metaeffectors, or "effectors of effectors". Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector-effector modulation, may be a common feature of many intracellular pathogens-with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Effector CD4 cell tolerization is mediated through functional inactivation and involves preferential impairment of TNF-α and IFN-γ expression potentials

PubMed Central

Long, Meixiao; Higgins, Amy D.; Mihalyo, Marianne A.; Adler, Adam J.

2010-01-01

It has recently been shown that effector/memory T cells can undergo peripheral tolerization in response to self-antigen. In the present study, we found that within 24 h self-antigen profoundly impairs the ability of CD4 effectors to express TNF-α (and to a lesser extent IFN-γ); however, several days of self-antigen exposure is required to impair non-effector functions such as IL-2 expression and proliferation. Since only half of the initial effector CD4 cell population expresses effector cytokines following brief antigenic stimulation, tolerization might have been mediated either through functional inactivation of effector-competent cells, or alternatively by the selective deletion of competent and expansion of non-competent cells. When briefly stimulated effectors were fractionated based on their expression of IFN-γ, the IFN-γ− sub-population was able to express IFN-γ following secondary stimulation, indicating that all effector CD4 cells are functionally competent. Furthermore, both IFN-γ+ and IFN-γ− sub-populations underwent tolerization in response to self-HA (although the former was slightly more prone to deletion at later time points). Thus, effector CD4 cell tolerization is mediated primarily through the functional inactivation of effector-competent cells. PMID:14609577

Colonization and effector functions of innate lymphoid cells in mucosal tissues.

PubMed

Kim, Myunghoo; Kim, Chang H

2016-10-01

Innate lymphoid cells (ILCs) protect mucosal barrier tissues to fight infection and maintain tissue integrity. ILCs and their progenitors are developmentally programmed to migrate, differentiate and populate various mucosal tissues and associated lymphoid tissues. Functionally mature ILC subsets respond to diverse pathogens such as bacteria, viruses, fungi and parasites in subset-specific manners. In this review, we will discuss how ILCs populate mucosal tissues and regulate immune responses to distinct pathogens to protect the host and maintain tissue integrity. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Potential of targeting TGF-β for organ transplant patients

PubMed Central

Iwashima, Makio; Love, Robert

2015-01-01

TGF-β was originally considered as an immunoregulatory cytokine, but accumulating data demonstrate that it also plays a critical role in development of effector immunity. Since TGF-β has a potent ability to alter immune responses, modulation of the TGF-β pathway for treatment of transplantation patients could be effective if carried out in a target selective manner. This review will focus on the role of TGF-β in T cell differentiation and discuss the prospect of TGF-β as the therapeutic target of lung transplantation acceptance. PMID:23464518

Bach2 Promotes Regulatory T-cell Development by Limiting Effector Differentiation | Center for Cancer Research

Cancer.gov

A number of allergic and autoimmune disorders, such as Crohn’s disease, asthma, type I diabetes and multiple sclerosis, are associated with polymorphisms in a gene encoding the transcription factor, BACH2. Despite this, the mechanism Bach2 uses to prevent immune-mediated diseases was not known. To function appropriately, the immune system relies on a delicate balance between immune-stimulating and immune-regulating cells. When immune-stimulating cells become too active, or immune-regulating cells become ineffective, autoimmune and allergic diseases can ensue.

Contribution of endothelial cells to organogenesis: a modern reappraisal of an old Aristotelian concept

PubMed Central

Crivellato, E; Nico, B; Ribatti, D

2007-01-01

It is well established that many tissue-derived factors are involved in blood vessel formation, but evidence is now emerging that endothelial cells themselves represent a crucial source of instructive signals to non-vascular tissue cells during organ development. Thus, endothelial cell signalling is currently believed to promote fundamental cues for cell fate specification, embryo patterning, organ differentiation and postnatal tissue remodelling. This review article summarizes some of the recent advances in our understanding of the role of endothelial cells as effector cells in organ formation. PMID:17683480

Mechanotransduction and the functional response of bone to mechanical strain

NASA Technical Reports Server (NTRS)

Duncan, R. L.; Turner, C. H.

1995-01-01

Mechanotransduction plays a crucial role in the physiology of many tissues including bone. Mechanical loading can inhibit bone resorption and increase bone formation in vivo. In bone, the process of mechanotransduction can be divided into four distinct steps: (1) mechanocoupling, (2) biochemical coupling, (3) transmission of signal, and (4) effector cell response. In mechanocoupling, mechanical loads in vivo cause deformations in bone that stretch bone cells within and lining the bone matrix and create fluid movement within the canaliculae of bone. Dynamic loading, which is associated with extracellular fluid flow and the creation of streaming potentials within bone, is most effective for stimulating new bone formation in vivo. Bone cells in vitro are stimulated to produce second messengers when exposed to fluid flow or mechanical stretch. In biochemical coupling, the possible mechanisms for the coupling of cell-level mechanical signals into intracellular biochemical signals include force transduction through the integrin-cytoskeleton-nuclear matrix structure, stretch-activated cation channels within the cell membrane, G protein-dependent pathways, and linkage between the cytoskeleton and the phospholipase C or phospholipase A pathways. The tight interaction of each of these pathways would suggest that the entire cell is a mechanosensor and there are many different pathways available for the transduction of a mechanical signal. In the transmission of signal, osteoblasts, osteocytes, and bone lining cells may act as sensors of mechanical signals and may communicate the signal through cell processes connected by gap junctions. These cells also produce paracrine factors that may signal osteoprogenitors to differentiate into osteoblasts and attach to the bone surface. Insulin-like growth factors and prostaglandins are possible candidates for intermediaries in signal transduction. In the effector cell response, the effects of mechanical loading are dependent upon the magnitude, duration, and rate of the applied load. Longer duration, lower amplitude loading has the same effect on bone formation as loads with short duration and high amplitude. Loading must be cyclic to stimulate new bone formation. Aging greatly reduces the osteogenic effects of mechanical loading in vivo. Also, some hormones may interact with local mechanical signals to change the sensitivity of the sensor or effector cells to mechanical load.

CD4+ virtual memory: Antigen-inexperienced T cells reside in the naïve, regulatory, and memory T cell compartments at similar frequencies, implications for autoimmunity.

PubMed

Marusina, Alina I; Ono, Yoko; Merleev, Alexander A; Shimoda, Michiko; Ogawa, Hiromi; Wang, Elizabeth A; Kondo, Kayo; Olney, Laura; Luxardi, Guillaume; Miyamura, Yoshinori; Yilma, Tilahun D; Villalobos, Itzel Bustos; Bergstrom, Jennifer W; Kronenberg, Daniel G; Soulika, Athena M; Adamopoulos, Iannis E; Maverakis, Emanual

2017-02-01

It is widely accepted that central and effector memory CD4 + T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4 + T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central "memory" cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4 + T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4 + T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4 + T cell maturation in which a specific clone can undergo a differentiation process to exhibit a "memory" or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4 + T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones. Published by Elsevier Ltd.

Acute Hepatopancreatic Necrosis Disease-Causing Vibrio parahaemolyticus Strains Maintain an Antibacterial Type VI Secretion System with Versatile Effector Repertoires.

PubMed

Li, Peng; Kinch, Lisa N; Ray, Ann; Dalia, Ankur B; Cong, Qian; Nunan, Linda M; Camilli, Andrew; Grishin, Nick V; Salomon, Dor; Orth, Kim

2017-07-01

Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. AHPND is caused by toxic strains of Vibrio parahaemolyticus that have acquired a "selfish plasmid" encoding the deadly binary toxins PirA vp /PirB vp To better understand the repertoire of virulence factors in AHPND-causing V. parahaemolyticus , we conducted a comparative analysis using the genome sequences of the clinical strain RIMD2210633 and of environmental non-AHPND and toxic AHPND isolates of V. parahaemolyticus Interestingly, we found that all of the AHPND strains, but none of the non-AHPND strains, harbor the antibacterial type VI secretion system 1 (T6SS1), which we previously identified and characterized in the clinical isolate RIMD2210633. This finding suggests that the acquisition of this T6SS might confer to AHPND-causing V. parahaemolyticus a fitness advantage over competing bacteria and facilitate shrimp infection. Additionally, we found highly dynamic effector loci in the T6SS1 of AHPND-causing strains, leading to diverse effector repertoires. Our discovery provides novel insights into AHPND-causing pathogens and reveals a potential target for disease control. IMPORTANCE Acute hepatopancreatic necrosis disease (AHPND) is a serious disease that has caused severe damage and significant financial losses to the global shrimp industry. To better understand and prevent this shrimp disease, it is essential to thoroughly characterize its causative agent, Vibrio parahaemolyticus Although the plasmid-encoded binary toxins PirA vp /PirB vp have been shown to be the primary cause of AHPND, it remains unknown whether other virulent factors are commonly present in V. parahaemolyticus and might play important roles during shrimp infection. Here, we analyzed the genome sequences of clinical, non-AHPND, and AHPND strains to characterize their repertoires of key virulence determinants. Our studies reveal that an antibacterial type VI secretion system is associated with the AHPND strains and differentiates them from non-AHPND strains, similar to what was seen with the PirA/PirB toxins. We propose that T6SS1 provides a selective advantage during shrimp infections. Copyright © 2017 American Society for Microbiology.

Acute Hepatopancreatic Necrosis Disease-Causing Vibrio parahaemolyticus Strains Maintain an Antibacterial Type VI Secretion System with Versatile Effector Repertoires

PubMed Central

Li, Peng; Kinch, Lisa N.; Ray, Ann; Dalia, Ankur B.; Nunan, Linda M.; Camilli, Andrew; Grishin, Nick V.

2017-01-01

ABSTRACT Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. AHPND is caused by toxic strains of Vibrio parahaemolyticus that have acquired a “selfish plasmid” encoding the deadly binary toxins PirAvp/PirBvp. To better understand the repertoire of virulence factors in AHPND-causing V. parahaemolyticus, we conducted a comparative analysis using the genome sequences of the clinical strain RIMD2210633 and of environmental non-AHPND and toxic AHPND isolates of V. parahaemolyticus. Interestingly, we found that all of the AHPND strains, but none of the non-AHPND strains, harbor the antibacterial type VI secretion system 1 (T6SS1), which we previously identified and characterized in the clinical isolate RIMD2210633. This finding suggests that the acquisition of this T6SS might confer to AHPND-causing V. parahaemolyticus a fitness advantage over competing bacteria and facilitate shrimp infection. Additionally, we found highly dynamic effector loci in the T6SS1 of AHPND-causing strains, leading to diverse effector repertoires. Our discovery provides novel insights into AHPND-causing pathogens and reveals a potential target for disease control. IMPORTANCE Acute hepatopancreatic necrosis disease (AHPND) is a serious disease that has caused severe damage and significant financial losses to the global shrimp industry. To better understand and prevent this shrimp disease, it is essential to thoroughly characterize its causative agent, Vibrio parahaemolyticus. Although the plasmid-encoded binary toxins PirAvp/PirBvp have been shown to be the primary cause of AHPND, it remains unknown whether other virulent factors are commonly present in V. parahaemolyticus and might play important roles during shrimp infection. Here, we analyzed the genome sequences of clinical, non-AHPND, and AHPND strains to characterize their repertoires of key virulence determinants. Our studies reveal that an antibacterial type VI secretion system is associated with the AHPND strains and differentiates them from non-AHPND strains, similar to what was seen with the PirA/PirB toxins. We propose that T6SS1 provides a selective advantage during shrimp infections. PMID:28432099

Utilizing Gene Tree Variation to Identify Candidate Effector Genes in Zymoseptoria tritici

PubMed Central

McDonald, Megan C.; McGinness, Lachlan; Hane, James K.; Williams, Angela H.; Milgate, Andrew; Solomon, Peter S.

2016-01-01

Zymoseptoria tritici is a host-specific, necrotrophic pathogen of wheat. Infection by Z. tritici is characterized by its extended latent period, which typically lasts 2 wks, and is followed by extensive host cell death, and rapid proliferation of fungal biomass. This work characterizes the level of genomic variation in 13 isolates, for which we have measured virulence on 11 wheat cultivars with differential resistance genes. Between the reference isolate, IPO323, and the 13 Australian isolates we identified over 800,000 single nucleotide polymorphisms, of which ∼10% had an effect on the coding regions of the genome. Furthermore, we identified over 1700 probable presence/absence polymorphisms in genes across the Australian isolates using de novo assembly. Finally, we developed a gene tree sorting method that quickly identifies groups of isolates within a single gene alignment whose sequence haplotypes correspond with virulence scores on a single wheat cultivar. Using this method, we have identified < 100 candidate effector genes whose gene sequence correlates with virulence toward a wheat cultivar carrying a major resistance gene. PMID:26837952

Liver-primed memory T cells generated under noninflammatory conditions provide anti-infectious immunity.

PubMed

Böttcher, Jan P; Schanz, Oliver; Wohlleber, Dirk; Abdullah, Zeinab; Debey-Pascher, Svenja; Staratschek-Jox, Andrea; Höchst, Bastian; Hegenbarth, Silke; Grell, Jessica; Limmer, Andreas; Atreya, Imke; Neurath, Markus F; Busch, Dirk H; Schmitt, Edgar; van Endert, Peter; Kolanus, Waldemar; Kurts, Christian; Schultze, Joachim L; Diehl, Linda; Knolle, Percy A

2013-03-28

Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1(+) memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Wnt and the Wnt signaling pathway in bone development and disease

PubMed Central

Wang, Yiping; Li, Yi-Ping; Paulson, Christie; Shao, Jian-Zhong; Zhang, Xiaoling; Wu, Mengrui; Chen, Wei

2014-01-01

Wnt signaling affects both bone modeling, which occurs during development, and bone remodeling, which is a lifelong process involving tissue renewal. Wnt signals are especially known to affect the differentiation of osteoblasts. In this review, we summarize recent advances in understanding the mechanisms of Wnt signaling, which is divided into two major branches: the canonical pathway and the noncanonical pathway. The canonical pathway is also called the Wnt/β-catenin pathway. There are two major noncanonical pathways: the Wnt-planar cell polarity pathway (Wnt-PCP pathway) and the Wnt-calcium pathway (Wnt-Ca2+ pathway). This review also discusses how Wnt ligands, receptors, intracellular effectors, transcription factors, and antagonists affect both the bone modeling and bone remodeling processes. We also review the role of Wnt ligands, receptors, intracellular effectors, transcription factors, and antagonists in bone as demonstrated in mouse models. Disrupted Wnt signaling is linked to several bone diseases, including osteoporosis, van Buchem disease, and sclerosteosis. Studying the mechanism of Wnt signaling and its interactions with other signaling pathways in bone will provide potential therapeutic targets to treat these bone diseases. PMID:24389191

Mast cells counteract regulatory T-cell suppression through interleukin-6 and OX40/OX40L axis toward Th17-cell differentiation.

PubMed

Piconese, Silvia; Gri, Giorgia; Tripodo, Claudio; Musio, Silvia; Gorzanelli, Andrea; Frossi, Barbara; Pedotti, Rosetta; Pucillo, Carlo E; Colombo, Mario P

2009-09-24

The development of inflammatory diseases implies inactivation of regulatory T (Treg) cells through mechanisms that still are largely unknown. Here we showed that mast cells (MCs), an early source of inflammatory mediators, are able to counteract Treg inhibition over effector T cells. To gain insight into the molecules involved in their interplay, we set up an in vitro system in which all 3 cellular components were put in contact. Reversal of Treg suppression required T cell-derived interleukin-6 (IL-6) and the OX40/OX40L axis. In the presence of activated MCs, concomitant abundance of IL-6 and paucity of Th1/Th2 cytokines skewed Tregs and effector T cells into IL-17-producing T cells (Th17). In vivo analysis of lymph nodes hosting T-cell priming in experimental autoimmune encephalomyelitis revealed activated MCs, Tregs, and Th17 cells displaying tight spatial interactions, further supporting the occurrence of an MC-mediated inhibition of Treg suppression in the establishment of Th17-mediated inflammatory responses.

Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hyatt, Dustin C.; Ceresa, Brian P.

2008-11-01

The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads canmore » stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.« less

T cell receptor-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac and Myosin IIA

PubMed Central

Manes, Thomas D.; Pober, Jordan S.

2013-01-01

Human effector memory (EM) CD4 T cells may be recruited from the blood into a site of inflammation in response either to inflammatory chemokines displayed on or specific antigen presented by venular endothelial cells (ECs), designated as chemokine-driven or TCR-driven transendothelial migration (TEM), respectively. We have previously described differences in the morphological appearance of transmigrating T cells as well as in the molecules that mediate T cell-EC interactions distinguishing these two pathways. Here we report that TCR-driven TEM requires ZAP-70-dependent activation of a pathway involving Vav, Rac and myosin IIA. Chemokine-driven TEM also utilizes ZAP-70, albeit in a quantitatively and spatially different manner of activation, and is independent of Vav, Rac and mysosin IIA, depending instead on an as yet unidentified GTP exchange factor that activates Cdc42. The differential use of small Rho family GTPases to activate the cytoskeleton is consistent with the morphological differences observed in T cells that undergo TEM in response to these distinct recruitment signals. PMID:23420881

Structural insights into the roles of the IcmS-IcmW complex in the type IVb secretion system of Legionella pneumophila.

PubMed

Xu, Jianpo; Xu, Dandan; Wan, Muyang; Yin, Li; Wang, Xiaofei; Wu, Lijie; Liu, Yanhua; Liu, Xiaoyun; Zhou, Yan; Zhu, Yongqun

2017-12-19

The type IVb secretion system (T4BSS) of Legionella pneumophila is a multiple-component apparatus that delivers ∼300 virulent effector proteins into host cells. The injected effectors modulate host cellular processes to promote bacterial infection and proliferation. IcmS and IcmW are two conserved small, acidic adaptor proteins that form a binary complex to interact with many effectors and facilitate their translocation. IcmS and IcmW can also interact with DotL, an ATPase of the type IV coupling protein complex (T4CP). However, how IcmS-IcmW recognizes effectors, and what the roles of IcmS-IcmW are in T4BSSs are unclear. In this study, we found that IcmS and IcmW form a 1:1 heterodimeric complex to bind effector substrates. Both IcmS and IcmW adopt new structural folds and have no structural similarities with known effector chaperones. IcmS has a compact global structure with an α/β fold, while IcmW adopts a fully α-folded, relatively loose architecture. IcmS stabilizes IcmW by binding to its two C-terminal α-helices. Photocrosslinking assays revealed that the IcmS-IcmW complex binds its cognate effectors via an extended hydrophobic surface, which can also interact with the C terminus of DotL. A crystal structure of the DotL-IcmS-IcmW complex reveals extensive and highly stable interactions between DotL and IcmS-IcmW. Moreover, IcmS-IcmW recruits LvgA to DotL and assembles a unique T4CP. These data suggest that IcmS-IcmW also functions as an inseparable integral component of the DotL-T4CP complex in the bacterial inner membrane. This study provides molecular insights into the dual roles of the IcmS-IcmW complex in T4BSSs.

Comprehensive assessment and performance improvement of effector protein predictors for bacterial secretion systems III, IV and VI.

PubMed

An, Yi; Wang, Jiawei; Li, Chen; Leier, André; Marquez-Lago, Tatiana; Wilksch, Jonathan; Zhang, Yang; Webb, Geoffrey I; Song, Jiangning; Lithgow, Trevor

2018-01-01

Bacterial effector proteins secreted by various protein secretion systems play crucial roles in host-pathogen interactions. In this context, computational tools capable of accurately predicting effector proteins of the various types of bacterial secretion systems are highly desirable. Existing computational approaches use different machine learning (ML) techniques and heterogeneous features derived from protein sequences and/or structural information. These predictors differ not only in terms of the used ML methods but also with respect to the used curated data sets, the features selection and their prediction performance. Here, we provide a comprehensive survey and benchmarking of currently available tools for the prediction of effector proteins of bacterial types III, IV and VI secretion systems (T3SS, T4SS and T6SS, respectively). We review core algorithms, feature selection techniques, tool availability and applicability and evaluate the prediction performance based on carefully curated independent test data sets. In an effort to improve predictive performance, we constructed three ensemble models based on ML algorithms by integrating the output of all individual predictors reviewed. Our benchmarks demonstrate that these ensemble models outperform all the reviewed tools for the prediction of effector proteins of T3SS and T4SS. The webserver of the proposed ensemble methods for T3SS and T4SS effector protein prediction is freely available at http://tbooster.erc.monash.edu/index.jsp. We anticipate that this survey will serve as a useful guide for interested users and that the new ensemble predictors will stimulate research into host-pathogen relationships and inspiration for the development of new bioinformatics tools for predicting effector proteins of T3SS, T4SS and T6SS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Real-time tracking of cell cycle progression during CD8+ effector and memory T-cell differentiation

PubMed Central

Kinjyo, Ichiko; Qin, Jim; Tan, Sioh-Yang; Wellard, Cameron J.; Mrass, Paulus; Ritchie, William; Doi, Atsushi; Cavanagh, Lois L.; Tomura, Michio; Sakaue-Sawano, Asako; Kanagawa, Osami; Miyawaki, Atsushi; Hodgkin, Philip D.; Weninger, Wolfgang

2015-01-01

The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8+ T cells. During influenza virus infection in vivo, naive T cells enter a CD62Lintermediate state of fast proliferation, which continues for at least nine generations. At the peak of the anti-viral immune response, a subpopulation of these cells markedly reduces their cycling speed and acquires a CD62Lhi central memory cell phenotype. Construction of T-cell family division trees in vitro reveals two patterns of proliferation dynamics. While cells initially divide rapidly with moderate stochastic variations of cycling times after each generation, a slow-cycling subpopulation displaying a CD62Lhi memory phenotype appears after eight divisions. Phenotype and cell cycle duration are inherited by the progeny of slow cyclers. We propose that memory precursors cell-intrinsically modulate their proliferative activity to diversify differentiation pathways. PMID:25709008

Distinct requirements for cranial ectoderm and mesenchyme-derived wnts in specification and differentiation of osteoblast and dermal progenitors.

PubMed

Goodnough, L Henry; Dinuoscio, Gregg J; Ferguson, James W; Williams, Trevor; Lang, Richard A; Atit, Radhika P

2014-02-01

The cranial bones and dermis differentiate from mesenchyme beneath the surface ectoderm. Fate selection in cranial mesenchyme requires the canonical Wnt effector molecule β-catenin, but the relative contribution of Wnt ligand sources in this process remains unknown. Here we show Wnt ligands are expressed in cranial surface ectoderm and underlying supraorbital mesenchyme during dermal and osteoblast fate selection. Using conditional genetics, we eliminate secretion of all Wnt ligands from cranial surface ectoderm or undifferentiated mesenchyme, to uncover distinct roles for ectoderm- and mesenchyme-derived Wnts. Ectoderm Wnt ligands induce osteoblast and dermal fibroblast progenitor specification while initiating expression of a subset of mesenchymal Wnts. Mesenchyme Wnt ligands are subsequently essential during differentiation of dermal and osteoblast progenitors. Finally, ectoderm-derived Wnt ligands provide an inductive cue to the cranial mesenchyme for the fate selection of dermal fibroblast and osteoblast lineages. Thus two sources of Wnt ligands perform distinct functions during osteoblast and dermal fibroblast formation.

Real-time tracking of cell cycle progression during CD8+ effector and memory T-cell differentiation.

PubMed

Kinjyo, Ichiko; Qin, Jim; Tan, Sioh-Yang; Wellard, Cameron J; Mrass, Paulus; Ritchie, William; Doi, Atsushi; Cavanagh, Lois L; Tomura, Michio; Sakaue-Sawano, Asako; Kanagawa, Osami; Miyawaki, Atsushi; Hodgkin, Philip D; Weninger, Wolfgang

2015-02-24

The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8(+) T cells. During influenza virus infection in vivo, naive T cells enter a CD62L(intermediate) state of fast proliferation, which continues for at least nine generations. At the peak of the anti-viral immune response, a subpopulation of these cells markedly reduces their cycling speed and acquires a CD62L(hi) central memory cell phenotype. Construction of T-cell family division trees in vitro reveals two patterns of proliferation dynamics. While cells initially divide rapidly with moderate stochastic variations of cycling times after each generation, a slow-cycling subpopulation displaying a CD62L(hi) memory phenotype appears after eight divisions. Phenotype and cell cycle duration are inherited by the progeny of slow cyclers. We propose that memory precursors cell-intrinsically modulate their proliferative activity to diversify differentiation pathways.

Leptin receptor signaling in T cells is required for Th17 differentiation.

PubMed

Reis, Bernardo S; Lee, Kihyun; Fanok, Melania H; Mascaraque, Cristina; Amoury, Manal; Cohn, Lillian B; Rogoz, Aneta; Dallner, Olof S; Moraes-Vieira, Pedro M; Domingos, Ana I; Mucida, Daniel

2015-06-01

The hormone leptin plays a key role in energy homeostasis, and the absence of either leptin or its receptor (LepR) leads to severe obesity and metabolic disorders. To avoid indirect effects and to address the cell-intrinsic role of leptin signaling in the immune system, we conditionally targeted LepR in T cells. In contrast with pleiotropic immune disorders reported in obese mice with leptin or LepR deficiency, we found that LepR deficiency in CD4(+) T cells resulted in a selective defect in both autoimmune and protective Th17 responses. Reduced capacity for differentiation toward a Th17 phenotype by lepr-deficient T cells was attributed to reduced activation of the STAT3 and its downstream targets. This study establishes cell-intrinsic roles for LepR signaling in the immune system and suggests that leptin signaling during T cell differentiation plays a crucial role in T cell peripheral effector function. Copyright © 2015 by The American Association of Immunologists, Inc.

Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants.

PubMed

Aung, Kyaw; Xin, Xiufang; Mecey, Christy; He, Sheng Yang

2017-01-01

Animal and plant pathogenic bacteria use type III secretion systems to translocate proteinaceous effectors to subvert innate immunity of their host organisms. Type III secretion/effector systems are a crucial pathogenicity factor in many bacterial pathogens of plants and animals. Pseudomonas syringae pv. tomato (Pst) DC3000 injects a total of 36 protein effectors that target a variety of host proteins. Studies of a subset of Pst DC3000 effectors demonstrated that bacterial effectors, once inside the host cell, are localized to different subcellular compartments, including plasma membrane, cytoplasm, mitochondria, chloroplast, and Trans-Golgi network, to carry out their virulence functions. Identifying the subcellular localization of bacterial effector proteins in host cells could provide substantial clues to understanding the molecular and cellular basis of the virulence activities of effector proteins. In this chapter, we present methods for transient or stable expression of bacterial effector proteins in tobacco and/or Arabidopsis thaliana for live cell imaging as well as confirming the subcellular localization in plants using fluorescent organelle markers or chemical treatment.

High-resolution definition of humoral immune response correlates of effective immunity against HIV.

PubMed

Alter, Galit; Dowell, Karen G; Brown, Eric P; Suscovich, Todd J; Mikhailova, Anastassia; Mahan, Alison E; Walker, Bruce D; Nimmerjahn, Falk; Bailey-Kellogg, Chris; Ackerman, Margaret E

2018-03-26

Defining correlates of immunity by comprehensively interrogating the extensive biological diversity in naturally or experimentally protected subjects may provide insights critical for guiding the development of effective vaccines and antibody-based therapies. We report advances in a humoral immunoprofiling approach and its application to elucidate hallmarks of effective HIV-1 viral control. Systematic serological analysis for a cohort of HIV-infected subjects with varying viral control was conducted using both a high-resolution, high-throughput biophysical antibody profiling approach, providing unbiased dissection of the humoral response, along with functional antibody assays, characterizing antibody-directed effector functions such as complement fixation and phagocytosis that are central to protective immunity. Profiles of subjects with varying viral control were computationally analyzed and modeled in order to deconvolute relationships among IgG Fab properties, Fc characteristics, and effector functions and to identify humoral correlates of potent antiviral antibody-directed effector activity and effective viral suppression. The resulting models reveal multifaceted and coordinated contributions of polyclonal antibodies to diverse antiviral responses, and suggest key biophysical features predictive of viral control. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Visualization of novel virulence activities of the Xanthomonas type III effectors AvrBs1, AvrBs3 and AvrBs4.

PubMed

Gürlebeck, Doreen; Jahn, Simone; Gürlebeck, Norman; Szczesny, Robert; Szurek, Boris; Hahn, Simone; Hause, Gerd; Bonas, Ulla

2009-03-01

Xanthomonas campestris pv. vesicatoria secretes at least 20 effector proteins through the type III secretion system directly into plant cells. In this study, we uncovered virulence activities of the effector proteins AvrBs1, AvrBs3 and AvrBs4 using Agrobacterium-mediated transient expression of the corresponding genes in Nicotiana benthamiana, followed by microscopic analyses. We showed that, in addition to the nuclear-localized AvrBs3, the effector AvrBs1, which localizes to the plant cell cytoplasm, also induces a morphological change in mesophyll cells. Comparative analyses revealed that avrBs3-expressing plant cells contain highly active nuclei. Furthermore, plant cells expressing avrBs3 or avrBs1 show a decrease in the starch content in chloroplasts and an increased number of vesicles, indicating an enlargement of the central vacuole and the cell wall. Both AvrBs1 and AvrBs3 cause an increased ion efflux when expressed in N. benthamiana. By contrast, expression of the avrBs3 homologue avrBs4 leads to large catalase crystals in peroxisomes, suggesting a possible virulence function of AvrBs4 in the suppression of the plant defence responses. Taken together, our data show that microscopic inspection can uncover subtle and novel virulence activities of type III effector proteins.

Effector-mediated discovery of a novel resistance gene against Bremia lactucae in a nonhost lettuce species.

PubMed

Giesbers, Anne K J; Pelgrom, Alexandra J E; Visser, Richard G F; Niks, Rients E; Van den Ackerveken, Guido; Jeuken, Marieke J W

2017-11-01

Candidate effectors from lettuce downy mildew (Bremia lactucae) enable high-throughput germplasm screening for the presence of resistance (R) genes. The nonhost species Lactuca saligna comprises a source of B. lactucae R genes that has hardly been exploited in lettuce breeding. Its cross-compatibility with the host species L. sativa enables the study of inheritance of nonhost resistance (NHR). We performed transient expression of candidate RXLR effector genes from B. lactucae in a diverse Lactuca germplasm set. Responses to two candidate effectors (BLR31 and BLN08) were genetically mapped and tested for co-segregation with disease resistance. BLN08 induced a hypersensitive response (HR) in 55% of the L. saligna accessions, but responsiveness did not co-segregate with resistance to Bl:24. BLR31 triggered an HR in 5% of the L. saligna accessions, and revealed a novel R gene providing complete B. lactucae race Bl:24 resistance. Resistant hybrid plants that were BLR31 nonresponsive indicated other unlinked R genes and/or nonhost QTLs. We have identified a candidate avirulence effector of B. lactucae (BLR31) and its cognate R gene in L. saligna. Concurrently, our results suggest that R genes are not required for NHR of L. saligna. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Distinct CD4+CD8+ Double-Positive T Cells in the Blood and Liver of Patients during Chronic Hepatitis B and C

PubMed Central

Nascimbeni, Michelina; Pol, Stanislas; Saunier, Bertrand

2011-01-01

CD4+ and CD8+ T cells, the main effectors of adaptive cellular immune responses, differentiate from immature, non-functional CD4+CD8+ double-positive T (DPT) cells in the thymus. Increased proportions of circulating DPT lymphocytes have been observed during acute viral infections; in chronic viral diseases, the role and repartition of extra-thymic DPT cells remain largely uncharacterized. We performed a phenotypic analysis of DPT cells in blood and liver from patients chronically infected by hepatitis C (HCV) or B (HBV) viruses. The highest percentages of DPT cells, predominantly CD4highCD8low, were observed in patients infected by HCV, while HBV-infected patients mostly displayed CD4lowCD8high and CD4highCD8high DPT cells. All proportions of DPT cells were higher in liver than in blood with, for each subpopulation referred to above, a correlation between their frequencies in these two compartments. In HCV patients, intra-hepatic DPT cells displayed more heterogeneous activation, differentiation and memory phenotypes than in the blood; most of them expressed CD1a, a marker of T cell development in the thymus. Ex vivo, the inoculation of liver slices with HCV produced in cell culture was accompanied by a disappearance of CD8high cells, suggesting a direct effect of the virus on the phenotype of DPT cells in the liver. Our results suggest that, in half of the patients, chronic HCV infection promotes the production of DPT cells, perhaps by their re-induction in the thymus and selection in the liver. PMID:21647449

Elevated FOXG1 and SOX2 in glioblastoma enforces neural stem cell identity through transcriptional control of cell cycle and epigenetic regulators.

PubMed

Bulstrode, Harry; Johnstone, Ewan; Marques-Torrejon, Maria Angeles; Ferguson, Kirsty M; Bressan, Raul Bardini; Blin, Carla; Grant, Vivien; Gogolok, Sabine; Gangoso, Ester; Gagrica, Sladjana; Ender, Christine; Fotaki, Vassiliki; Sproul, Duncan; Bertone, Paul; Pollard, Steven M

2017-04-15

Glioblastoma multiforme (GBM) is an aggressive brain tumor driven by cells with hallmarks of neural stem (NS) cells. GBM stem cells frequently express high levels of the transcription factors FOXG1 and SOX2. Here we show that increased expression of these factors restricts astrocyte differentiation and can trigger dedifferentiation to a proliferative NS cell state. Transcriptional targets include cell cycle and epigenetic regulators (e.g., Foxo3 , Plk1 , Mycn , Dnmt1 , Dnmt3b , and Tet3 ). Foxo3 is a critical repressed downstream effector that is controlled via a conserved FOXG1/SOX2-bound cis -regulatory element. Foxo3 loss, combined with exposure to the DNA methylation inhibitor 5-azacytidine, enforces astrocyte dedifferentiation. DNA methylation profiling in differentiating astrocytes identifies changes at multiple polycomb targets, including the promoter of Foxo3 In patient-derived GBM stem cells, CRISPR/Cas9 deletion of FOXG1 does not impact proliferation in vitro; however, upon transplantation in vivo, FOXG1 -null cells display increased astrocyte differentiation and up-regulate FOXO3. In contrast, SOX2 ablation attenuates proliferation, and mutant cells cannot be expanded in vitro. Thus, FOXG1 and SOX2 operate in complementary but distinct roles to fuel unconstrained self-renewal in GBM stem cells via transcriptional control of core cell cycle and epigenetic regulators. © 2017 Bulstrode et al.; Published by Cold Spring Harbor Laboratory Press.

Evaluation of Secretion Prediction Highlights Differing Approaches Needed for Oomycete and Fungal Effectors.

PubMed

Sperschneider, Jana; Williams, Angela H; Hane, James K; Singh, Karam B; Taylor, Jennifer M

2015-01-01

The steadily increasing number of sequenced fungal and oomycete genomes has enabled detailed studies of how these eukaryotic microbes infect plants and cause devastating losses in food crops. During infection, fungal and oomycete pathogens secrete effector molecules which manipulate host plant cell processes to the pathogen's advantage. Proteinaceous effectors are synthesized intracellularly and must be externalized to interact with host cells. Computational prediction of secreted proteins from genomic sequences is an important technique to narrow down the candidate effector repertoire for subsequent experimental validation. In this study, we benchmark secretion prediction tools on experimentally validated fungal and oomycete effectors. We observe that for a set of fungal SwissProt protein sequences, SignalP 4 and the neural network predictors of SignalP 3 (D-score) and SignalP 2 perform best. For effector prediction in particular, the use of a sensitive method can be desirable to obtain the most complete candidate effector set. We show that the neural network predictors of SignalP 2 and 3, as well as TargetP were the most sensitive tools for fungal effector secretion prediction, whereas the hidden Markov model predictors of SignalP 2 and 3 were the most sensitive tools for oomycete effectors. Thus, previous versions of SignalP retain value for oomycete effector prediction, as the current version, SignalP 4, was unable to reliably predict the signal peptide of the oomycete Crinkler effectors in the test set. Our assessment of subcellular localization predictors shows that cytoplasmic effectors are often predicted as not extracellular. This limits the reliability of secretion predictions that depend on these tools. We present our assessment with a view to informing future pathogenomics studies and suggest revised pipelines for secretion prediction to obtain optimal effector predictions in fungi and oomycetes.

Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptors

PubMed Central

Hintersteiner, Beate; Lingg, Nico; Zhang, Peiqing; Woen, Susanto; Hoi, Kong Meng; Stranner, Stefan; Wiederkum, Susanne; Mutschlechner, Oliver; Schuster, Manfred; Loibner, Hans; Jungbauer, Alois

2016-01-01

ABSTRACT We identified active isoforms of the chimeric anti-GD2 antibody, ch14.18, a recombinant antibody produced in Chinese hamster ovary cells, which is already used in clinical trials.1,2,3 We separated the antibody by high resolution ion-exchange chromatography with linear pH gradient elution into acidic, main and basic charge variants on a preparative scale yielding enough material for an in-depth study of the sources and the effects of microheterogeneity. The binding affinity of the charge variants toward the antigen and various cell surface receptors was studied by Biacore. Effector functions were evaluated using cellular assays for antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. Basic charge variants showed increased binding to cell surface receptor FcγRIIIa, which plays a major role in regulating effector functions. Furthermore, increased binding of the basic fractions to the neonatal receptor was observed. As this receptor mediates the prolonged half-life of IgG in human serum, this data may well hint at an increased serum half-life of these basic variants compared to their more acidic counterparts. Different glycoform patterns, C-terminal lysine clipping and N-terminal pyroglutamate formation were identified as the main structural sources for the observed isoform pattern. Potential differences in structural stability between individual charge variant fractions by nano differential scanning calorimetry could not been detected. Our in-vitro data suggests that the connection between microheterogeneity and the biological activity of recombinant antibody therapeutics deserves more attention than commonly accepted. PMID:27559765

Pi3kcb links Hippo-YAP and PI3K-AKT signaling pathways to promote cardiomyocyte proliferation and survival.

PubMed

Lin, Zhiqiang; Zhou, Pingzhu; von Gise, Alexander; Gu, Fei; Ma, Qing; Chen, Jinghai; Guo, Haidong; van Gorp, Pim R R; Wang, Da-Zhi; Pu, William T

2015-01-02

Yes-associated protein (YAP), the nuclear effector of Hippo signaling, regulates cellular growth and survival in multiple organs, including the heart, by interacting with TEA (transcriptional enhancer activator)-domain sequence-specific DNA-binding proteins. Recent studies showed that YAP stimulates cardiomyocyte proliferation and survival. However, the direct transcriptional targets through which YAP exerts its effects are poorly defined. To identify direct YAP targets that mediate its mitogenic and antiapoptotic effects in the heart. We identified direct YAP targets by combining differential gene expression analysis in YAP gain- and loss-of-function with genome-wide identification of YAP-bound loci using chromatin immunoprecipitation and high throughput sequencing. This screen identified Pik3cb, encoding p110β, a catalytic subunit of phosphoinositol-3-kinase, as a candidate YAP effector that promotes cardiomyocyte proliferation and survival. YAP and TEA-domain occupied a conserved enhancer within the first intron of Pik3cb, and this enhancer drove YAP-dependent reporter gene expression. Yap gain- and loss-of-function studies indicated that YAP is necessary and sufficient to activate the phosphoinositol-3-kinase-Akt pathway. Like Yap, Pik3cb gain-of-function stimulated cardiomyocyte proliferation, and Pik3cb knockdown dampened YAP mitogenic activity. Reciprocally, impaired heart function in Yap loss-of-function was significantly rescued by adeno-associated virus-mediated Pik3cb expression. Pik3cb is a crucial direct target of YAP, through which the YAP activates phosphoinositol-3-kinase-AKT pathway and regulates cardiomyocyte proliferation and survival. © 2014 American Heart Association, Inc.

Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.

PubMed Central

Shoelson, S E; Sivaraja, M; Williams, K P; Hu, P; Schlessinger, J; Weiss, M A

1993-01-01

SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism. Images PMID:8382612

QFT Multi-Input, Multi-Output Design with Non-Diagonal, Non-Square Compensation Matrices

NASA Technical Reports Server (NTRS)

Hess, R. A.; Henderson, D. K.

1996-01-01

A technique for obtaining a non-diagonal compensator for the control of a multi-input, multi-output plant is presented. The technique, which uses Quantitative Feedback Theory, provides guaranteed stability and performance robustness in the presence of parametric uncertainty. An example is given involving the lateral-directional control of an uncertain model of a high-performance fighter aircraft in which redundant control effectors are in evidence, i.e. more control effectors than output variables are used.

Plant-Pathogen Effectors: Cellular Probes Interfering with Plant Defenses in Spatial and Temporal Manners

PubMed Central

Toruño, Tania Y.; Stergiopoulos, Ioannis; Coaker, Gitta

2017-01-01

Plants possess large arsenals of immune receptors capable of recognizing all pathogen classes. To cause disease, pathogenic organisms must be able to overcome physical barriers, suppress or evade immune perception, and derive nutrients from host tissues. Consequently, to facilitate some of these processes, pathogens secrete effector proteins that promote colonization. This review covers recent advances in the field of effector biology, focusing on conserved cellular processes targeted by effectors from diverse pathogens. The ability of effectors to facilitate pathogen entry into the host interior, suppress plant immune perception, and alter host physiology for pathogen benefit is discussed. Pathogens also deploy effectors in a spatial and temporal manner, depending on infection stage. Recent advances have also enhanced our understanding of effectors acting in specific plant organs and tissues. Effectors are excellent cellular probes that facilitate insight into biological processes as well as key points of vulnerability in plant immune signaling networks. PMID:27359369

Behind the lines–actions of bacterial type III effector proteins in plant cells

PubMed Central

Büttner, Daniela

2016-01-01

Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

Role of effector cells (CCR7(-)CD27(-)) and effector-memory cells (CCR7(-)CD27(+)) in drug-induced maculopapular exanthema.

PubMed

Fernandez, T D; Torres, M J; Lopez, S; Antunez, C; Gomez, E; Del Prado, M F; Canto, G; Blanca, M; Mayorga, C

2010-01-01

Maculopapular exanthema (MPE) induced by drugs is a T-cell mediated reaction and effector cells may play an important role in its development. We assessed the effector and cutaneous homing phenotype in peripheral blood cells from allergic patients after drug stimulation. This study included 10 patients and 10 controls. The effector phenotype (CCR7(-)CD27(+/-)), chemokine receptors (CCR4 and CCR10), and activation (CD25(low)) and regulatory markers (CD25(high)) were measured by flow cytometry in both peripheral blood mononuclear cells (PBMCs) and CD4-T-lymphocytes. Proliferation was determined by 5-(-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) assay and the migratory capacity by a chemotaxis assay using CCL17 and CCL27. Compared to controls, CCR7(-)CD27(-) cells were increased in patients without (p=0.003) and with drug stimulation (p less than 0.001) and had significantly higher proliferation (p=0.010). CCR10 expression was increased in patients after drug stimulation in total and memory CD27(+) T-cells. Lymphocyte migration with CCL27 was higher in patients with drug stimulation (p=0.048), with a decrease in CCR7(-)CD27(-) (p less than 0.0001) and an increase in CCR7(-)CD27(+) (p=0.017). In patients, CD4-T-lymphocytes were significantly activated after drug stimulation (p less than 0.001). In conclusion, we show that effector memory CD4(+) T-cells (CCR7(-)CD27(+)) respond specifically to the drug responsible for MPE and confirm previous data about the involvement of CCR10 in cell trafficking to the skin.

Desirable cytolytic immune effector cell recruitment by interleukin-15 dendritic cells.

PubMed

Van Acker, Heleen H; Beretta, Ottavio; Anguille, Sébastien; De Caluwé, Lien; Papagna, Angela; Van den Bergh, Johan M; Willemen, Yannick; Goossens, Herman; Berneman, Zwi N; Van Tendeloo, Viggo F; Smits, Evelien L; Foti, Maria; Lion, Eva

2017-02-21

Success of dendritic cell (DC) therapy in treating malignancies is depending on the DC capacity to attract immune effector cells, considering their reciprocal crosstalk is partially regulated by cell-contact-dependent mechanisms. Although critical for therapeutic efficacy, immune cell recruitment is a largely overlooked aspect regarding optimization of DC vaccination. In this paper we have made a head-to-head comparison of interleukin (IL)-15-cultured DCs and conventional IL-4-cultured DCs with regard to their proficiency in the recruitment of (innate) immune effector cells. Here, we demonstrate that IL-4 DCs are suboptimal in attracting effector lymphocytes, while IL15 DCs provide a favorable chemokine milieu for recruiting CD8+ T cells, natural killer (NK) cells and gamma delta (γδ) T cells. Gene expression analysis revealed that IL-15 DCs exhibit a high expression of chemokines involved in antitumor immune effector cell attraction, while IL-4 DCs display a more immunoregulatory profile characterized by the expression of Th2 and regulatory T cell-attracting chemokines. This is confirmed by functional data indicating an enhanced recruitment of granzyme B+ effector lymphocytes by IL-15 DCs, as compared to IL-4 DCs, and subsequent superior killing of tumor cells by the migrated lymphocytes. Elevated CCL4 gene expression in IL-15 DCs and lowered CCR5 expression on both migrated γδ T cells and NK cells, led to validation of increased CCL4 secretion by IL15 DCs. Moreover, neutralization of CCR5 prior to migration resulted in an important inhibition of γδ T cell and NK cell recruitment by IL-15 DCs. These findings further underscore the strong immunotherapeutic potential of IL-15 DCs.

In Planta Processing and Glycosylation of a Nematode CLAVATA3/ENDOSPERM SURROUNDING REGION-Like Effector and Its Interaction with a Host CLAVATA2-Like Receptor to Promote Parasitism1[OPEN

PubMed Central

Chen, Shiyan; Lang, Ping; Chronis, Demosthenis; Zhang, Sheng; De Jong, Walter S.; Mitchum, Melissa G.

2015-01-01

Like other biotrophic plant pathogens, plant-parasitic nematodes secrete effector proteins into host cells to facilitate infection. Effector proteins that mimic plant CLAVATA3/ENDOSPERM SURROUNDING REGION-related (CLE) proteins have been identified in several cyst nematodes, including the potato cyst nematode (PCN); however, the mechanistic details of this cross-kingdom mimicry are poorly understood. Plant CLEs are posttranslationally modified and proteolytically processed to function as bioactive ligands critical to various aspects of plant development. Using ectopic expression coupled with nanoliquid chromatography-tandem mass spectrometry analysis, we show that the in planta mature form of proGrCLE1, a multidomain CLE effector secreted by PCN during infection, is a 12-amino acid arabinosylated glycopeptide (named GrCLE1-1Hyp4,7g) with striking structural similarity to mature plant CLE peptides. This glycopeptide is more resistant to hydrolytic degradation and binds with higher affinity to a CLAVATA2-like receptor (StCLV2) from potato (Solanum tuberosum) than its nonglycosylated forms. We further show that StCLV2 is highly up-regulated at nematode infection sites and that transgenic potatoes with reduced StCLV2 expression are less susceptible to PCN infection, indicating that interference of the CLV2-mediated signaling pathway confers nematode resistance in crop plants. These results strongly suggest that phytonematodes have evolved to utilize host cellular posttranslational modification and processing machinery for the activation of CLE effectors following secretion into plant cells and highlight the significance of arabinosylation in regulating nematode CLE effector activity. Our finding also provides evidence that multidomain CLEs are modified and processed similarly to single-domain CLEs, adding new insight into CLE maturation in plants. PMID:25416475

Active Plasma Kallikrein Localizes to Mast Cells and Regulates Epithelial Cell Apoptosis, Adipocyte Differentiation, and Stromal Remodeling during Mammary Gland Involution*

PubMed Central

Lilla, Jennifer N.; Joshi, Ravi V.; Craik, Charles S.; Werb, Zena

2009-01-01

The plasminogen cascade of serine proteases directs both development and tumorigenesis in the mammary gland. Plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (PKal). The dominant plasminogen activator for mammary involution is PKal, a serine protease that participates in the contact activation system of blood coagulation. We observed that the prekallikrein gene (Klkb1) is expressed highly in the mammary gland during stromal remodeling periods including puberty and postlactational involution. We used a variant of ecotin (ecotin-PKal), a macromolecular inhibitor of serine proteases engineered to be highly specific for active PKal, to demonstrate that inhibition of PKal with ecotin-PKal delays alveolar apoptosis, adipocyte replenishment, and stromal remodeling in the involuting mammary gland, producing a phenotype resembling that resulting from plasminogen deficiency. Using biotinylated ecotin-PKal, we localized active PKal to connective tissue-type mast cells in the mammary gland. Taken together, these results implicate PKal as an effector of the plasminogen cascade during mammary development. PMID:19297327

Transcription activator-like (TAL) effectors targeting OsSWEET genes enhance virulence on diverse rice (Oryza sativa) varieties when expressed individually in a TAL effector-deficient strain of Xanthomonas oryzae.

PubMed

Verdier, Valérie; Triplett, Lindsay R; Hummel, Aaron W; Corral, Rene; Cernadas, R Andres; Schmidt, Clarice L; Bogdanove, Adam J; Leach, Jan E

2012-12-01

Genomes of the rice (Oryza sativa) xylem and mesophyll pathogens Xanthomonas oryzae pv. oryzae (Xoo) and pv. oryzicola (Xoc) encode numerous secreted transcription factors called transcription activator-like (TAL) effectors. In a few studied rice varieties, some of these contribute to virulence by activating corresponding host susceptibility genes. Some activate disease resistance genes. The roles of X. oryzae TAL effectors in diverse rice backgrounds, however, are poorly understood. Xoo TAL effectors that promote infection by activating SWEET sucrose transporter genes were expressed in TAL effector-deficient X. oryzae strain X11-5A, and assessed in 21 rice varieties. Some were also tested in Xoc on variety Nipponbare. Several Xoc TAL effectors were tested in X11-5A on four rice varieties. Xoo TAL effectors enhanced X11-5A virulence on most varieties, but to varying extents depending on the effector and variety. SWEET genes were activated in all tested varieties, but increased virulence did not correlate with activation level. SWEET activators also enhanced Xoc virulence on Nipponbare. Xoc TAL effectors did not alter X11-5A virulence. SWEET-targeting TAL effectors contribute broadly and non-tissue-specifically to virulence in rice, and their function is affected by host differences besides target sequences. Further, the utility of X11-5A for characterizing individual TAL effectors in rice was established. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Transcriptional profiling of predator-induced phenotypic plasticity in Daphnia pulex.

PubMed

Rozenberg, Andrey; Parida, Mrutyunjaya; Leese, Florian; Weiss, Linda C; Tollrian, Ralph; Manak, J Robert

2015-01-01

Predator-induced defences are a prominent example of phenotypic plasticity found from single-celled organisms to vertebrates. The water flea Daphnia pulex is a very convenient ecological genomic model for studying predator-induced defences as it exhibits substantial morphological changes under predation risk. Most importantly, however, genetically identical clones can be transcriptionally profiled under both control and predation risk conditions and be compared due to the availability of the sequenced reference genome. Earlier gene expression analyses of candidate genes as well as a tiled genomic microarray expression experiment have provided insights into some genes involved in predator-induced phenotypic plasticity. Here we performed the first RNA-Seq analysis to identify genes that were differentially expressed in defended vs. undefended D. pulex specimens in order to explore the genetic mechanisms underlying predator-induced defences at a qualitatively novel level. We report 230 differentially expressed genes (158 up- and 72 down-regulated) identified in at least two of three different assembly approaches. Several of the differentially regulated genes belong to families of paralogous genes. The most prominent classes amongst the up-regulated genes include cuticle genes, zinc-metalloproteinases and vitellogenin genes. Furthermore, several genes from this group code for proteins recruited in chromatin-reorganization or regulation of the cell cycle (cyclins). Down-regulated gene classes include C-type lectins, proteins involved in lipogenesis, and other families, some of which encode proteins with no known molecular function. The RNA-Seq transcriptome data presented in this study provide important insights into gene regulatory patterns underlying predator-induced defences. In particular, we characterized different effector genes and gene families found to be regulated in Daphnia in response to the presence of an invertebrate predator. These effector genes are mostly in agreement with expectations based on observed phenotypic changes including morphological alterations, i.e., expression of proteins involved in formation of protective structures and in cuticle strengthening, as well as proteins required for resource re-allocation. Our findings identify key genetic pathways associated with anti-predator defences.

Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges

PubMed Central

Selin, Carrie; de Kievit, Teresa R.; Belmonte, Mark F.; Fernando, W. G. Dilantha

2016-01-01

Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as “effectors” is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR/Cas9 technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function. PMID:27199930

Fighting Viral Infections and Virus-Driven Tumors with Cytotoxic CD4+ T Cells

PubMed Central

Muraro, Elena; Merlo, Anna; Martorelli, Debora; Cangemi, Michela; Dalla Santa, Silvia; Dolcetti, Riccardo; Rosato, Antonio

2017-01-01

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors. PMID:28289418

Oral lichen planus: An overview

PubMed Central

Krupaa, R. Jayasri; Sankari, S. Leena; Masthan, K. M. K.; Rajesh, E.

2015-01-01

Lichen planus is an immunologically mediated mucocutaneous disease that is triggered by varied etiological agents. The oral lichenoid reaction is considered a variant of the disease that needs to be clearly diagnosed as a separate entity from oral lichen planus and treated. They follow a strict cause-effector relationship, protocols that suggest the differentiation. Lichen planus has varied clinical forms in the oral mucosa and cutaneously that has different prognosis. This condition also arises in association with various other systemic conditions such as hypertension, diabetes mellitus. There have been cases reported in the esophagus, larynx, scalp, nail, cutaneous areas, especially arms and wrists, trunk. There is reported malignant transformation that essentiates careful examination, treatment protocol and regular follow-up sessions. This article throws light on the disease condition of oral lichen planus and oral lichenoid reaction that is essential for the differentiation and treatment. PMID:26015696

Submembraneous microtubule cytoskeleton: regulation of microtubule assembly by heterotrimeric G proteins

PubMed Central

Roychowdhury, Sukla; Rasenick, Mark. M

2009-01-01

Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. G proteins also interact with microtubules and participate in microtubule-dependent centrosome/chromosome movement during cell division, as well as neuronal differentiation. In recent years, significant progress has been made in our understanding of the biochemical/functional interactions between G protein subunits (α and βγ) and microtubules, and the molecular details emerging from these studies suggest that α and βγ subunits of G proteins interact with tubulin/microtubules to regulate assembly/dynamics of microtubules, providing a novel mechanism for hormone or neurotransmitter induced rapid remodeling of cytoskeleton, regulation of mitotic spindle for centrosome/chromosome movements in cell division, and neuronal differentiation where structural plasticity mediated by microtubules is important for appropriate synaptic connections and signal transmission. PMID:18754776

Assaying effector function in planta using double-barreled particle bombardment.

PubMed

Kale, Shiv D; Tyler, Brett M

2011-01-01

The biolistic transient gene expression assay is a beneficial tool for studying gene function in vivo. However, biolistic transient assay systems have inherent pitfalls that often cause experimental inaccuracies such as poor transformation efficiency, which can be confused with biological phenomena. The double-barreled gene gun device is an inexpensive and highly effective attachment that enables statistically significant data to be obtained with one-tenth the number of experimental replicates compared to conventional biolistic assays. The principle behind the attachment is to perform two simultaneous bombardments with control and test DNA preparations onto the same leaf. The control bombardment measures the efficiency of the transformation while the ratio of the test bombardment to the control bombardment measures the activity of the gene of interest. With care, the ratio between the pair of bombardments can be highly reproducible from bombardment to bombardment. The double-barreled attachment has been used to study plant resistance (R) gene-mediated responses to effectors, induction and suppression of cell death by a wide variety of pathogen and host molecules, and the role of oömycete effector RXLR motifs in cell reentry.

PTSD is associated with an increase in aged T cell phenotypes in adults living in Detroit

PubMed Central

Aiello, Allison E.; Dowd, Jennifer B.; Jayabalasingham, Bamini; Feinstein, Lydia; Uddin, Monica; Simanek, Amanda M.; Cheng, Caroline K.; Galea, Sandro; Wildman, Derek E.; Koenen, Karestan; Pawelec, Graham

2016-01-01

Background Psychosocial stress is thought to play a key role in the acceleration of immunological aging. This study investigated the relationship between lifetime and past-year history of post-traumatic stress disorder (PTSD) and the distribution of T cell phenotypes thought to be characteristic of immunological aging. Methods Data were from 85 individuals who participated in the community-based Detroit Neighborhood Health Study. Immune markers assessed included the CD4:CD8 ratio, the ratio of late-differentiated effector (CCR7-CD45RA+CD27-CD28-) to naïve (CCR7+CD45RA+CD27+CD28+) T cells, the percentage of KLRG1-expressing cells, and the percentage of CD57-expressing cells. Results In models adjusted for age, gender, race/ethnicity, education, smoking status, and medication use, we found that past-year PTSD was associated with statistically significant differences in the CD8+ T cell population, including a higher ratio of late-differentiated effector to naïve T cells, a higher percentage of KLRG1+ cells, and a higher percentage of CD57+ cells. The percentage of CD57+ cells in the CD4 subset was also significantly higher and the CD4:CD8 ratio significantly lower among individuals who had experienced past-year PTSD. Lifetime PTSD was also associated with differences in several parameters of immune aging. Conclusions PTSD is associated with an aged immune phenotype and should be evaluated as a potential catalyzer of accelerated immunological aging in future studies. PMID:26894484

CD40L Expression Allows CD8+ T Cells to Promote Their Own Expansion and Differentiation through Dendritic Cells

PubMed Central

Tay, Neil Q.; Lee, Debbie C. P.; Chua, Yen Leong; Prabhu, Nayana; Gascoigne, Nicholas R. J.; Kemeny, David M.

2017-01-01

CD8+ T cells play an important role in providing protective immunity against a wide range of pathogens, and a number of different factors control their activation. Although CD40L-mediated CD40 licensing of dendritic cells (DCs) by CD4+ T cells is known to be necessary for the generation of a robust CD8+ T cell response, the contribution of CD8+ T cell-expressed CD40L on DC licensing is less clear. We have previously shown that CD8+ T cells are able to induce the production of IL-12 p70 by DCs in a CD40L-dependent manner, providing some evidence that CD8+ T cell-mediated activation of DCs is possible. To better understand the role of CD40L on CD8+ T cell responses, we generated and characterized CD40L-expressing CD8+ T cells both in vitro and in vivo. We found that CD40L was expressed on 30–50% of effector CD8+ T cells when stimulated and that this expression was transient. The expression of CD40L on CD8+ T cells promoted the proliferation and differentiation of both the CD40L-expressing CD8+ T cells and the bystander effector CD8+ T cells. This process occurred via a cell-extrinsic manner and was mediated by DCs. These data demonstrate the existence of a mechanism where CD8+ T cells and DCs cooperate to maximize CD8+ T cell responses. PMID:29163545