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  1. Dynamic Regulation of TCR-Microclusters and the Microsynapse for T Cell Activation.

    PubMed

    Hashimoto-Tane, Akiko; Saito, Takashi

    2016-01-01

    The interaction between a T cell and an antigen-presenting cell is the initiating event in T cell-mediated adaptive immunity. The Immunological Synapse (IS) is formed at the interface between these two cell types, and is the site where antigen (Ag)-specific recognition and activation are induced through the T cell receptor (TCR). This occurs at the center of the IS, and cell adhesion is supported through integrins in the area surrounding the TCR. Recently, this model has been revised based on data indicating that the initial Ag-specific activation signal is triggered prior to IS formation at TCR-microclusters (MCs), sites where TCR, kinases and adaptors of TCR proximal downstream signaling molecules accumulate as an activation signaling cluster. TCR-MCs then move into the center of the cell-cell interface to generate the cSMAC. This translocation of TCR-MCs is mediated initially by the actin cytoskeleton and then by dynein-induced movement along microtubules. The translocation of TCR-MCs and cSMAC formation is induced upon strong TCR stimulation through the assembly of a TCR-dynein super complex with microtubules. The Ag-specific activation signal is induced at TCR-MCs, but the adhesion signal is now shown to be induced by generating a "microsynapse," which is composed of a core of TCR-MCs and the surrounding adhesion ring of integrin and focal adhesion molecules. Since the microsynapse is critical for activation, particularly under weak TCR stimulation, this structure supports a weak TCR signal through a cell-cell adhesion signal. The microsynapse has a structure similar to the IS but on a micro-scale and regulates Ag-specific activation as well as cell-cell adhesion. We describe here the dynamic regulation of TCR-MCs, responsible for inducing Ag-specific activation signals, and the microsynapse, responsible for adhesion signals critical for cell-cell interactions, and their interrelationship.

  2. Caveolin-1 regulates TCR signal strength and regulatory T-cell differentiation into alloreactive T cells.

    PubMed

    Schönle, Anne; Hartl, Frederike A; Mentzel, Jan; Nöltner, Theresa; Rauch, Katharina S; Prestipino, Alessandro; Wohlfeil, Sebastian A; Apostolova, Petya; Hechinger, Anne-Kathrin; Melchinger, Wolfgang; Fehrenbach, Kerstin; Guadamillas, Marta C; Follo, Marie; Prinz, Gabriele; Ruess, Ann-Katrin; Pfeifer, Dietmar; del Pozo, Miguel Angel; Schmitt-Graeff, Annette; Duyster, Justus; Hippen, Keli I; Blazar, Bruce R; Schachtrup, Kristina; Minguet, Susana; Zeiser, Robert

    2016-04-14

    Caveolin-1 (Cav-1) is a key organizer of membrane specializations and a scaffold protein that regulates signaling in multiple cell types. We found increased Cav-1 expression in human and murine T cells after allogeneic hematopoietic cell transplantation. Indeed, Cav-1(-/-)donor T cells caused less severe acute graft-versus-host disease (GVHD) and yielded higher numbers of regulatory T cells (Tregs) compared with controls. Depletion of Tregs from the graft abrogated this protective effect. Correspondingly, Treg frequencies increased when Cav-1(-/-)T cells were exposed to transforming growth factor-β/T-cell receptor (TCR)/CD28 activation or alloantigen stimulation in vitro compared with wild-type T cells. Mechanistically, we found that the phosphorylation of Cav-1 is dispensable for the control of T-cell fate by using a nonphosphorylatable Cav-1 (Y14F/Y14F) point-mutation variant. Moreover, the close proximity of lymphocyte-specific protein tyrosine kinase (Lck) to the TCR induced by TCR-activation was reduced in Cav-1(-/-)T cells. Therefore, less TCR/Lck clustering results in suboptimal activation of the downstream signaling events, which correlates with the preferential development into a Treg phenotype. Overall, we report a novel role for Cav-1 in TCR/Lck spatial distribution upon TCR triggering, which controls T-cell fate toward a regulatory phenotype. This alteration translated into a significant increase in the frequency of Tregs and reduced GVHD in vivo.

  3. PP6 controls T cell development and homeostasis by negatively regulating distal TCR signaling.

    PubMed

    Ye, Jian; Shi, Hao; Shen, Ye; Peng, Chao; Liu, Yan; Li, Chenyu; Deng, Kejing; Geng, Jianguo; Xu, Tian; Zhuang, Yuan; Zheng, Biao; Tao, Wufan

    2015-02-15

    T cell development and homeostasis are both regulated by TCR signals. Protein phosphorylation and dephosphorylation, which are catalyzed by protein kinases and phosphatases, respectively, serve as important switches controlling multiple downstream pathways triggered by TCR recognition of Ags. It has been well documented that protein tyrosine phosphatases are involved in negative regulation of proximal TCR signaling. However, how TCR signals are terminated or attenuated in the distal TCR signaling pathways is largely unknown. We investigated the function of Ser/Thr protein phosphatase (PP) 6 in TCR signaling. T cell lineage-specific ablation of PP6 in mice resulted in enhanced thymic positive and negative selection, and preferential expansion of fetal-derived, IL-17-producing Vγ6Vδ1(+) T cells. Both PP6-deficient peripheral CD4(+) helper and CD8(+) cytolytic cells could not maintain a naive state and became fast-proliferating and short-lived effector cells. PP6 deficiency led to profound hyperactivation of multiple distal TCR signaling molecules, including MAPKs, AKT, and NF-κB. Our studies demonstrate that PP6 acts as a critical negative regulator, not only controlling both αβ and γδ lineage development, but also maintaining naive T cell homeostasis by preventing their premature activation before Ag stimulation.

  4. The Allostery Model of TCR Regulation.

    PubMed

    Schamel, Wolfgang W A; Alarcon, Balbino; Höfer, Thomas; Minguet, Susana

    2017-01-01

    The activity of the αβ TCR is controlled by conformational switches. In the resting conformation, the TCR is not phosphorylated and is inactive. Binding of multivalent peptide-MHC to the TCR stabilizes the active conformation, leading to TCR signaling. These two conformations allow the TCRs to be allosterically regulated. We review recent data on heterotropic allostery where peptide-MHC and membrane cholesterol serve opposing functions as positive and negative allosteric regulators, respectively. In resting T cells cholesterol keeps TCRs in the resting conformation that otherwise would become spontaneously active. This regulation is well described by the classical Monod-Wyman-Changeux model of allostery. Moreover, the observation that TCRs assemble into nanoclusters might allow for homotropic allostery, in which individual TCRs could positively cooperate and thus enhance the sensitivity of T cell activation. This new view of TCR regulation will contribute to a better understanding of TCR functioning.

  5. TCR Signaling in T Cell Memory.

    PubMed

    Daniels, Mark A; Teixeiro, Emma

    2015-01-01

    T cell memory plays a critical role in our protection against pathogens and tumors. The antigen and its interaction with the T cell receptor (TCR) is one of the initiating elements that shape T cell memory together with inflammation and costimulation. Over the last decade, several transcription factors and signaling pathways that support memory programing have been identified. However, how TCR signals regulate them is still poorly understood. Recent studies have shown that the biochemical rules that govern T cell memory, strikingly, change depending on the TCR signal strength. Furthermore, TCR signal strength regulates the input of cytokine signaling, including pro-inflammatory cytokines. These highlight how tailoring antigenic signals can improve immune therapeutics. In this review, we focus on how TCR signaling regulates T cell memory and how the quantity and quality of TCR-peptide-MHC interactions impact the multiple fates a T cell can adopt in the memory pool.

  6. TCR Signaling in T Cell Memory

    PubMed Central

    Daniels, Mark A.; Teixeiro, Emma

    2015-01-01

    T cell memory plays a critical role in our protection against pathogens and tumors. The antigen and its interaction with the T cell receptor (TCR) is one of the initiating elements that shape T cell memory together with inflammation and costimulation. Over the last decade, several transcription factors and signaling pathways that support memory programing have been identified. However, how TCR signals regulate them is still poorly understood. Recent studies have shown that the biochemical rules that govern T cell memory, strikingly, change depending on the TCR signal strength. Furthermore, TCR signal strength regulates the input of cytokine signaling, including pro-inflammatory cytokines. These highlight how tailoring antigenic signals can improve immune therapeutics. In this review, we focus on how TCR signaling regulates T cell memory and how the quantity and quality of TCR–peptide–MHC interactions impact the multiple fates a T cell can adopt in the memory pool. PMID:26697013

  7. Regulation of immunological disorders by invariant Vα19-Jα33 TCR-bearing cells.

    PubMed

    Shimamura, Michio; Huang, Yi-Ying; Goji, Hiroshi; Endo, Shin; Migishima, Rika; Yokoyama, Minesuke

    2011-03-01

    We have previously shown that over-expression of the invariant Vα19-Jα33 TCR α transgene (Tg) using a natural TCR α promoter in mice induces the development of NK1.1(+) T cells (Vα19 NKT cells) in lymphoid organs, including the liver and intestine. These cells produce different spectra of immunoregulatory cytokines such as IL-4, IL-10, IL-17, and IFN-γ depending on the duration and intensity of the invariant TCR stimulation. In this study, we examined the effects of over-expression of invariant Vα19-Jα33 TCR-bearing cells on disease progress in the models of immunological disorders. The introduction of invariant Vα19 TCR Tg into non-obese diabetic mice delayed the onset of the disease. In addition, delayed-type hypersensitivity (DTH) to sheep erythrocytes was suppressed in the Vα19 Tg mice. DTH was also suppressed in the wild type mice previously transferred with Vα19 Tg(+) but not non-Tg cells. Thus, invariant Vα19 TCR-bearing cells are suggested to participate in the homeostasis of immunity to suppress disease progression resulting from Th1-immunity excess.

  8. TCR-engineered T cells: a model of inducible TCR expression to dissect the interrelationship between two TCRs.

    PubMed

    Reuß, Simone; Sebestyén, Zsolt; Heinz, Niels; Loew, Rainer; Baum, Christopher; Debets, Reno; Uckert, Wolfgang

    2014-01-01

    TCR gene modified T cells for adoptive therapy simultaneously express the Tg TCR and the endogenous TCR, which might lead to mispaired TCRs with harmful unknown specificity and to a reduced function of TCR-Tg T cells. We generated dual TCR T cells in two settings in which either TCR was constitutively expressed by a retroviral promoter while the second TCR expression was regulable by a Tet-on system. Constitutively expressed TCR molecules were reduced on the cell surface depending on the induced TCR expression leading to strongly hampered function. Besides that, using fluorescence resonance energy transfer we detected mispaired TCR dimers and different pairing behaviors of individual TCR chains with a mutual influence on TCR chain expression. The loss of function and mispairing could not be avoided by changing the TCR expression level or by introduction of an additional cysteine bridge. However, in polyclonal T cells, optimized TCR formats (cysteineization, codon optimization) enhanced correct pairing and function. We conclude from our data that (i) the level of mispairing depends on the individual TCRs and is not reduced by increasing the level of one TCR, and (ii) modifications (cysteineization, codon optimization) improve correct pairing but do not completely exclude mispairing (cysteineization).

  9. Innate signals overcome acquired TCR signaling pathway regulation and govern the fate of human CD161(hi) CD8α⁺ semi-invariant T cells.

    PubMed

    Turtle, Cameron J; Delrow, Jeff; Joslyn, Rochelle C; Swanson, Hillary M; Basom, Ryan; Tabellini, Laura; Delaney, Colleen; Heimfeld, Shelly; Hansen, John A; Riddell, Stanley R

    2011-09-08

    Type 17 programmed CD161(hi)CD8α(+) T cells contribute to mucosal immunity to bacteria and yeast. In early life, microbial colonization induces proliferation of CD161(hi) cells that is dependent on their expression of a semi-invariant Vα7.2(+) TCR. Although prevalent in adults, CD161(hi)CD8α(+) cells exhibit weak proliferative and cytokine responses to TCR ligation. The mechanisms responsible for the dichotomous response of neonatal and adult CD161(hi) cells, and the signals that enable their effector function, have not been established. We describe acquired regulation of TCR signaling in adult memory CD161(hi)CD8α(+) T cells that is absent in cord CD161(hi) cells and adult CD161(lo) cells. Regulated TCR signaling in CD161(hi) cells was due to profound alterations in TCR signaling pathway gene expression and could be overcome by costimulation through CD28 or innate cytokine receptors, which dictated the fate of their progeny. Costimulation with IL-1β during TCR ligation markedly increased proinflammatory IL-17 production, while IL-12-induced Tc1-like function and restored the response to TCR ligation without costimulation. CD161(hi) cells from umbilical cord blood and granulocyte colony stimulating factor-mobilized leukaphereses differed in frequency and function, suggesting future evaluation of the contribution of CD161(hi) cells in hematopoietic stem cell grafts to transplant outcomes is warranted.

  10. Recruitment of calcineurin to the TCR positively regulates T cell activation.

    PubMed

    Dutta, Debjani; Barr, Valarie A; Akpan, Itoro; Mittelstadt, Paul R; Singha, Laishram I; Samelson, Lawrence E; Ashwell, Jonathan D

    2017-02-01

    Calcineurin is a phosphatase whose primary targets in T cells are NFAT transcription factors, and inhibition of calcineurin activity by treatment with cyclosporin A (CsA) or FK506 is a cornerstone of immunosuppressive therapies. Here we found that calcineurin was recruited to the T cell antigen receptor (TCR) signaling complex, where it reversed inhibitory phosphorylation of the tyrosine kinase Lck on Ser59 (Lck(S59)). Loss of calcineurin activity impaired phosphorylation of Tyr493 of the tyrosine kinase ZAP-70 (ZAP-70(Y493)), as well as some downstream pathways in a manner consistent with signaling in cells expressing Lck(S59A) (Lck that cannot be phosphorylated) or Lck(S59E) (a phosphomimetic mutant). Notably, CsA inhibited integrin-LFA-1-dependent and NFAT-independent adhesion of T cells to the intercellular adhesion molecule ICAM-1, with little effect on cells expressing mutant Lck. These results provide new understanding of how widely used immunosuppressive drugs interfere with essential processes in the immune response.

  11. Analysis of CD2 and TCR-{beta} gene expression in jurkat cell mutants suggests a cis regulation of gene transcription

    SciTech Connect

    Kamoun, M.; Woods, J.S.; Sano, N.

    1995-10-15

    Thirty CD2{sup -} J32 stable clones, derived by mutagenesis and subsequent immunoselection with anti-CD2 Ab, were used to study the regulation of CD2 and TCR gene expression. Analysis of RNA expression revealed that the loss of surface expression of CD2 was due to a lack of expression of CD2 mRNA and was associated with a lack of expression of VDJ TCR-{beta} transcripts in 12 of these mutants, sparing the expression of DJ TCR-{beta}, TCR-{alpha}, CD3{gamma}, {delta}, {epsilon}, and {zeta} RNA. The expression of other differentiation molecules was unaffected, except for CD1, CD4, and CD5, which were either decreased or absent in most of these mutants. A gain in the expression of TCR-{gamma} transcripts was observed in each of these mutants, while, as expected, no TCR-{gamma} transcripts were detected in wild-type J32 cells. Several mutants were able to use the human CD2 enhancer and the murine TCR-{beta} enhancer and promoter to activate transcription from reporter genes in the context of heterologous promoters, indicating that the mutation(s) does not affect transcription pathways. Consistent with this finding is the adequate expression in these mutants of several lineage-specific transcription factors. The expression of CD2 in several of these mutants was rescued by gene transfer using a genomic 28.5-kb CD2 fragment, suggesting that the enchancer function of this gene may be dependent on the enhancer site. These observations suggest that the coordinate expressions of CD2 and TCR-{beta} genes share common regulatory mechanisms involving factors regulating chromatin structure and accessibility. 51 refs., 6 figs., 3 tabs.

  12. Role of TCR-induced extracellular signal-regulated kinase activation in the regulation of early IL-4 expression in naive CD4+ T cells.

    PubMed

    Jorritsma, Patricia J; Brogdon, Jennifer L; Bottomly, Kim

    2003-03-01

    Although extracellular signal-regulated kinase (Erk) activation influences IL-4 production in various experimental systems, its role during Th differentiation is unclear. In this study, we show that Erk plays a critical role in IL-4 expression during TCR-induced Th differentiation of naive CD4(+) T cells. Stimulation of CD4(+) T cells with a high affinity peptide resulted in sustained Erk activation and Th1 differentiation. However, reduction of Erk activity led to a dramatic increase in IL-4 production and Th2 generation. Analysis of RNA and nuclear proteins of CD4(+) T cells 48 h after stimulation revealed that this was due to early IL-4 expression. Interestingly, transient Erk activation resulted in altered AP-1 DNA binding activity and the induction of an AP-1 complex that was devoid of Fos protein and consisted of Jun-Jun dimers. These data show that in the presence of a strong TCR signal, IL-4 expression can be induced in naive CD4(+) T cells by altering the strength of Erk activation. In addition, these data suggest that TCR-induced Erk activation is involved in the regulation of IL-4 expression by altering the composition of the AP-1 complex and its subsequent DNA binding activity.

  13. Maintenance of TCR clonality in T cells expressing genes for two TCR heterodimers.

    PubMed

    Sant'Angelo, D B; Cresswell, P; Janeway, C A; Denzin, L K

    2001-06-05

    T cell receptor (TCR) allelic exclusion is believed to be primarily mediated by suppression of further recombination at the TCR locus after the expression of a functional TCR protein. Genetic allelic exclusion has been shown to be leaky for the beta chain and, more commonly, for the alpha chain. Here, we demonstrate an additional mechanism by which T cells can maintain monoclonality. T cells from double TCR transgenic mice express only one or the other of the two available TCRs at the cell surface. This "functional allelic exclusion" is apparently due to control of the TCR assembly process because these T cells express RNA and protein for all four transgenic TCR proteins. Lack of cell surface expression of the second TCR may be controlled by a failure to assemble the TCR heterodimer.

  14. Immunotherapy with TCR-redirected T cells: comparison of TCR-transduced and TCR-engineered hematopoietic stem cell-derived T cells.

    PubMed

    Stärck, Lilian; Popp, Katja; Pircher, Hanspeter; Uckert, Wolfgang

    2014-01-01

    Redirecting Ag specificity by transfer of TCR genes into PBLs is an attractive method to generate large numbers of cytotoxic T cells for immunotherapy of cancer and viral diseases. However, transferred TCR chains can pair with endogenous TCR chains, resulting in the formation of mispaired TCR dimers and decreased or unspecific reactivity. TCR gene transfer into hematopoietic stem cells (HSCs) is an alternative to create T cells with desired Ag specificity, because in this case expression of endogenous TCR chains is then less likely owing to allelic exclusion. We generated TCR-transduced T cells from peripheral T cells using the lymphocytic choriomeningitis virus-specific P14 TCR. After transfer of the P14 TCR genes into HSCs and subsequent reconstitution of irradiated mice, TCR-engineered HSC-derived T cells were produced. We then compared the Ag-specific T cell populations with P14 TCR-transgenic T cells for their therapeutic efficiency in three in vivo models. In this study, we demonstrate that TCR-transduced T cells and TCR-engineered HSC-derived T cells are comparable in controlling lymphocytic choriomeningitis virus infection in mice and suppress growth of B16 tumor cells expressing the cognate Ag in a comparable manner.

  15. Regulatory T cells require TCR signaling for their suppressive function.

    PubMed

    Schmidt, Amanda M; Lu, Wen; Sindhava, Vishal J; Huang, Yanping; Burkhardt, Janis K; Yang, Enjun; Riese, Matthew J; Maltzman, Jonathan S; Jordan, Martha S; Kambayashi, Taku

    2015-05-01

    Regulatory T cells (Tregs) are a subset of CD4(+) T cells that maintain immune tolerance in part by their ability to inhibit the proliferation of conventional CD4(+) T cells (Tconvs). The role of the TCR and the downstream signaling pathways required for this suppressive function of Tregs are not fully understood. To yield insight into how TCR-mediated signals influence Treg suppressive function, we assessed the ability of Tregs with altered TCR-mediated signaling capacity to inhibit Tconv proliferation. Mature Tregs deficient in Src homology 2 domain containing leukocyte protein of 76 kDa (SLP-76), an adaptor protein that nucleates the proximal signaling complex downstream of the TCR, were unable to inhibit Tconv proliferation, suggesting that TCR signaling is required for Treg suppressive function. Moreover, Tregs with defective phospholipase C γ (PLCγ) activation due to a Y145F mutation of SLP-76 were also defective in their suppressive function. Conversely, enhancement of diacylglycerol-mediated signaling downstream of PLCγ by genetic ablation of a negative regulator of diacylglycerol kinase ζ increased the suppressive ability of Tregs. Because SLP-76 is also important for integrin activation and signaling, we tested the role of integrin activation in Treg-mediated suppression. Tregs lacking the adaptor proteins adhesion and degranulation promoting adapter protein or CT10 regulator of kinase/CT10 regulator of kinase-like, which are required for TCR-mediated integrin activation, inhibited Tconv proliferation to a similar extent as wild-type Tregs. Together, these data suggest that TCR-mediated PLCγ activation, but not integrin activation, is required for Tregs to inhibit Tconv proliferation.

  16. Predominant role of T cell receptor (TCR)-alpha chain in forming preimmune TCR repertoire revealed by clonal TCR reconstitution system.

    PubMed

    Yokosuka, Tadashi; Takase, Kan; Suzuki, Misao; Nakagawa, Yohko; Taki, Shinsuke; Takahashi, Hidemi; Fujisawa, Takehiko; Arase, Hisashi; Saito, Takashi

    2002-04-15

    The CDR3 regions of T cell receptor (TCR)-alpha and -beta chains play central roles in the recognition of antigen (Ag)-MHC complex. TCR repertoire is created on the basis of Ag recognition specificity by CDR3s. To analyze the potential spectrum of TCR-alpha and -beta to exhibit Ag specificity and generate TCR repertoire, we established hundreds of TCR transfectants bearing a single TCR-alpha or -beta chain derived from a cytotoxic T cell (CTL) clone, RT-1, specific for HIVgp160 peptide, and randomly picked up TCR-beta or -alpha chains. Surprisingly, one-third of such TCR-beta containing random CDR3 beta from naive T cells of normal mice could reconstitute the antigen-reactive TCR coupling with RT-1 TCR-alpha. A similar dominant function of TCR-alpha in forming Ag-specific TCR, though low-frequency, was obtained for lymphocytic choriomeningitis virus-specific TCR. Subsequently, we generated TCR-alpha and/or -beta transgenic (Tg) mice specific for HIVgp160 peptide, and analyzed the TCR repertoire of Ag-specific CTLs. Similar to the results from TCR reconstitution, TCR-alpha Tg generated CTLs with heterogeneous TCR-beta, whereas TCR-beta Tg-induced CTLs bearing a single TCR-alpha. These findings of Ag recognition with minimum involvement of CDR3 beta expand our understanding regarding the flexibility of the spectrum of TCR and suggest a predominant role of TCR-alpha chain in determining the preimmune repertoire of Ag-specific TCR.

  17. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  18. Negative regulation of TCR signaling by ubiquitination of Zap-70 Lys-217.

    PubMed

    Ivanova, Elitza; Carpino, Nick

    2016-05-01

    The tyrosine kinase Zap-70 is a key regulator of T cell receptor (TCR) signaling downstream of antigen presentation, with coordinated regulation of Zap-70 kinase activity critical for proper T cell proliferation, differentiation, and effector function during an immune response. Zap-70 is cytosolic in unstimulated T cells, but is rapidly recruited to the TCR complex following receptor stimulation. Its activity is regulated both by binding to subunits of the TCR and by phosphorylation on multiple tyrosine residues. Zap-70 also has been reported to be ubiquitinated following TCR stimulation. Herein, we confirm the ubiquitination of Zap-70 in T cell lines and in primary human and mouse T cells, and report the identification of nine novel Zap-70 ubiquitination sites. Three sites, including Lys-193, Lys-217, and Lys-376, displayed greater than 20-fold increase in modification levels following TCR stimulation. Abrogation of Lys-217 ubiquitination results in increased kinase activation, enhanced activation of downstream signaling pathways, and elevated IL-2 production following TCR stimulation. These data suggest that Zap-70 ubiquitination contributes to the regulation of Zap-70 signaling following TCR stimulation.

  19. Requirements for effective antitumor responses of TCR transduced T cells.

    PubMed

    de Witte, Moniek A; Jorritsma, Annelies; Kaiser, Andrew; van den Boom, Marly D; Dokter, Maarten; Bendle, Gavin M; Haanen, John B A G; Schumacher, Ton N M

    2008-10-01

    Adoptive transfer of TCR gene-modified T cells has been proposed as an attractive approach to target tumors for which it is difficult or impossible to induce strong tumor-specific T cell responses by vaccination. Whereas the feasibility of generating tumor Ag-specific T cells by gene transfer has been demonstrated, the factors that determine the in vivo effectiveness of TCR-modified T cells are largely unknown. We have analyzed the value of a number of clinically feasible strategies to enhance the antitumor potential of TCR modified T cells. These experiments reveal three factors that contribute greatly to the in vivo potency of TCR-modified T cells. First, irradiation-induced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR sequences has a profound effect on in vivo antitumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Tumors that ultimately progress in animals treated with this optimized regimen for TCR-based adoptive cell transfer invariably display a reduced expression of the target Ag. This suggests TCR gene therapy can achieve a sufficiently strong selective pressure to warrant the simultaneous targeting of multiple Ags. The strategies outlined in this study should be of value to enhance the antitumor activity of TCR-modified T cells in clinical trials.

  20. TCR repertoires of intratumoral T-cell subsets.

    PubMed

    Linnemann, Carsten; Mezzadra, Riccardo; Schumacher, Ton N M

    2014-01-01

    The infiltration of human tumors by T cells is a common phenomenon, and over the past decades, it has become increasingly clear that the nature of such intratumoral T-cell populations can predict disease course. Furthermore, intratumoral T cells have been utilized therapeutically in clinical studies of adoptive T-cell therapy. In this review, we describe how novel methods that are either based on T-cell receptor (TCR) sequencing or on cancer exome analysis allow the analysis of the tumor reactivity and antigen-specificity of the intratumoral TCR repertoire with unprecedented detail. Furthermore, we discuss studies that have started to utilize these techniques to probe the link between cancer exomes and the intratumoral TCR pool. Based on the observation that both the cancer epitope repertoire and intratumoral TCR repertoire appear highly individual, we outline strategies, such as 'autologous TCR gene therapy', that exploit the tumor-resident TCR repertoire for the development of personalized immunotherapy.

  1. γδTCR immunoglobulin constant region domain exchange in human αβTCRs improves TCR pairing without altering TCR gene-modified T cell function.

    PubMed

    Tao, Changli; Shao, Hongwei; Zhang, Wenfeng; Bo, Huaben; Wu, Fenglin; Shen, Han; Huang, Shulin

    2017-02-15

    The adoptive genetic transfer of T cell receptors (TCRs) has been shown to be overall feasible and offer clinical potential as a treatment for different types of cancer. However, this promising clinical approach is limited by the serious potential consequence that exogenous TCR mispairing with endogenous TCR chains may lead to the risk of self-reactivity. In the present study, domain‑exchange and three‑dimensional modeling strategies were used to create a set of chimeric TCR variants, which were used to exchange the partial or complete constant region of αβTCR with corresponding γδTCR domains. The expression, assembly and function of the chimeric TCR variants were examined in Jurkat T cells and peripheral mononuclear blood cells (PBMCs). Genetically‑encoded chimeras were fused with a pair of fluorescent proteins (ECFP/EYFP) to monitor expression and the pairing between chimeric TCRα chains and TCRβ chains. The fluorescence energy transfer based on confocal laser scanning microscopy showed that the introduction of γδTCR constant sequences into the αβTCR did not result in a global reduction of mispairing with endogenous TCR. However, the TCR harboring the immunoglobulin‑like domain of the γδTCR constant region (i.e., TCR∆IgC), showed a higher expression and preferential pairing, compared with wild‑type (wt)TCR. The function analysis showed that TCR∆IgC exhibited the same levels of interferon-γ production and cytotoxic activity, compared with wtTCR. Furthermore, these modified TCR-transduced T cells retained the classic human leukocyte antigen restriction of the original TCR. The other two chimeric TCRs, had either exchange of the cp+tm+ic domain or exchange of the whole C domain (Fig. 1). Ultimately, exchange of these domains demonstrated defective function in the transduced T cells. Taken together, these findings may provide further understanding of the γδTCR constant domain with implications for the improvement of TCR gene transfer

  2. Requirement of full TCR repertoire for regulatory T cells to maintain intestinal homeostasis.

    PubMed

    Nishio, Junko; Baba, Minato; Atarashi, Koji; Tanoue, Takeshi; Negishi, Hideo; Yanai, Hideyuki; Habu, Sonoko; Hori, Shohei; Honda, Kenya; Taniguchi, Tadatsugu

    2015-10-13

    The regulation of intestinal homeostasis by the immune system involves the dynamic interplay between gut commensal microbiota and resident immune cells. It is well known that a large and diverse lymphocyte antigen receptor repertoire enables the immune system to recognize and respond to a wide range of invading pathogens. There is also an emerging appreciation for a critical role the T-cell receptor (TCR) repertoire serves in the maintenance of peripheral tolerance by regulatory T cells (Tregs). Nevertheless, how the diversity of the TCR repertoire in Tregs affects intestinal homeostasis remains unknown. To address this question, we studied mice whose T cells express a restricted TCR repertoire. We observed the development of spontaneous colitis, accompanied by the induction of T-helper type 17 cells in the colon that is driven by gut commensal microbiota. We provide further evidence that a restricted TCR repertoire causes a loss of tolerogenicity to microbiota, accompanied by a paucity of peripherally derived, Helios(-) Tregs and hyperactivation of migratory dendritic cells. These results thus reveal a new facet of the TCR repertoire in which Tregs require a diverse TCR repitoire for intestinal homeostasis, suggesting an additional driving force in the evolutional significance of the TCR repertoire.

  3. Exposure of Human CD4 T Cells to IL-12 Results in Enhanced TCR-Induced Cytokine Production, Altered TCR Signaling, and Increased Oxidative Metabolism.

    PubMed

    Vacaflores, Aldo; Chapman, Nicole M; Harty, John T; Richer, Martin J; Houtman, Jon C D

    2016-01-01

    Human CD4 T cells are constantly exposed to IL-12 during infections and certain autoimmune disorders. The current paradigm is that IL-12 promotes the differentiation of naïve CD4 T cells into Th1 cells, but recent studies suggest IL-12 may play a more complex role in T cell biology. We examined if exposure to IL-12 alters human CD4 T cell responses to subsequent TCR stimulation. We found that IL-12 pretreatment increased TCR-induced IFN-γ, TNF-α, IL-13, IL-4 and IL-10 production. This suggests that prior exposure to IL-12 potentiates the TCR-induced release of a range of cytokines. We observed that IL-12 mediated its effects through both transcriptional and post-transcriptional mechanisms. IL-12 pretreatment increased the phosphorylation of AKT, p38 and LCK following TCR stimulation without altering other TCR signaling molecules, potentially mediating the increase in transcription of cytokines. In addition, the IL-12-mediated enhancement of cytokines that are not transcriptionally regulated was partially driven by increased oxidative metabolism. Our data uncover a novel function of IL-12 in human CD4 T cells; specifically, it enhances the release of a range of cytokines potentially by altering TCR signaling pathways and by enhancing oxidative metabolism.

  4. Distinct TCR signaling pathways drive proliferation and cytokine production in T cells.

    PubMed

    Guy, Clifford S; Vignali, Kate M; Temirov, Jamshid; Bettini, Matthew L; Overacre, Abigail E; Smeltzer, Matthew; Zhang, Hui; Huppa, Johannes B; Tsai, Yu-Hwai; Lobry, Camille; Xie, Jianming; Dempsey, Peter J; Crawford, Howard C; Aifantis, Iannis; Davis, Mark M; Vignali, Dario A A

    2013-03-01

    The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.

  5. Strong TCR-mediated signals suppress integrated stress responses induced by KDELR1 deficiency in naive T cells.

    PubMed

    Kamimura, Daisuke; Arima, Yasunobu; Tsuruoka, Mineko; Jiang, Jing-Jing; Bando, Hidenori; Meng, Jie; Sabharwal, Lavannya; Stofkova, Andrea; Nishikawa, Naoki; Higuchi, Kotaro; Ogura, Hideki; Atsumi, Toru; Murakami, Masaaki

    2016-03-01

    KDEL receptor 1 (KDELR1) regulates integrated stress responses (ISR) to promote naive T-cell survival in vivo. In a mouse line having nonfunctional KDELR1, T-Red (naive T-cell reduced) mice, polyclonal naive T cells show excessive ISR and eventually undergo apoptosis. However, breeding T-Red mice with TCR-transgenic mice bearing relatively high TCR affinity rescued the T-Red phenotype, implying a link between ISR-induced apoptosis and TCR-mediated signaling. Here, we showed that strong TCR stimulation reduces ISR in naive T cells. In mice lacking functional KDELR1, surviving naive T cells expressed significantly higher levels of CD5, a surrogate marker of TCR self-reactivity. In addition, higher TCR affinity/avidity was confirmed using a tetramer dissociation assay on the surviving naive T cells, suggesting that among the naive T-cell repertoire, those that receive relatively stronger TCR-mediated signals via self-antigens survive enhanced ISR. Consistent with this observation, weak TCR stimulation with altered peptide ligands decreased the survival and proliferation of naive T cells, whereas stimulation with ligands having higher affinity had no such effect. These results suggest a novel role of TCR-mediated signals in the attenuation of ISR in vivo.

  6. Fli-1 regulates the DN2 to DN3 thymocyte transition and promotes γδ T-cell commitment by enhancing TCR signal strength.

    PubMed

    Smeets, Monique F M A; Wiest, David L; Izon, David J

    2014-09-01

    Friend leukemia integration 1 (Fli-1) is a member of the Ets transcription factor family and is expressed during T-cell development; however, the role Fli-1 plays in early T-cell differentiation has not been elucidated. In this report, we demonstrate that in mouse, Fli-1 overexpression retards the CD4(-) CD8(-) double-negative (DN) to CD4(+) CD8(+) double-positive (DP) transition by deregulating normal DN thymocyte development. Specifically, Fli-1 expression moderates the DN2 and DN3 developmental transitions. We further show that Fli-1 overexpression partially mimics strong TCR signals in developing DN thymocytes and thereby enhances γδ T-cell development. Conversely, Fli-1 knockdown by small hairpin RNA reverses the lineage bias from γδ T cells and directs DN cells to the αβ lineage by attenuating TCR signaling. Therefore, Fli-1 plays a critical role in both the DN2 to DN3 transition and αβ/γδ lineage commitment.

  7. Altered production of immunoregulatory cytokines by invariant Valpha19 TCR-bearing cells dependent on the duration and intensity of TCR engagement.

    PubMed

    Shimamura, Michio; Huang, Yi-Ying; Kobayashi, Masumi; Goji, Hiroshi

    2009-02-01

    Cells bearing invariant Valpha19-Jalpha33 TCR alpha chains are believed to participate in the regulation of inflammatory autoimmune diseases. In this study, the potential to produce immunoregulatory cytokines by these cells was characterized in order to find the mechanism underlying their immunoregulatory functions. Serum levels of IL-4, IL-10, transforming growth factor-beta, IFN-gamma and IL-17 increased in mice over-expressing an invariant Valpha19-Jalpha33 TCR alpha transgene (Valpha19 Tg) in response to anti-CD3 antibody injection. NK1.1(+) Valpha19 Tg(+), but not NK1.1(-) Valpha19 Tg(+) cells, promptly produced immunoregulatory IL-4, IFN-gamma and IL-17 upon invariant TCR engagement with immobilized anti-CD3 antibody in culture. The activation of Valpha19 Tg(+) cells then triggered the production of pro-inflammatory cytokines by bystander cells. Interestingly, the ratio of T(h)2 cytokines such as IL-4, IL-5 and IL-10, but not pro-inflammatory IL-17, to IFN-gamma was increased when the intensity of the stimulation to invariant TCR was attenuated. Collectively, these findings suggest that invariant Valpha19 TCR(+) cells have the potential to participate in the regulation of inflammatory autoimmunity by producing T(h)2-biased cytokines in certain circumstances.

  8. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

    PubMed

    Shorter, Shayla K; Schnell, Frederick J; McMaster, Sean R; Pinelli, David F; Andargachew, Rakieb; Evavold, Brian D

    2016-01-01

    T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

  9. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

    PubMed Central

    Shorter, Shayla K.; Schnell, Frederick J.; McMaster, Sean R.; Pinelli, David F.; Andargachew, Rakieb; Evavold, Brian D.

    2016-01-01

    T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant. PMID:26915099

  10. WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.

    PubMed

    Stauss, Hans J; Thomas, Sharyn; Cesco-Gaspere, Michela; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; King, Judy; Wright, Graham; Perro, Mario; Pospori, Constantina; Morris, Emma

    2008-01-01

    Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies.

  11. BAP31 is involved in T cell activation through TCR signal pathways

    PubMed Central

    Niu, Kunwei; Xu, Jialin; Cao, Yuhua; Hou, Yue; Shan, Mu; Wang, Yanqing; Xu, Yang; Sun, Mingyi; Wang, Bing

    2017-01-01

    BAP31 is a ubiquitously expressed endoplasmic reticulum (ER) membrane protein. The functions of BAP31 in the immune system have not been investigated due to the lack of animal models. Therefore we created a BAP31 conditional knockdown mouse by performing a knockdown of BAP31 in the thymus. In doing so, we demonstrate that the maturation of T cells is normal but the number of T cells is less in the thymus of the knockout mouse. In addition, the spleen and lymph nodes of peripheral immune organs contained a lesser proportion of the mature T cells in the thymus specific BAP31 knockout mice. The BAP31 knockout T cells decreased the proliferation activated by TCR signal pathways. Further studies clarified that BAP31 affects the phosphorylation levels of both Zap70/Lck/Lat of the upstream members and Akt/GSK/Jnk/Erk of the downstream members of TCR signal pathways. Furthermore, BAP31 can regulate the expression of some markers such as CD3/TCRα/TCRβ and some cytokines like IL-2/IFN-γ/IL-6/TNF-α which are important for T cell activation. Taken together, these results demonstrate that BAP31 may play an important role in T cell activation by regulating TCR signaling. PMID:28333124

  12. Accumulation of raft lipids in T-cell plasma membrane domains engaged in TCR signalling

    PubMed Central

    Zech, Tobias; Ejsing, Christer S; Gaus, Katharina; de Wet, Ben; Shevchenko, Andrej; Simons, Kai; Harder, Thomas

    2009-01-01

    Activating stimuli for T lymphocytes are transmitted through plasma membrane domains that form at T-cell antigen receptor (TCR) signalling foci. Here, we determined the molecular lipid composition of immunoisolated TCR activation domains. We observed that they accumulate cholesterol, sphingomyelin and saturated phosphatidylcholine species as compared with control plasma membrane fragments. This provides, for the first time, direct evidence that TCR activation domains comprise a distinct molecular lipid composition reminiscent of liquid-ordered raft phases in model membranes. Interestingly, TCR activation domains were also enriched in plasmenyl phosphatidylethanolamine and phosphatidylserine. Modulating the T-cell lipidome with polyunsaturated fatty acids impaired the plasma membrane condensation at TCR signalling foci and resulted in a perturbed molecular lipid composition. These results correlate the accumulation of specific molecular lipid species with the specific plasma membrane condensation at sites of TCR activation and with early TCR activation responses. PMID:19177148

  13. NKT cell-TCR expression activates conventional T cells in vivo, but is largely dispensable for mature NKT cell biology.

    PubMed

    Vahl, J Christoph; Heger, Klaus; Knies, Nathalie; Hein, Marco Y; Boon, Louis; Yagita, Hideo; Polic, Bojan; Schmidt-Supprian, Marc

    2013-01-01

    Natural killer T (NKT) cell development depends on recognition of self-glycolipids via their semi-invariant Vα14i-TCR. However, to what extent TCR-mediated signals determine identity and function of mature NKT cells remains incompletely understood. To address this issue, we developed a mouse strain allowing conditional Vα14i-TCR expression from within the endogenous Tcrα locus. We demonstrate that naïve T cells are activated upon replacement of their endogenous TCR repertoire with Vα14i-restricted TCRs, but they do not differentiate into NKT cells. On the other hand, induced TCR ablation on mature NKT cells did not affect their lineage identity, homeostasis, or innate rapid cytokine secretion abilities. We therefore propose that peripheral NKT cells become unresponsive to and thus are independent of their autoreactive TCR.

  14. Domain-swapped T cell receptors improve the safety of TCR gene therapy.

    PubMed

    Bethune, Michael T; Gee, Marvin H; Bunse, Mario; Lee, Mark S; Gschweng, Eric H; Pagadala, Meghana S; Zhou, Jing; Cheng, Donghui; Heath, James R; Kohn, Donald B; Kuhns, Michael S; Uckert, Wolfgang; Baltimore, David

    2016-11-08

    T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy.

  15. Domain-swapped T cell receptors improve the safety of TCR gene therapy

    PubMed Central

    Bethune, Michael T; Gee, Marvin H; Bunse, Mario; Lee, Mark S; Gschweng, Eric H; Pagadala, Meghana S; Zhou, Jing; Cheng, Donghui; Heath, James R; Kohn, Donald B; Kuhns, Michael S; Uckert, Wolfgang; Baltimore, David

    2016-01-01

    T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy. DOI: http://dx.doi.org/10.7554/eLife.19095.001 PMID:27823582

  16. TCR ITAM multiplicity is required for the generation of follicular helper T-cells.

    PubMed

    Hwang, SuJin; Palin, Amy C; Li, LiQi; Song, Ki-Duk; Lee, Jan; Herz, Jasmin; Tubo, Noah; Chu, Hamlet; Pepper, Marion; Lesourne, Renaud; Zvezdova, Ekaterina; Pinkhasov, Julia; Jenkins, Marc K; McGavern, Dorian; Love, Paul E

    2015-05-11

    The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3ζ subunits. We generated 'knock-in' mice that express non-signalling CD3ζ chains in lieu of wild-type CD3ζ. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR-ligand interactions, but is not essential for 'general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire.

  17. Enforcement of γδ-lineage commitment by the pre-T-cell receptor in precursors with weak γδ-TCR signals.

    PubMed

    Zarin, Payam; Wong, Gladys W; Mohtashami, Mahmood; Wiest, David L; Zúñiga-Pflücker, Juan Carlos

    2014-04-15

    Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-β and γδ-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.

  18. T cell receptor (TCR) gene transfer with lentiviral vectors allows efficient redirection of tumor specificity in naive and memory T cells without prior stimulation of endogenous TCR.

    PubMed

    Circosta, Paola; Granziero, Luisa; Follenzi, Antonia; Vigna, Elisa; Stella, Stefania; Vallario, Antonella; Elia, Angela Rita; Gammaitoni, Loretta; Vitaggio, Katiuscia; Orso, Francesca; Geuna, Massimo; Sangiolo, Dario; Todorovic, Maja; Giachino, Claudia; Cignetti, Alessandro

    2009-12-01

    We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

  19. Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity.

    PubMed

    Levine, Andrew G; Hemmers, Saskia; Baptista, Antonio P; Schizas, Michail; Faire, Mehlika B; Moltedo, Bruno; Konopacki, Catherine; Schmidt-Supprian, Marc; Germain, Ronald N; Treuting, Piper M; Rudensky, Alexander Y

    2017-03-06

    The regulatory T cell (T reg cell) T cell receptor (TCR) repertoire is highly diverse and skewed toward recognition of self-antigens. TCR expression by T reg cells is continuously required for maintenance of immune tolerance and for a major part of their characteristic gene expression signature; however, it remains unknown to what degree diverse TCR-mediated interactions with cognate self-antigens are required for these processes. In this study, by experimentally switching the T reg cell TCR repertoire to a single T reg cell TCR, we demonstrate that T reg cell function and gene expression can be partially uncoupled from TCR diversity. An induced switch of the T reg cell TCR repertoire to a random repertoire also preserved, albeit to a limited degree, the ability to suppress lymphadenopathy and T helper cell type 2 activation. At the same time, these perturbations of the T reg cell TCR repertoire led to marked immune cell activation, tissue inflammation, and an ultimately severe autoimmunity, indicating the importance of diversity and specificity for optimal T reg cell function.

  20. Functional comparison of engineered T cells carrying a native TCR versus TCR-like antibody-based chimeric antigen receptors indicates affinity/avidity thresholds.

    PubMed

    Oren, Ravit; Hod-Marco, Moran; Haus-Cohen, Maya; Thomas, Sharyn; Blat, Dan; Duvshani, Nerri; Denkberg, Galit; Elbaz, Yael; Benchetrit, Fabrice; Eshhar, Zelig; Stauss, Hans; Reiter, Yoram

    2014-12-01

    Adoptive transfer of Ag-specific T lymphocytes is an attractive form of immunotherapy for cancers. However, acquiring sufficient numbers of host-derived tumor-specific T lymphocytes by selection and expansion is challenging, as these cells may be rare or anergic. Using engineered T cells can overcome this difficulty. Such engineered cells can be generated using a chimeric Ag receptor based on common formats composed from Ag-recognition elements such as αβ-TCR genes with the desired specificity, or Ab variable domain fragments fused with T cell-signaling moieties. Combining these recognition elements are Abs that recognize peptide-MHC. Such TCR-like Abs mimic the fine specificity of TCRs and exhibit both the binding properties and kinetics of high-affinity Abs. In this study, we compared the functional properties of engineered T cells expressing a native low affinity αβ-TCR chains or high affinity TCR-like Ab-based CAR targeting the same specificity. We isolated high-affinity TCR-like Abs recognizing HLA-A2-WT1Db126 complexes and constructed CAR that was transduced into T cells. Comparative analysis revealed major differences in function and specificity of such CAR-T cells or native TCR toward the same antigenic complex. Whereas the native low-affinity αβ-TCR maintained potent cytotoxic activity and specificity, the high-affinity TCR-like Ab CAR exhibited reduced activity and loss of specificity. These results suggest an upper affinity threshold for TCR-based recognition to mediate effective functional outcomes of engineered T cells. The rational design of TCRs and TCR-based constructs may need to be optimized up to a given affinity threshold to achieve optimal T cell function.

  1. Higher Sensitivity of Foxp3+ Treg Compared to Foxp3- Conventional T Cells to TCR-Independent Signals for CD69 Induction.

    PubMed

    Bremser, Anna; Brack, Maria; Izcue, Ana

    2015-01-01

    T lymphocytes elicit specific responses after recognizing cognate antigen. However, antigen-experienced T cells can also respond to non-cognate stimuli, such as cytokines. CD4+ Foxp3+ regulatory T cells (Treg) exhibit an antigen-experienced-like phenotype. Treg can regulate T cell responses in an antigen-specific or bystander way, and it is still unclear as to which extent they rely on T cell receptor (TCR) signals. The study of the antigen response of Treg has been hampered by the lack of downstream readouts for TCR stimuli. Here we assess the effects of TCR signals on the expression of a classical marker of early T cell activation, CD69. Although it can be induced following cytokine exposure, CD69 is commonly used as a readout for antigen response on T cells. We established that upon in vitro TCR stimulation CD69 induction on Foxp3+ Treg cells was more dependent on signaling via soluble factors than on TCR activation. By contrast, expression of the activation marker Nur77 was only induced after TCR stimulation. Our data suggest that Treg are more sensitive to TCR-independent signals than Foxp3- cells, which could contribute to their bystander activity.

  2. Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy.

    PubMed

    Joshi, Sunil K; Lang, Gillian A; Larabee, Jason L; Devera, T Scott; Aye, Lindsay M; Shah, Hemangi B; Ballard, Jimmy D; Lang, Mark L

    2009-09-01

    Exogenous CD1d-binding glycolipid (alpha-Galactosylceramide, alpha-GC) stimulates TCR signaling and activation of type-1 natural killer-like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

  3. Optimization of T-cell Reactivity by Exploiting TCR Chain Centricity for the Purpose of Safe and Effective Antitumor TCR Gene Therapy.

    PubMed

    Ochi, Toshiki; Nakatsugawa, Munehide; Chamoto, Kenji; Tanaka, Shinya; Yamashita, Yuki; Guo, Tingxi; Fujiwara, Hiroshi; Yasukawa, Masaki; Butler, Marcus O; Hirano, Naoto

    2015-09-01

    Adoptive transfer of T cells redirected by a high-affinity antitumor T-cell receptor (TCR) is a promising treatment modality for cancer patients. Safety and efficacy depend on the selection of a TCR that induces minimal toxicity and elicits sufficient antitumor reactivity. Many, if not all, TCRs possess cross-reactivity to unrelated MHC molecules in addition to reactivity to target self-MHC/peptide complexes. Some TCRs display chain centricity, in which recognition of MHC/peptide complexes is dominated by one of the TCR hemi-chains. In this study, we comprehensively studied how TCR chain centricity affects reactivity to target self-MHC/peptide complexes and alloreactivity using the TCR, clone TAK1, which is specific for human leukocyte antigen-A*24:02/Wilms tumor 1(235-243) (A24/WT1(235)) and cross-reactive with B*57:01 (B57). The TAK1β, but not the TAK1α, hemi-chain possessed chain centricity. When paired with multiple clonotypic TCRα counter-chains encoding TRAV12-2, 20, 36, or 38-2, the de novo TAK1β-containing TCRs showed enhanced, weakened, or absent reactivity to A24/WT1(235) and/or to B57. T cells reconstituted with these TCRα genes along with TAK1β possessed a very broad range (>3 log orders) of functional and structural avidities. These results suggest that TCR chain centricity can be exploited to enhance desired antitumor TCR reactivity and eliminate unwanted TCR cross-reactivity. TCR reactivity to target MHC/peptide complexes and cross-reactivity to unrelated MHC molecules are not inextricably linked and are separable at the TCR sequence level. However, it is still mandatory to carefully monitor for possible harmful toxicities caused by adoptive transfer of T cells redirected by thymically unselected TCRs.

  4. Pretreatment of activated human CD8 T cells with IL-12 leads to enhanced TCR-induced signaling and cytokine production.

    PubMed

    Vacaflores, Aldo; Freedman, Samantha N; Chapman, Nicole M; Houtman, Jon C D

    2017-01-01

    During the immune response to pathogens and autoantigens, CD8T cells are exposed to numerous inflammatory agents including the cytokine IL-12. Previous studies have focused on how IL-12 regulates T cell functions when present during or after the activation of the T cell receptor (TCR). However, recent studies suggest that prior exposure to IL-12 also alters the TCR responsiveness of murine T cells. Whether similar phenomena occur in human activated CD8T cells and the mechanisms mediating these effects remain unexplored. In this study, we observed that pretreatment of human activated CD8T cells with IL-12 results in increased cytokine mRNA and protein production following subsequent TCR challenge. The potentiation of TCR-mediated cytokine release was transient and required low doses of IL-12 for at least 24h. Mechanistically, prior exposure to IL-12 increased the TCR induced activation of select MAPKs and AKT without altering the activation of more proximal TCR signaling molecules, suggesting that the IL-12 mediated changes in TCR signaling are responsible for the increased production of cytokines. Our data suggest that prior treatment with IL-12 potentiates human CD8T cell responses at sites of infection and inflammation, expanding our understanding of the function of this clinically important cytokine.

  5. Antigen Specificity of Type I NKT Cells Is Governed by TCR β-Chain Diversity.

    PubMed

    Cameron, Garth; Pellicci, Daniel G; Uldrich, Adam P; Besra, Gurdyal S; Illarionov, Petr; Williams, Spencer J; La Gruta, Nicole L; Rossjohn, Jamie; Godfrey, Dale I

    2015-11-15

    NKT cells recognize lipid-based Ags presented by CD1d. Type I NKT cells are often referred to as invariant owing to their mostly invariant TCR α-chain usage (Vα14-Jα18 in mice, Vα24-Jα18 in humans). However, these cells have diverse TCR β-chains, including Vβ8, Vβ7, and Vβ2 in mice and Vβ11 in humans, joined to a range of TCR Dβ and Jβ genes. In this study, we demonstrate that TCR β-chain composition can dramatically influence lipid Ag recognition in an Ag-dependent manner. Namely, the glycolipids α-glucosylceramide and isoglobotrihexosylceramide were preferentially recognized by Vβ7(+) NKT cells from mice, whereas the α-galactosylceramide analog OCH, with a truncated sphingosine chain, was preferentially recognized by Vβ8(+) NKT cells from mice. We show that the influence of the TCR β-chain is due to a combination of Vβ-, Jβ-, and CDR3β-encoded residues and that these TCRs can recapitulate the selective Ag reactivity in TCR-transduced cell lines. Similar observations were made with human NKT cells where different CDR3β-encoded residues determined Ag preference. These findings indicate that NKT TCR β-chain diversity results in differential and nonhierarchical Ag recognition by these cells, which implies that some Ags can preferentially activate type I NKT cell subsets.

  6. Discrete TCR Binding Kinetics Control Invariant NKT Cell Selection and Central Priming.

    PubMed

    Cruz Tleugabulova, Mayra; Escalante, Nichole K; Deng, Shenglou; Fieve, Stephanie; Ereño-Orbea, June; Savage, Paul B; Julien, Jean-Philippe; Mallevaey, Thierry

    2016-11-15

    Invariant NKT (iNKT) cells develop and differentiate in the thymus, segregating into iNKT1/2/17 subsets akin to Th1/2/17 classical CD4(+) T cells; however, iNKT TCRs recognize Ags in a fundamentally different way. How the biophysical parameters of iNKT TCRs influence signal strength in vivo and how such signals affect the development and differentiation of these cells are unknown. In this study, we manipulated TCRs in vivo to generate clonotypic iNKT cells using TCR retrogenic chimeras. We report that the biophysical properties of CD1d-lipid-TCR interactions differentially impacted the development and effector differentiation of iNKT cells. Whereas selection efficiency strongly correlated with TCR avidity, TCR signaling, cell-cell conjugate formation, and iNKT effector differentiation correlated with the half-life of CD1d-lipid-TCR interactions. TCR binding properties, however, did not modulate Ag-induced iNKT cytokine production. Our work establishes that discrete TCR interaction kinetics influence iNKT cell development and central priming.

  7. Contribution of TCR signaling strength to CD8+ T cell peripheral tolerance mechanisms.

    PubMed

    Smith, Trevor R F; Verdeil, Gregory; Marquardt, Kristi; Sherman, Linda A

    2014-10-01

    Peripheral tolerance mechanisms are in place to prevent T cells from mediating aberrant immune responses directed against self and environmental Ags. Mechanisms involved in the induction of peripheral tolerance include T cell-intrinsic pathways, such as anergy or deletion, or exogenous tolerance mediated by regulatory T cells. We have previously shown that the density of peptide-MHC class I recognized by the TCR determines whether CD8(+) T cells undergo anergy or deletion. Specifically, using a TCR-transgenic CD8(+) T cell model, we demonstrated that persistent peripheral exposure to low- or high-dose peptides in the absence of inflammatory signals resulted in clonal deletion or anergy of the T cell, respectively. In this study, by altering the affinity of the peptide-MHC tolerogen for TCR, we have confirmed that this mechanism is dependent on the level of TCR signaling that the CD8(+) T cell receives. Using altered peptide ligands (APLs) displaying high TCR affinities, we show that increasing the TCR signaling favors anergy induction. Conversely, using APLs displaying a decreased TCR affinity tilted our system in the direction of deletional tolerance. We demonstrate how differential peripheral CD8(+) T cell tolerance mechanisms are controlled by both the potency and density of MHC class I-peptide tolerogen.

  8. COMPARATIVE ANALYSES OF DIFFERENTIALLY-INDUCED TCR-MEDIATED PHOSPHORYLATION PATHWAYS IN T LYMPHOMA CELLS

    PubMed Central

    Ortiz, Serina; Lee, Wenhui; Smith, David; Forman, Stephen J.; Lee, Terry D.; Liu, Chih-Pin

    2011-01-01

    Activation of T lymphoma cells expressing Syk, but not ZAP-70 tyrosine kinase, has been shown to negatively regulate cell activation and activation induced cell death (AICD), perhaps due to differential induction of tyrosine phosphorylation modified proteins. To better understand the role of these proteins and their associated molecules/pathways, we studied a previously described model of T lymphoma cells expressing either a kinase-activated chimeric Syk or ZAP-70 genetically linked to TCR ζ chain (Z/Syk or Z/ZAP cells, respectively). To help identify molecules and pathways linked to cell activation or AICD, a comparative semi-quantitative proteomics-based approach was utilized to analyze tyrosine phosphorylated protein immunoprecipitates from 2 min short-term activated Z/Syk or Z/ZAP cells. Using the resulting bioinformatics datasets, we identified several differentially immunoprecipitated proteins that could be validated biochemically. More tyrosine-phosphorylated and phosphotyrosine-associated proteins were found in Z/Syk than in Z/ZAP cells. Proteins involved in different unique functional pathways were induced in these cells and showed altered intermolecular interactions in varied pathways. Remarkably, 41% of differentially identified proteins in Z/Syk cells belonged to cell cycle or vesicle/trafficking pathways. In contrast, 21% of such proteins in Z/ZAP cells belonged to metabolism pathways. Therefore, molecular pathways involved in post-translational modifications linked to distinct cellular/physiological functions are differentially activated, which may contribute to varied activation and AICD responses of these cells. In summary, we identified proteins belonging to novel differentially activated pathways involved in TCR-mediated signaling, which may be targets for regulating activation and AICD of T lymphoma cells and for potential cancer therapy. PMID:21127342

  9. IL-2 Modulates the TCR Signaling Threshold for CD8 but Not CD4 T Cell Proliferation on a Single-Cell Level.

    PubMed

    Au-Yeung, Byron B; Smith, Geoffrey Alexander; Mueller, James L; Heyn, Cheryl S; Jaszczak, Rebecca Garrett; Weiss, Arthur; Zikherman, Julie

    2017-03-15

    Lymphocytes integrate Ag and cytokine receptor signals to make cell fate decisions. Using a specific reporter of TCR signaling that is insensitive to cytokine signaling, Nur77-eGFP, we identify a sharp, minimal threshold of cumulative TCR signaling required for proliferation in CD4 and CD8 T cells that is independent of both Ag concentration and affinity. Unexpectedly, IL-2 reduces this threshold in CD8 but not CD4 T cells, suggesting that integration of multiple mitogenic inputs may alter the minimal requirement for TCR signaling in CD8 T cells. Neither naive CD4 nor naive CD8 T cells are responsive to low doses of IL-2. We show that activated CD8 T cells become responsive to low doses of IL-2 more quickly than CD4 T cells, and propose that this relative delay in turn accounts for the differential effects of IL-2 on the minimal TCR signaling threshold for proliferation in these populations. In contrast to Nur77-eGFP, c-Myc protein expression integrates mitogenic signals downstream of both IL-2 and the TCR, yet marks an invariant minimal threshold of cumulative mitogenic stimulation required for cell division. Our work provides a conceptual framework for understanding the regulation of clonal expansion of CD8 T cells by subthreshold TCR signaling in the context of mitogenic IL-2 signals, thereby rendering CD8 T cells exquisitely dependent upon environmental cues. Conversely, CD4 T cell proliferation requires an invariant minimal intensity of TCR signaling that is not modulated by IL-2, thereby restricting responses to low-affinity or low-abundance self-antigens even in the context of an inflammatory milieu.

  10. Therapeutic vaccination with a trivalent T-cell receptor (TCR) peptide vaccine restores deficient FoxP3 expression and TCR recognition in subjects with multiple sclerosis.

    PubMed

    Vandenbark, Arthur A; Culbertson, Nicole E; Bartholomew, Richard M; Huan, Jianya; Agotsch, Marci; LaTocha, Dorian; Yadav, Vijayshree; Mass, Michele; Whitham, Ruth; Lovera, Jesus; Milano, June; Theofan, Georgia; Chou, Yuan K; Offner, Halina; Bourdette, Dennis N

    2008-01-01

    Therapeutic vaccination using T-cell receptor (TCR) peptides from V genes commonly expressed by potentially pathogenic T cells remains an approach of interest for treatment of multiple sclerosis (MS) and other autoimmune diseases. We developed a trivalent TCR vaccine containing complementarity determining region (CDR) 2 peptides from BV5S2, BV6S5 and BV13S1 emulsified in incomplete Freund's adjuvant that reliably induced high frequencies of TCR-specific T cells. To evaluate induction of regulatory T-cell subtypes, immunological and clinical parameters were followed in 23 treatment-naïve subjects with relapsing-remitting or progressive MS who received 12 monthly injections of the trivalent peptide vaccine over 1 year in an open-label study design. Prior to vaccination, subjects had reduced expression of forkhead box (Fox) P3 message and protein, and reduced recognition of the expressed TCR repertoire by TCR-reactive cells compared with healthy control donors. After three or four injections, most vaccinated MS subjects developed high frequencies of circulating interleukin (IL)-10-secreting T cells specific for the injected TCR peptides and significantly enhanced expression of FoxP3 by regulatory T cells present in both 'native' CD4+ CD25+ and 'inducible' CD4+ CD25- peripheral blood mononuclear cells (PBMC). At the end of the trial, PBMC from vaccinated MS subjects retained or further increased FoxP3 expression levels, exhibited significantly enhanced recognition of the TCR V gene repertoire apparently generated by perturbation of the TCR network, and significantly suppressed neuroantigen but not recall antigen responses. These findings demonstrate that therapeutic vaccination using only three commonly expressed BV gene determinants can induce an expanded immunoregulatory network in vivo that may optimally control complex autoreactive responses that characterize the inflammatory phase of MS.

  11. GADS is Required for TCR-Mediated Calcium Influx and Cytokine Release, but not Cellular Adhesion, in Human T Cells

    PubMed Central

    Bilal, Mahmood Y.; Zhang, Elizabeth Y.; Dinkel, Brittney; Hardy, Daimon; Yankee, Thomas M.; Houtman, Jon C.D.

    2015-01-01

    GRB2 related adaptor protein downstream of Shc (GADS) is a member of the GRB2 family of adaptors and is critical for TCR-induced signaling. The current model is that GADS recruits SLP-76 to the LAT complex, which facilitates the phosphorylation of SLP-76, the activation of PLC-γ1, T cell adhesion and cytokine production. However, this model is largely based on studies of disruption of the GADS/SLP-76 interaction and murine T cell differentiation in GADS deficient mice. The role of GADS in mediating TCR-induced signals in human CD4+ T cells has not been thoroughly investigated. In this study, we have suppressed the expression of GADS in human CD4+ HuT78 T cells. GADS deficient HuT78 T cells displayed similar levels of TCR-induced SLP-76 and PLC-γ1 phosphorylation but exhibited substantial decrease in TCR-induced IL-2 and IFN-γ release. The defect in cytokine production occurred because of impaired calcium mobilization due to reduced recruitment of SLP-76 and PLC-γ1 to the LAT complex. Surprisingly, both GADS deficient HuT78 and GADS deficient primary murine CD8+ T cells had similar TCR-induced adhesion when compared to control T cells. Overall, our results show that GADS is required for calcium influx and cytokine production, but not cellular adhesion, in human CD4+ T cells, suggesting that the current model for T cell regulation by GADS is incomplete. PMID:25636200

  12. T-Cell Receptor (TCR) Clonotype-Specific Differences in Inhibitory Activity of HIV-1 Cytotoxic T-Cell Clones Is Not Mediated by TCR Alone.

    PubMed

    Flerin, Nina C; Chen, Huabiao; Glover, Tynisha D; Lamothe, Pedro A; Zheng, Jian Hua; Fang, Justin W; Ndhlovu, Zaza M; Newell, Evan W; Davis, Mark M; Walker, Bruce D; Goldstein, Harris

    2017-03-15

    Functional analysis of T-cell responses in HIV-infected individuals has indicated that virus-specific CD8(+) T cells with superior antiviral efficacy are well represented in HIV-1 controllers but are rare or absent in HIV-1 progressors. To define the role of individual T-cell receptor (TCR) clonotypes in differential antiviral CD8(+) T-cell function, we performed detailed functional and mass cytometric cluster analysis of multiple CD8(+) T-cell clones recognizing the identical HLA-B*2705-restricted HIV-1 epitope KK10 (KRWIILGLNK). Effective and ineffective CD8(+) T-cell clones segregated based on responses to HIV-1-infected and peptide-loaded target cells. Following cognate peptide stimulation, effective HIV-specific clones displayed significantly more rapid TCR signal propagation, more efficient initial lytic granule release, and more sustained nonlytic cytokine and chemokine secretion than ineffective clones. To evaluate the TCR clonotype contribution to CD8(+) T-cell function, we cloned the TCR α and β chain genes from one effective and two ineffective CD8(+) T-cell clones from an elite controller into TCR-expressing lentivectors. We show that Jurkat/MA cells and primary CD8(+) T cells transduced with lentivirus expressing TCR from one of the ineffective clones exhibited a level of activation by cognate peptide and inhibition of in vitro HIV-1 infection, respectively, that were comparable to those of the effective clonotype. Taken together, these data suggest that the potent antiviral capacity of some HIV-specific CD8(+) T cells is a consequence of factors in addition to TCR sequence that modulate functionality and contribute to the increased antiviral capacity of HIV-specific CD8(+) T cells in elite controllers to inhibit HIV infection.IMPORTANCE The greater ex vivo antiviral inhibitory activity of CD8(+) T cells from elite controllers than from HIV-1 progressors supports the crucial role of effective HIV-specific CD8(+) T cells in controlling HIV-1

  13. Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells

    PubMed Central

    Giannoni, Francesca; Hardee, Cinnamon L; Wherley, Jennifer; Gschweng, Eric; Senadheera, Shantha; Kaufman, Michael L; Chan, Rebecca; Bahner, Ingrid; Gersuk, Vivian; Wang, Xiaoyan; Gjertson, David; Baltimore, David; Witte, Owen N; Economou, James S; Ribas, Antoni; Kohn, Donald B

    2013-01-01

    Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34+ HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8+ and CD4+ T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4–5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8+ and CD4+ T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34+ HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma. PMID:23380815

  14. Generation of V α13/β21+T cell specific target CML cells by TCR gene transfer.

    PubMed

    Zha, Xianfeng; Xu, Ling; Chen, Shaohua; Yang, Lijian; Zhang, Yikai; Lu, Yuhong; Yu, Zhi; Li, Bo; Wu, Xiuli; Zheng, Wenjie; Li, Yangqiu

    2016-12-20

    Adoptive immunotherapy with antigen-specific T cells can be effective for treating melanoma and chronic myeloid leukemia (CML). However, to obtain sufficient antigen-specific T cells for treatment, the T cells have to be cultured for several weeks in vitro, but in vitro T cell expansion is difficult to control. Alternatively, the transfer of T cell receptors (TCRs) with defined antigen specificity into recipient T cells may be a simple solution for generating antigen-specific T cells. The objective of this study was to identify CML-associated, antigen-specific TCR genes and generate CML-associated, antigen-specific T cells with T cell receptor (TCR) gene transfer. Our previous study has screened an oligoclonal Vβ21 with a different oligoclonal Vα partner in peripheral blood mononuclear cells (PBMCs) derived from patients with CML. In this study, oligoclonally expanded TCR α genes, which pair with TCR Vβ21, were cloned into the pIRES eukaryotic expression vector (TCR Vα-IRES-Vβ21). Next, two recombinant plasmids, TCR Vα13-IRES-Vβ21 and TCR Vα18-IRES-Vβ21, were successfully transferred into T cells, and the TCR gene-modified T cells acquired CML-specific cytotoxicity with the best cytotoxic effects for HLA-A11+ K562 cells observed for the TCR Vα13/Vβ21 gene redirected T cells. In summary, our data confirmed TCRVα13/Vβ21 as a CML-associated, antigen-specific TCR. This study provided new evidence that genetically engineered antigen-specific TCR may become a druggable approach for gene therapy of CML.

  15. Differential Requirements of TCR Signaling in Homeostatic Maintenance and Function of Dendritic Epidermal T Cells.

    PubMed

    Zhang, Baojun; Wu, Jianxuan; Jiao, Yiqun; Bock, Cheryl; Dai, Meifang; Chen, Benny; Chao, Nelson; Zhang, Weiguo; Zhuang, Yuan

    2015-11-01

    Dendritic epidermal T cells (DETCs) are generated exclusively in the fetal thymus and maintained in the skin epithelium throughout postnatal life of the mouse. DETCs have restricted antigenic specificity as a result of their exclusive usage of a canonical TCR. Although the importance of the TCR in DETC development has been well established, the exact role of TCR signaling in DETC homeostasis and function remains incompletely defined. In this study, we investigated TCR signaling in fully matured DETCs by lineage-restricted deletion of the Lat gene, an essential signaling molecule downstream of the TCR. We found that Lat deletion impaired TCR-dependent cytokine gene activation and the ability of DETCs to undergo proliferative expansion. However, linker for activation of T cells-deficient DETCs were able to maintain long-term population homeostasis, although with a reduced proliferation rate. Mice with Lat deletion in DETCs exhibited delayed wound healing accompanied by impaired clonal expansion within the wound area. Our study revealed differential requirements for TCR signaling in homeostatic maintenance of DETCs and in their effector function during wound healing.

  16. T cell receptor (TCR) usage determines disease susceptibility in experimental autoimmune encephalomyelitis: studies with TCR V beta 8.2 transgenic mice

    PubMed Central

    1994-01-01

    Experimental allergic encephalomyelitis (EAE) is an autoimmune disease that can be induced in laboratory animals by immunization with the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP). We analyzed the role of the T cell receptor (TCR) repertoire in susceptibility to EAE induced by these two autoantigens. Autoreactive T cells induced after immunization with MBP use a limited set of TCR. In contrast, we demonstrate that T cell clones that recognize the encephalitogenic PLP epitope (PLP 139-151) use diverse TCR genes. When the TCR repertoire is limited by introduction of a novel rearranged TCR V beta 8.2 chain in transgenic SJL mice, EAE could be induced in the transgenic mice by immunization with the encephalitogenic epitopes of PLP, but not with the encephalitogenic epitope of MBP. Thus, skewing the TCR repertoire affects the susceptibility to EAE by immunization with MBP but not with PLP. These data demonstrate the biological consequences of the usage of a more diverse T cell repertoire in the development of an autoimmune disease. PMID:8163944

  17. Direct single molecule measurement of TCR triggering by agonist pMHC in living primary T cells.

    PubMed

    O'Donoghue, Geoff P; Pielak, Rafal M; Smoligovets, Alexander A; Lin, Jenny J; Groves, Jay T

    2013-07-03

    T cells discriminate between self and foreign antigenic peptides, displayed on antigen presenting cell surfaces, via the TCR. While the molecular interactions between TCR and its ligands are well characterized in vitro, quantitative measurements of these interactions in living cells are required to accurately resolve the physical mechanisms of TCR signaling. We report direct single molecule measurements of TCR triggering by agonist pMHC in hybrid junctions between live primary T cells and supported lipid membranes. Every pMHC:TCR complex over the entire cell is tracked while simultaneously monitoring the local membrane recruitment of ZAP70, as a readout of TCR triggering. Mean dwell times for pMHC:TCR molecular binding of 5 and 54 s were measured for two different pMHC:TCR systems. Single molecule measurements of the pMHC:TCR:ZAP70 complex indicate that TCR triggering is stoichiometric with agonist pMHC in a 1:1 ratio. Thus any signal amplification must occur downstream of TCR triggering. DOI:http://dx.doi.org/10.7554/eLife.00778.001.

  18. High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire.

    PubMed

    Hou, Xianliang; Lu, Chong; Chen, Sisi; Xie, Qian; Cui, Guangying; Chen, Jianing; Chen, Zhi; Wu, Zhongwen; Ding, Yulong; Ye, Ping; Dai, Yong; Diao, Hongyan

    2016-03-01

    The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals.Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR.Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides.Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination.

  19. High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire

    PubMed Central

    Hou, Xianliang; Lu, Chong; Chen, Sisi; Xie, Qian; Cui, Guangying; Chen, Jianing; Chen, Zhi; Wu, Zhongwen; Ding, Yulong; Ye, Ping; Dai, Yong; Diao, Hongyan

    2016-01-01

    Abstract The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals. Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR. Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides. Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination. PMID:26962778

  20. CD45-mediated control of TCR tuning in naïve and memory CD8(+) T cells.

    PubMed

    Cho, Jae-Ho; Kim, Hee-Ok; Ju, Young-Jun; Kye, Yoon-Chul; Lee, Gil-Woo; Lee, Sung-Woo; Yun, Cheol-Heui; Bottini, Nunzio; Webster, Kylie; Goodnow, Christopher C; Surh, Charles D; King, Cecile; Sprent, Jonathan

    2016-11-14

    Continuous contact with self-major histocompatibility complex (MHC) ligands is essential for survival of naïve T cells but not memory cells. This surprising finding implies that T cell subsets may vary in their relative T-cell receptor (TCR) sensitivity. Here we show that in CD8(+)T cells TCR sensitivity correlates inversely with levels of CD5, a marker for strong self-MHC reactivity. We also show that TCR sensitivity is lower in memory CD8(+) T cells than naïve cells. In both situations, TCR hypo-responsiveness applies only to short-term TCR signalling events and not to proliferation, and correlates directly with increased expression of a phosphatase, CD45 and reciprocal decreased expression of activated LCK. Inhibition by high CD45 on CD8(+) T cells may protect against overt TCR auto-MHC reactivity, while enhanced sensitivity to cytokines ensures strong responses to foreign antigens.

  1. CD45-mediated control of TCR tuning in naïve and memory CD8+ T cells

    PubMed Central

    Cho, Jae-Ho; Kim, Hee-Ok; Ju, Young-Jun; Kye, Yoon-Chul; Lee, Gil-Woo; Lee, Sung-Woo; Yun, Cheol-Heui; Bottini, Nunzio; Webster, Kylie; Goodnow, Christopher C.; Surh, Charles D.; King, Cecile; Sprent, Jonathan

    2016-01-01

    Continuous contact with self-major histocompatibility complex (MHC) ligands is essential for survival of naïve T cells but not memory cells. This surprising finding implies that T cell subsets may vary in their relative T-cell receptor (TCR) sensitivity. Here we show that in CD8+T cells TCR sensitivity correlates inversely with levels of CD5, a marker for strong self-MHC reactivity. We also show that TCR sensitivity is lower in memory CD8+ T cells than naïve cells. In both situations, TCR hypo-responsiveness applies only to short-term TCR signalling events and not to proliferation, and correlates directly with increased expression of a phosphatase, CD45 and reciprocal decreased expression of activated LCK. Inhibition by high CD45 on CD8+ T cells may protect against overt TCR auto-MHC reactivity, while enhanced sensitivity to cytokines ensures strong responses to foreign antigens. PMID:27841348

  2. Prior TLR5 induction in human T cells results in a transient potentiation of subsequent TCR-induced cytokine production.

    PubMed

    Tremblay, Mikaela M; Bilal, Mahmood Y; Houtman, Jon C D

    2014-02-01

    Activation of TLRs by components required for pathogen viability results in increased inflammation and an enhanced immune response to infection. Unlike their effects on other immune cells, TLR activation in the absence of T cell antigen receptor (TCR) induction has little effect on T cell activity. Instead, the simultaneous induction of TLR and TCR results in increased cytokine release compared to TCR treatment alone. Thus, the current model states that TLRs alter T cell function only if activated at the same time as the TCR. In this study, we tested the novel hypothesis that prior TLR induction can also alter TCR-mediated functions. We found that human T cells responded to ligands for TLR2 and TLR5. However, only prior TLR5 induction potentiated subsequent TCR-mediated cytokine production in human T cells. This response required at least 24h of TLR5 induction and lasted for approximately 24-36h after removal of a TLR5 ligand. Interestingly, prior TLR5 induction enhanced TCR-mediated activation of Akt without increasing Lck, LAT or ERK kinase phosphorylation. Together, our studies show that TLR5 induction leads to a transient increase in the sensitivity of T cells to TCR stimulation by selectively enhancing TCR-mediated Akt function, highlighting that timeframe when TLR5 can potentiate TCR-induced downstream functions are significantly longer that previously appreciated.

  3. Nanoclusters of the resting T cell antigen receptor (TCR) localize to non-raft domains.

    PubMed

    Beck-García, Katharina; Beck-García, Esmeralda; Bohler, Sheila; Zorzin, Carina; Sezgin, Erdinc; Levental, Ilya; Alarcón, Balbino; Schamel, Wolfgang W A

    2015-04-01

    In the last decade an increasing number of plasma membrane (PM) proteins have been shown to be non-randomly distributed but instead forming submicron-sized oligomers called nanoclusters. Nanoclusters exist independently of the ligand-bound state of the receptors and their existence implies a high degree of lateral organisation of the PM and its proteins. The mechanisms that drive receptor nanoclustering are largely unknown. One well-defined example of a transmembrane receptor that forms nanoclusters is the T cell antigen receptor (TCR), a multisubunit protein complex whose nanoclustering influences its activity. Membrane lipids, namely cholesterol and sphingomyelin, have been shown to contribute to TCR nanoclustering. However, the identity of the membrane microdomain in which the TCR resides remains controversial. Using a GFP-labeled TCR we show here that the resting TCR localized in the disordered domain of giant PM vesicles (GPMVs) and PM spheres (PMSs) and that single and nanoclustered TCRs are found in the high-density fractions in sucrose gradients. Both findings are indicative of non-raft localization. We discuss possible mechanisms of TCR nanoclustering in T cells. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

  4. Phosphoantigen Presentation to TCR γδ Cells, a Conundrum Getting Less Gray Zones.

    PubMed

    De Libero, Gennaro; Lau, Sze-Yi; Mori, Lucia

    2014-01-01

    The mechanistic requirements of antigen recognition by T cells expressing a γδ TCR has revealed important differences with those of αβ TCR cells and, despite impressive new data generated in the very recent years, they remain poorly understood. Based on the structure of the TCR chains and the tissue distribution, γδ cells are represented in a variety of populations. The major subset of human peripheral blood γδ cells express Vγ9Vδ2 TCR heterodimers and are all stimulated by phosphorylated metabolites (commonly called phosphoantigens). Phosphoantigens are molecules with a very small mass and only stimulate Vγ9Vδ2 cells in the presence of antigen-presenting cells, suggesting a strict requirement for dedicated antigen-presenting molecules. Recent studies have identified butyrophilin (BTN) 3A1 as the molecule necessary to stimulate Vγ9Vδ2 cells. BTN3A1 extracellular, transmembrane, and cytoplasmic domains have different functions, including cognate interaction with the Vγ9Vδ2 TCR, binding of the phosphoantigens, and interaction with cytoplasmic proteins. This review mainly discusses the known molecular mechanisms of BTN3A1-mediated antigen presentation to γδ cells and proposes a model of phosphoantigen presentation, which integrates past and recent studies.

  5. TCR bias and affinity define two compartments of the CD1b-glycolipid-specific T Cell repertoire.

    PubMed

    Van Rhijn, Ildiko; Gherardin, Nicholas A; Kasmar, Anne; de Jager, Wilco; Pellicci, Daniel G; Kostenko, Lyudmila; Tan, Li Lynn; Bhati, Mugdha; Gras, Stephanie; Godfrey, Dale I; Rossjohn, Jamie; Moody, D Branch

    2014-05-01

    Current views emphasize TCR diversity as a key feature that differentiates the group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 systems. Whereas TCR sequence motifs define CD1d-reactive NKT cells, the available data do not allow a TCR-based organization of the group 1 CD1 repertoire. The observed TCR diversity might result from donor-to-donor differences in TCR repertoire, as seen for MHC-restricted T cells. Alternatively, diversity might result from differing CD1 isoforms, Ags, and methods used to identify TCRs. Using CD1b tetramers to isolate clones recognizing the same glycolipid, we identified a previously unknown pattern of V gene usage (TRAV17, TRBV4-1) among unrelated human subjects. These TCRs are distinct from those present on NKT cells and germline-encoded mycolyl lipid-reactive T cells. Instead, they resemble the TCR of LDN5, one of the first known CD1b-reactive clones that was previously thought to illustrate the diversity of the TCR repertoire. Interdonor TCR conservation was observed in vitro and ex vivo, identifying LDN5-like T cells as a distinct T cell type. These data support TCR-based organization of the CD1b repertoire, which consists of at least two compartments that differ in TCR sequence motifs, affinity, and coreceptor expression.

  6. Regulation of TCR Signaling to NF-kB

    DTIC Science & Technology

    2013-03-20

    occurs at lysines 31 and 63 (82). Furthermore, blockade of Bcl10 polyubiquitination by mutation of these residues to arginines dramatically inhibits...16 hr; MG132 in DMSO (20μM), 1 hr. Antibodies are detailed in Supplemental Methods.    37   CD8+ Tcm and CD4+ Th2 cell...and burn , have limited correlations with the human immune responses (65). Thus before we try to seek out human applications of this study

  7. Adoptive transfer of tracer alloreactive CD4(+) TCR-transgenic T cells alters the endogenous immune response to an allograft.

    PubMed

    Miller, Michelle L; Chen, Jianjun; Daniels, Melvin D; McKeague, Matthew G; Wang, Ying; Yin, Dengping; Vu, Vinh; Chong, Anita S; Alegre, Maria-Luisa

    2016-04-11

    T cell receptor transgenic (TCR-Tg) T cells are often used as tracer populations of antigen-specific responses to extrapolate findings to endogenous T cells. The extent to which TCR-Tg T cells behave purely as tracer cells or modify the endogenous immune response is not clear. To test the impact of TCR-Tg T cell transfer on endogenous alloimmunity, recipient mice were seeded with CD4(+) or CD8(+) TCR-Tg or polyclonal T cells at the time of cardiac allograft transplantation. Only CD4(+) TCR-Tg T cells accelerated rejection, and unexpectedly led to a dose-dependent decrease in both transferred and endogenous T cells infiltrating the graft. In contrast, recipients of CD4(+) TCR-Tg cell exhibited enhanced endogenous donor-specific CD8(+) T-cell activation in the spleen and accelerated alloantibody production. Introduction of CD4(+) TCR-Tg T cells also perturbed the intra-graft accumulation of innate cell populations. Thus, transferred CD4(+) TCR-Tg T cells alter many aspects of endogenous alloimmunity, suggesting that caution should be used when interpreting experiments utilizing these adoptively-transferred cells, as the overall nature of allograft rejection may be altered. These results may also have implications for adoptive CD4(+) T cell immunotherapy in tumor and infectious clinical settings as cell infusion may have additional effects on natural immune responses. This article is protected by copyright. All rights reserved.

  8. TCR clonotypes: molecular determinants of T-cell efficacy against HIV.

    PubMed

    Lissina, Anna; Chakrabarti, Lisa A; Takiguchi, Masafumi; Appay, Victor

    2016-02-01

    Because of the enormous complexity and breadth of the overall HIV-specific CD8(+) T-cell response, invaluable information regarding important aspects of T-cell efficacy against HIV can be sourced from studies performed on individual clonotypes. Data gathered from ex vivo and in vitro analyses of T-cell responses and viral evolution bring us one step closer towards deciphering the correlates of protection against HIV. HIV-responsive CD8(+) T-cell populations are characterized by specific clonotypic immunodominance patterns and public TCRs. The TCR endows T-cells with two key features, important for the effective control of HIV: avidity and crossreactivity. While TCR avidity is a major determinant of CD8(+) T-cell functional efficacy against the virus, crossreactivity towards wildtype and mutant viral epitopes is crucial for adaptation to HIV evolution. The properties of CD4(+) T-cell responses in HIV controllers appear also to be shaped by high avidity public TCR clonotypes. The molecular nature of the TCR, together with the clonotypic composition of the HIV-specific T-cell response, emerge as major determinants of anti-viral efficacy.

  9. Obesity increases the production of proinflammatory mediators from adipose tissue T cells and compromises TCR repertoire diversity: implications for systemic inflammation and insulin resistance.

    PubMed

    Yang, Hyunwon; Youm, Yun-Hee; Vandanmagsar, Bolormaa; Ravussin, Anthony; Gimble, Jeffrey M; Greenway, Frank; Stephens, Jacqueline M; Mynatt, Randall L; Dixit, Vishwa Deep

    2010-08-01

    Emerging evidence suggests that increases in activated T cell populations in adipose tissue may contribute toward obesity-associated metabolic syndrome. The present study investigates three unanswered questions: 1) Do adipose-resident T cells (ARTs) from lean and obese mice have altered cytokine production in response to TCR ligation?; 2) Do the extralymphoid ARTs possess a unique TCR repertoire compared with lymphoid-resident T cells and whether obesity alters the TCR diversity in specific adipose depots?; and 3) Does short-term elimination of T cells in epididymal fat pad without disturbing the systemic T cell homeostasis regulate inflammation and insulin-action during obesity? We found that obesity reduced the frequency of naive ART cells in s.c. fat and increased the effector-memory populations in visceral fat. The ARTs from diet-induced obese (DIO) mice had a higher frequency of IFN-gamma(+), granzyme B(+) cells, and upon TCR ligation, the ARTs from DIO mice produced increased levels of proinflammatory mediators. Importantly, compared with splenic T cells, ARTs exhibited markedly restricted TCR diversity, which was further compromised by obesity. Acute depletion of T cells from epididymal fat pads improved insulin action in young DIO mice but did not reverse obesity-associated feed forward cascade of chronic systemic inflammation and insulin resistance in middle-aged DIO mice. Collectively, these data establish that ARTs have a restricted TCR-Vbeta repertoire, and T cells contribute toward the complex proinflammatory microenvironment of adipose tissue in obesity. Development of future long-term T cell depletion protocols specific to visceral fat may represent an additional strategy to manage obesity-associated comorbidities.

  10. Obesity Increases the Production of Proinflammatory Mediators from Adipose Tissue T Cells and Compromises TCR Repertoire Diversity: Implications for Systemic Inflammation and Insulin Resistance

    PubMed Central

    Yang, Hyunwon; Youm, Yun-Hee; Vandanmagsar, Bolormaa; Ravussin, Anthony; Gimble, Jeffrey M.; Greenway, Frank; Stephens, Jacqueline M.; Mynatt, Randall L.; Dixit, Vishwa Deep

    2016-01-01

    Emerging evidence suggests that increases in activated T cell populations in adipose tissue may contribute toward obesity-associated metabolic syndrome. The present study investigates three unanswered questions: 1) Do adipose-resident T cells (ARTs) from lean and obese mice have altered cytokine production in response to TCR ligation?; 2) Do the extralymphoid ARTs possess a unique TCR repertoire compared with lymphoid-resident T cells and whether obesity alters the TCR diversity in specific adipose depots?; and 3) Does short-term elimination of T cells in epididymal fat pad without disturbing the systemic T cell homeostasis regulate inflammation and insulin-action during obesity? We found that obesity reduced the frequency of naive ART cells in s.c. fat and increased the effector-memory populations in visceral fat. The ARTs from diet-induced obese (DIO) mice had a higher frequency of IFN-γ+, granzyme B+ cells, and upon TCR ligation, the ARTs from DIO mice produced increased levels of proinflammatory mediators. Importantly, compared with splenic T cells, ARTs exhibited markedly restricted TCR diversity, which was further compromised by obesity. Acute depletion of T cells from epididymal fat pads improved insulin action in young DIO mice but did not reverse obesity-associated feed forward cascade of chronic systemic inflammation and insulin resistance in middle-aged DIO mice. Collectively, these data establish that ARTs have a restricted TCR-Vβ repertoire, and T cells contribute toward the complex proinflammatory microenvironment of adipose tissue in obesity. Development of future long-term T cell depletion protocols specific to visceral fat may represent an additional strategy to manage obesity-associated comorbidities. PMID:20581149

  11. RNAi-mediated TCR knockdown prevents autoimmunity in mice caused by mixed TCR dimers following TCR gene transfer.

    PubMed

    Bunse, Mario; Bendle, Gavin M; Linnemann, Carsten; Bies, Laura; Schulz, Stephan; Schumacher, Ton N; Uckert, Wolfgang

    2014-11-01

    Genetically modified T cells that express a transduced T cell receptor (TCR) α/β heterodimer in addition to their endogenous TCR are used in clinical studies to treat cancer. These cells express two TCR-α and two TCR-β chains that do not only compete for CD3 proteins but also form potentially self-reactive mixed TCR dimers, composed of endogenous and transferred chains. To overcome these deficits, we developed an RNAi-TCR replacement vector that simultaneously silences the endogenous TCR and expresses an RNAi-resistant TCR. Transduction of the virus-specific P14 TCR without RNAi resulted in unequal P14 TCR-α and -β chain surface levels, indicating heterodimerization with endogenous TCR chains. Such unequal expression was also observed following TCR gene optimization. Equal surface levels of the introduced TCR chains were however achieved by silencing the endogenous TCR. Importantly, all mice that received cells transduced with the native or optimized P14 TCR developed lethal TCR gene transfer-induced graft-versus-host-disease (TI-GVHD) due to formation of mixed TCR dimers. In contrast, TI-GVHD was almost completely prevented when using the RNAi-TCR replacement vector. Our data demonstrate that RNAi-assisted TCR replacement reduces the formation of mixed TCR dimers, and thereby significantly reduces the risk of TI-GVHD in TCR gene therapy.

  12. Oligoclonality and innate-like features in the TCR repertoire of type II NKT cells reactive to a β-linked self-glycolipid

    PubMed Central

    Arrenberg, Philomena; Halder, Ramesh; Dai, Yang; Maricic, Igor; Kumar, Vipin

    2010-01-01

    TCR-mediated recognition of β-linked self-glycolipids bound to CD1d is poorly understood. Here, we have characterized the TCR repertoire of a CD1d-restricted type II NKT cell subset reactive to sulfatide involved in the regulation of autoimmunity and antitumor immunity. The sulfatide/CD1d-tetramer+ cells isolated from naïve mice show an oligoclonal TCR repertoire with predominant usage of the Vα3/Vα1-Jα7/Jα9 and Vβ8.1/Vβ3.1-Jβ2.7 gene segments. The CDR3 regions of both the α- and β-chains are encoded by either germline or nongermline gene segments of limited lengths containing several conserved residues. Presence of dominant clonotypes with limited TCR gene usage for both TCR α- and β-chains in type II NKT cells reflects specific antigen recognition not found in the type I NKT cells but similar to the MHC-restricted T cells. Although potential CD1d-binding tyrosine residues in the CDR2β region are conserved between most type I and type II NKT TCRs, CDR 1α and 3α regions differ significantly between the two subsets. Collectively, the TCR repertoire of sulfatide-reactive type II NKT cells exhibits features of both antigen-specific conventional T cells and innate-like cells, and these findings provide important clues to the recognition of β-linked glycolipids by CD1d-restricted T cells in general. PMID:20534460

  13. Essential role of Rap signal in pre-TCR-mediated beta-selection checkpoint in alphabeta T-cell development.

    PubMed

    Kometani, Kohei; Moriyama, Masaki; Nakashima, Yasuhiro; Katayama, Yoshinori; Wang, Shu-Fang; Yamasaki, Sho; Saito, Takashi; Hattori, Masakazu; Minato, Nagahiro

    2008-12-01

    We demonstrate that lck promoter-driven conditional expression of transgenic SPA-1, a Rap GTPase-activation protein, causes a profound defect of alphabeta T-cell development at the CD4/CD8 double-negative (DN) stage due to enhanced cell death without affecting gammadelta T-cell development. The effect was specific to the DN stage, because CD4 promoter-driven SPA-1 expression hardly affected T-cell development. Rap1A17, a dominant-negative Rap mutant, interfered with the generation of double-positive (DP) cells from Rag2(-/-) fetal thymocytes in vitro in the presence of anti-CD3epsilon antibody and Notch ligand. Rap GTPases were activated in a DN cell line by the expression of self-oligomerizing CD3 (CD8:CD3epsilon chimera), which substituted autonomous pre-T-cell receptor (TCR) signal, inducing CD69 expression and CD25 down-regulation. Reciprocally, expression of C3G, a Rap guanine nucleotide exchange factor, in both normal and Rag2(-/-) DN cells markedly enhanced Notch-dependent generation and expansion of DP cells without additional anti-CD3epsilon antibody, thus bypassing pre-TCR. Defective alphabeta T-cell development in the conditional SPA-1-transgenic mice was restored completely by introducing a p53(-/-) mutation. These results suggest that endogenous Rap GTPases downstream of pre-TCR play an essential role in rescuing pre-T cells from the p53-mediated checkpoint response, thus allowing Notch-mediated expansion and differentiation.

  14. A CD22-reactive TCR from the T-cell allorepertoire for the treatment of acute lymphoblastic leukemia by TCR gene transfer.

    PubMed

    Jahn, Lorenz; Hagedoorn, Renate S; van der Steen, Dirk M; Hombrink, Pleun; Kester, Michel G D; Schoonakker, Marjolein P; de Ridder, Daniëlle; van Veelen, Peter A; Falkenburg, J H Frederik; Heemskerk, Mirjam H M

    2016-11-01

    CD22 is currently evaluated as a target-antigen for the treatment of B-cell malignancies using chimeric antigen receptor (CAR)-engineered T-cells or monoclonal antibodies (mAbs). CAR- and mAbs-based immunotherapies have been successfully applied targeting other antigens, however, occurrence of refractory disease to these interventions urges the identification of additional strategies. Here, we identified a TCR recognizing the CD22-derived peptide RPFPPHIQL (CD22RPF) presented in human leukocyte antigen (HLA)-B*07:02. To overcome tolerance to self-antigens such as CD22, we exploited the immunogenicity of allogeneic HLA. CD22RPF-specific T-cell clone 9D4 was isolated from a healthy HLA-B*07:02neg individual, efficiently produced cytokines upon stimulation with primary acute lymphoblastic leukemia and healthy B-cells, but did not react towards healthy hematopoietic and nonhematopoietic cell subsets, including dendritic cells (DCs) and macrophages expressing low levels of CD22. Gene transfer of TCR-9D4 installed potent CD22-specificity onto recipient CD8+ T-cells that recognized and lysed primary B-cell leukemia. TCR-transduced T-cells spared healthy CD22neg hematopoietic cell subsets but weakly lysed CD22low-expressing DCs and macrophages. CD22-specific TCR-engineered T-cells could form an additional immunotherapeutic strategy with a complementary role to CAR- and antibody-based interventions in the treatment of B-cell malignancies. However, CD22 expression on non-B-cells may limit the attractiveness of CD22 as target-antigen in cellular immunotherapy.

  15. Phospholipase Cγ1 is required for pre-TCR signal transduction and pre-T cell development.

    PubMed

    Fu, Guoping; Yu, Mei; Chen, Yuhong; Zheng, Yongwei; Zhu, Wen; Newman, Debra K; Wang, Demin; Wen, Renren

    2017-01-01

    Pre-T cell receptor (TCR) signaling is required for pre-T cell survival, proliferation, and differentiation from the CD4 and CD8 double negative (DN) to the double positive (DP) stage. However, the pre-TCR signal transduction pathway is not fully understood and the signaling molecules involved have not been completely identified. Phospholipase Cγ (PLCγ) 1 is an important signaling molecule that generates two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, that are important to mediate PKC activation and intracellular Ca(2+) flux in many signaling pathways. Previously, we have shown that PLCγ1 is important for TCR-mediated signaling, development and T-cell activation, but the role of PLCγ1 in pre-TCR signal transduction and pre-T cell development is not known. In this study, we demonstrated that PLCγ1 expression level in pre-T cells was comparable to that in mature T cells. Deletion of PLCγ1 prior to the pre-TCR signaling stage partially blocked the DN3 to DN4 transition and reduced thymic cellularity. We also demonstrated that deletion of PLCγ1 impaired pre-T cell proliferation without affecting cell survival. Further study showed that deficiency of PLCγ1 impaired pre-TCR mediated Ca(2+) flux and Erk activation. Thus our studies demonstrate that PLCγ1 is important for pre-TCR mediated signal transduction and pre-T cell development.

  16. Distinct temporal programming of naive CD4+ T cells for cell division versus TCR-dependent death susceptibility by antigen-presenting macrophages.

    PubMed

    Schrum, Adam G; Palmer, Ed; Turka, Laurence A

    2005-02-01

    Naive T cells become programmed for clonal expansion and contraction during the early hours of antigenic signaling. Recent studies support an 'autopilot' model, wherein the commitment to proliferate and the magnitude of the proliferative response are simultaneously determined during a single, brief period of antigen exposure. Here, we have examined whether the proliferation of naive CD4+ T cells must occur on 'autopilot', or whether extended periods of antigenic signaling can impact primary proliferative responses to antigen-presenting macrophages (macrophage APC). We found that a single exposure to antigen (18 h) simultaneously committed T cells to (1) up-regulate surface TCR above the level expressed on naive T cells, (2) undergo minimal cell division, and (3) acquire susceptibility to TCR-dependent activation-induced cell death. However, continued antigenic signaling between 18 and 72 h was required to amplify the number of daughter cells derived from the already committed T cells. Thus, a discrete commitment time was followed by a 'tuning' period, where extended antigenic signaling determined the volume of the proliferative response. We conclude that T cell commitment to full clonal expansion versus TCR-dependent death susceptibility represent two separate programming events whose timing can be segregated by macrophage APC.

  17. Discovery of invariant T cells by next-generation sequencing of the human TCR α-chain repertoire.

    PubMed

    van Schaik, Barbera; Klarenbeek, Paul; Doorenspleet, Marieke; van Kampen, Antoine; Moody, D Branch; de Vries, Niek; Van Rhijn, Ildiko

    2014-11-15

    During infection and autoimmune disease, activation and expansion of T cells take place. Consequently, the TCR repertoire contains information about ongoing and past diseases. Analysis and interpretation of the human TCR repertoire are hampered by its size and stochastic variation and by the diversity of Ags and Ag-presenting molecules encoded by the MHC, but are highly desirable and would greatly impact fundamental and clinical immunology. A subset of the TCR repertoire is formed by invariant T cells. Invariant T cells express interdonor-conserved TCRs and recognize a limited set of Ags, presented by nonpolymorphic Ag-presenting molecules. Discovery of the three known invariant T cell populations has been a tedious and slow process, identifying them one by one. Because conservation of the TCR α-chain of invariant T cells is much higher than the β-chain, and because the TCR α-chain V gene segment TRAV1-2 is used by two of the three known invariant TCRs, we employed next-generation sequencing of TCR α-chains that contain the TRAV1-2 gene segment to identify 16 invariant TCRs shared among many blood donors. Frequency analysis of individual clones indicates these T cells are expanded in many donors, implying an important role in human immunity. This approach extends the number of known interdonor-conserved TCRs and suggests that many more exist and that these TCR patterns can be used to systematically evaluate human Ag exposure.

  18. T cell receptor (TCR) V gene usage in patients with systemic necrotizing vasculitis.

    PubMed Central

    Giscombe, R; Grunewald, J; Nityanand, S; Lefvert, A K

    1995-01-01

    Wegener's granulomatosis (WG) and polyarteritis nodosa (PAN) are systemic necrotizing vasculitides of unknown etiology. These disorders run a fatal course if untreated. T lymphocytes are implicated in the pathogenesis of WG, since they have been found to infiltrate affected organs, and sIL-2R correlates with disease activity. To elucidate further the role of T cells in necrotizing vasculitis, we have used a panel of 12 TCR V-specific MoAbs to investigate the number of cells expressing certain V alpha and V beta gene segments in the CD4+ and CD8+ subsets of altogether 11 patients with WG or PAN. In the group of patients, we found abnormal expansions of T cells using particular TCR V alpha or V beta gene products. These T cell expansions were more numerous, of a dramatically higher magnitude, and frequently more often found in the CD4 subset, compared with T cell expansions identified in healthy individuals. In long-term studies of the T cell expansions for up to 18 months, a heterogeneous pattern was revealed, with no obvious correlation to clinical features such as disease activity or treatment. Studies of TCR V gene usage in this group of patients may help in understanding the pathogenesis of necrotizing vasculitis, and in the identification of unknown antigens, and may open the possibility to a highly selective immunotherapy by targeting disease-mediating T cells. PMID:7648706

  19. TCR-based therapy for multiple myeloma and other B-cell malignancies targeting intracellular transcription factor BOB1.

    PubMed

    Jahn, Lorenz; Hombrink, Pleun; Hagedoorn, Renate S; Kester, Michel G D; van der Steen, Dirk M; Rodriguez, Tania; Pentcheva-Hoang, Tsvetelina; de Ru, Arnoud H; Schoonakker, Marjolein P; Meeuwsen, Miranda H; Griffioen, Marieke; van Veelen, Peter A; Falkenburg, J H Frederik; Heemskerk, Mirjam H M

    2017-03-09

    Immunotherapy for hematological malignancies or solid tumors by administration of monoclonal antibodies or T cells engineered to express chimeric antigen receptors or T-cell receptors (TCRs) has demonstrated clinical efficacy. However, antigen-loss tumor escape variants and the absence of currently targeted antigens on several malignancies hamper the widespread application of immunotherapy. We have isolated a TCR targeting a peptide of the intracellular B cell-specific transcription factor BOB1 presented in the context of HLA-B*07:02. TCR gene transfer installed BOB1 specificity and reactivity onto recipient T cells. TCR-transduced T cells efficiently lysed primary B-cell leukemia, mantle cell lymphoma, and multiple myeloma in vitro. We also observed recognition and lysis of healthy BOB1-expressing B cells. In addition, strong BOB1-specific proliferation could be demonstrated for TCR-modified T cells upon antigen encounter. Furthermore, clear in vivo antitumor reactivity was observed of BOB1-specific TCR-engineered T cells in a xenograft mouse model of established multiple myeloma. Absence of reactivity toward a broad panel of BOB1(-) but HLA-B*07:02(+) nonhematopoietic and hematopoietic cells indicated no off-target toxicity. Therefore, administration of BOB1-specific TCR-engineered T cells may provide novel cellular treatment options to patients with B-cell malignancies, including multiple myeloma.

  20. TCR signal strength controls thymic differentiation of discrete proinflammatory γδ T cell subsets

    PubMed Central

    Muñoz-Ruiz, Miguel; Ribot, Julie C.; Grosso, Ana R.; Gonçalves-Sousa, Natacha; Pamplona, Ana; Pennington, Daniel J.; Regueiro, José R.

    2016-01-01

    The murine thymus produces discrete γδ T cell subsets making either interferon-γ (IFN--γ) or interleukin 17 (IL-17), but the role of the TCR in this developmental process remains controversial. Here we show that mice haploinsufficient for both Cd3g and Cd3d (CD3DH, for CD3 double haploinsufficient) have reduced TCR expression and signaling strength selectively on γδ T cells. CD3DH mice had normal numbers and phenotype of αβ thymocyte subsets but impaired differentiation of fetal Vγ6+ (but not Vγ4+) IL-17-producing γδ T cells and a marked depletion of IFN-γ-producing CD122+ NK1.1+ γδ T cells throughout ontogeny. Adult CD3DH mice showed reduced peripheral IFN-γ+ γδ T cells and were resistant to experimental cerebral malaria. Thus, TCR signal strength within specific thymic developmental windows is a major determinant of the generation of proinflammatory γδ T cell subsets and their impact on pathophysiology. PMID:27043412

  1. Affinity and dose of TCR engagement yield proportional enhancer and gene activity in CD4+ T cells

    PubMed Central

    Allison, Karmel A; Sajti, Eniko; Collier, Jana G; Gosselin, David; Troutman, Ty Dale; Stone, Erica L; Hedrick, Stephen M; Glass, Christopher K

    2016-01-01

    Affinity and dose of T cell receptor (TCR) interaction with antigens govern the magnitude of CD4+ T cell responses, but questions remain regarding the quantitative translation of TCR engagement into downstream signals. We find that while the response of mouse CD4+ T cells to antigenic stimulation is bimodal, activated cells exhibit analog responses proportional to signal strength. Gene expression output reflects TCR signal strength, providing a signature of T cell activation. Expression changes rely on a pre-established enhancer landscape and quantitative acetylation at AP-1 binding sites. Finally, we show that graded expression of activation genes depends on ERK pathway activation, suggesting that an ERK-AP-1 axis plays an important role in translating TCR signal strength into proportional activation of enhancers and genes essential for T cell function. DOI: http://dx.doi.org/10.7554/eLife.10134.001 PMID:27376549

  2. Altered differentiation, diminished pathogenicity, and regulatory activity of myelin-specific T cells expressing an enhanced affinity TCR

    PubMed Central

    Alli, Rajshekhar; Nguyen, Phuong; Geiger, Terrence L.

    2011-01-01

    Whereas increased affinity enhances T cell competitiveness after immunization, the role of affinity in modulating the pathogenicity of self-reactive T cells is less established. To assess this, we generated two myelin-specific, class II MHC-restricted TCR that differ only in a buried hydroxymethyl that forms a common TRBV variant. The variation, predicted to increase TCR stability, resulted in a ~3log10 difference in TCR sensitivity with preserved fine specificity. The high affinity TCR markedly diminished T cell pathogenicity. T cells were not deleted, did not upregulate Foxp3, and barring disease induction were predominantly naïve. However, high affinity CD4+ T cells showed an altered cytokine profile characterized by the production of protective cytokines prior to experimental allergic encephalomyelitis induction and decreased effector cytokines after. Further, the high affinity TCR promoted the development of CD4−CD8− and CD8+ T cells that possessed low intrinsic pathogenicity, were protective even in small numbers when transferred into wild type mice and in mixed chimeras, and outcompete CD4+ T cells during disease development. Therefore TCR affinities exceeding an upper affinity threshold may impede the development of autoimmunity through altered development and functional maturation of T cells, including diminished intrinsic CD4+ T-cell pathogenicity and the development of CD4− Foxp3− regulatory populations. PMID:22025553

  3. TCRs genetically linked to CD28 and CD3ε do not mispair with endogenous TCR chains and mediate enhanced T cell persistence and anti-melanoma activity.

    PubMed

    Govers, Coen; Sebestyén, Zsolt; Roszik, János; van Brakel, Mandy; Berrevoets, Cor; Szöőr, Árpád; Panoutsopoulou, Konstantina; Broertjes, Marieke; Van, Tan; Vereb, György; Szöllősi, János; Debets, Reno

    2014-11-15

    Adoptive transfer of T cells that are gene engineered to express a defined TCR represents a feasible and promising therapy for patients with tumors. However, TCR gene therapy is hindered by the transient presence and effectiveness of transferred T cells, which are anticipated to be improved by adequate T cell costimulation. In this article, we report the identification and characterization of a novel two-chain TCR linked to CD28 and CD3ε (i.e., TCR:28ε). This modified TCR demonstrates enhanced binding of peptide-MHC and mediates enhanced T cell function following stimulation with peptide compared with wild-type TCR. Surface expression of TCR:28ε depends on the transmembrane domain of CD28, whereas T cell functions depend on the intracellular domains of both CD28 and CD3ε, with IL-2 production showing dependency on CD28:LCK binding. TCR:28ε, but not wild-type TCR, induces detectable immune synapses in primary human T cells, and such immune synapses show significantly enhanced accumulation of TCR transgenes and markers of early TCR signaling, such as phosphorylated LCK and ERK. Importantly, TCR:28ε does not show signs of off-target recognition, as evidenced by lack of TCR mispairing, as well as preserved specificity. Notably, when testing TCR:28ε in immune-competent mice, we observed a drastic increase in T cell survival, which was accompanied by regression of large melanomas with limited recurrence. Our data argue that TCR transgenes that contain CD28, and, thereby, may provide T cell costimulation in an immune-suppressive environment, represent candidate receptors to treat patients with tumors.

  4. Physical mapping of the human T-cell antigen receptor (TCR) {beta}-chain gene complex

    SciTech Connect

    Yashim, Y.; So, A.K.

    1994-09-01

    The genetic variation of the TCR loci and their contribution to autoimmune diseases is poorly defined, in direct contrast to the clear examples of disease association with the Class I and II alleles of the major histocompatibility complex. We have therefore started to determine the gene organization and polymorphism of the TCR {beta} locus. Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human TCR {beta}-chain gene complex. Variable gene (V{beta}) sequences for the 25 known V{beta} subfamilies were amplified by PCR and were used as probes to screen a YAC library. Five positive YACs were identified. YACs designated B3, E11 and H11 of sizes 820, 400 and 600 kbp, respectively, were analyzed for their V{beta} content by pulse-field gel electrophoresis (PFGE). YAC B3 was found to contain all 25 V{beta} subfamilies, E11 for 14 and H11 for 7. B3 was also positive for the constant region genes. Restriction enzyme mapping of B3 located V{beta} and C{beta} gene regions to four Sfi I fragments of 280, 110, 90 and 125 kbp, and was in accordance with published data. The data thus showed that YAC B3 encoded a complete and unrearranged TCR {beta}-gene locus. The map was further resolved by locating restriction sites for Sal I and Bssll II on B3. Fluorescent in situ hybridization to human metaphase chromosomes localized B3 to chromosome 7q35. However, two additional signals were obtained: one attributable to V{beta} orphon cluster on chromosome 9q21; the second to the long arm of chromosome 2. PCR amplification of a chromosome 2 somatic cell hybrid using primers for all 25 V{beta} gene families revealed the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCR {beta} locus flanked by chromosome 2 sequences. The determination of the TCR genomic organization will help extend studies of the role T-cells play in autoimmune diseases.

  5. Hard wiring of T cell receptor specificity for the major histocompatibility complex is underpinned by TCR adaptability

    SciTech Connect

    Burrows, Scott R.; Chen, Zhenjun; Archbold, Julia K.; Tynan, Fleur E.; Beddoe, Travis; Kjer-Nielsen, Lars; Miles, John J.; Khanna, Rajiv; Moss, Denis J.; Liu, Yu Chih; Gras, Stephanie; Kostenko, Lyudmila; Brennan, Rebekah M.; Clements, Craig S.; Brooks, Andrew G.; Purcell, Anthony W.; McCluskey, James; Rossjohn, Jamie

    2010-07-07

    {alpha}{beta} T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR-pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR-pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR-pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I.

  6. Single TCR-Vβ2 evaluation discloses the circulating T cell clone in Sezary syndrome: one family fits all!

    PubMed

    Scala, Enrico; Abeni, Damiano; Pomponi, Debora; Russo, Nicoletta; Russo, Giandomenico; Narducci, Maria Grazia

    2015-08-01

    Sézary Syndrome (SS/L-CTCL) is a rare but aggressive variant of cutaneous T cell lymphoma (CTCL), characterized by erythroderma, lymphadenopathy, and the presence of a circulating memory CD4(+) T cell malignant clone with a skin homing behavior, lacking CD26 and CD49d and over-expressing CD60. The availability of a panel of monoclonal antibodies recognizing distinct TCR-Vβ families, allows to typify the clone by flow cytometry in about 70 % of cases. The TCR-Vβ repertoire of 533 individuals, comprising 308 patients affected by CTCL, 50 healthy donors, and subjects affected by various non-neoplastic dermatological affections was evaluated by flow cytometry. Statistical analyses were performed using the SPSS statistical software package for Microsoft Windows (SPSS, version 21, Chicago, IL). TCR-Vβ2 levels below 5.4 % or above 39.5 %, within total CD4(+) T cells, showed the best balance between sensitivity (98.1 %) and specificity (96 %) to identify the presence of a clone in the peripheral blood of patients affected by SS. Based on this observation, a "two-step" procedure in the detection of the malignant T cell clone in CTCLs is herein suggested. TCR-Vβ2 assessment in all cases (first step). In the case of TCR-Vβ2 levels above 39.5 %, the presence of a clonal expansion of this family is suggested, deserving further confirmation by means of T cell gene rearrangement evaluation. In patients having a TCR-Vβ2 reactivity below 5.4 % (second step), the entire TCR-Vβ repertoire should be evaluated to typify the expanded clone. In conclusion, the single TCR-Vβ2 expression check, instead of the entire repertoire assessment, represents an easy and cost-effective method for the recognition of CTCL aggressive leukemic variant.

  7. TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL

    PubMed Central

    O'Malley, John T.; Williamson, David W.; Scott, Laura-Louise; Elco, Christopher P.; Teague, Jessica E.; Gehad, Ahmed; Lowry, Elizabeth L.; LeBoeuf, Nicole R.; Krueger, James G.; Robins, Harlan S.; Kupper, Thomas S.; Clark, Rachael A.

    2016-01-01

    Early diagnosis of CTCL is difficult and takes on average six years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL but the TCRγ PCR analysis in current clinical use detect clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46/46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting a niche of finite size may exist for benign T cells in skin. Lastly, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases and provided insights into the cell of origin and location of malignant CTCL cells in skin. PMID:26446955

  8. Quantitative TCR:pMHC Dissociation Rate Assessment by NTAmers Reveals Antimelanoma T Cell Repertoires Enriched for High Functional Competence.

    PubMed

    Gannon, Philippe O; Wieckowski, Sébastien; Baumgaertner, Petra; Hebeisen, Michaël; Allard, Mathilde; Speiser, Daniel E; Rufer, Nathalie

    2015-07-01

    Experimental models demonstrated that therapeutic induction of CD8 T cell responses may offer protection against tumors or infectious diseases providing that T cells have sufficiently high TCR/CD8:pMHC avidity for efficient Ag recognition and consequently strong immune functions. However, comprehensive characterization of TCR/CD8:pMHC avidity in clinically relevant situations has remained elusive. In this study, using the novel NTA-His tag-containing multimer technology, we quantified the TCR:pMHC dissociation rates (koff) of tumor-specific vaccine-induced CD8 T cell clones (n = 139) derived from seven melanoma patients vaccinated with IFA, CpG, and the native/EAA or analog/ELA Melan-A(MART-1)(26-35) peptide, binding with low or high affinity to MHC, respectively. We observed substantial correlations between koff and Ca(2+) mobilization (p = 0.016) and target cell recognition (p < 0.0001), with the latter independently of the T cell differentiation state. Our strategy was successful in demonstrating that the type of peptide impacted on TCR/CD8:pMHC avidity, as tumor-reactive T cell clones derived from patients vaccinated with the low-affinity (native) peptide expressed slower koff rates than those derived from patients vaccinated with the high-affinity (analog) peptide (p < 0.0001). Furthermore, we observed that the low-affinity peptide promoted the selective differentiation of tumor-specific T cells bearing TCRs with high TCR/CD8:pMHC avidity (p < 0.0001). Altogether, TCR:pMHC interaction kinetics correlated strongly with T cell functions. Our study demonstrates the feasibility and usefulness of TCR/CD8:pMHC avidity assessment by NTA-His tag-containing multimers of naturally occurring polyclonal T cell responses, which represents a strong asset for the development of immunotherapy.

  9. Expression of TCR-Vβ peptides by murine bone marrow cells does not identify T-cell progenitors.

    PubMed

    Abbey, Janice L; Karsunky, Holger; Serwold, Thomas; Papathanasiou, Peter; Weissman, Irving L; O'Neill, Helen C

    2015-08-01

    Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vβ8.2 in absence of anti-Cβ antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vβ8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vβ8.2(+) Cβ(-) are found in several lymphoid sites, and among the lineage-negative (Lin(-)) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin(-) Vβ8.2(+) Cβ(-) BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin(-) Vβ8.2(+) Cβ(-) BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.

  10. Chimeric Antigen Receptor- and TCR-Modified T Cells Enter Main Street and Wall Street.

    PubMed

    Barrett, David M; Grupp, Stephan A; June, Carl H

    2015-08-01

    The field of adoptive cell transfer (ACT) is currently comprised of chimeric Ag receptor (CAR)- and TCR-engineered T cells and has emerged from principles of basic immunology to paradigm-shifting clinical immunotherapy. ACT of T cells engineered to express artificial receptors that target cells of choice is an exciting new approach for cancer, and it holds equal promise for chronic infection and autoimmunity. Using principles of synthetic biology, advances in immunology, and genetic engineering have made it possible to generate human T cells that display desired specificities and enhanced functionalities. Clinical trials in patients with advanced B cell leukemias and lymphomas treated with CD19-specific CAR T cells have induced durable remissions in adults and children. The prospects for the widespread availability of engineered T cells have changed dramatically given the recent entry of the pharmaceutical industry to this arena. In this overview, we discuss some of the challenges and opportunities that face the field of ACT.

  11. Targeting of HPV-16+ epithelial cancer cells by TCR gene engineered T cells directed against E6

    PubMed Central

    Draper, Lindsey M.; Kwong, Mei Li; Gros, Alena; Stevanović, Sanja; Tran, Eric; Kerkar, Sid; Raffeld, Mark; Rosenberg, Steven A.; Hinrichs, Christian S.

    2015-01-01

    Purpose The E6 and E7 oncoproteins of HPV-associated epithelial cancers are in principle ideal immunotherapeutic targets, but evidence that T cells specific for these antigens can recognize and kill HPV+ tumor cells is limited. We sought to determine if TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV+ tumor cells. Experimental design T cell responses against the HPV-16 oncoproteins were investigated in a patient with an ongoing 22-month disease-free interval after her second resection of distant metastatic anal cancer. T cells genetically engineered to express an oncoprotein-specific TCR from this patient’s tumor-infiltrating T cells were tested for specific reactivity against HPV+ epithelial tumor cells. Results We identified, from an excised metastatic anal cancer tumor, T cells that recognized an HLA-A*02:01-restricted epitope of HPV-16 E6. The frequency of the dominant T cell clonotype from these cells was approximately 400-fold greater in the patient’s tumor than in her peripheral blood. T cells genetically engineered to express the TCR from this clonotype displayed high avidity for an HLA-A*02:01-restricted epitope of HPV-16, and they showed specific recognition and killing of HPV-16+ cervical, and head and neck cancer cell lines. Conclusion These findings demonstrate that HPV-16+ tumors can be targeted by E6-specific TCR gene engineered T cells, and they provide the foundation for a novel cellular therapy directed against HPV-16+ malignancies including cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers. PMID:26429982

  12. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    PubMed

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.

  13. Cognate peptide-MHC complexes are expressed as tightly apposed nanoclusters in virus-infected cells to allow TCR crosslinking.

    PubMed

    Ferez, María; Castro, Mario; Alarcon, Balbino; van Santen, Hisse M

    2014-01-01

    Antigenic T cell stimulation requires interaction between the TCR of the T cell and cognate peptide-MHC molecules presented by the APC. Although studies with TCR-specific Abs and soluble peptide-MHC ligands have shown that the TCR needs to be crosslinked by two or more ligands to induce T cell stimulation, it is not understood how several MHC molecules loaded with the cognate antigenic peptide can produce crosslinking under physiological conditions. We show at the molecular level that large clusters of cognate peptide-MHC are formed at the surface of murine professional and nonprofessional APCs upon virus infection and that these clusters impinge on the stimulatory capacity of the APC. These clusters are formed by tight apposition of cognate peptide-MHC complexes in a configuration that is compatible with simultaneous engagement of two or more TCRs. This suggests that physiological expression of Ag allows formation of multivalent ligands for the TCR that permit TCR crosslinking and T cell activation.

  14. Activating mutations in genes related to TCR signaling in angioimmunoblastic and other follicular helper T-cell-derived lymphomas.

    PubMed

    Vallois, David; Dobay, Maria Pamela D; Morin, Ryan D; Lemonnier, François; Missiaglia, Edoardo; Juilland, Mélanie; Iwaszkiewicz, Justyna; Fataccioli, Virginie; Bisig, Bettina; Roberti, Annalisa; Grewal, Jasleen; Bruneau, Julie; Fabiani, Bettina; Martin, Antoine; Bonnet, Christophe; Michielin, Olivier; Jais, Jean-Philippe; Figeac, Martin; Bernard, Olivier A; Delorenzi, Mauro; Haioun, Corinne; Tournilhac, Olivier; Thome, Margot; Gascoyne, Randy D; Gaulard, Philippe; de Leval, Laurence

    2016-09-15

    Angioimmunoblastic T-cell lymphoma (AITL) and other lymphomas derived from follicular T-helper cells (TFH) represent a large proportion of peripheral T-cell lymphomas (PTCLs) with poorly understood pathogenesis and unfavorable treatment results. We investigated a series of 85 patients with AITL (n = 72) or other TFH-derived PTCL (n = 13) by targeted deep sequencing of a gene panel enriched in T-cell receptor (TCR) signaling elements. RHOA mutations were identified in 51 of 85 cases (60%) consisting of the highly recurrent dominant negative G17V variant in most cases and a novel K18N in 3 cases, the latter showing activating properties in in vitro assays. Moreover, half of the patients carried virtually mutually exclusive mutations in other TCR-related genes, most frequently in PLCG1 (14.1%), CD28 (9.4%, exclusively in AITL), PI3K elements (7%), CTNNB1 (6%), and GTF2I (6%). Using in vitro assays in transfected cells, we demonstrated that 9 of 10 PLCG1 and 3 of 3 CARD11 variants induced MALT1 protease activity and increased transcription from NFAT or NF-κB response element reporters, respectively. Collectively, the vast majority of variants in TCR-related genes could be classified as gain-of-function. Accordingly, the samples with mutations in TCR-related genes other than RHOA had transcriptomic profiles enriched in signatures reflecting higher T-cell activation. Although no correlation with presenting clinical features nor significant impact on survival was observed, the presence of TCR-related mutations correlated with early disease progression. Thus, targeting of TCR-related events may hold promise for the treatment of TFH-derived lymphomas.

  15. Evaluation of TCR Vbeta subfamily T cell expansion in NOD/SCID mice transplanted with human cord blood hematopoietic stem cells.

    PubMed

    Lin, Chen; Chen, Shaohua; Yang, Lijian; Tan, Yubo; Bai, Xue; Li, Yangqiu

    2007-08-01

    Examination of the T cell receptor (TCR) gene repertoire is important in the analysis of the immune status of models, because clonal expansion of T cells permits the identification of specific antigen responses of T cells. Little is known about T-cell immunity in the humanized NOD/SCID mouse model. TCR Vbeta repertoire usage and clonality were analyzed to investigate the distribution and clonal expansion of TCR Vbeta subfamily T cells in NOD/SCID mice transplanted with human cord blood (CB) hematopoietic stem cells. The NOD/SCID mice were sublethally irradiated ((60)Co, 300cGy) to eliminate residual innate immunity in the host. The experimental mice were transplanted intravenously with CB CD34(+) cells sorted by MACS. After 6 weeks, RNA was obtained from peripheral blood, bone marrow and thymus of the study animals. The gene expression and clonality of the TCR Vbeta repertoire were determined by RT-PCR and GeneScan techniques. A restricted range of TCR Vbeta usage was exhibited in the bone marrow of mice, which included TCR Vbeta 1, 2, 9, 13 and 19. Further, oligoclonal expression of some TCR Vbeta subfamilies (Vbeta9, 13, 19) was identified by GeneScan technique. To investigate the reason for oligoclonal expansion of the TCR Vbeta subfamily T cells from CB in mouse models, the T-cell culture with tissue-antigen of NOD/SCID mouse was performed in vitro. The cells from peripheral blood mononuclear cells and bone marrow, spleen, thymus in NOD/SCID mice were frozen and thawed, and used as tissue-antigen. CB mononuclear cells were separately cultured with the component from those murine cells for 15-20 days. Oligoclonal expression or oligoclonal trend of some TCR Vbeta subfamilies (Vbeta10, 11 and Vbeta2, 15, 16, 19) was detected in T cells after stimulation with tissue-antigen of NOD/SCID mouse. Interestingly, a similar clonal expansion of the TCR Vbeta11 subfamily was found in T cells cultured with peripheral blood, bone marrow and spleen respectively. The TCR Vbeta

  16. USP2a positively regulates TCR-induced NF-κB activation by bridging MALT1-TRAF6.

    PubMed

    Li, Yi; He, Xiao; Wang, Shuai; Shu, Hong-Bing; Liu, Yu

    2013-01-01

    The paracaspase MALT1 is essential for the activation of NF-κB in response to T cell receptor (TCR) stimulation. It recruits downstream TRAF6 and activates the E3 ligase activity of TRAF6 to polyubiquitinate several targets, which ultimately leads to NF-κB activation. Here we identified ubiquitin-specific protease 2a (USP2a) as a MALT1-associated protein by biochemical affinity purification. Endogenous USP2a constitutively interacted with TRAF6, but dynamically interacted with MALT1 and CARMA1 in a stimulation-dependent manner. RNA interference (RNAi)-mediated silencing of USP2a attenuated TCR-induced NF-κB activation and production of interleukin-2 (IL-2). In addition, the ubiquitination of MALT1 and TRAF6 were both suppressed by USP2a knockdown. By knockdown and reconstitution assays, we found that USP2a mediated the interaction between MALT1 and TRAF6 in a catalytic activity-dependent manner. Furthermore, USP2a deSUMOylated TRAF6. Our findings implicate that USP2a plays an important role in TCR signaling by deSUMOylating TRAF6 and mediating TRAF6-MALT1 interaction.

  17. High-throughput sequencing of TCR repertoires in multiple sclerosis reveals intrathecal enrichment of EBV-reactive CD8+ T cells.

    PubMed

    Lossius, Andreas; Johansen, Jorunn N; Vartdal, Frode; Robins, Harlan; Jūratė Šaltytė, Benth; Holmøy, Trygve; Olweus, Johanna

    2014-11-01

    Epstein-Barr virus (EBV) has long been suggested as a pathogen in multiple sclerosis (MS). Here, we used high-throughput sequencing to determine the diversity, compartmentalization, persistence, and EBV-reactivity of the T-cell receptor (TCR) repertoires in MS. TCR-β genes were sequenced in paired samples of cerebrospinal fluid (CSF) and blood from patients with MS and controls with other inflammatory neurological diseases. The TCR repertoires were highly diverse in both compartments and patient groups. Expanded T-cell clones, represented by TCR-β sequences >0.1%, were of different identity in CSF and blood of MS patients, and persisted for more than a year. Reference TCR-β libraries generated from peripheral blood T cells reactive against autologous EBV-transformed B cells were highly enriched for public EBV-specific sequences and were used to quantify EBV-reactive TCR-β sequences in CSF. TCR-β sequences of EBV-reactive CD8+ T cells, including several public EBV-specific sequences, were intrathecally enriched in MS patients only, whereas those of EBV-reactive CD4+ T cells were also enriched in CSF of controls. These data provide evidence for a clonally diverse, yet compartmentalized and persistent, intrathecal T-cell response in MS. The presented strategy links TCR sequence to intrathecal T-cell specificity, demonstrating enrichment of EBV-reactive CD8+ T cells in MS.

  18. IL-12-mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8+ T cells.

    PubMed

    Stark, Regina; Hartung, Anett; Zehn, Dietmar; Frentsch, Marco; Thiel, Andreas

    2013-06-01

    CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4(+) T cells have been regarded as the major T-cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8(+) helper T-cell subset expressing CD40L is induced in human and murine CD8(+) T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8(+) T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8(+) T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8(+) T cells regulated by IL-12 and TCR signaling may enable CD8(+) T cells to respond autonomously of CD4(+) T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs.

  19. Generating HPV specific T helper cells for the treatment of HPV induced malignancies using TCR gene transfer

    PubMed Central

    2011-01-01

    Background Infection with high risk Human Papilloma Virus (HPV) is associated with cancer of the cervix, vagina, penis, vulva, anus and some cases of head and neck carcinomas. The HPV derived oncoproteins E6 and E7 are constitutively expressed in tumor cells and therefore potential targets for T cell mediated adoptive immunotherapy. Effective immunotherapy is dependent on the presence of both CD4+ and CD8+ T cells. However, low precursor frequencies of HPV16 specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer purposes. An alternative to generate HPV specific CD4+ and CD8+ T cells is TCR gene transfer. Methods HPV specific CD4+ T cells were generated using either a MHC class I or MHC class II restricted TCR (from clones A9 and 24.101 respectively) directed against HPV16 antigens. Functional analysis was performed by interferon-γ secretion, proliferation and cytokine production assays. Results Introduction of HPV16 specific TCRs into blood derived CD4+ recipient T cells resulted in recognition of the relevant HPV16 epitope as determined by IFN-γ secretion. Importantly, we also show recognition of the endogenously processed and HLA-DP1 presented HPV16E6 epitope by 24.101 TCR transgenic CD4+ T cells and recognition of the HLA-A2 presented HPV16E7 epitope by A9 TCR transgenic CD4+ T cells. Conclusion Our data indicate that TCR transfer is feasible as an alternative strategy to generate human HPV16 specific CD4+ T helper cells for the treatment of patients suffering from cervical cancer and other HPV16 induced malignancies. PMID:21892941

  20. A Functional γδTCR/CD3 Complex Distinct from γδT Cells Is Expressed by Human Eosinophils

    PubMed Central

    Woerly, Gaëtane; Loiseau, Sylvie; Hermann, Emmanuel; Fournié, Jean-Jacques; Héliot, Laurent; Mattot, Virginie; Soncin, Fabrice; Gougeon, Marie-Lise; Dombrowicz, David; Capron, Monique

    2009-01-01

    Background Eosinophils are effector cells during parasitic infections and allergic responses. However, their contribution to innate immunity has been only recently unravelled. Methodology/Principal Findings Here we show that human eosinophils express CD3 and γδ T Cell Receptor (TCR) but not αβ TCR. Surface expression of γδTCR/CD3 is heterogeneous between eosinophil donors and inducible by mycobacterial ligands. Surface immunoprecipitation revealed expression of the full γδTCR/CD3 complex. Real-time PCR amplification for CD3, γ and δ TCR constant regions transcripts showed a significantly lower expression in eosinophils than in γδT cells. Limited TCR rearrangements occur in eosinophils as shown by spectratyping analysis of CDR3 length profiles and in situ hybridization. Release by eosinophils of Reactive Oxygen Species, granule proteins, Eosinophil Peroxidase and Eosinophil-Derived Neurotoxin and cytokines (IFN-γ and TNF-α) was observed following activation by γδTCR-specific agonists or by mycobacteria. These effects were inhibited by anti-γδTCR blocking antibodies and antagonists. Moreover, γδTCR/CD3 was involved in eosinophil cytotoxicity against tumor cells. Conclusions/Significance Our results provide evidence that human eosinophils express a functional γδTCR/CD3 with similar, but not identical, characteristics to γδTCR from γδT cells. We propose that this receptor contributes to eosinophil innate responses against mycobacteria and tumors and may represent an additional link between lymphoid and myeloid lineages. PMID:19536290

  1. TCR hypervariable regions expressed by T cells that respond to effective tumor vaccines.

    PubMed

    Jordan, Kimberly R; Buhrman, Jonathan D; Sprague, Jonathan; Moore, Brandon L; Gao, Dexiang; Kappler, John W; Slansky, Jill E

    2012-10-01

    A major goal of immunotherapy for cancer is the activation of T cell responses against tumor-associated antigens (TAAs). One important strategy for improving antitumor immunity is vaccination with peptide variants of TAAs. Understanding the mechanisms underlying the expansion of T cells that respond to the native tumor antigen is an important step in developing effective peptide-variant vaccines. Using an immunogenic mouse colon cancer model, we compare the binding properties and the TCR genes expressed by T cells elicited by peptide variants that elicit variable antitumor immunity directly ex vivo. The steady-state affinity of the natural tumor antigen for the T cells responding to effective peptide vaccines was higher relative to ineffective peptides, consistent with their improved function. Ex vivo analysis showed that T cells responding to the effective peptides expressed a CDR3β motif, which was also shared by T cells responding to the natural antigen and not those responding to the less effective peptide vaccines. Importantly, these data demonstrate that peptide vaccines can expand T cells that naturally respond to tumor antigens, resulting in more effective antitumor immunity. Future immunotherapies may require similar stringent analysis of the responding T cells to select optimal peptides as vaccine candidates.

  2. Mushroom acidic glycosphingolipid induction of cytokine secretion from murine T cells and proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in vitro

    SciTech Connect

    Nozaki, Hirofumi; Itonori, Saki; Sugita, Mutsumi; Nakamura, Kimihide; Ohba, Kiyoshi; Suzuki, Akemi; Kushi, Yasunori

    2008-08-29

    Interferon (IFN)-{gamma} and interleukin (IL)-4 regulate many types of immune responses. Here we report that acidic glycosphingolipids (AGLs) of Hypsizigus marmoreus and Pleurotus eryngii induced secretion of IFN- {gamma} and IL-4 from T cells in a CD11c-positive cell-dependent manner similar to that of {alpha}-galactosylceramide ({alpha}-GalCer) and isoglobotriaosylceramide (iGb3), although activated T cells by AGLs showed less secretion of cytokine than those activated by {alpha}-GalCer. In addition, stimulation of these mushroom AGLs induced proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in splenocytes. Administration of a mixture of {alpha}-GalCer and AGLs affected the stimulation of {alpha}-GalCer and generally induced a subtle Th1 bias for splenocytes but induced an extreme Th2 bias for thymocytes. These results suggested that edible mushroom AGLs contribute to immunomodulation.

  3. Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain

    PubMed Central

    1993-01-01

    CD4-CD8- (double negative [DN]) alpha/beta T cells are a largely uncharacterized subpopulation of unknown function. To investigate whether these cells are selected to recognize particular antigens or antigen-presenting molecules, DN alpha/beta T cells were purified from the peripheral blood of five normal donors and their T cell receptor (TCR) alpha and beta chains were examined. Random cloning of TCR alpha chains by single-sided polymerase chain reaction (PCR) amplification identified an invariant rearrangement between V alpha 24 and J alpha Q, with no N region diversity, which was expressed preferentially by DN alpha/beta T cells from all donors. Random cloning also identified a precise V alpha 7.2-J alpha (IGRJa14) rearrangement, with two variable amino acids encoded in the V-J junction, which was enriched in the DN alpha/beta T cell preparations from some, but not all, donors. Analysis of TCR beta chains by quantitative PCR amplification demonstrated that the expression of four V beta gene families, V beta 2, 8, 11, and 13, was markedly increased in these DN alpha/beta T cell preparations. The expression of particular TCRs by DN alpha/beta T cells from multiple donors indicates that these cells, or at least a subpopulation of cells with this phenotype, recognize a limited spectrum of antigens and suggests that they may use nonpolymorphic antigen-presenting molecules. PMID:8391057

  4. Tcr-like antibody drug conjugates mediate killing of tumor cells with low peptide/hla targets.

    PubMed

    Lowe, Devin B; Bivens, Camille K; Mobley, Alexis S; Herrera, Christian E; McCormick, Amanda L; Wichner, Timea; Sabnani, Manoj K; Wood, Laurence M; Weidanz, Jon A

    2017-03-08

    The currently marketed antibody-drug conjugates (ADC) destabilize microtubule assembly in cancer cells and initiate apoptosis in patients. However, few tumor antigens (TA) are expressed at high densities on cancer lesions, potentially minimizing the therapeutic index of current ADC regimens. The peptide/human leukocyte antigen (HLA) complex can be specifically targeted by therapeutic antibodies (designated T cell receptor [TCR]-like antibodies) and adequately distinguish malignant cells, but has not been the focus of ADC development. We analyzed the killing potential of TCR-like ADCs when cross-linked to the DNA alkylating compound duocarmycin. Our data comprise proof-of-principle results that TCR-like ADCs mediate potent tumor cytotoxicity, particularly under common scenarios of low TA/HLA density, and support their continued development alongside agents that disrupt DNA replication. Additionally, TCR-like antibody ligand binding appears to play an important role in ADC functionality and should be addressed during therapy development to avoid binding patterns that negate ADC killing efficacy.

  5. Unique ζ-chain motifs mediate a direct TCR-actin linkage critical for immunological synapse formation and T-cell activation.

    PubMed

    Klieger, Yair; Almogi-Hazan, Osnat; Ish-Shalom, Eliran; Pato, Aviad; Pauker, Maor H; Barda-Saad, Mira; Wang, Lynn; Baniyash, Michal

    2014-01-01

    TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance.

  6. IRF2BP2 transcriptional repressor restrains naive CD4 T cell activation and clonal expansion induced by TCR triggering.

    PubMed

    Sécca, Cristiane; Faget, Douglas V; Hanschke, Steffi C; Carneiro, Mayra S; Bonamino, Martin H; de-Araujo-Souza, Patricia S; Viola, João P B

    2016-11-01

    CD4 T cell activation and differentiation mechanisms constitute a complex and intricate signaling network involving several regulatory proteins. IRF2BP2 is a transcriptional repressor that is involved in gene-expression regulation in very diverse biologic contexts. Information regarding the IRF2BP2 regulatory function in CD4 T lymphocytes is very limited and suggests a role for this protein in repressing the expression of different cytokine genes. Here, we showed that Irf2bp2 gene expression was decreased in CD4 T cells upon activation. To investigate the possible regulatory roles for IRF2BP2 in CD4 T cell functions, this protein was ectopically expressed in murine primary-activated CD4 T lymphocytes through retroviral transduction. Interestingly, ectopic expression of IRF2BP2 led to a reduction in CD25 expression and STAT5 phosphorylation, along with an impaired proliferative capacity. The CD69 expression was also diminished in IRF2BP2-overexpressing cells, whereas CD44 and CD62L levels were not altered. In vivo, transferred, IRF2BP2-overexpressing, transduced cells displayed an impaired expansion capacity compared with controls. Furthermore, overexpression of IRF2BP2 in differentiated Th cells resulted in slightly reduced IL-4 and pro-TGF-β production in Th2 and iTregs but had no effect on IFN-γ or IL-17 expression in Th1 and Th17 cells, respectively. Taken together, our data suggest a role for IRF2BP2 in regulating CD4 T cell activation by repressing proliferation and the expression of CD25 and CD69 induced by TCR stimuli.

  7. Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR

    PubMed Central

    Munson, Daniel J.; Egelston, Colt A.; Chiotti, Kami E.; Parra, Zuly E.; Bruno, Tullia C.; Moore, Brandon L.; Nakano, Taizo A.; Simons, Diana L.; Jimenez, Grecia; Yim, John H.; Rozanov, Dmitri V.; Falta, Michael T.; Fontenot, Andrew P.; Reynolds, Paul R.; Leach, Sonia M.; Borges, Virginia F.; Kappler, John W.; Spellman, Paul T.; Slansky, Jill E.

    2016-01-01

    Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha–beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients’ tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer. PMID:27307436

  8. Sensing of cell stress by human γδ TCR-dependent recognition of annexin A2

    PubMed Central

    Pappalardo, Angela; Kaminski, Hannah; Willcox, Carrie R.; Pitard, Vincent; Netzer, Sonia; Khairallah, Camille; Lomenech, Anne-Marie; Harly, Christelle; Bonneville, Marc; Moreau, Jean-François; Scotet, Emmanuel; Willcox, Benjamin E.; Faustin, Benjamin; Déchanet-Merville, Julie

    2017-01-01

    Human γδ T cells comprise a first line of defense through T-cell receptor (TCR) recognition of stressed cells. However, the molecular determinants and stress pathways involved in this recognition are largely unknown. Here we show that exposure of tumor cells to various stress situations led to tumor cell recognition by a Vγ8Vδ3 TCR. Using a strategy that we previously developed to identify antigenic ligands of γδ TCRs, annexin A2 was identified as the direct ligand of Vγ8Vδ3 TCR, and was found to be expressed on tumor cells upon the stress situations tested in a reactive oxygen species-dependent manner. Moreover, purified annexin A2 was able to stimulate the proliferation of a Vδ2neg γδ T-cell subset within peripheral blood mononuclear cells and other annexin A2-specific Vδ2neg γδ T-cell clones could be derived from peripheral blood mononuclear cells. We thus propose membrane exposure of annexin A2 as an oxidative stress signal for some Vδ2neg γδ T cells that could be involved in an adaptive stress surveillance. PMID:28270598

  9. T cell expansion is the limiting factor of virus control in mice with attenuated TCR signaling: implications for human immunodeficiency.

    PubMed

    Hillen, Kristina M; Gather, Ruth; Enders, Anselm; Pircher, Hanspeter; Aichele, Peter; Fisch, Paul; Blumenthal, Britta; Schamel, Wolfgang W; Straub, Tobias; Goodnow, Christopher C; Ehl, Stephan

    2015-03-15

    Defining the minimal thresholds for effective antiviral T cell immunity is important for clinical decisions in immunodeficient patients. TCR signaling is critical for T cell development, activation, and effector functions. In this article, we analyzed which of these TCR-mediated processes is limiting for antiviral immunity in a mouse strain with reduced expression of SLP-76 (twp mice). Despite severe T cell activation defects in vitro, twp mice generated a normal proportion of antiviral effector T cells postinfection with lymphocytic choriomeningitis virus (LCMV). Twp CD8(+) T cells showed impaired polyfunctional cytokine production, whereas cytotoxicity as the crucial antiviral effector function for LCMV control was normal. The main limiting factor in the antiviral response of twp mice was impaired T cell proliferation and survival, leading to a 5- to 10-fold reduction of antiviral T cells at the peak of the immune response. This was still sufficient to control infection with the LCMV Armstrong strain, but the more rapidly replicating LCMV-WE induced T cell exhaustion and viral persistence. Thus, under conditions of impaired TCR signaling, reduced T cell expansion was the limiting factor in antiviral immunity. These findings have implications for understanding antiviral immunity in patients with T cell deficiencies.

  10. Activation of human naïve Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters.

    PubMed

    Villanueva-Cabello, Tania M; Mollicone, Rosella; Cruz-Muñoz, Mario E; López-Guerrero, Delia V; Martínez-Duncker, Iván

    2015-12-01

    CD4+ T helper lymphocytes (Th) orchestrate the immune response after their activation by antigen-presenting cells. Activation of naïve Th cells is reported to generate the reduction in surface epitopes of sialic acid (Sia) in α2,3 and α2,6 linkages. In this work, we report that in spite of this glycophenotype, anti-CD3/anti-CD28-activated purified human naïve Th cells show a significant increase in surface Sia, as assessed by metabolic labeling, compared with resting naïve Th cells, suggesting an increased flux of Sia toward Siaα2,8 glycoconjugates. To understand this increase as a result of ganglioside up-regulation, we observed that very early after activation, human naïve Th cells show an increased expression in surface GD3 and neoexpression of surface GD2 gangliosides, the latter clustering with the T cell receptor (TCR). Also, we report that in contrast to GM2/GD2 synthase null mice, lentiviral vector-mediated silencing of the GM2/GD2 synthase in activated human naïve Th cells reduced efficient TCR clustering and downstream signaling, as assessed by proliferation assays and IL-2 and IL-2R expression, pointing to an important role of this enzyme in activation of human naive Th cells.

  11. Engagement of the T-cell receptor during positive selection in the thymus down-regulates RAG-1 expression.

    PubMed Central

    Brändle, D; Müller, C; Rülicke, T; Hengartner, H; Pircher, H

    1992-01-01

    We have examined the expression of the recombination activating gene RAG-1 by in situ hybridization to thymi from mice bearing transgenes for the T-cell receptor (TCR) alpha chain, TCR beta chain, or both TCR alpha and beta chains. RAG-1 transcription was found in the thymic cortex of transgenic mice carrying a single TCR alpha- or TCR beta-chain transgene, comparable to normal mice. However, RAG-1 transcription was strikingly reduced in the thymic cortex from transgenic mice carrying both TCR alpha- and beta-chain genes and expressing major histocompatibility complex (MHC) class I (H-2b) molecules necessary for positive selection of the transgenic TCR. In contrast, thymi of transgenic mice also carrying both TCR alpha- and beta-chain genes but expressing MHC molecules (H-2d) that did not positively select the transgenic TCR displayed high levels of RAG-1 transcription. The low thymic RAG-1 expression coincided with high transgenic TCR alpha-chain surface expression and with inhibition of endogenous TCR alpha-chain rearrangement. Our findings suggest that binding of the TCR to self MHC molecules during positive selection down-regulates RAG-1 transcription in cortical thymocytes and thereby prevents further TCR alpha-chain rearrangements. Images PMID:1329099

  12. Accumulation of serial forces on TCR and CD8 frequently applied by agonist antigenic peptides embedded in MHC molecules triggers calcium in T cells.

    PubMed

    Pryshchep, Sergey; Zarnitsyna, Veronika I; Hong, Jinsung; Evavold, Brian D; Zhu, Cheng

    2014-07-01

    T cell activation by Ag is one of the key events in adaptive immunity. It is triggered by interactions of the TCR and coreceptor (CD8 or CD4) with antigenic peptides embedded in MHC (pMHC) molecules expressed on APCs. The mechanism of how signal is initiated remains unclear. In this article, we complement our two-dimensional kinetic analysis of TCR-pMHC-CD8 interaction with concurrent calcium imaging to examine how ligand engagement of TCR with and without the coengagement of CD8 initiates signaling. We found that accumulation of frequently applied forces on the TCR via agonist pMHC triggered calcium, which was further enhanced by CD8 cooperative binding. Prolonging the intermission between sequential force applications impaired calcium signals. Our data support a model where rapid accumulation of serial forces on TCR-pMHC-CD8 bonds triggers calcium in T cells.

  13. An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

    PubMed Central

    Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

    2016-01-01

    Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells. PMID:27028870

  14. An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells.

    PubMed

    Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

    2016-04-19

    Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells.

  15. Human TCR-αβ+ CD4− CD8− T Cells Can Derive from CD8+ T Cells and Display an Inflammatory Effector Phenotype1

    PubMed Central

    Crispín, José C.; Tsokos, George C.

    2010-01-01

    The origin and function of human double negative (DN) TCR-αβ+ T cells is unknown. They are thought to contribute to the pathogenesis of systemic lupus erythematosus because they expand and accumulate in inflamed organs. In this study, we provide evidence that human TCR-αβ+ CD4− CD8− DN T cells can derive from activated CD8+ T cells. Freshly isolated TCR-αβ+ DN T cells display a distinct gene expression and cytokine production profile. DN cells isolated from peripheral blood as well as DN cells derived in vitro from CD8+ T cells produce a defined array of proinflammatory mediators that includes IL-1β, IL-17, IFN-γ, CXCL3, and CXCL2. These results indicate that, upon activation, CD8+ T cells have the capacity to acquire a distinct phenotype that grants them inflammatory capacity. PMID:19734235

  16. TCR contact residue hydrophobicity is a hallmark of immunogenic CD8+ T cell epitopes

    PubMed Central

    Chowell, Diego; Krishna, Sri; Cocita, Clément; Shu, Jack; Tan, Xuefang; Greenberg, Philip D.; Klavinskis, Linda S.; Blattman, Joseph N.; Anderson, Karen S.

    2015-01-01

    Despite the availability of major histocompatibility complex (MHC)-binding peptide prediction algorithms, the development of T-cell vaccines against pathogen and tumor antigens remains challenged by inefficient identification of immunogenic epitopes. CD8+ T cells must distinguish immunogenic epitopes from nonimmunogenic self peptides to respond effectively against an antigen without endangering the viability of the host. Because this discrimination is fundamental to our understanding of immune recognition and critical for rational vaccine design, we interrogated the biochemical properties of 9,888 MHC class I peptides. We identified a strong bias toward hydrophobic amino acids at T-cell receptor contact residues within immunogenic epitopes of MHC allomorphs, which permitted us to develop and train a hydrophobicity-based artificial neural network (ANN-Hydro) to predict immunogenic epitopes. The immunogenicity model was validated in a blinded in vivo overlapping epitope discovery study of 364 peptides from three HIV-1 Gag protein variants. Applying the ANN-Hydro model on existing peptide-MHC algorithms consistently reduced the number of candidate peptides across multiple antigens and may provide a correlate with immunodominance. Hydrophobicity of TCR contact residues is a hallmark of immunogenic epitopes and marks a step toward eliminating the need for empirical epitope testing for vaccine development. PMID:25831525

  17. The Vα14 invariant natural killer T cell TCR forces microbial glycolipids and CD1d into a conserved binding mode

    PubMed Central

    Li, Yali; Girardi, Enrico; Wang, Jing; Yu, Esther Dawen; Painter, Gavin F.; Kronenberg, Mitchell

    2010-01-01

    Invariant natural killer T cells (iNKT cells) rapidly produce effector cytokines. In this study, we report the first crystal structures of the iNKT cell T cell receptor (TCR) bound to two natural, microbial glycolipids presented by CD1d. Binding of the TCR induced CDR3-α–dependent structural changes in the F′ roof of CD1d; these changes resemble those occurring in the absence of TCR engagement when the highly potent synthetic antigen α-galactosylceramide (α-GalCer) binds CD1d. Furthermore, in the Borrelia burgdorferi α–galactosyl diacylglycerol–CD1d complex, TCR binding caused a marked repositioning of the galactose sugar into an orientation that closely resembles α-GalCer. The TCR-dependent reorientation of the sugar, together with the induced CD1d fit, may explain the weaker potency of the microbial antigens compared with α-GalCer. We propose that the TCR of iNKT cells binds with a conserved footprint onto CD1d, regardless of the bound glycolipid antigen, and that for microbial antigens this unique binding mode requires TCR-initiated conformational changes. PMID:20921281

  18. Clonal selection in the human Vδ1 T cell repertoire indicates γδ TCR-dependent adaptive immune surveillance

    PubMed Central

    Davey, Martin S.; Willcox, Carrie R.; Joyce, Stephen P.; Ladell, Kristin; Kasatskaya, Sofya A.; McLaren, James E.; Hunter, Stuart; Salim, Mahboob; Mohammed, Fiyaz; Price, David A.; Chudakov, Dmitriy M.; Willcox, Benjamin E.

    2017-01-01

    γδ T cells are considered to be innate-like lymphocytes that respond rapidly to stress without clonal selection and differentiation. Here we use next-generation sequencing to probe how this paradigm relates to human Vδ2neg T cells, implicated in responses to viral infection and cancer. The prevalent Vδ1 T cell receptor (TCR) repertoire is private and initially unfocused in cord blood, typically becoming strongly focused on a few high-frequency clonotypes by adulthood. Clonal expansions have differentiated from a naive to effector phenotype associated with CD27 downregulation, retaining proliferative capacity and TCR sensitivity, displaying increased cytotoxic markers and altered homing capabilities, and remaining relatively stable over time. Contrastingly, Vδ2+ T cells express semi-invariant TCRs, which are present at birth and shared between individuals. Human Vδ1+ T cells have therefore evolved a distinct biology from the Vδ2+ subset, involving a central, personalized role for the γδ TCR in directing a highly adaptive yet unconventional form of immune surveillance. PMID:28248310

  19. Lck, Membrane Microdomains, and TCR Triggering Machinery: Defining the New Rules of Engagement

    PubMed Central

    Filipp, Dominik; Ballek, Ondrej; Manning, Jasper

    2012-01-01

    In spite of a comprehensive understanding of the schematics of T cell receptor (TCR) signaling, the mechanisms regulating compartmentalization of signaling molecules, their transient interactions, and rearrangement of membrane structures initiated upon TCR engagement remain an outstanding problem. These gaps in our knowledge are exemplified by recent data demonstrating that TCR triggering is largely dependent on a preactivated pool of Lck concentrated in T cells in a specific type of membrane microdomains. Our current model posits that in resting T cells all critical components of TCR triggering machinery including TCR/CD3, Lck, Fyn, CD45, PAG, and LAT are associated with distinct types of lipid-based microdomains which represent the smallest structural and functional units of membrane confinement able to negatively control enzymatic activities and substrate availability that is required for the initiation of TCR signaling. In addition, the microdomains based segregation spatially limits the interaction of components of TCR triggering machinery prior to the onset of TCR signaling and allows their rapid communication and signal amplification after TCR engagement, via the process of their coalescence. Microdomains mediated compartmentalization thus represents an essential membrane organizing principle in resting T cells. The integration of these structural and functional aspects of signaling into a unified model of TCR triggering will require a deeper understanding of membrane biology, novel interdisciplinary approaches and the generation of specific reagents. We believe that the fully integrated model of TCR signaling must be based on membrane structural network which provides a proper environment for regulatory processes controlling TCR triggering. PMID:22701458

  20. A Molecular Switch Abrogates Glycoprotein 100 (gp100) T-cell Receptor (TCR) Targeting of a Human Melanoma Antigen*

    PubMed Central

    Bianchi, Valentina; Bulek, Anna; Fuller, Anna; Lloyd, Angharad; Attaf, Meriem; Rizkallah, Pierre J.; Dolton, Garry; Sewell, Andrew K.; Cole, David K.

    2016-01-01

    Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280–288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination. PMID:26917722

  1. Nck Binds to the T Cell Antigen Receptor Using Its SH3.1 and SH2 Domains in a Cooperative Manner, Promoting TCR Functioning.

    PubMed

    Paensuwan, Pussadee; Hartl, Frederike A; Yousefi, O Sascha; Ngoenkam, Jatuporn; Wipa, Piyamaporn; Beck-Garcia, Esmeralda; Dopfer, Elaine P; Khamsri, Boonruang; Sanguansermsri, Donruedee; Minguet, Susana; Schamel, Wolfgang W; Pongcharoen, Sutatip

    2016-01-01

    Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning.

  2. Regulator T cells: specific for antigen and/or antigen receptors?

    PubMed

    Rubin, B; de Durana, Y Diaz; Li, N; Sercarz, E E

    2003-05-01

    Adaptive immune responses are regulated by many different molecular and cellular effectors. Regulator T cells are coming to their rights again, and these T cells seem to have ordinary alpha/beta T-cell receptors (TCRs) and to develop in the thymus. Autoimmune responses are tightly regulated by such regulatory T cells, a phenomenon which is beneficial to the host in autoimmune situations. However, the regulation of autoimmune responses to tumour cells is harmful to the host, as this regulation delays the defence against the outgrowth of neoplastic cells. In the present review, we discuss whether regulatory T cells are specific for antigen and/or for antigen receptors. Our interest in these phenomena comes from the findings that T cells produce many more TCR-alpha and TCR-beta chains than are necessary for surface membrane expression of TCR-alphabeta heterodimers with CD3 complexes. Excess TCR chains are degraded by the proteasomes, and TCR peptides thus become available to the assembly pathway of major histocompatibility complex class I molecules. Consequently, do T cells express two different identification markers on the cell membrane, the TCR-alphabeta clonotype for recognition by B-cell receptors and clonotypic TCR-alphabeta peptides for recognition by T cells?

  3. Immune selection of tumor cells in TCR β-chain transgenic mice.

    PubMed

    Silaeva, Yulia Yu; Grinenko, Tatyana S; Vagida, Murad S; Kalinina, Anastasia A; Khromykh, Ludmila M; Kazansky, Dmitry B

    2014-10-01

    The concept of immunological surveillance implies that immunogenic variants of tumor cells arising in the organism can be recognized by the immune system. Tumor progression is provided by somatic evolution of tumor cells under the pressure of the immune system. The loss of MHC Class I molecules on the surface of tumor cells is one of the most known outcomes of immune selection. This study developed a model of immune selection based on the immune response of TCR 1d1 single β-chain transgenic B10.D2(R101) (K(d)I(d)D(b)) mice to allogeneic EL4 (H-2(b)) thymoma cells. In wild-type B10.D2(R101) mice, immunization with EL4 cells induced a vigorous CTL response targeted to the H-2K(b) molecule and results in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in mixed-lymphocyte response assays and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, CD8(+) T-lymphocytes with endogenous β-chains accumulated predominantly in the spleen of transgenic mice and only a small part of the T-lymphocytes expressing transgenic β-chains became CD8(+)CD44(+)CD62L(-) effectors. Then, instead of a full elimination of tumor cells as in wild-type mice, a reproducible prolonged equilibrium phase and subsequent escape was observed in transgenic mice that resulted in death of 90% of the mice in 40-60 days after grafting. Prolonged exposure of tumor cells to the pressure of the immune system in transgenic mice in vivo resulted in a stable loss of H-2K(b) molecules on the EL4 cell surface. Genetic manipulation of the T-lymphocyte repertoire was sufficient to reproduce the classic pattern of interactions between tumor cells and the immune system, usually observed in reliable syngeneic models of anti-tumor immunity. This newly-developed model could be used in further studies of immunoregulatory circuits common for transplantational and anti-tumor immune responses.

  4. Regulatory T cells play a role in T-cell receptor CDR2 peptide regulation of experimental autoimmune encephalomyelitis.

    PubMed

    Buenafe, Abigail C; Andrew, Shayne; Offner, Halina; Vandenbark, Arthur A

    2012-02-01

    Eliciting T-cell receptor (TCR) -specific responsiveness has been known to provide an effective autoregulatory mechanism for limiting inflammation mediated by T effector cells. Our previous use of TCR peptides derived from the CDR3 regions of a pathogenic TCR effectively reversed ongoing experimental autoimmune encephalomyelitis (EAE) in a humanized TCR transgenic model. In this study, we use the TCR BV8S2 CDR2 peptide in the non-transgenic C57BL/6 EAE model to down-regulate the heterogeneous TCR BV8S2(+)  MOG-35-55-specific pathogenic T-cell population and demonstrate successful treatment of EAE after disease onset. Suppression of disease was associated with reduced MOG-35-55-specific and non-specific T-cell production of interleukin-17a and interferon-γ in the central nervous system, as well as reduced numbers of CD4(+) and Foxp3(+) T cells in the central nervous system. With the use of Foxp3-GFP and Foxp3 conditional knockout mice, we demonstrate that the TCR CDR2 peptide treatment effect is dependent on the presence of Foxp3(+) regulatory T cells and that regulatory T cell numbers are significantly expanded in the periphery of treated mice. Hence, TCR CDR2 peptide therapy is effective in regulating heterogeneous, pathogenic T-cell populations through the activity of the Foxp3(+) regulatory T cell population.

  5. TCR-induced, PKC-θ-mediated NF-κB Activation Is Regulated by a Caspase-8-Caspase-9-Caspase-3 Cascade

    PubMed Central

    Zhao, Yixia; Lei, Minxiang; Wang, Zhaoyuan; Qiao, Guilin; Yang, Tianlun; Zhang, Jian

    2014-01-01

    It has been documented that caspase-8, a central player in apoptosis, is also crucial for TCR-mediated NF-κB activation. However, whether other caspases are also involved this process is unknown. In this report, we showed that in addition to caspase-8, caspase-9 is required for TCR-mediated NF-κB activation. Caspase-9 induces activation of PKC-θ, phosphorylation of Bcl10 and NF-κB activation in a caspase-3-dependent manner, but it appears that Bcl10 phosphorylation is uncoupled from NF-κB activation. Furthermore, caspase-8 lies upstream of caspase-9 during T cell activation. Therefore, TCR ligation elicits a caspase cascade involving caspase-8, caspase-9 and caspase-3 which initiates PKC-θ-dependent pathway leading to NF-κB activation and PKC-θ-independent Bcl10 phosphorylation which limits NF-kB activity. PMID:24924627

  6. Kinetics of early TCR signaling regulate the pathway of lytic granule delivery to the secretory domain

    PubMed Central

    Beal, Allison M.; Anikeeva, Nadia; Varma, Rajat; Cameron, Thomas O.; Vasiliver-Shamis, Gaia; Norris, Philip J.; Dustin, Michael L.; Sykulev, Yuri

    2009-01-01

    SUMMARY Cytolytic granule mediated killing of virus-infected cells is an essential function of cytotoxic T lymphocytes. Analysis of lytic granule delivery shows that the granules can take long or short paths to the secretory domain where they are released. Both paths utilize the same intracellular molecular events, which have different spatial and temporal arrangements in each path and are regulated by the kinetics of downstream Ca2+ mediated signaling. Rapid and robust signaling causes swift granule concentration near the MTOC and subsequent delivery by the polarized MTOC directly to the secretory domain - the shortest and fastest path. Indolent signaling leads to late recruitment of granules that move along microtubules to the periphery of the synapse and then move tangentially to fuse at the outer edge of the secretory domain - a longer path. The short pathway is associated with faster granule release and more efficient killing than the long pathway. PMID:19833088

  7. [Development of Tax-redirected T-cell immunotherapy using TCR gene transduction in patients with ATL].

    PubMed

    Tanaka, Yukie; Kanda, Yoshinobu

    2015-07-01

    ATL is an aggressive T-cell malignancy caused by HTLV-1 virus infection. Tax, which is the most important regulatory protein of HTLV-1, is associated with aggressive proliferation of host cells and is also a major target antigen for CD8⁺ cytotoxic T-cells (CTLs). Recently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) has proven effective for ATL, and donor-derived Tax-specific CTL might contribute to graft-versus-ATL effects in some recipients who maintained complete remission after allo-HSCT. We, for the first time, analyzed the Tax-specific T-cell receptor (TCR) repertoire, phenotypes and functions of Tax-specific CTLs at single-cell levels in HLA-A24⁺ ATL patients who underwent allo-HSCT. We found that 1) a particular amino acid sequence motif (PDR) in the CDR3 region of TCR-β was conserved in different patients and also within the same patient before and after allo-HSCT, and 2) the PDR⁺ Tax-specific CTL clone selectively expanded in ATL long-term survivors as less-differentiated effector memory CTLs. Actually, the PDR⁺ CTL showed not only strong binding activity for the Tax-tetramer but also strong killing activity against patients' HTLV-1-infected T-cells without any reaction against normal cells. We are presently evaluating the killing activities of PDR⁺ TCR-transduced T-cells against Tax in immunodeficient mice, with the aim of developing a new immunotherapy for ATL.

  8. Modulation of TCR responsiveness by the Grb2-family adaptor, Gads.

    PubMed

    Lugassy, Jennie; Corso, Jasmin; Beach, Dvora; Petrik, Thomas; Oellerich, Thomas; Urlaub, Henning; Yablonski, Deborah

    2015-01-01

    T cell antigen receptor (TCR) signaling depends on three interacting adaptor proteins: SLP-76, Gads, and LAT. Their mechanisms of signaling have been extensively explored, with the aid of fortuitously isolated LAT- and SLP-76-deficient T cell lines, but no such tools were available for Gads, a Grb2-family adaptor that bridges the TCR-inducible interaction between SLP-76 and LAT. TALEN-directed genome editing was applied to disrupt the first coding exon of human Gads in the Jurkat T cell line. Gads was dispensable for TCR-induced phosphorylation of SLP-76, but was a dose-dependent amplifier of TCR-induced CD69 expression. Gads conferred responsiveness to weak TCR stimuli, leading to PLC-γ1 phosphorylation and calcium flux. TALEN-derived, Gads-deficient T cell lines provide a uniquely tractable genetic platform for exploring its regulatory features, such as Gads phosphorylation at T262, which we observed by mass spectrometry. Upon mutation of this site, TCR responsiveness and sensitivity to weak TCR stimuli were increased. This study demonstrates the feasibility of TALEN-based reverse genetics in Jurkat T cells, while enriching our understanding of Gads as a regulated modulator of TCR sensitivity.

  9. TCR Affinity Associated with Functional Differences between Dominant and Subdominant SIV Epitope-Specific CD8+ T Cells in Mamu-A*01+ Rhesus Monkeys

    PubMed Central

    Osuna, Christa E.; Gonzalez, Ana Maria; Chang, Hsun-Hsien; Hung, Amy Shi; Ehlinger, Elizabeth; Anasti, Kara; Alam, S. Munir; Letvin, Norman L.

    2014-01-01

    Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design. PMID:24743648

  10. TCR affinity associated with functional differences between dominant and subdominant SIV epitope-specific CD8+ T cells in Mamu-A*01+ rhesus monkeys.

    PubMed

    Osuna, Christa E; Gonzalez, Ana Maria; Chang, Hsun-Hsien; Hung, Amy Shi; Ehlinger, Elizabeth; Anasti, Kara; Alam, S Munir; Letvin, Norman L

    2014-04-01

    Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design.

  11. Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes

    PubMed Central

    1996-01-01

    The Src-family and Syk/ZAP-70 family of protein tyrosine kinases (PTK) are required for T cell receptor (TCR) functions. We provide evidence that the Src-family PTK Lck is responsible for regulating the constitutive tyrosine phosphorylation of the TCR zeta subunit in murine thymocytes. Moreover, ligation of the TCR expressed on thymocytes from Lck-deficient mice largely failed to induce the phosphorylation of TCR- zeta, CD3 epsilon, or ZAP-70. In contrast, we find that the TCR-zeta subunit is weakly constitutively tyrosine phosphorylated in peripheral T cells isolated from Lck-null mice. These data suggest that Lck has a functional role in regulation of TCR signal transduction in thymocytes. In peripheral T cells, other Src-family PTKs such as Fyn may partially compensate for the absence of Lck. PMID:8642247

  12. NY-ESO-1 specific TCR engineered T-cells mediate sustained antigen-specific antitumor effects in myeloma

    PubMed Central

    Goloubeva, Olga; Vogl, Dan T.; Lacey, Simon F.; Badros, Ashraf Z.; Garfall, Alfred; Weiss, Brendan; Finklestein, Jeffrey; Kulikovskaya, Irina; Sinha, Sanjoy K.; Kronsberg, Shari; Gupta, Minnal; Bond, Sarah; Melchiori, Luca; Brewer, Joanna E.; Bennett, Alan D.; Gerry, Andrew B.; Pumphrey, Nicholas J.; Williams, Daniel; Tayton-Martin, Helen K.; Ribeiro, Lilliam; Holdich, Tom; Yanovich, Saul; Hardy, Nancy; Yared, Jean; Kerr, Naseem; Philip, Sunita; Westphal, Sandra; Siegel, Don L.; Levine, Bruce L.; Jakobsen, Bent K.; Kalos, Michael; June, Carl H.

    2015-01-01

    Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Herein we report results of a phase I/II trial to evaluate the safety and activity of autologous T-cells engineered to express an affinity-enhanced T-cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4×109 engineered T cells two days after autologous stem cell transplant (ASCT). Infusions were well-tolerated without clinically apparent cytokine release syndrome, despite high IL-6 levels. Engineered T-cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, consistent with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression free survival of 19.1 months. NY-ESO-1/LAGE-1 TCR-engineered T-cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma. PMID:26193344

  13. Preliminary study of the clonal characteristics of the TCR BV subfamilies in T cells in the peripheral blood from patients with uveitis.

    PubMed

    Zou, H-Y; Yu, W-Z; Zhang, Q; Yang, H-C; Huang, H-Y; Jiao, M

    2014-02-28

    The aim of this study was to investigate the characteristics and polymorphisms of the T-cell receptor BV complementarity-determining region 3 (TCR BV CDR3) gene in peripheral blood mononuclear cells (PBMCs) from patients with uveitis to provide an experimental basis for studying the pathogenesis of this disease. RT-PCR amplification of 26 subfamilies of the TCR BV CDR3 gene and immune spectratyping analysis were used to study the pedigree drift of TCR BV CDR3 in PBMCs from the uveitis patients. The following results were obtained: 1) the vast majority of the TCR BV CDR3 spectra in PBMCs in 5 healthy subjects fit the normal (or Gaussian) distribution. The distributions of the TCR BV CDR3 spectra in 4 patients with uveitis were non-normal and showed an abnormal peak including a widowed peak trend, a partial peak, and an irregular abnormal peak. 2) In the 26 TCR BV subfamilies, the abnormal peak frequency was different in the various subfamilies. The BV2 and BV17 (both 3/4) subfamilies had higher frequencies of the non-normally distributed abnormal peak. The BV5.2, BV6, BV15, and BV18 subfamilies showed no abnormal peaks. 3) TCR BV2 and BV17 yielded an abnormal peak in 3 HLA-B27-negative patients; however, no such abnormalities were detected in HLA-B27-positive patients. The abnormal expression of some TCR BV subfamilies in PBMCs from patients with uveitis may be associated with the immune pathogenesis of the disease. Our study provides the basis for further investigations into the pathogenesis of uveitis.

  14. Tripartite motif-containing protein 30 modulates TCR-activated proliferation and effector functions in CD4+ T cells.

    PubMed

    Choi, Un Yung; Hur, Ji Yeon; Lee, Myeong Sup; Zhang, Quanri; Choi, Won Young; Kim, Lark Kyun; Lee, Wook-Bin; Oh, Goo Taeg; Kim, Young-Joon

    2014-01-01

    To avoid excessive activation, immune signals are tightly controlled by diverse inhibitory proteins. TRIM30, a tripartite motif (TRIM)-containing protein is one of such inhibitors known to function in macrophages. To define the roles of TRIM30, we generated Trim30 knockout (Trim30-/-) mice. Trim30 deletion caused no major developmental defects in any organs, nor showed any discernable defect in the activation of macrophages. But, Trim30-/- mice showed increased CD4/CD8 ratio when aged and Trim30-/- CD4+ T cells exhibited an abnormal response upon TCR activation, in particular in the absence of a costimulatory signal. Adoptive transfer of wild-type and Trim30-/- CD4+ T cells together into lymphopenic hosts confirmed higher proliferation of the Trim30-/- CD4+ T cells in vivo. Despite the enhanced proliferation, Trim30-/- T cells showed decreased levels of NF-κB activation and IL-2 production compared to wild-type cells. These results indicate a distinct requirement for TRIM30 in modulation of NF-κB activation and cell proliferation induced by TCR stimulation.

  15. MHC-I restricted Melanoma Antigen Specific TCR Engineered Human CD4+ T Cells Exhibit Multifunctional Effector and Helper Responses, In Vitro

    PubMed Central

    Ray, Swagatam; Chhabra, Arvind; Chakraborty, Nitya G.; Hegde, Upendra; Dorsky, David I.; Chodon, Thinle; von Euw, Erika; Comin-Anduix, Begonya; Koya, Richard C.; Ribas, Antoni; Economou, James S.; Rosenberg, Steven A.; Mukherji, Bijay

    2010-01-01

    MHC class 1-restricted human melanoma epitope MART-127–35 specific TCR engineered CD4+CD25− T cells synthesize Th1 type cytokines and exhibit cytolytic effector function upon cognate stimulation. A detailed characterization of such TCR-engineered CD4+CD25− T cells now reveals that they are multifunctional. For example, they undergo multiple rounds of division, synthesize cytokines (IFN-γ, TNF-α, IL-2, MIP1ß), lyse target cells, and “help” the expansion of the MART-127–35 specific CD8+ T cells when stimulated by the MART-127–35 peptide pulsed DC. Multiparametric analyses reveal that a single TCR-engineered CD4+ T cell can perform as many as five different functions. Nearly 100% MART-127–35 specific TCR expressing CD4+ T cells can be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR-transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies. PMID:20547105

  16. TCR-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac, and myosin IIA.

    PubMed

    Manes, Thomas D; Pober, Jordan S

    2013-04-01

    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 Ag 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. In this study, we report that TCR-driven TEM requires ZAP-70-dependent activation of a pathway involving Vav, Rac, and myosin IIA. Chemokine-driven TEM also uses 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.

  17. Revisiting the putative TCR Cα dimerization model through structural analysis.

    PubMed

    Wang, Jia-Huai; Reinherz, Ellis L

    2013-01-01

    Despite major advances in T cell receptor (TCR) biology and structure, how peptide-MHC complex (pMHC) ligands trigger αβ TCR activation remains unresolved. Two views exist. One model postulates that monomeric TCR-pMHC ligation events are sufficient while a second proposes that TCR-TCR dimerization in cis via Cα domain interaction plus pMHC binding is critical. We scrutinized 22 known TCR/pMHC complex crystal structures, and did not find any predicted molecular Cα-Cα contacts in these crystals that would allow for physiological TCR dimerization. Moreover, the presence of conserved glycan adducts on the outer face of the Cα domain preclude the hypothesized TCR dimerization through the Cα domain. Observed functional consequences of Cα mutations are likely indirect, with TCR microclusters at the immunological synapse driven by TCR transmembrane/cytoplasmic interactions via signaling molecules, scaffold proteins, and/or cytoskeletal elements.

  18. Selective phosphorylation of the Dlg1AB variant is critical for TCR-induced p38 activation and induction of proinflammatory cytokines in CD8+ T cells.

    PubMed

    Crocetti, Jillian; Silva, Oscar; Humphries, Lisa A; Tibbs, Michelle D; Miceli, M Carrie

    2014-09-15

    CD8(+) T cells respond to TCR stimulation by producing proinflammatory cytokines, and destroying infected or malignant cells through the production and release of cytotoxic granules. Scaffold protein Discs large homolog 1 (Dlg1) specifies TCR-dependent functions by channeling proximal signals toward the activation of p38-dependent proinflammatory cytokine gene expression and/or p38-independent cytotoxic granule release. Two Dlg1 variants are expressed in CD8(+) T cells via alternative splicing, Dlg1AB and Dlg1B, which have differing abilities coordinate TCR-dependent functions. Although both variants facilitate p38-independent cytotoxicity, only Dlg1AB coordinates p38-dependent proinflammatory cytokine expression. In this study, we identify TCR-induced Dlg1 tyrosine phosphorylation as a key regulatory step required for Dlg1AB-mediated p38-dependent functions, including proinflammatory cytokine expression. We find that Dlg1AB but not Dlg1B is tyrosine phosphorylated by proximal tyrosine kinase Lck in response to TCR stimulation. Furthermore, we identify Dlg1 tyrosine 222 (Y222) as a major site of Dlg1 phosphorylation required for TCR-triggered p38 activation and NFAT-dependent expression of proinflammatory cytokines, but not for p38-independent cytotoxicity. Taken together, our data support a model where TCR-induced phosphorylation of Dlg1 Y222 is a key point of control that endows Dlg1AB with the ability to coordinate p38 activation and proinflammatory cytokine production. We propose blocking Dlg1AB phosphorylation as a novel therapeutic target to specifically block proinflammatory cytokine production but not cytotoxicity.

  19. Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor-independent TCR signaling hub.

    PubMed

    Roncagalli, Romain; Hauri, Simon; Fiore, Fréderic; Liang, Yinming; Chen, Zhi; Sansoni, Amandine; Kanduri, Kartiek; Joly, Rachel; Malzac, Aurélie; Lähdesmäki, Harri; Lahesmaa, Riitta; Yamasaki, Sho; Saito, Takashi; Malissen, Marie; Aebersold, Ruedi; Gstaiger, Matthias; Malissen, Bernard

    2014-04-01

    T cell antigen receptor (TCR)-mediated activation of T cells requires the interaction of dozens of proteins. Here we used quantitative mass spectrometry and activated primary CD4(+) T cells from mice in which a tag for affinity purification was knocked into several genes to determine the composition and dynamics of multiprotein complexes that formed around the kinase Zap70 and the adaptors Lat and SLP-76. Most of the 112 high-confidence time-resolved protein interactions we observed were previously unknown. The surface receptor CD6 was able to initiate its own signaling pathway by recruiting SLP-76 and the guanine nucleotide-exchange factor Vav1 regardless of the presence of Lat. Our findings provide a more complete model of TCR signaling in which CD6 constitutes a signaling hub that contributes to the diversification of TCR signaling.

  20. Quantitative proteomic analysis of signalosome dynamics in primary T cells identifies the CD6 surface receptor as a Lat-independent TCR signaling hub

    PubMed Central

    Fiore, Fréderic; Liang, Yinming; Chen, Zhi; Sansoni, Amandine; Kanduri, Kartiek; Joly, Rachel; Malzac, Aurélie; Lähdesmäki, Harri; Lahesmaa, Riitta; Yamasaki, Sho; Saito, Takashi; Malissen, Marie; Aebersold, Ruedi; Gstaiger, Matthias; Malissen, Bernard

    2014-01-01

    T cell antigen receptor (TCR)-mediated T cell activation requires the interaction of dozens of proteins. We used quantitative mass spectrometry and activated primary CD4+ T cells from mice in which a tag for affinity purification was knocked into several genes to determine the composition and dynamics of multiprotein complexes forming around the kinase Zap70 and the adaptors Lat and SLP-76. Most of the 112 high confidence time-resolved protein interactions we observed were novel. The CD6 surface receptor was found capable of initiating its own signaling pathway by recruiting SLP-76 and Vav1, irrespective of the presence of Lat. Our findings provide a more complete model of TCR signaling in which CD6 constitutes a signaling hub contributing to TCR signal diversification. PMID:24584089

  1. Flow cytometry-based TCR-ligand Koff -rate assay for fast avidity screening of even very small antigen-specific T cell populations ex vivo.

    PubMed

    Nauerth, Magdalena; Stemberger, Christian; Mohr, Fabian; Weißbrich, Bianca; Schiemann, Matthias; Germeroth, Lothar; Busch, Dirk H

    2016-09-01

    High epitope-specific sensitivity of CD8(+) T cells is required for optimal immune protection against intracellular pathogens as well as certain malignancies. The quality of antigen recognition of CD8(+) T cells is usually described as "avidity" to its cognate peptide MHCI complex. T cell avidity is mainly dependent on the structural qualities of the T cell receptor (TCR), as convincingly demonstrated by recombinant TCR re-expression experiments. Based on reversible MHCI multimer staining and koff -rate measurements of monomeric peptide MHCI complexes, we recently established a microscopic assay for determining the structural avidity of individual CD8(+) T cells. Here we demonstrate that this assay can be adapted for rapid flow-cytometric avidity screening of epitope-specific T cell populations. Furthermore, we show that-in combination with conventional nonreversible MHCI multimer staining-even very small epitope-specific CD8(+) T cell populations can be analyzed directly ex vivo without the need for previous TCR cloning or T cell sorting. This simplified approach provides highly accurate mean TCR-ligand koff -rate values for poly- or oligoclonal T cell populations and is ideally suited for high-throughput applications in basic research as well as clinical settings. © 2016 International Society for Advancement of Cytometry.

  2. New Insights into How Trafficking Regulates T Cell Receptor Signaling

    PubMed Central

    Lou, Jieqiong; Rossy, Jérémie; Deng, Qiji; Pageon, Sophie V.; Gaus, Katharina

    2016-01-01

    There is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR) signaling. The trafficking molecules involved in lytic granule (LG) secretion in cytotoxic T lymphocytes (CTL) have been well-studied due to the immune disorder known as familial hemophagocytic lymphohistiocytosis (FHLH). However, the knowledge of trafficking machineries regulating the exocytosis of receptors and signaling molecules remains quite limited. In this review, we summarize the reported trafficking molecules involved in the transport of the TCR and downstream signaling molecules to the cell surface. By combining this information with the known knowledge of LG exocytosis and general exocytic trafficking machinery, we attempt to draw a more complete picture of how the TCR signaling network and exocytic trafficking matrix are interconnected to facilitate T cell activation. This also highlights how membrane compartmentalization facilitates the spatiotemporal organization of cellular responses that are essential for immune functions. PMID:27508206

  3. Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T cell response.

    PubMed

    Reichstetter, S; Ettinger, R A; Liu, A W; Gebe, J A; Nepom, G T; Kwok, W W

    2000-12-15

    The polyclonal nature of T cells expanding in an ongoing immune response results in a range of disparate affinities and activation potential. Recently developed human class II tetramers provide a means to analyze this diversity by direct characterization of the trimolecular TCR-peptide-MHC interaction in live cells. Two HSV-2 VP16(369-379)-specific, DQA1*0102/DQB1*0602 (DQ0602)-restricted T cell clones were compared by means of T cell proliferation assay and HLA-DQ0602 tetramer staining. These two clones were obtained from the same subject, but show different TCR gene usage. Clone 48 was 10-fold more sensitive to VP16(369-379) peptide stimulation than clone 5 as assayed by proliferation assays, correlating with differences in MHC tetramer binding. Clone 48 gave positive staining with the DQ0602/VP16(369-379) tetramer at either 23 or 37 degrees C. Weak staining was also observed at 4 degrees C. Clone 5 showed weaker staining compared with clone 48 at 37 degrees C, and no staining was observed at 23 degrees C or on ice. Receptor internalization was not required for positive staining. Competitive binding indicates that the cell surface TCR of clone 48 has higher affinity for the DQ0602/VP16(369-379) complex than clone 5. The higher binding affinity of clone 48 for the peptide-MHC complex also correlates with a slower dissociation rate compared with clone 5.

  4. Long-term Persistence of CD4+ but Rapid Disappearance of CD8+ T Cells Expressing an MHC Class I-restricted TCR of Nanomolar Affinity

    PubMed Central

    Engels, Boris; Chervin, Adam S; Sant, Andrea J; Kranz, David M; Schreiber, Hans

    2012-01-01

    Most T cells have T cell receptors (TCR) of micromolar affinity for peptide-major histocompatibility complex (MHC) ligands, but genetic engineering can generate TCRs of nanomolar affinity. The affinity of the TCR used, m33, for its cognate non-self peptide–MHC-I complex (SIYRYYGL-Kb) is 1,000-fold higher than of the wild-type TCR 2C. The affinity of m33 for the self-peptide dEV-8 on Kb is only twofold higher. Mouse CD8+ T cells transduced with an m33-encoding retrovirus showed binding of SIY-Kb and potent function in vitro, but in vivo these T cells disappeared within hours after transfer into syngeneic hosts without causing graft-versus-host disease (GVHD). Accordingly, in cases where such CD8-dependent self-reactivity might occur in human adoptive T cell therapies, our results show that a peripheral T-cell deletion mechanism could operate to avoid reactions with the host. In contrast to CD8+ T cells, we show that CD4+ T cells expressing m33 survived for months in vivo. Furthermore, the m33-transduced CD4+ T cells were able to mediate antigen-specific rejection of 6-day-old tumors. Together, we show that CD8+ T cell expressing a MHC class I-restricted high-affinity TCR were rapidly deleted whereas CD4+ T cells expressing the same TCR survived and provided function while being directed against a class I-restricted antigen. PMID:22233579

  5. TCR sequences and tissue distribution discriminate the subsets of naïve and activated/memory Treg cells in mice.

    PubMed

    Bergot, Anne-Sophie; Chaara, Wahiba; Ruggiero, Eliana; Mariotti-Ferrandiz, Encarnita; Dulauroy, Sophie; Schmidt, Manfred; von Kalle, Christof; Six, Adrien; Klatzmann, David

    2015-05-01

    Analyses of the regulatory T (Treg) cell TCR repertoire should help elucidate the nature and diversity of their cognate antigens and thus how Treg cells protect us from autoimmune diseases. We earlier identified CD44(hi) CD62L(low) activated/memory (am) Treg cells as a Treg-cell subset with a high turnover and possible self-specificity. We now report that amTreg cells are predominantly distributed in lymph nodes (LNs) draining deep tissues. Multivariate analyses of CDR3 spectratyping first revealed that amTreg TCR repertoire is different from that of naïve Treg cells (nTreg cells) and effector T (Teff) cells. Furthermore, in deep- versus superficial LNs, TCR-β deep sequencing further revealed diversified nTreg-cell and amTreg-cell repertoires, although twofold less diverse than that of Teff cells, and with repertoire richness significantly lower in deep-LN versus superficial-LN Treg cells. Importantly, expanded clonotypes were mostly detected in deep-LN amTreg cells, some accounting for 20% of the repertoire. Strikingly, these clonotypes were absent from nTreg cells, but found at low frequency in Teff cells. Our results, obtained in nonmanipulated mice, indicate different antigenic targets for naïve and amTreg cells and that amTreg cells are self-specific. The data we present are consistent with an instructive component in Treg-cell differentiation.

  6. The Sts Proteins Target Tyrosine Phosphorylated, Ubiquitinated Proteins within TCR Signaling Pathways

    SciTech Connect

    Carpino, N.; Chen, Y; Nassar, N; Oh, H

    2009-01-01

    The T cell receptor (TCR) detects the presence of infectious pathogens and activates numerous intracellular signaling pathways. Protein tyrosine phosphorylation and ubiquitination serve as key regulatory mechanisms downstream of the TCR. Negative regulation of TCR signaling pathways is important in controlling the immune response, and the Suppressor of TCR Signaling proteins (Sts-1 and Sts-2) have been shown to function as critical negative regulators of TCR signaling. Although their mechanism of action has yet to be fully uncovered, it is known that the Sts proteins possess intrinsic phosphatase activity. Here, we demonstrate that Sts-1 and Sts-2 are instrumental in down-modulating proteins that are dually modified by both protein tyrosine phosphorylation and ubiquitination. Specifically, both naive and activated T cells derived from genetically engineered mice that lack the Sts proteins display strikingly elevated levels of tyrosine phosphorylated, ubiquitinated proteins following TCR stimulation. The accumulation of the dually modified proteins is transient, and in activated T cells but not naive T cells is significantly enhanced by co-receptor engagement. Our observations hint at a novel regulatory mechanism downstream of the T cell receptor.

  7. Efficient Nef-mediated downmodulation of TCR-CD3 and CD28 is associated with high CD4+ T cell counts in viremic HIV-2 infection.

    PubMed

    Khalid, Mohammad; Yu, Hangxing; Sauter, Daniel; Usmani, Shariq M; Schmokel, Jan; Feldman, Jerome; Gruters, Rob A; van der Ende, Marchina E; Geyer, Matthias; Rowland-Jones, Sarah; Osterhaus, Albert D; Kirchhoff, Frank

    2012-05-01

    The role of the multifunctional accessory Nef protein in the immunopathogenesis of HIV-2 infection is currently poorly understood. Here, we performed comprehensive functional analyses of 50 nef genes from 21 viremic (plasma viral load, >500 copies/ml) and 16 nonviremic (<500) HIV-2-infected individuals. On average, nef alleles from both groups were equally active in modulating CD4, TCR-CD3, CD28, MHC-I, and Ii cell surface expression and in enhancing virion infectivity. Thus, many HIV-2-infected individuals efficiently control the virus in spite of efficient Nef function. However, the potency of nef alleles in downmodulating TCR-CD3 and CD28 to suppress the activation and apoptosis of T cells correlated with high numbers of CD4(+) T cells in viremic patients. No such correlations were observed in HIV-2-infected individuals with undetectable viral load. Further functional analyses showed that the Nef-mediated downmodulation of TCR-CD3 suppressed the induction of Fas, Fas-L, PD-1, and CTLA-4 cell surface expression as well as the secretion of gamma interferon (IFN-γ) by primary CD4(+) T cells. Moreover, we identified a single naturally occurring amino acid variation (I132T) in the core domain of HIV-2 Nef that selectively disrupts its ability to downmodulate TCR-CD3 and results in functional properties highly reminiscent of HIV-1 Nef proteins. Taken together, our data suggest that the efficient Nef-mediated downmodulation of TCR-CD3 and CD28 help viremic HIV-2-infected individuals to maintain normal CD4(+) T cell homeostasis by preventing T cell activation and by suppressing the induction of death receptors that may affect the functionality and survival of both virally infected and uninfected bystander cells.

  8. Human effector T cells derived from central memory cells rather than CD8(+)T cells modified by tumor-specific TCR gene transfer possess superior traits for adoptive immunotherapy.

    PubMed

    Wu, Fenglin; Zhang, Wenfeng; Shao, Hongwei; Bo, Huaben; Shen, Han; Li, Jiandong; Liu, Yichen; Wang, Teng; Ma, Wenli; Huang, Shulin

    2013-10-10

    Adoptive cell therapy provides an attractive treatment of cancer, and our expanding capacity to target tumor antigens is driven by genetically engineered human T lymphocytes that express genes encoding tumor-specific T cell receptors (TCRs). The intrinsic properties of cultured T cells used for therapy were reported to have tremendous influences on their persistence and antitumor efficacy in vivo. In this study, we isolated CD8(+) central memory T cells from peripheral blood lymphocytes of healthy donors, and then transferred with the gene encoding TCR specific for tumor antigen using recombinant adenovirus vector Ad5F35-TRAV-TRBV. We found effector T cells derived from central memory T cells improved cell viability, maintained certain level of CD62L expression, and reacquired the CD62L(+)CD44(high) phenotype of central memory T cells after effector T cells differentiation. We then compared the antitumor reactivity of central memory T cells and CD8(+)T cells after TCR gene transferred. The results indicated that tumor-specific TCR gene being transferred to central memory T cells effectively increased the specific killing of antigen positive tumor cells and the expression of cytolytic granule protein. Furthermore, TCR gene transferred central memory T cells were more effective than TCR gene transferred CD8(+)T cells in CTL activity and effector cytokine secretion. These results implicated that isolating central memory T cells rather than CD8(+)T cells for insertion of gene encoding tumor-specific TCR may provide a superior tumor-reactive T cell population for adoptive transfer.

  9. The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

    PubMed Central

    Alarcón, B; Ley, S C; Sánchez-Madrid, F; Blumberg, R S; Ju, S T; Fresno, M; Terhorst, C

    1991-01-01

    The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development. Images PMID:1826255

  10. Inhibition of programmed cell death by cyclosporin A; preferential blocking of cell death induced by signals via TCR/CD3 complex and its mode of action.

    PubMed Central

    Yasutomi, D; Odaka, C; Saito, S; Niizeki, H; Kizaki, H; Tadakuma, T

    1992-01-01

    Cyclosporin A (CsA) is reported to inhibit programmed cell death. We confirmed this by using T-cell hybridomas which are inducible to programmed cell death by activation with immobilized anti-CD3 antibody or with anti-Thy 1.2 antibody. Cell death and DNA fragmentation, characteristic features of programmed cell death, were almost completely blocked by CsA or FK506. To investigate whether CsA inhibits only the cell death through the signals via the TCR/CD3 complex or all of the programmed cell death induced by various reagents, we further established CD4+8+ thymic lymphomas which result in programmed cell death after activation with calcium ionophore, dexamethasone, cyclic AMP or anti-CD3 antibody. It was revealed that CsA could block only the cell death mediated by the TCR/CD3 complex. For the clarification of the site of action of CsA, Ca2+ influx and endocytosis of receptors after stimulation with anti-CD3 antibody were monitored in the presence of CsA, and no significant effects of CsA were observed. Furthermore, prevention of cell death was examined by adding CsA at various periods of time after initiation of culture. CsA was found to exert its effect even when added after 4 h of cultivation, and the kinetic pattern of suppression was similar to that of the suppressive effect on IL-2 production. These observations indicate that in the events of programmed cell death, the major site of action of CsA will not be the inhibition of the immediate membrane events after activation of the TCR/CD3 complex but rather the interference in the function of molecules that transmit signals between membrane events and the activation of genes in the nucleus. Images Figure 2 Figure 3 PMID:1383138

  11. An {alpha}{beta} T cell receptor structure at 2.5 {angstrom} and its orientation in the TCR-MHC complex

    SciTech Connect

    Garcia, K.C.; Degano, M.; Stanfield, R.L.

    1996-10-11

    The central event in the cellular immune response to invading microorganisms is the specific recognition of foreign peptides bound to major histocompatibility complex (MHC) molecules by the {alpha}{beta} T cell receptor (TCR). The x-ray structure of the complete extracellular fragment of a glycosylated {alpha}{beta} TCR was determined at 2.5 angstrom, and its orientation bound to a class 1 MHC- peptide (pMHC) complex was elucidated from crystals of the TCR-pMHC complex. The TCR resembles an antibody in the variable V{alpha} and V{beta} domains but deviates in the constant C{alpha} domain and in the interdomain pairing of C{alpha} with C{beta}. Four of seven possible asparagine-linked glycosylation sites have ordered carbohydrate moieties, one of which lies in the C{alpha}-C{beta} interface. The TCR combining site is relatively flat except for a deep hydrophobic cavity between the hypervariable CDR3d (complementarity-determining regions) of the {alpha} and {beta} chains. The 2C TCR covers the class l MHC H-2K{sup b} binding groove so that the V{alpha} CDRs 1 and 2 are positioned over the amino-terminal region of the bound dEV8 peptide, the V{beta} chain CDRs 1 and 2 are over the carboxyl-terminal region of the peptide, and the V{alpha} and V{beta} CDR3s straddle the peptide between the helices around the central position of the peptide. 61 refs., 9 refs., 1 tab.

  12. Monoclonal TCR-Vbeta13.1+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis: evidence for an antigen-driven chronic T-cell stimulation origin.

    PubMed

    Garrido, Pilar; Ruiz-Cabello, Francisco; Bárcena, Paloma; Sandberg, Yorick; Cantón, Julia; Lima, Margarida; Balanzategui, Ana; González, Marcos; López-Nevot, Miguel Angel; Langerak, Anton W; García-Montero, Andrés C; Almeida, Julia; Orfao, Alberto

    2007-06-01

    Monoclonal TCRalphabeta(+)/CD4+ T-large granular lymphocyte (T-LGL) lymphocytosis is a T-cell disorder with a restricted TCR-Vbeta repertoire. In the present study we explored the potential association between the expanded TCR-Vbeta families, the CDR3 sequences of the TCR-Vbeta gene, and the HLA genotype of patients with monoclonal TCRalphabeta(+)/CD4+ T-LGL lymphocytosis. For that purpose, 36 patients with monoclonal TCRalphabeta(+)/CD4+ T-LGL lymphocytosis (15 TCR-Vbeta13.1 versus 21 non-TCR-Vbeta13.1) were selected. For each patient, both the HLA (class I and II) genotype and the DNA sequences of the VDJ-rearranged TCR-Vbeta were analyzed. Our results show a clear association between the TCR-Vbeta repertoire and the HLA genotype, all TCR-Vbeta13.1(+) cases being HLA-DRB1*0701 (P = .004). Interestingly, the HLA-DR7/TCR-Vbeta13.1-restricted T-cell expansions displayed a highly homogeneous and strikingly similar TCR arising from the use of common TCR-Vbeta gene segments, which shared (1) unique CDR3 structural features with a constantly short length, (2) similar combinatorial gene rearrangements with frequent usage of the Jbeta1.1 gene, and (3) a homolog consensus protein sequence at recombination junctions. Overall, these findings strongly support the existence of a common antigen-driven origin for monoclonal CD4+ T-LGL lymphocytosis, with the identification of the exact peptides presented to the expanded T cells deserving further investigations.

  13. Transduction of SIV-Specific TCR Genes into Rhesus Macaque CD8+ T Cells Conveys the Ability to Suppress SIV Replication

    PubMed Central

    Barsov, Eugene V.; Trivett, Matthew T.; Minang, Jacob T.; Sun, Haosi; Ohlen, Claes; Ott, David E.

    2011-01-01

    Background The SIV/rhesus macaque model for HIV/AIDS is a powerful system for examining the contribution of T cells in the control of AIDS viruses. To better our understanding of CD8+ T-cell control of SIV replication in CD4+ T cells, we asked whether TCRs isolated from rhesus macaque CD8+ T-cell clones that exhibited varying abilities to suppress SIV replication could convey their suppressive properties to CD8+ T cells obtained from an uninfected/unvaccinated animal. Principal Findings We transferred SIV-specific TCR genes isolated from rhesus macaque CD8+ T-cell clones with varying abilities to suppress SIV replication in vitro into CD8+ T cells obtained from an uninfected animal by retroviral transduction. After sorting and expansion, transduced CD8+ T-cell lines were obtained that specifically bound their cognate SIV tetramer. These cell lines displayed appropriate effector function and specificity, expressing intracellular IFNγ upon peptide stimulation. Importantly, the SIV suppression properties of the transduced cell lines mirrored those of the original TCR donor clones: cell lines expressing TCRs transferred from highly suppressive clones effectively reduced wild-type SIV replication, while expression of a non-suppressing TCR failed to reduce the spread of virus. However, all TCRs were able to suppress the replication of an SIV mutant that did not downregulate MHC-I, recapitulating the properties of their donor clones. Conclusions Our results show that antigen-specific SIV suppression can be transferred between allogenic T cells simply by TCR gene transfer. This advance provides a platform for examining the contributions of TCRs versus the intrinsic effector characteristics of T-cell clones in virus suppression. Additionally, this approach can be applied to develop non-human primate models to evaluate adoptive T-cell transfer therapy for AIDS and other diseases. PMID:21886812

  14. Naive CD8⁺ T-cell precursors display structured TCR repertoires and composite antigen-driven selection dynamics.

    PubMed

    Neller, Michelle A; Ladell, Kristin; McLaren, James E; Matthews, Katherine K; Gostick, Emma; Pentier, Johanne M; Dolton, Garry; Schauenburg, Andrea J A; Koning, Dan; Fontaine Costa, Ana Isabel C A; Watkins, Thomas S; Venturi, Vanessa; Smith, Corey; Khanna, Rajiv; Miners, Kelly; Clement, Mathew; Wooldridge, Linda; Cole, David K; van Baarle, Debbie; Sewell, Andrew K; Burrows, Scott R; Price, David A; Miles, John J

    2015-08-01

    Basic parameters of the naive antigen (Ag)-specific T-cell repertoire in humans remain poorly defined. Systematic characterization of this 'ground state' immunity in comparison with memory will allow a better understanding of clonal selection during immune challenge. Here, we used high-definition cell isolation from umbilical cord blood samples to establish the baseline frequency, phenotype and T-cell antigen receptor (TCR) repertoire of CD8(+) T-cell precursor populations specific for a range of viral and self-derived Ags. Across the board, these precursor populations were phenotypically naive and occurred with hierarchical frequencies clustered by Ag specificity. The corresponding patterns of TCR architecture were highly ordered and displayed partial overlap with adult memory, indicating biased structuring of the T-cell repertoire during Ag-driven selection. Collectively, these results provide new insights into the complex nature and dynamics of the naive T-cell compartment.

  15. TCR variable gene involvement in chromosome inversion between 14q11 and 14q24 in adult T-cell leukemia.

    PubMed

    Haider, Shawkat; Hayakawa, Kousuke; Itoyama, Takahiro; Sadamori, Naoki; Kurosawa, Nobuyuki; Isobe, Masaharu

    2006-01-01

    Chromosomal translocations in T-cell malignancies frequently involve the T-cell receptor (TCR)alpha/delta locus at chromosome 14q11. Although 14q11 abnormalities are found in about 10% of adult T-cell leukemia (ATL) cases, until now there has been no direct evidence showing involvement of the TCR locus in ATL-a malignancy closely associated with HTLV-1 infection. The breakpoints of T-cell malignancies most commonly occur within the Jalpha or Jdelta region of the TCR locus. In ATL, however, despite extensive searching no breakpoint has yet been found in that region. Using fluorescence in situ hybridization with a panel of cosmid and bacterial artificial chromosome probes derived from chromosome 14, including the variable region of the TCRalpha locus, comprehensive analysis of an ATL patient carrying inv(14)(q11q32) revealed that the TCR locus was indeed involved in this inversion. Molecular cloning of the breakpoint revealed the juxtaposition of TCR Valpha to the 14q24 region as a result of two consecutive inversions: inv(14)(q11q32) and inv(14)(q11q24). We also found a gene near the breakpoint at the 14q24 region that is downregulated in this ATL patient and is assigned in the database as a pseudogene of ADAM21 (a disintegrin and metalloproteinase domain 21). Our expression analysis, however, showed that this pseudogene was actually expressed and was capable of encoding a protein similar to ADAM21; thus we have named this gene ADAM21-like (ADAM21-L).

  16. Myeloid-derived suppressor cells are associated with viral persistence and downregulation of TCR ζ chain expression on CD8(+) T cells in chronic hepatitis C patients.

    PubMed

    Zeng, Qing-Lei; Yang, Bin; Sun, Hong-Qi; Feng, Guo-Hua; Jin, Lei; Zou, Zheng-Sheng; Zhang, Zheng; Zhang, Ji-Yuan; Wang, Fu-Sheng

    2014-01-01

    Myeloid-derived suppressor cells (MDSCs) play an important role in impairing the function of T cells. We characterized MDSCs in two chronic hepatitis C (CHC) cohorts: a cross-sectional group that included 61 treatment-naive patients with CHC, 14 rapid virologic response (RVR) cases and 22 early virologic response (EVR) cases; and a longitudinal group of 13 cases of RVR and 10 cases of EVR after pegylated-interferon-α/ribavirin treatment for genotype 1b HCV infection. Liver samples from 32 CHC patients and six healthy controls were subjected to immunohistochemical analysis. MDSCs frequency in treatment-naive CHC was significantly higher than in RVR, EVR, or healthy subjects and was positively correlated with HCV RNA. Patients infected with HCV genotype 2a had a significantly higher frequency of MDSCs than those infected with genotype 1b. Decreased T cell receptor (TCR) ζ expression on CD8(+) T cells was significantly associated with an increased frequency of MDSCs in treatment-naive CHC patients and was restored by L-arginine treatment in vitro. Increased numbers of liver arginase-1(+) cells were closely associated with the histological activity index in CHC. The TCR ζ chain was significantly downregulated on hepatic CD8(+) T cells in CHC. During antiviral follow up, MDSCs frequency in peripheral blood mononuclear cells was directly correlated with the HCV RNA load in the plasma and inversely correlated with TCR ζ chain expression in CD8(+) T cells in both RVR and EVR cases. Notably, the RVR group had a higher frequency of MDSCs at baseline than the EVR group. Collectively, this study provides evidence that MDSCs might be associated with HCV persistence and downregulation of CD8 ζ chain expression.

  17. Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells.

    PubMed

    MacLeod, Daniel T; Antony, Jeyaraj; Martin, Aaron J; Moser, Rachel J; Hekele, Armin; Wetzel, Keith J; Brown, Audrey E; Triggiano, Melissa A; Hux, Jo Ann; Pham, Christina D; Bartsevich, Victor V; Turner, Caitlin A; Lape, Janel; Kirkland, Samantha; Beard, Clayton W; Smith, Jeff; Hirsch, Matthew L; Nicholson, Michael G; Jantz, Derek; McCreedy, Bruce

    2017-04-05

    Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19(+) hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19(+) tumors in an in vivo mouse model.

  18. A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis.

    PubMed

    Carpenter, Stephen M; Nunes-Alves, Cláudio; Booty, Matthew G; Way, Sing Sing; Behar, Samuel M

    2016-01-01

    T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

  19. A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis

    PubMed Central

    Carpenter, Stephen M.; Nunes-Alves, Cláudio; Booty, Matthew G.; Way, Sing Sing; Behar, Samuel M.

    2016-01-01

    T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection. PMID:26745507

  20. Extracellular domains of CD8α and CD8ß subunits are sufficient for HLA class I restricted helper functions of TCR-engineered CD4(+) T cells.

    PubMed

    van Loenen, Marleen M; Hagedoorn, Renate S; de Boer, Renate; Falkenburg, J H Frederik; Heemskerk, Mirjam H M

    2013-01-01

    By gene transfer of HLA-class I restricted T-cell receptors (TCRs) (HLA-I-TCR) into CD8(+) as well as CD4(+) T-cells, both effector T-cells as well as helper T-cells can be generated. Since most HLA-I-TCRs function best in the presence of the CD8 co-receptor, the CD8αß molecule has to be co-transferred into the CD4(+) T-cells to engineer optimal helper T-cells. In this study, we set out to determine the minimal part of CD8αβ needed for optimal co-receptor function in HLA-I-TCR transduced CD4(+) T-cells. For this purpose, we transduced human peripheral blood derived CD4(+) T-cells with several HLA-class I restricted TCRs either with or without co-transfer of different CD8 subunits. We demonstrate that the co-transduced CD8αβ co-receptor in HLA-I-TCR transduced CD4(+) T-cells behaves as an adhesion molecule, since for optimal antigen-specific HLA class I restricted CD4(+) T-cell reactivity the extracellular domains of the CD8α and ß subunits are sufficient.

  1. [Homology modeling and eukaryotic expression of a modified αβ TCR harboring the immunoglobulin-like domain of γδ TCR].

    PubMed

    Tao, Changli; Shao, Hongwei; Shen, Han; Huang, Shulin

    2016-08-01

    Objective To design, construct and express a chimeric αβ TCR harboring the immunoglobulin-like (Ig) domain of γδ TCR in Jurkat T cells. Methods The fusion sites of TCR δIg were determined by bioinformatics analysis. Then the protein structures of TCR α δIg and TCR β δIg were predicted by homology modeling. Furthermore, the structures of TCR α δIg and TCR β δIg were compared with the wild type (wt) TCR α and TCR β respectively by combinatorial extension (CE). After that, the TCR α δIg and TCR β δIg were fused to fluorescent protein ECFP and EYFP respectively via the overlap PCR, and then the fusion genes (TCR α δIg-ECFP and TCR β δIg-EYFP) were cloned into pIRES2-EGFP vector and respectively located at the upstream and downstream of an internal ribosome entry site (IRES). The recombinant prokaryotic expression vector pIRES-TCR βδIg-EYFP/TCR αδIg-ECFP was transferred into Jurkat T cells. Finally, the expression of TCR δIg in Jurkat T cells was monitored by confocal laser scanning microscopy (CLSM). Results The variable region structure of the TCR δIg did not change and the antigen recognition active regions remained stable compared to the wtTCR. The recombinant expression plasmid was successfully constructed as confirmed by PCR identification and sequencing analysis. CLSM showed that TCR δIg was expressed and located at the plasma membrane of Jurkat T cells. Conclusion The design of TCR δIg was reasonable and the TCR δIg could be expressed on Jurkat T cell surface.

  2. TCR repertoire sequencing identifies synovial Treg cell clonotypes in the bloodstream during active inflammation in human arthritis

    PubMed Central

    Spreafico, Roberto; Consolaro, Alessandro; Leong, Jing Yao; Chua, Camillus; Massa, Margherita; Saidin, Suzan; Magni-Manzoni, Silvia; Arkachaisri, Thaschawee; Wallace, Carol A; Gattorno, Marco; Martini, Alberto; Lovell, Daniel J; Albani, Salvatore

    2017-01-01

    Objectives The imbalance between effector and regulatory T (Treg) cells is crucial in the pathogenesis of autoimmune arthritis. Immune responses are often investigated in the blood because of its accessibility, but circulating lymphocytes are not representative of those found in inflamed tissues. This disconnect hinders our understanding of the mechanisms underlying disease. Our goal was to identify Treg cells implicated in autoimmunity at the inflamed joints, and also readily detectable in the blood upon recirculation. Methods We compared Treg cells of patients with juvenile idiopathic arthritis responding or not to therapy by using: (i) T cell receptor (TCR) sequencing, to identify clonotypes shared between blood and synovial fluid; (ii) FOXP3 Treg cell-specific demethylated region DNA methylation assays, to investigate their stability and (iii) flow cytometry and suppression assays to probe their tolerogenic functions. Results We found a subset of synovial Treg cells that recirculated into the bloodstream of patients with juvenile idiopathic and adult rheumatoid arthritis. These inflammation-associated (ia)Treg cells, but not other blood Treg cells, expanded during active disease and proliferated in response to their cognate antigens. Despite the typical inflammatory-skewed balance of immune mechanisms in arthritis, iaTreg cells were stably committed to the regulatory lineage and fully suppressive. A fraction of iaTreg clonotypes were in common with pathogenic effector T cells. Conclusions Using an innovative antigen-agnostic approach, we uncovered a population of bona fide synovial Treg cells readily accessible from the blood and selectively expanding during active disease, paving the way to non-invasive diagnostics and better understanding of the pathogenesis of autoimmunity. PMID:27311837

  3. Time-Dependent Regulation of IL-2R α-Chain (CD25) Expression by TCR Signal Strength and IL-2-Induced STAT5 Signaling in Activated Human Blood T Lymphocytes

    PubMed Central

    Shatrova, Alla N.; Mityushova, Elena V.; Vassilieva, Irina O.; Aksenov, Nikolay D.; Zenin, Valery V.; Nikolsky, Nikolay N.; Marakhova, Irina I.

    2016-01-01

    The expression of the IL-2R α-chain (IL-2Rα) is regulated at the transcriptional level via TCR- and IL-2R-signaling. The question is how to precede in time the activation signals to induce the IL-2Rα expression in native primary T cells. By comparing the effects of selective drugs on the dynamics of CD25 expression during the mitogen stimulation of human peripheral blood lymphocytes, we identified distinct Src- and JAK-dependent stages of IL-2Rα upregulation. PP2, a selective inhibitor of TCR-associated Src kinase, prevents CD25 expression at initial stages of T cell activation, prior to the cell growth. This early IL-2Rα upregulation underlies the T cell competence and the IL-2 responsiveness. We found that the activated with “weak” mitogen, the population of blood lymphocytes has some pool of competent CD25+ cells bearing a high affinity IL-2R. A distinct pattern of IL-2R signaling in resting and competent T lymphocytes has been shown. Based on the inhibitory effect of WHI-P131, a selective drug of JAK3 kinase activity, we concluded that in quiescent primary T lymphocytes, the constitutive STAT3 and the IL-2-induced prolonged STAT5 activity (assayed by tyrosine phosphorylation) is mostly JAK3-independent. In competent T cells, in the presence of IL-2 JAK3/STAT5 pathway is switched to maintain the higher and sustained IL-2Rα expression as well as cell growth and proliferation. We believe that understanding the temporal coordination of antigen- and cytokine-evoked signals in primary T cells may be useful for improving immunotherapeutic strategies. PMID:27936140

  4. Otud7b facilitates T cell activation and inflammatory responses by regulating Zap70 ubiquitination.

    PubMed

    Hu, Hongbo; Wang, Hui; Xiao, Yichuan; Jin, Jin; Chang, Jae-Hoon; Zou, Qiang; Xie, Xiaoping; Cheng, Xuhong; Sun, Shao-Cong

    2016-03-07

    Signal transduction from the T cell receptor (TCR) is crucial for T cell-mediated immune responses and, when deregulated, also contributes to the development of autoimmunity. How TCR signaling is regulated is incompletely understood. In this study, we demonstrate a ubiquitin-dependent mechanism in which the deubiquitinase Otud7b has a crucial role in facilitating TCR signaling. Upon TCR ligation, Otud7b is rapidly recruited to the tyrosine kinase Zap70, a central mediator of TCR-proximal signaling. Otud7b deficiency attenuates the activation of Zap70 and its downstream pathways and impairs T cell activation and differentiation, rendering mice refractory to T cell-mediated autoimmune and inflammatory responses. Otud7b facilitated Zap70 activation by deubiquitinating Zap70, thus preventing the association of Zap70 with the negative-regulatory phosphatases Sts1 and Sts2. These findings establish Otud7b as a positive regulator of TCR-proximal signaling and T cell activation, highlighting the importance of deubiquitination in regulating Zap70 function.

  5. Pulmonary TCR γδ T cells induce the early inflammation of granuloma formation by a glycolipid trehalose 6,6'-dimycolate (TDM) isolated from Mycobacterium tuberculosis.

    PubMed

    Saitoh, Takayoshi; Yano, Ikuya; Kumazawa, Yoshio; Takimoto, Hiroaki

    2012-10-01

    We previously showed that formation of pulmonary granulomas in mice in response to a mycobacterial glycolipid, trehalose 6,6'-dimycolate (TDM) is due to the action of TNF-α and not of IFN-γ. However, the mechanisms of formation and maintenance of pulmonary granulomas are not yet clear. The purpose of the present study is to evaluate the mechanisms of granuloma formation by TDM at the early phase. Histological analysis showed that inflammatory cells infiltrated the murine pulmonary interstitium on day 2 after an intravenous injection with TDM as a w/o/w emulsion. Clear granuloma formation was observed on day 7 after the injection. The mRNA expression of IL-17, IFN-γ and macrophage inflammatory protein 2 was found in lung mononuclear cells at the day after TDM injection. The major IL-17-producing cells were T-cell receptor (TCR) γδ T cells expressing Vγ6. In mice depleted of γδ T cells by treatment with anti-TCR γδ monoclonal antibody, the number of TDM-induced granuloma was decreased, but the size of granuloma was not affected. Our results suggest that the mycobacterial glycolipid TDM causes activation of IL-17-producing TCR γδ T cells and stimulates chemotaxis of inflammatory cells including neutrophils in to lung.

  6. Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities

    PubMed Central

    Van Caeneghem, Yasmine; De Munter, Stijn; Tieppo, Paola; Goetgeluk, Glenn; Weening, Karin; Verstichel, Greet; Bonte, Sarah; Taghon, Tom; Leclercq, Georges; Kerre, Tessa; Debets, Reno; Vermijlen, David; Abken, Hinrich; Vandekerckhove, Bart

    2017-01-01

    ABSTRACT Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5+CD7+ T-lineage precursors, to CD4+ CD8+ double positive cells and finally to mature AR+ T cells. The AR+ T cells were largely naive CD45RA+CD62L+ T cells. These T cells had mostly germline TCRα and TCRβ loci and therefore lacked surface-expressed CD3/TCRαβ complexes. The CD3− AR-transgenic cells were mono-specific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3− AR+ T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD.

  7. NSOM/QD-based direct visualization of CD3-induced and CD28-enhanced nanospatial coclustering of TCR and coreceptor in nanodomains in T cell activation.

    PubMed

    Zhong, Liyun; Zeng, Gucheng; Lu, Xiaoxu; Wang, Richard C; Gong, Guangming; Yan, Lin; Huang, Dan; Chen, Zheng W

    2009-06-17

    Direct molecular imaging of nano-spatial relationship between T cell receptor (TCR)/CD3 and CD4 or CD8 co-receptor before and after activation of a primary T cell has not been reported. We have recently innovated application of near-field scanning optical microscopy (NSOM) and immune-labeling quantum dots (QD) to image Ag-specific TCR response during in vivo clonal expansion, and now up-graded the NSOM/QD-based nanotechnology through dipole-polarization and dual-color imaging. Using this imaging system scanning cell-membrane molecules at a best-optical lateral resolution, we demonstrated that CD3, CD4 or CD8 molecules were distinctly distributed as single QD-bound molecules or nano-clusters equivalent to 2-4 QD fluorescence-intensity/size on cell-membrane of un-stimulated primary T cells, and approximately 6-10% of CD3 were co-clustering with CD4 or CD8 as 70-110 nm nano-clusters without forming nano-domains. The ligation of TCR/CD3 on CD4 or CD8 T cells led to CD3 nanoscale co-clustering or interaction with CD4 or CD8 co-receptors forming 200-500 nm nano-domains or >500 nm micro-domains. Such nano-spatial co-clustering of CD3 and CD4 or CD3 and CD8 appeared to be an intrinsic event of TCR/CD3 ligation, not purely limited to MHC engagement, and be driven by Lck phosphorylation. Importantly, CD28 co-stimulation remarkably enhanced TCR/CD3 nanoscale co-clustering or interaction with CD4 co-receptor within nano- or micro-domains on the membrane. In contrast, CD28 co-stimulation did not enhance CD8 clustering or CD3-CD8 co-clustering in nano-domains although it increased molecular number and density of CD3 clustering in the enlarged nano-domains. These nanoscale findings provide new insights into TCR/CD3 interaction with CD4 or CD8 co-receptor in T-cell activation.

  8. Otud7b facilitates T cell activation and inflammatory responses by regulating Zap70 ubiquitination

    PubMed Central

    Hu, Hongbo; Wang, Hui; Xiao, Yichuan; Jin, Jin; Chang, Jae-Hoon; Zou, Qiang; Xie, Xiaoping; Cheng, Xuhong

    2016-01-01

    Signal transduction from the T cell receptor (TCR) is crucial for T cell–mediated immune responses and, when deregulated, also contributes to the development of autoimmunity. How TCR signaling is regulated is incompletely understood. In this study, we demonstrate a ubiquitin-dependent mechanism in which the deubiquitinase Otud7b has a crucial role in facilitating TCR signaling. Upon TCR ligation, Otud7b is rapidly recruited to the tyrosine kinase Zap70, a central mediator of TCR-proximal signaling. Otud7b deficiency attenuates the activation of Zap70 and its downstream pathways and impairs T cell activation and differentiation, rendering mice refractory to T cell–mediated autoimmune and inflammatory responses. Otud7b facilitated Zap70 activation by deubiquitinating Zap70, thus preventing the association of Zap70 with the negative-regulatory phosphatases Sts1 and Sts2. These findings establish Otud7b as a positive regulator of TCR-proximal signaling and T cell activation, highlighting the importance of deubiquitination in regulating Zap70 function. PMID:26903241

  9. TCR stimulation without co-stimulatory signals induces expression of "tolerogenic" genes in memory CD4 T cells but does not compromise cell proliferation.

    PubMed

    Xie, Aini; Zheng, Xiong; Khattar, Mithun; Schroder, Paul; Stepkowski, Stanislaw; Xia, Jiahong; Chen, Wenhao

    2015-02-01

    Memory T cells resist co-stimulatory blockade and present a unique therapeutic challenge in transplantation and autoimmune diseases. Herein, we determined whether memory T cells express less "tolerogenic" genes than naïve T cells to reinforce a proliferative response under the deprivation of co-stimulatory signals. The expression of ∼40 tolerogenic genes in memory and naïve CD4(+) T cells was thus assessed during an in vitro TCR stimulation without co-stimulation. Briefly, upon TCR stimulation with an anti-CD3 mAb alone, memory CD4(+) T cells exhibited more proliferation than naïve CD4(+) T cells. To our surprise, at 24h upon anti-CD3 mAb stimulation, memory CD4(+) T cells expressed more than a 5-fold higher level of the transcription factor Egr2 and a 20-fold higher level of the transmembrane E3 ubiquitin ligase GRAIL than those in naïve T cells. Hence, the high-level expression of tolerogenic genes, Egr2 and GRAIL, in memory CD4(+) T cells does not prevent cell proliferation. Importantly, anti-CD3 mAb-stimulated memory CD4(+) T cells expressed high protein/gene levels of phosphorylated STAT5, Nedd4, Bcl-2, and Bcl-XL. Therefore, co-stimulation-independent proliferation of memory CD4(+) T cells may be due to elevated expression of molecules that support cell proliferation and survival, but not lack of tolerogenic molecules.

  10. Generation of functional, antigen-specific CD8+ human T cells from cord blood stem cells using exogenous Notch and tetramer-TCR signaling.

    PubMed

    Fernandez, Irina; Ooi, Tracy P; Roy, Krishnendu

    2014-01-01

    In vitro differentiation of mouse and human stem cells into early T cells has been successfully demonstrated using artificial Notch signaling systems. However, generation of mature, antigen-specific, functional T cells, directly from human stem cells has remained elusive, except when using stromal coculture of stem cells retrovirally transfected with antigen-specific T cell receptors (TCRs). Here we show that human umbilical cord blood (UCB)-derived CD34+CD38-/low hematopoietic stem cells can be successfully differentiated into functional, antigen-specific cytotoxic CD8+ T cells without direct stromal coculture or retroviral TCR transfection. Surface-immobilized Notch ligands (DLL1) and stromal cell conditioned medium successfully induced the development of CD1a+CD7+ and CD4+CD8+ early T cells. These cells, upon continued culture with cytomegalovirus (CMV) or influenza-A virus M1 (GIL) epitope-loaded human leukocyte antigen (HLA)-A*0201 tetramers, resulted in the generation of a polyclonal population of CMV-specific or GIL-specific CD8+ T cells, respectively. Upon further activation with antigen-loaded target cells, these antigen-specific, stem cell-derived T cells exhibited cytolytic functionality, specifically CD107a surface mobilization, interferon gamma (IFNg) production, and Granzyme B secretion. Such scalable, in vitro generation of functional, antigen-specific T cells from human stem cells could eventually provide a readily available cell source for adoptive transfer immunotherapies and also allow better understanding of human T cell development.

  11. Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors.

    PubMed

    McCormack, Emmet; Adams, Katherine J; Hassan, Namir J; Kotian, Akhil; Lissin, Nikolai M; Sami, Malkit; Mujić, Maja; Osdal, Tereza; Gjertsen, Bjørn Tore; Baker, Deborah; Powlesland, Alex S; Aleksic, Milos; Vuidepot, Annelise; Morteau, Olivier; Sutton, Deborah H; June, Carl H; Kalos, Michael; Ashfield, Rebecca; Jakobsen, Bent K

    2013-04-01

    NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157-165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157-165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1157-165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.

  12. Generation of CD20-specific TCRs for TCR gene therapy of CD20low B-cell malignancies insusceptible to CD20-targeting antibodies.

    PubMed

    Jahn, Lorenz; van der Steen, Dirk M; Hagedoorn, Renate S; Hombrink, Pleun; Kester, Michel G D; Schoonakker, Marjolein P; de Ridder, Daniëlle; van Veelen, Peter A; Falkenburg, J H Frederik; Heemskerk, Mirjam H M

    2016-11-22

    Immunotherapy of B-cell leukemia and lymphoma with CD20-targeting monoclonal antibodies (mAbs) has demonstrated clinical efficacy. However, the emergence of unresponsive disease due to low or absent cell surface CD20 urges the need to develop additional strategies. In contrast to mAbs, T-cells via their T-cell receptor (TCR) can recognize not only extracellular but also intracellular antigens in the context of HLA molecules. We hypothesized that T-cells equipped with high affinity CD20-targeting TCRs would be able to recognize B-cell malignancies even in the absence of extracellular CD20. We isolated CD8+ T-cell clones binding to peptide-MHC-tetramers composed of HLA-A*02:01 and CD20-derived peptide SLFLGILSV (CD20SLF) from HLA-A*02:01neg healthy individuals to overcome tolerance towards self-antigens such as CD20. High avidity T-cell clones were identified that readily recognized and lysed primary HLA-A2pos B-cell leukemia and lymphoma in the absence of reactivity against CD20-negative but HLA-A2pos healthy hematopoietic and nonhematopoietic cells. The T-cell clone with highest avidity efficiently lysed malignant cell-lines that had insufficient extracellular CD20 to be targeted by CD20 mAbs. Transfer of this TCR installed potent CD20-specificity onto recipient T-cells and led to lysis of CD20low malignant cell-lines. Moreover, our approach facilitates the generation of an off-the-shelf TCR library with broad applicability by targeting various HLA alleles. Using the same methodology, we isolated a T-cell clone that efficiently lysed primary HLA-B*07:02pos B-cell malignancies by targeting another CD20-derived peptide. TCR gene transfer of high affinity CD20-specific TCRs can be a valuable addition to current treatment options for patients suffering from CD20low B-cell malignancies.

  13. Generation of CD20-specific TCRs for TCR gene therapy of CD20low B-cell malignancies insusceptible to CD20-targeting antibodies

    PubMed Central

    Jahn, Lorenz; van der Steen, Dirk M.; Hagedoorn, Renate S.; Hombrink, Pleun; Kester, Michel G.D.; Schoonakker, Marjolein P.; de Ridder, Daniëlle; van Veelen, Peter A.; Falkenburg, J.H. Frederik; Heemskerk, Mirjam H.M.

    2016-01-01

    Immunotherapy of B-cell leukemia and lymphoma with CD20-targeting monoclonal antibodies (mAbs) has demonstrated clinical efficacy. However, the emergence of unresponsive disease due to low or absent cell surface CD20 urges the need to develop additional strategies. In contrast to mAbs, T-cells via their T-cell receptor (TCR) can recognize not only extracellular but also intracellular antigens in the context of HLA molecules. We hypothesized that T-cells equipped with high affinity CD20-targeting TCRs would be able to recognize B-cell malignancies even in the absence of extracellular CD20. We isolated CD8+ T-cell clones binding to peptide-MHC-tetramers composed of HLA-A*02:01 and CD20-derived peptide SLFLGILSV (CD20SLF) from HLA-A*02:01neg healthy individuals to overcome tolerance towards self-antigens such as CD20. High avidity T-cell clones were identified that readily recognized and lysed primary HLA-A2pos B-cell leukemia and lymphoma in the absence of reactivity against CD20-negative but HLA-A2pos healthy hematopoietic and nonhematopoietic cells. The T-cell clone with highest avidity efficiently lysed malignant cell-lines that had insufficient extracellular CD20 to be targeted by CD20 mAbs. Transfer of this TCR installed potent CD20-specificity onto recipient T-cells and led to lysis of CD20low malignant cell-lines. Moreover, our approach facilitates the generation of an off-the-shelf TCR library with broad applicability by targeting various HLA alleles. Using the same methodology, we isolated a T-cell clone that efficiently lysed primary HLA-B*07:02pos B-cell malignancies by targeting another CD20-derived peptide. TCR gene transfer of high affinity CD20-specific TCRs can be a valuable addition to current treatment options for patients suffering from CD20low B-cell malignancies. PMID:27776339

  14. A unique unresponsive CD4+ T cell phenotype post TCR antagonism

    PubMed Central

    Edwards, Lindsay J.; Evavold, Brian D.

    2010-01-01

    The functional outcomes of the T cell’s interaction with the peptide:MHC complex can be dramatically altered by the introduction of a single amino acid substitution. Previous studies have described the varied effects of these altered peptide ligands (APL) on T cell responses. These outcomes of T cell interaction with an APL include the induction of clonal unresponsiveness (anergy) and inhibition of T cell responses (antagonism). The phenotype of peptide-induced anergy, i.e. low proliferation and low IL-2 production, has been extensively described, and a number of groups have demonstrated antagonism. However, the response of T cells to an agonist ligand after encountering an antagonistic stimulus has not been previously characterized. Here, we show that T cells post-antagonism fail to proliferate but produce large quantities of IL-2 upon stimulation with their wild type ligand. This unique phenotype is not due to differences in IL-2 receptor expression or rates of apoptosis, and cannot be overcome by the addition of recombinant IL-2. The response of CD4 T cells to agonist stimulation after encountering an antagonist is a novel phenotype, and is distinct from previously described forms of anergy. PMID:20031121

  15. The cellular environment regulates in situ kinetics of T-cell receptor interaction with peptide major histocompatibility complex.

    PubMed

    Liu, Baoyu; Chen, Wei; Natarajan, Kannan; Li, Zhenhai; Margulies, David H; Zhu, Cheng

    2015-07-01

    T cells recognize antigens at the two-dimensional (2D) interface with antigen-presenting cells (APCs), which trigger T-cell effector functions. T-cell functional outcomes correlate with 2D kinetics of membrane-embedded T-cell receptors (TCRs) binding to surface-tethered peptide-major histocompatibility complex molecules (pMHCs). However, most studies have measured TCR-pMHC kinetics for recombinant TCRs in 3D by surface plasmon resonance, which differs drastically from 2D measurements. Here, we compared pMHC dissociation from native TCR on the T-cell surface to recombinant TCR immobilized on glass surface or in solution. Force on TCR-pMHC bonds regulated their lifetimes differently for native than recombinant TCRs. Perturbing the cellular environment suppressed 2D on-rates but had no effect on 2D off-rate regardless of whether force was applied. In contrast, for the TCR interacting with its monoclonal antibody, the 2D on-rate was insensitive to cellular perturbations and the force-dependent off-rates were indistinguishable for native and recombinant TCRs. These data present novel features of TCR-pMHC kinetics that are regulated by the cellular environment, underscoring the limitations of 3D kinetics in predicting T-cell functions and calling for further elucidation of the underlying molecular and cellular mechanisms that regulate 2D kinetics in physiological settings.

  16. Structure of Staphylococcal Enterotoxin E in Complex with TCR Defines the Role of TCR Loop Positioning in Superantigen Recognition.

    PubMed

    Rödström, Karin E J; Regenthal, Paulina; Lindkvist-Petersson, Karin

    2015-01-01

    T cells are crucial players in cell-mediated immunity. The specificity of their receptor, the T cell receptor (TCR), is central for the immune system to distinguish foreign from host antigens. Superantigens are bacterial toxins capable of inducing a toxic immune response by cross-linking the TCR and the major histocompatibility complex (MHC) class II and circumventing the antigen specificity. Here, we present the structure of staphylococcal enterotoxin E (SEE) in complex with a human T cell receptor, as well as the unligated T cell receptor structure. There are clear structural changes in the TCR loops upon superantigen binding. In particular, the HV4 loop moves to circumvent steric clashes upon complex formation. In addition, a predicted ternary model of SEE in complex with both TCR and MHC class II displays intermolecular contacts between the TCR α-chain and the MHC, suggesting that the TCR α-chain is of importance for complex formation.

  17. Inverted repeats in the promoter as an autoregulatory sequence for TcrX in Mycobacterium tuberculosis

    SciTech Connect

    Bhattacharya, Monolekha; Das, Amit Kumar

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer The regulatory sequences recognized by TcrX have been identified. Black-Right-Pointing-Pointer The regulatory region comprises of inverted repeats segregated by 30 bp region. Black-Right-Pointing-Pointer The mode of binding of TcrX with regulatory sequence is unique. Black-Right-Pointing-Pointer In silico TcrX-DNA docked model binds one of the inverted repeats. Black-Right-Pointing-Pointer Both phosphorylated and unphosphorylated TcrX binds regulatory sequence in vitro. -- Abstract: TcrY, a histidine kinase, and TcrX, a response regulator, constitute a two-component system in Mycobacterium tuberculosis. tcrX, which is expressed during iron scarcity, is instrumental in the survival of iron-dependent M. tuberculosis. However, the regulator of tcrX/Y has not been fully characterized. Crosslinking studies of TcrX reveal that it can form oligomers in vitro. Electrophoretic mobility shift assays (EMSAs) show that TcrX recognizes two regions in the promoter that are comprised of inverted repeats separated by {approx}30 bp. The dimeric in silico model of TcrX predicts binding to one of these inverted repeat regions. Site-directed mutagenesis and radioactive phosphorylation indicate that D54 of TcrX is phosphorylated by H256 of TcrY. However, phosphorylated and unphosphorylated TcrX bind the regulatory sequence with equal efficiency, which was shown with an EMSA using the D54A TcrX mutant.

  18. MHC-derived allopeptide activates TCR-biased CD8+ Tregs and suppresses organ rejection

    PubMed Central

    Picarda, Elodie; Bézie, Séverine; Venturi, Vanessa; Echasserieau, Klara; Mérieau, Emmanuel; Delhumeau, Aurélie; Renaudin, Karine; Brouard, Sophie; Bernardeau, Karine; Anegon, Ignacio; Guillonneau, Carole

    2014-01-01

    In a rat heart allograft model, preventing T cell costimulation with CD40Ig leads to indefinite allograft survival, which is mediated by the induction of CD8+CD45RClo regulatory T cells (CD8+CD40Ig Tregs) interacting with plasmacytoid dendritic cells (pDCs). The role of TCR-MHC-peptide interaction in regulating Treg activity remains a topic of debate. Here, we identified a donor MHC class II–derived peptide (Du51) that is recognized by TCR-biased CD8+CD40Ig Tregs and activating CD8+CD40Ig Tregs in both its phenotype and suppression of antidonor alloreactive T cell responses. We generated a labeled tetramer (MHC-I RT1.Aa/Du51) to localize and quantify Du51-specific T cells within rat cardiac allografts and spleen. RT1.Aa/Du51-specific CD8+CD40Ig Tregs were the most suppressive subset of the total Treg population, were essential for in vivo tolerance induction, and expressed a biased, restricted Vβ11-TCR repertoire in the spleen and the graft. Finally, we demonstrated that treatment of transplant recipients with the Du51 peptide resulted in indefinite prolongation of allograft survival. These results show that CD8+CD40Ig Tregs recognize a dominant donor antigen, resulting in TCR repertoire alterations in the graft and periphery. Furthermore, this allopeptide has strong therapeutic activity and highlights the importance of TCR-peptide-MHC interaction for Treg generation and function. PMID:24789907

  19. CD4+CD25− T cells transduced to express MHC class I-restricted epitope specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model

    PubMed Central

    Chhabra, Arvind; Yang, Lili; Wang, Pin; Comin-Anduix, Begoña; Das, Raja; Chakraborty, Nitya G.; Ray, Swagatam; Mehrotra, Shikhar; Yang, Haiguang; Hardee, Cinnamon L.; Hollis, Roger; Dorsky, David I.; Koya, Richard; Kohn, Donald B.; Ribas, Antoni; Economou, James S.; Baltimore, David; Mukherji, Bijay

    2009-01-01

    Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4+ T cells. Considering the difficulties in simultaneously engaging CD4+ and CD8+ T cells in tumor immunotherapy -- especially in an antigen specific manner -- “redirecting” CD4+ T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope specific T cell receptors (TCR) in CD4+ T cells has emerged as a strategic consideration. Such TCR engineered CD4+ T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have carried out a critical examination of functional characteristics of CD4+ T cells engineered to express the α and β chains of a high functional avidity TCR specific for the melanoma epitope, MART-127–35 (M1), as a prototypic human tumor antigen system. We found that unpolarized CD4+CD25− T cells engineered to express the M1 TCR selectively synthesize Th1 cytokines and exhibit a potent antigen-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8+ CTL. Such TCR engineered CD4+ T cells, therefore, might be useful in clinical immunotherapy. PMID:18606658

  20. Visualization of the human CD4{sup +} T-cell response in humanized HLA-DR4-expressing NOD/Shi-scid/γc{sup null} (NOG) mice by retrogenic expression of the human TCR gene

    SciTech Connect

    Takahashi, Takeshi Katano, Ikumi; Ito, Ryoji; Ito, Mamoru

    2015-01-02

    Highlights: • β-Lactoglobulin (BLG) specific TCR genes were introduced to human HSC by retrovirus. • Human HSC with BLG-specific TCR were transplanted into NOG-HLA-DR4 I-A{sup −/−} mice. • BLG-specific TCR induced positive selection of thymocytes. • BLG-specific TCR positive CD4{sup +} T cells mediated immune responses in humanized mice. - Abstract: The development of severe immunodeficient mouse strains containing various human genes, including cytokines or HLA, has enabled the reconstitution of functional human immune systems after transplantation of human hematopoietic stem cells (HSC). Accumulating evidence has suggested that HLA-restricted antigen-specific human T-cell responses can be generated in these humanized mice. To directly monitor immune responses of human CD4{sup +} T cells, we introduced β-lactoglobulin (BLG)-specific T cell receptor (TCR) genes derived from CD4{sup +} T-cell clones of cow-milk allergy patients into HSCs, and subsequently transplanted them into NOG-HLA-DR4 transgenic/I-Aβ deficient mice (NOG-DR4/I-A{sup o}). In the thymus, thymocytes with BLG-specific TCR preferentially differentiated into CD4{sup +}CD8{sup −} single-positive cells. Adoptive transfer of mature CD4{sup +} T cells expressing the TCR into recipient NOG-DR4/I-A{sup o} mice demonstrated that human CD4{sup +} T cells proliferated in response to antigenic stimulation and produced IFN-γ in vivo, suggesting that functional T-cell reactions (especially Th1-skewed responses) were induced in humanized mice.

  1. TCR-ligand koff rate correlates with the protective capacity of antigen-specific CD8+ T cells for adoptive transfer.

    PubMed

    Nauerth, Magdalena; Weißbrich, Bianca; Knall, Robert; Franz, Tobias; Dössinger, Georg; Bet, Jeannette; Paszkiewicz, Paulina J; Pfeifer, Lukas; Bunse, Mario; Uckert, Wolfgang; Holtappels, Rafaela; Gillert-Marien, Dorothea; Neuenhahn, Michael; Krackhardt, Angela; Reddehase, Matthias J; Riddell, Stanley R; Busch, Dirk H

    2013-07-03

    Adoptive immunotherapy is a promising therapeutic approach for the treatment of chronic infections and cancer. T cells within a certain range of high avidity for their cognate ligand are believed to be most effective. T cell receptor (TCR) transfer experiments indicate that a major part of avidity is hardwired within the structure of the TCR. Unfortunately, rapid measurement of structural avidity of TCRs is difficult on living T cells. We developed a technology where dissociation (koff rate) of truly monomeric peptide-major histocompatibility complex (pMHC) molecules bound to surface-expressed TCRs can be monitored by real-time microscopy in a highly reliable manner. A first evaluation of this method on distinct human cytomegalovirus (CMV)-specific T cell populations revealed unexpected differences in the koff rates. CMV-specific T cells are currently being evaluated in clinical trials for efficacy in adoptive immunotherapy; therefore, determination of koff rates could guide selection of the most effective donor cells. Indeed, in two different murine infection models, we demonstrate that T cell populations with lower koff rates confer significantly better protection than populations with fast koff rates. These data indicate that koff rate measurements can improve the predictability of adoptive immunotherapy and provide diagnostic information on the in vivo quality of T cells.

  2. Soluble OX40L and JAG1 Induce Selective Proliferation of Functional Regulatory T-Cells Independent of canonical TCR signaling

    PubMed Central

    Kumar, Prabhakaran; Alharshawi, Khaled; Bhattacharya, Palash; Marinelarena, Alejandra; Haddad, Christine; Sun, Zuoming; Chiba, Shigeru; Epstein, Alan L.; Prabhakar, Bellur S.

    2017-01-01

    Regulatory T-cells (Tregs) play a pivotal role in maintaining peripheral tolerance. Increasing Treg numbers/functions has been shown to ameliorate autoimmune diseases. However, common Treg expansion approaches use T-Cell Receptor (TCR)-mediated stimulation which also causes proliferation of effector T-cells (Teff). To overcome this limitation, purified patient-specific Tregs are expanded ex vivo and transfused. Although promising, this approach is not suitable for routine clinical use. Therefore, an alternative approach to selectively expand functional Tregs in vivo is highly desired. We report a novel TCR-independent strategy for the selective proliferation of Foxp3+Tregs (without Teff proliferation), by co-culturing CD4+ T-cells with OX40 L+Jagged(JAG)-1+ bone marrow-derived DCs differentiated with GM-CSF or treating them with soluble OX40 L and JAG1 in the presence of exogenous IL-2. Tregs expanded using soluble OX40 L and JAG1 were of suppressive phenotype and delayed the onset of diabetes in NOD mice. Ligation of OX40 L and JAG1 with their cognate-receptors OX40 and Notch3, preferentially expressed on Tregs but not on Teff cells, was required for selective Treg proliferation. Soluble OX40L-JAG1-induced NF-κB activation as well as IL-2-induced STAT5 activation were essential for the proliferation of Tregs with sustained Foxp3 expression. Altogether, these findings demonstrate the utility of soluble OX40 L and JAG1 to induce TCR-independent Treg proliferation. PMID:28045060

  3. Cytoskeletal polarization of T cells is regulated by an immunoreceptor tyrosine-based activation motif-dependent mechanism.

    PubMed

    Lowin-Kropf, B; Shapiro, V S; Weiss, A

    1998-02-23

    Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell-cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex-dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-zeta and CD3epsilon, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-zeta and once in CD3epsilon, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling

  4. OAS/PKR Pathways and α/β TCR+ T Cells are Required for Ad: IFN-γ Inhibition of HSV-1 in Cornea1

    PubMed Central

    Austin, Bobbie Ann; Halford, William P.; Williams, Bryan R. G.; Carr, Daniel J. J.

    2007-01-01

    An adenoviral vector containing the muIFN-γ transgene (Ad:IFN-γ) was evaluated for its capacity to inhibit HSV-1. To measure effectiveness, viral titers were analyzed in cornea and trigeminal ganglia (TG) during acute ocular HSV-1 infection. Ad: IFN-γ potently suppressed HSV-1 replication in a dose-dependent fashion, requiring IFN-γ R. Moreover, Ad:IFN-γ was effective when delivered -72 and -24 h prior to infection as well as 24 h post infection. Associated with anti-viral opposition, TG from Ad: IFN-γ transduced mice harbored fewer T cells. Also related to T cell involvement, Ad:IFN-γ was effective but attenuated in TG from α/β TCR deficient mice. In corneas, α/β TCR+ T cells were obligatory for protection against viral multiplication. Type I IFN involvement amid anti-viral efficacy of Ad: IFN-γ was further investigated because type I and II IFN pathways have synergistic anti-HSV-1 activity. Ad:IFN-γ inhibited viral reproduction in corneas and TG from IFN-α/β R deficient (CD118 −/−) mice, although viral titers were 2–3 fold higher in cornea and TG, compared to wild type. The absence of IFN-stimulated anti-viral proteins, 2’-5’ oligoadenylate synthetase/RNase L and ds RNA dependent protein kinase R, completely eliminated the anti-viral effectiveness of Ad:IFN-γ. Collectively, the results demonstrate: (1) nonexistence of type I IFN R does not abolish defense of Ad:IFN-γ against HSV-1; (2) anti-viral pathways, OAS/RNase L and PKR are mandatory; and (3) α/β TCR+ T cells are compulsory for Ad: IFN-γ effectiveness against HSV-1 in cornea but not in TG. PMID:17404299

  5. CD43 REGULATES THE THRESHOLD FOR T CELL ACTIVATION BY TARGETING CBL FUNCTIONS

    PubMed Central

    Pedraza-Alva, Gustavo; Lilia, B. Mérida; del Rio, Roxana; Nora, A. Fierro; Cruz-Muñoz, Mario E.; Olivares, Norma; Melchy, Erika; Igras, Vivian; Georg, A. Holländer; Steven, J. Burakoff; Rosenstein, Yvonne

    2013-01-01

    SUMMARY T cell (TC) activation requires the coordinated signaling of the T cell receptor (TCR) and co-receptor molecules, allowing TCs to respond to lower degrees of TCR occupancy. Co-receptor molecules set the threshold for TC activation by controlling different regulatory signaling loops. The Cbl family members prevent undesired activation of TCs by regulating TCR signals. In this report we show that TC pre-stimulation by the CD43 co-receptor molecule before TCR engagement inhibits TCR-dependent c-Cbl tyrosine phosphorylation, c-Cbl interaction with the adapter molecule Crk-L and promotes Cbl-b degradation in a PKCθ–dependent manner. Consequently, the prolonged tyrosine phosphorylation and delayed degradation of ZAP-70 and of the ζ chain lead to enhanced MAPK activation and robust TC response. These data indicates that CD43-mediated signals lower the threshold for TC activation by restricting the c-Cbl and Cbl-b inhibitory effects on TCR signaling. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune TC signal quality, and ultimately immune function. PMID:21905200

  6. Selective T-cell Ablation with Bismuth-213 Labeled Anti-TCR Alpha Beta as Nonmyeloablative Conditionaing for Allogeneic Canine Marrow Transplantion

    SciTech Connect

    Bethge, W. A.; Wilbur, D. Scott; Storb, R.; Hamlin, Donald K.; Santos, E. B.; Brechbiel, M. W.; Fisher, Darrell R.; Sandmaier, B. M.

    2003-06-15

    Two major immunological barriers, the host versus graft (HVG) and the graft versus host (GVH) reaction, must be overcome for successful allogeneic hematopoietic stem cell transplantation. T-cells are involved in these barriers in the major histocompatibility complex-identical settings. We hypothesized that selective ablation of T-cells using radioimmunotherapy, together with postgrafting immunosuppression, would ensure stable allogeneic engraftment. We developed a canine model of nonmyeloablative marrow transplantation in which host immune reactions are impaired by a single dose of 2 Gy total body irradiation (TBI), and where both GVH and residual HVG reactions are controlled by postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP). We substituted the alpha-emitter bismuth-213 linked to a monoclonal antibody against TCR(alpha,beta)using the metal-binding chelate CHX-A”-DTPA, for 2 Gy TBI. Biodistribution studies using a gamma-emitting indium-111-labeled anti-TCR mAb showed uptake primarily in blood, marrow, lymph nodes, spleen and liver. In a dosimetry study, 4 dogs were treated with 0.13-0.46 mg/kg TCR mAb labeled with 3.7-5.6 mCi/kg (137-207 MBq/kg) Bi-213. The treatment was administered in 6 injections on days -3 and -2 followed by transplantion of dog leukocyte antigen-identical marrow on day 0 and postgrafting immunosuppression with MMF and CSP. Therapy was well tolerated except for elevations of transaminases, which were transient in all but one dog. No other organ toxicities or signs of graft-versus-host-disease were noted. The dogs had prompt allogeneic hematopoietic engraftment and achieved stable mixed donor-host hematopoietic chimerism with donor contributions ranging from 5-55 % with >30 weeks follow up.

  7. Analyzing the CDR3 Repertoire with respect to TCR-Beta Chain V-D-J and V-J Rearrangements in Peripheral T Cells using HTS.

    PubMed

    Ma, Long; Yang, Liwen; Bin Shi; He, Xiaoyan; Peng, Aihua; Li, Yuehong; Zhang, Teng; Sun, Suhong; Ma, Rui; Yao, Xinsheng

    2016-07-12

    V-D-J rearrangement of the TCR-beta chain follows the 12/23 rule and the beyond 12/23 restriction. Currently, the proportion and characteristics of TCR-beta chain V-J rearrangement is unclear. We used high-throughput sequencing to compare and analyze TCR-beta chain V-J rearrangement and V-D-J rearrangement in the CDR3 repertoires of T cells from the PBMCs of six volunteers and six BALB/c mice. The results showed that the percentage of V-J rearrangement of the volunteers was approximately 0.7%, whereas that of the mice was 2.2%. The clonality of mice V-J rearrangement was significantly reduced compared with the V-D-J rearrangement, whereas the clonality of human V-J rearrangement was slightly reduced compared with the V-D-J rearrangement. V-J rearrangement in CDR3 involved the significant usage of N, S, F and L, whereas V-D-J rearrangement in CDR3 involved the significant usage of R and G. The levels of V deletion and J deletion in V-J rearrangement were significantly reduced compared with V-D-J rearrangement. TRBD and TRBJ usage in V-J rearrangement differed from that of V-D-J rearrangement, including dominant usage of TRBV and TRBJ and their pairing. Taken together, these results provide new ideas and technology for studies of V-D-J rearrangement and V-J rearrangement in the CDR3 repertoire.

  8. A Phosphatase Activity of Sts-1 Contributes to the Suppression of TCR Signaling

    SciTech Connect

    Mikhailik,A.; Ford, B.; Keller, J.; Chen, Y.; Nassar, N.; Carpino, N.

    2007-01-01

    Precise signaling by the T cell receptor (TCR) is crucial for a proper immune response. To ensure that T cells respond appropriately to antigenic stimuli, TCR signaling pathways are subject to multiple levels of regulation. Sts-1 negatively regulates signaling pathways downstream of the TCR by an unknown mechanism(s). Here, we demonstrate that Sts-1 is a phosphatase that can target the tyrosine kinase Zap-70 among other proteins. The X-ray structure of the Sts-1 C terminus reveals that it has homology to members of the phosphoglycerate mutase/acid phosphatase (PGM/AcP) family of enzymes, with residues known to be important for PGM/AcP catalytic activity conserved in nature and position in Sts-1. Point mutations that impair Sts-1 phosphatase activity in vitro also impair the ability of Sts-1 to regulate TCR signaling in T cells. These observations reveal a PGM/AcP-like enzyme activity involved in the control of antigen receptor signaling.

  9. A phosphatase activity of Sts-1 contributes to the suppression TCR signaling

    PubMed Central

    Mikhailik, Anatoly; Ford, Bradley; Keller, James; Chen, Yunting; Nassar, Nicolas; Carpino, Nick

    2009-01-01

    Summary Precise signaling by the T cell receptor (TCR) is crucial for a proper immune response. To ensure that T cells respond appropriately to antigenic stimuli, TCR signaling pathways are subject to multiple levels of regulation. Sts-1 negatively regulates signaling pathways downstream of the TCR by an unknown mechanism(s). Here, we demonstrate that Sts-1 is a phosphatase that can target the tyrosine kinase Zap-70 among other proteins. The x-ray structure of the Sts-1 C-terminus reveals that it has homology to members of the phosphoglycerate mutase/acid phosphatase (PGM/AcP) family of enzymes, with residues known to be important for PGM/AcP catalytic activity conserved in nature and position in Sts-1. Point mutations that impair Sts-1 phosphatase activity in vitro also impair the ability of Sts-1 to regulate TCR signaling in T cells. These observations reveal a PGM/AcP-like enzyme activity involved in the control of antigen receptor signaling. PMID:17679096

  10. Elongated TCR alpha chain CDR3 favors an altered CD4 cytokine profile

    PubMed Central

    2014-01-01

    Background CD4 T lymphocyte activation requires T cell receptor (TCR) engagement by peptide/MHC (major histocompatibility complex) (pMHC). The TCR complementarity-determining region 3 (CDR3) contains variable α and β loops critical for pMHC recognition. During any immune response, tuning of TCR usage through progressive clonal selection occurs. Th1 and Th2 cells operate at different avidities for activation and display distinct transcriptional programs, although polarization may be plastic, influenced by pathogens and cytokines. We therefore hypothesized that CDR3αβ sequence features may intrinsically influence CD4 phenotype during progression of a response. Results We show that CD4 polarization involves distinct CDR3α usage: Th1 and Th17 cells favored short TCR CDR3α sequences of 12 and 11 amino acids, respectively, while Th2 cells favored elongated CDR3α loops of 14 amino acids, with lower predicted affinity. The dominant Th2- and Th1-derived TCRα sequences with14 amino acid CDR3 loops and 12 amino acid CDR3 loops, respectively, were expressed in TCR transgenics. The functional impact of these TCRα transgenes was assessed after in vivo priming with a peptide/adjuvant. The short, Th1-derived receptor transgenic T cell lines made IFNγ, but not IL-4, 5 or 13, while the elongated, Th2-derived receptor transgenic T cell lines made little or no IFNγ, but increased IL-4, 5 and 13 with progressive re-stimulations, mirrored by GATA-3 up-regulation. T cells from primed Th2 TCRα transgenics selected dominant TCR Vβ expansions, allowing us to generate TCRαβ transgenics carrying the favored, Th2-derived receptor heterodimer. Primed T cells from TCRαβ transgenics made little or no IL-17 or IFNγ, but favored IL-9 after priming with Complete Freund’s adjuvant and IL-4, 5, 9, 10 and 13 after priming with incomplete Freund’s. In tetramer-binding studies, this transgenic receptor showed low binding avidity for pMHC and polarized T cell lines show TCR avidity

  11. Quantifying Distribution of Flow Cytometric TCR-Vβ Usage with Economic Statistics.

    PubMed

    van der Geest, Kornelis S M; Abdulahad, Wayel H; Horst, Gerda; Lorencetti, Pedro G; Bijzet, Johan; Arends, Suzanne; van der Heiden, Marieke; Buisman, Anne-Marie; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M H

    2015-01-01

    Measuring changes of the T cell receptor (TCR) repertoire is important to many fields of medicine. Flow cytometry is a popular technique to study the TCR repertoire, as it quickly provides insight into the TCR-Vβ usage among well-defined populations of T cells. However, the interpretation of the flow cytometric data remains difficult, and subtle TCR repertoire changes may go undetected. Here, we introduce a novel means for analyzing the flow cytometric data on TCR-Vβ usage. By applying economic statistics, we calculated the Gini-TCR skewing index from the flow cytometric TCR-Vβ analysis. The Gini-TCR skewing index, which is a direct measure of TCR-Vβ distribution among T cells, allowed us to track subtle changes of the TCR repertoire among distinct populations of T cells. Application of the Gini-TCR skewing index to the flow cytometric TCR-Vβ analysis will greatly help to gain better understanding of the TCR repertoire in health and disease.

  12. Quantifying Distribution of Flow Cytometric TCR-Vβ Usage with Economic Statistics

    PubMed Central

    van der Geest, Kornelis S. M.; Abdulahad, Wayel H.; Horst, Gerda; Lorencetti, Pedro G.; Bijzet, Johan; Arends, Suzanne; van der Heiden, Marieke; Buisman, Anne-Marie; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M. H.

    2015-01-01

    Measuring changes of the T cell receptor (TCR) repertoire is important to many fields of medicine. Flow cytometry is a popular technique to study the TCR repertoire, as it quickly provides insight into the TCR-Vβ usage among well-defined populations of T cells. However, the interpretation of the flow cytometric data remains difficult, and subtle TCR repertoire changes may go undetected. Here, we introduce a novel means for analyzing the flow cytometric data on TCR-Vβ usage. By applying economic statistics, we calculated the Gini-TCR skewing index from the flow cytometric TCR-Vβ analysis. The Gini-TCR skewing index, which is a direct measure of TCR-Vβ distribution among T cells, allowed us to track subtle changes of the TCR repertoire among distinct populations of T cells. Application of the Gini-TCR skewing index to the flow cytometric TCR-Vβ analysis will greatly help to gain better understanding of the TCR repertoire in health and disease. PMID:25923356

  13. IL-23 Promotes TCR-mediated Negative Selection of Thymocytes through the Upregulation of IL-23 Receptor and RORγt

    PubMed Central

    Li, Hao; Hsu, Hui-Chen; Wu, Qi; Yang, PingAr; Li, Jun; Luo, Bao; Oukka, Mohamed; Steele, Claude H.; Cua, Daniel J; Grizzle, William E.; Mountz, John D.

    2014-01-01

    Summary Transient thymic involution is frequently found during inflammation, yet the mode of action of inflammatory cytokines is not well defined. Here we report that interleukin-23 (IL-23) production by the thymic dendritic cells (DCs) promotes apoptosis of the CD4hiCD8hi double positive (DP) thymocytes. A deficiency in IL-23 signaling interferes with negative selection in the male Db/H-Y T-cell receptor (TCR) transgenic mice. IL-23 plus TCR signaling results in significant up-regulation of IL-23 receptor (IL-23R) expressed predominantly on CD4hiCD8hiCD3+αβTCR+ DP thymocytes, and leads to RORγt dependent apoptosis. These results extend the action of IL-23 beyond its peripheral effects to a unique role in TCR mediated negative selection including elimination of natural T regulatory cells in the thymus. PMID:25001511

  14. CD4+CD25- T cells transduced to express MHC class I-restricted epitope-specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model.

    PubMed

    Chhabra, Arvind; Yang, Lili; Wang, Pin; Comin-Anduix, Begoña; Das, Raja; Chakraborty, Nitya G; Ray, Swagatam; Mehrotra, Shikhar; Yang, Haiguang; Hardee, Cinnamon L; Hollis, Roger; Dorsky, David I; Koya, Richard; Kohn, Donald B; Ribas, Antoni; Economou, James S; Baltimore, David; Mukherji, Bijay

    2008-07-15

    Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4(+) T cells. Considering the difficulties in simultaneously engaging CD4(+) and CD8(+) T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4(+) T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4(+) T cells has emerged as a strategic consideration. Such TCR-engineered CD4(+) T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4(+) T cells engineered to express the alpha- and beta-chains of a high functional avidity TCR specific for the melanoma epitope, MART-1(27-35), as a prototypic human tumor Ag system. We found that unpolarized CD4(+)CD25(-) T cells engineered to express the MART-1(27-35) TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8(+) CTL. Such TCR engineered CD4(+) T cells, therefore, might be useful in clinical immunotherapy.

  15. Studying the Dynamics of TCR Internalization at the Immune Synapse.

    PubMed

    Calleja, Enrique; Alarcón, Balbino; Oeste, Clara L

    2017-01-01

    Establishing a stable interaction between a T cell and an antigen presenting cell (APC) involves the formation of an immune synapse (IS). It is through this structure that the T cell can integrate all the signals provided by the APC. The IS also serves as a mechanism for TCR downregulation through internalization. Here, we describe methods for visualizing MHC-engaged T cell receptor (TCR) internalization from the IS in human cell lines and mouse primary T cells by confocal fluorescence microscopy techniques.

  16. CD25 and CD69 induction by α4β1 outside-in signalling requires TCR early signalling complex proteins.

    PubMed

    Cimo, Ann-Marie; Ahmed, Zamal; McIntyre, Bradley W; Lewis, Dorothy E; Ladbury, John E

    2013-08-15

    Distinct signalling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the TCR (T-cell receptor) ESC (early signalling complex) are also involved in interferon-α receptor signalling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signalling networks with one another. We investigated the contributions of the TCR ESC proteins Lck (lymphocyte-specific kinase), ZAP-70 (ζ-chain-associated protein of 70 kDa), Vav1, SLP-76 [SH2 (Src homology 2)-domain-containing leukocyte protein of 76 kDa] and LAT (linker for activation of T-cells) to integrin outside-in signalling in human T-cells. Lck, ZAP-70, SLP-76, Vav1 and LAT were activated by α4β1 outside-in signalling, but in a manner different from TCR signalling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase ERK (extracellular-signal-regulated kinase). α4β1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4β1 outside-in signalling induced CD25 and co-stimulated CD69 and this was dependent on TCR ESC proteins. TCR and α4β1 outside-in signalling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the cross-talk between integrin outside-in and TCR signalling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T-cell deregulation.

  17. CD27 cooperates with the pre-T cell receptor in the regulation of murine T cell development

    PubMed Central

    1996-01-01

    CD27 is a lymphocyte-specific member of the TNF receptor family and has a TNF-related transmembrane ligand, CD70. The CD27/CD70 receptor-ligand pair cooperates with the TCR in the regulation of the peripheral T cell response. The study presented here reveals that CD27 may play a similar role in thymic pre-T cell development. We have previously cloned the cDNA encoding murine CD27, prepared specific mAbs and observed that murine CD27 is expressed on virtually all thymocytes, with the exception of a subpopulation of CD4-8- precursor T cells. It is shown here that induction of murine CD27 expression occurs at the transition from the CD4-8-25+ to the CD4-8-25- precursor T cell stage and is regulated by the pre-TCR. Therefore, we investigated whether CD27 contributes to pre-TCR-mediated thymocyte development. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombination activating gene (RAG)-deficient mice. This in vivo anti- CD3 epsilon mAb treatment induces an about fifty fold numerical expansion of CD4-8-25+ thymocytes and their differentiation to the CD4+8+25- stage. Co-injection of anti-CD27 mAb inhibited the CD3- mediated expansion and differentiation of the CD4-8-25+ precursor population. Also, injection of anti-CD27 mAb in TCR alpha-/- mutant mice led to a reduction in the absolute number of CD4+8+25- thymocytes. We present evidence that in these in vivo systems, anti-CD27 mAb inhibits CD27-ligand interaction. Therefore, we conclude that CD27 may contribute to normal murine T cell development by synergizing with the pre-TCR-mediated signal. PMID:8760821

  18. Identification of CMS as a cytosolic adaptor of the human pTalpha chain involved in pre-TCR function.

    PubMed

    Navarro, María N; Nusspaumer, Gretel; Fuentes, Patricia; González-García, Sara; Alcain, Juan; Toribio, María L

    2007-12-15

    The T-cell receptor beta (TCRbeta)/pre-TCRalpha (pTalpha) pre-TCR complex (pre-TCR) signals the expansion and differentiation of de-veloping thymocytes. Functional pro-perties of the pre-TCR rely on its unique pTalpha chain, which suggests the participation of specific intracellular adaptors. However, pTalpha-interacting molecules remain unknown. Here, we identified a polyproline-arginine sequence in the human pTalpha cytoplasmic tail that interacted in vitro with SH3 domains of the CIN85/CMS family of adaptors, and mediated the recruitment of multiprotein complexes involving all (CMS, CIN85, and CD2BP3) members. Supporting the physiologic relevance of this interaction, we found that 1 such adaptor, CMS, interacted in vivo with human pTalpha, and its expression was selectively up-regulated during human thymopoiesis in pre-TCR-activated thymocytes. Upon activation, pre-TCR clustering was induced, and CMS and polymerized actin were simultaneously recruited to the pre-TCR activation site. CMS also associated via its C-terminal region to the actin cytoskeleton in the endocytic compartment, where it colocalized with internalized pTalpha in traffic to lysosomal degradation. Notably, deletion of the pTalpha CIN85/CMS-binding motif impaired pre-TCR-mediated Ca(2+) mobilization and NFAT transcriptional activity, and precluded activation induced by overexpression of a CMS-SH3 N-terminal mutant. These results provide the first molecular evidence for a pTalpha intracellular adaptor involved in pre-TCR function.

  19. Antigen-specific CD4{sup +} effector T cells: Analysis of factors regulating clonal expansion and cytokine production

    SciTech Connect

    Ohnuki, Kazunobu; Watanabe, Yuri; Takahashi, Yusuke; Kobayashi, Sakiko; Watanabe, Shiho; Ogawa, Shuhei; Kotani, Motoko; Kozono, Haruo; Tanabe, Kazunari; Abe, Ryo

    2009-03-20

    In order to fully understand T cell-mediated immunity, the mechanisms that regulate clonal expansion and cytokine production by CD4{sup +} antigen-specific effector T cells in response to a wide range of antigenic stimulation needs clarification. For this purpose, panels of antigen-specific CD4{sup +} T cell clones with different thresholds for antigen-induced proliferation were generated by repeated stimulation with high- or low-dose antigen. Differences in antigen sensitivities did not correlate with expression of TCR, CD4, adhesion or costimulatory molecules. There was no significant difference in antigen-dependent cytokine production by TG40 cells transfected with TCR obtained from either high- or low-dose-responding T cell clones, suggesting that the affinity of TCRs for their ligands is not primary determinant of T cell antigen reactivity. The proliferative responses of all T cell clones to both peptide stimulation and to TCR{beta} crosslinking revealed parallel dose-response curves. These results suggest that the TCR signal strength of effector T cells and threshold of antigen reactivity is determined by an intrinsic property, such as the TCR signalosome and/or intracellular signaling machinery. Finally, the antigen responses of high- and low-peptide-responding T cell clones reveal that clonal expansion and cytokine production of effector T cells occur independently of antigen concentration. Based on these results, the mechanisms underlying selection of high 'avidity' effector and memory T cells in response to pathogen are discussed.

  20. Id1 expression promotes peripheral CD4{sup +} T cell proliferation and survival upon TCR activation without co-stimulation

    SciTech Connect

    Liu, Chen; Jin, Rong; Wang, Hong-Cheng; Tang, Hui; Liu, Yuan-Feng; Qian, Xiao-Ping; Sun, Xiu-Yuan; Ge, Qing; Sun, Xiao-Hong; Zhang, Yu

    2013-06-21

    Highlights: •Id1 expression enables naïve T cell proliferation without anti-CD28 co-stimulation. •Id1 expression facilitates T cells survival when stimulated with anti-CD3. •Elevation of IL-2 production by Id1 contributes increased proliferation and survival. •Id1 potentiates NF-κB activation by anti-CD3 stimulation. -- Abstract: Although the role of E proteins in the thymocyte development is well documented, much less is known about their function in peripheral T cells. Here we demonstrated that CD4 promoter-driven transgenic expression of Id1, a naturally occurring dominant-negative inhibitor of E proteins, can substitute for the co-stimulatory signal delivered by CD28 to facilitate the proliferation and survival of naïve CD4{sup +} cells upon anti-CD3 stimulation. We next discovered that IL-2 production and NF-κB activity after anti-CD3 stimulation were significantly elevated in Id1-expressing cells, which may be, at least in part, responsible for the augmentation of their proliferation and survival. Taken together, results from this study suggest an important role of E and Id proteins in peripheral T cell activation. The ability of Id proteins to by-pass co-stimulatory signals to enable T cell activation has significant implications in regulating T cell immunity.

  1. HLA-DPB1*05: 01-restricted WT1332-specific TCR-transduced CD4+ T lymphocytes display a helper activity for WT1-specific CTL induction and a cytotoxicity against leukemia cells.

    PubMed

    Lin, Yuhung; Fujiki, Fumihiro; Katsuhara, Akiko; Oka, Yoshihiro; Tsuboi, Akihiro; Aoyama, Nao; Tanii, Satoe; Nakajima, Hiroko; Tatsumi, Naoya; Morimoto, Soyoko; Tamanaka, Taichi; Tachino, Sho; Hosen, Naoki; Nishida, Sumiyuki; Oji, Yusuke; Kumanogoh, Atsushi; Sugiyama, Haruo

    2013-04-01

    Wilms tumor gene 1 (WT1) is overexpressed in various malignant neoplasms, and has been demonstrated as an attractive target for cancer immunotherapy. We previously reported the identification of a WT1 protein-derived, 16-mer helper peptide WT1332 that could elicit Th1-type CD4+ T-cell response and bind to multiple HLA class II molecules. In this study, we examined the feasibility of adoptive therapy using CD4+ T cells that were transduced an HLA-DPB1*05:01-restricted, WT1332-specific T-cell receptor (TCR). HLA-DPB1*05:01-restricted, WT1332-specific TCR-transduced CD4+ T cells were successfully generated using lentiviral vector and exhibited strong proliferative response and Th1-type cytokine production in response to WT1332 peptide, WT1 protein, or WT1-expressing tumor cell lysate. Furthermore, the WT1332-specific TCR-transduced CD4+ T cells lysed HLA-DPB1*05:01-positive, WT1-expressing human leukemia cells through granzyme B/perforin pathway. Furthermore, stimulation of peripheral blood mononuclear cells with both HLA-A*24:02-restricted cytotoxic T lymphocytes-epitope peptide (modified 9-mer WT1235 peptide, WT1235m) and WT1332 helper peptide in the presence of WT1332-specific TCR-transduced CD4+ T cells strikingly enhanced the induction of WT1235m-specific cytotoxic T lymphocytes. Thus, these results demonstrated the feasibility of immunotherapy based on adoptive transfer of WT1332-specific TCR-transduced CD4+ T cells for the treatment of leukemia.

  2. Regulation of T Cell Receptor Signaling by DENND1B in TH2 Cells and Allergic Disease.

    PubMed

    Yang, Chiao-Wen; Hojer, Caroline D; Zhou, Meijuan; Wu, Xiumin; Wuster, Arthur; Lee, Wyne P; Yaspan, Brian L; Chan, Andrew C

    2016-01-14

    The DENN domain is an evolutionary conserved protein module found in all eukaryotes and serves as an exchange factor for Rab-GTPases to regulate diverse cellular functions. Variants in DENND1B are associated with development of childhood asthma and other immune disorders. To understand how DENND1B may contribute to human disease, Dennd1b(-/-) mice were generated and exhibit hyper-allergic responses following antigen challenge. Dennd1b(-/-) TH2, but not other TH cells, exhibit delayed receptor-induced T cell receptor (TCR) downmodulation, enhanced TCR signaling, and increased production of effector cytokines. As DENND1B interacts with AP-2 and Rab35, TH2 cells deficient in AP-2 or Rab35 also exhibit enhanced TCR-mediated effector functions. Moreover, human TH2 cells carrying asthma-associated DENND1B variants express less DENND1B and phenocopy Dennd1b(-/-) TH2 cells. These results provide a molecular basis for how DENND1B, a previously unrecognized regulator of TCR downmodulation in TH2 cells, contributes to asthma pathogenesis and how DENN-domain-containing proteins may contribute to other human disorders.

  3. Vav1 regulates T cell activation through a feedback mechanism and crosstalk between the T cell receptor and CD28

    PubMed Central

    Helou, Ynes A.; Petrashen, Anna P.; Salomon, Arthur R.

    2015-01-01

    Vavl, a Rac/Rho guanine nucleotide exchange factor and a critical component of the T cell receptor (TCR) signaling cascade, is rapidly tyrosine phosphorylated in response to T cell activation. Vav1 has established roles in proliferation, cytokine secretion, Ca2+ responses, and actin cytoskeleton regulation, however, its function in the regulation of phosphorylation of TCR components, including the ζ chain, the CD3 δ, ε, γ chains, and the associated kinases Lck, and ZAP-70 is not well established. To obtain a more comprehensive picture of the role of Vav1 in receptor proximal signaling, we performed a wide-scale characterization of Vav1-dependent tyrosine phosphorylation events using quantitative phosphoproteomic analysis of Vav1-deficient T cells across a time course of TCR stimulation. Importantly, this study revealed a new function for Vav1 in the negative feedback regulation of the phosphorylation of immunoreceptor tyrosine-based activation motifs within the ζ chains, CD3 δ, ε, γ chains, as well as activation sites on the critical T cell tyrosine kinases Itk, Lck, and ZAP-70. Our study also uncovered a previously unappreciated role for Vav1 in crosstalk between the CD28 and TCR signaling pathways. PMID:26043137

  4. Crystal Structure of a Complete Ternary Complex of TCR, Superantigen and Peptide-MHC

    SciTech Connect

    Wang,L.; Zhao, Y.; Li, Z.; Guo, Y.; Jones, L.; Kranz, D.; Mourad, W.; Li, H.

    2007-01-01

    'Superantigens' (SAgs) trigger the massive activation of T cells by simultaneous interactions with MHC and TCR receptors, leading to human diseases. Here we present the first crystal structure, at 2.5-{angstrom} resolution, of a complete ternary complex between a SAg and its two receptors, HLA-DR1/HA and TCR. The most striking finding is that the SAg Mycoplasma arthritidis mitogen, unlike others, has direct contacts not only with TCR V{beta} but with TCR V{alpha}.

  5. Ubiquitylation as a Rheostat for TCR Signaling: From Targeted Approaches Toward Global Profiling.

    PubMed

    O'Leary, Claire E; Lewis, Emma L; Oliver, Paula M

    2015-01-01

    cells. These methods provide an exciting opportunity for further defining how TCR signals are regulated and for identifying new targets for therapeutic modulation.

  6. Ubiquitylation as a Rheostat for TCR Signaling: From Targeted Approaches Toward Global Profiling

    PubMed Central

    O’Leary, Claire E.; Lewis, Emma L.; Oliver, Paula M.

    2015-01-01

    primary T cells. These methods provide an exciting opportunity for further defining how TCR signals are regulated and for identifying new targets for therapeutic modulation. PMID:26732666

  7. Immune Tolerance Maintained by Cooperative Interactions between T Cells and Antigen Presenting Cells Shapes a Diverse TCR Repertoire

    PubMed Central

    Best, Katharine; Chain, Benny; Watkins, Chris

    2015-01-01

    The T cell population in an individual needs to avoid harmful activation by self peptides while maintaining the ability to respond to an unknown set of foreign peptides. This property is acquired by a combination of thymic and extra-thymic mechanisms. We extend current models for the development of self/non-self discrimination to consider the acquisition of self-tolerance as an emergent system level property of the overall T cell receptor repertoire. We propose that tolerance is established at the level of the antigen presenting cell/T cell cluster, which facilitates and integrates cooperative interactions between T cells of different specificities. The threshold for self-reactivity is therefore imposed at a population level, and not at the level of the individual T cell/antigen encounter. Mathematically, the model can be formulated as a linear programing optimization problem that can be implemented as a multiplicative update algorithm, which shows a rapid convergence to a stable state. The model constrains self-reactivity within a predefined threshold, but maintains repertoire diversity and cross reactivity which are key characteristics of human T cell immunity. We show further that the size of individual clones in the model repertoire becomes heterogeneous, and that new clones can establish themselves even when the repertoire has stabilized. Our study combines the salient features of the “danger” model of self/non-self discrimination with the concepts of quorum sensing, and extends repertoire generation models to encompass the establishment of tolerance. Furthermore, the dynamic and continuous repertoire reshaping, which underlies tolerance in this model, suggests opportunities for therapeutic intervention to achieve long-term tolerance following transplantation. PMID:26300880

  8. Characterization of human TCR Vbeta gene promoter. Role of the dodecamer motif in promoter activity.

    PubMed

    Deng, X; Sun, G R; Zheng, Q; Li, Y

    1998-09-11

    During T-lymphocyte development, the T-cell antigen receptor (TCR) gene expression is controlled by its promoter and enhancer elements and regulated in tissue- and development stage-specific manner. To uncover the promoter function and to define positive and negative regulatory elements in TCR gene promoters, the promoter activities from 13 human TCR Vbeta genes were determined by the transient transfection system and luciferase reporter assay. Although most of the TCR Vbeta gene promoters that we tested are inactive by themselves, some promoters were found to be constitutively strong. Among them, Vbeta6.7 is the strongest. 5'-Deletion and fragmentation experiments have narrowed the full promoter activity of Vbeta6.7 to a fragment of 147 base pairs immediately 5' to the transcription initiation site. A decanucleotide motif with the consensus sequence AGTGAYRTCA has been found to be conserved in most TCR Vbeta gene promoters. There are three such decamer motifs in the promoter region of Vbeta6.7, and the contribution of each such motif to the promoter activity has been examined. Further site-directed mutagenesis analyses showed that: 1) when two Ts in the decamer were mutated, the promoter activity was totally abolished; 2) when two additional nucleotides 3' to the end of decamer were mutated, the promoter activity was decreased to two-thirds of the full level; and 3) when the element with the sequence AGTGATGTCACT was inserted into other promoters, the original weak promoters become very strong. Taken together, our data suggest that the positive regulatory element in Vbeta6.7 should be considered a dodecamer rather than a decamer and that it confers strong basal transcriptional activity on TCR Vbeta genes.

  9. A20 negatively regulates T cell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitin chains.

    PubMed

    Düwel, Michael; Welteke, Verena; Oeckinghaus, Andrea; Baens, Mathijs; Kloo, Bernhard; Ferch, Uta; Darnay, Bryant G; Ruland, Jürgen; Marynen, Peter; Krappmann, Daniel

    2009-06-15

    The Carma1-Bcl10-Malt1 signaling module bridges TCR signaling to the canonical IkappaB kinase (IKK)/NF-kappaB pathway. Covalent attachment of regulatory ubiquitin chains to Malt1 paracaspase directs TCR signaling to IKK activation. Further, the ubiquitin-editing enzyme A20 was recently suggested to suppress T cell activation, but molecular targets for A20 remain elusive. In this paper, we show that A20 regulates the strength and duration of the IKK/NF-kappaB response upon TCR/CD28 costimulation. By catalyzing the removal of K63-linked ubiquitin chains from Malt1, A20 prevents sustained interaction between ubiquitinated Malt1 and the IKK complex and thus serves as a negative regulator of inducible IKK activity. Upon T cell stimulation, A20 is rapidly removed and paracaspase activity of Malt1 has been suggested to cleave A20. Using antagonistic peptides or reconstitution of Malt1(-/-) T cells, we show that Malt1 paracaspase activity is required for A20 cleavage and optimal IL-2 production, but dispensable for initial IKK/NF-kappaB signaling in CD4(+) T cells. However, proteasomal inhibition impairs A20 degradation and impedes TCR/CD28-induced IKK activation. Taken together, A20 functions as a Malt1 deubiquitinating enzyme and proteasomal degradation and de novo synthesis of A20 contributes to balance TCR/CD28-induced IKK/NF-kappaB signaling.

  10. Domains of the TCR beta-chain required for early thymocyte development

    PubMed Central

    1996-01-01

    The T cell receptor beta (TCR beta) chain controls the developmental transition from CD4-CD8- to CD4+8+thymocytes. We show that the extracellular constant region and the transmembrane region, but not the variable domain or cytoplasmic tail of the TCR beta chain are required for this differentiation step. TCR beta mutant chains lacking the cytoplasmic tail can be found at the cell surface both in functional TCR/CD3 complexes and in a GPI-anchored monomeric form indicating that the cytoplasmic tail of the TCR beta chain functions as an ER retention signal. The concordance between cell surface expression of the mutant chains as TCR/CD3 complexes and their capacity to mediate thymocyte differentiation supports the CD3 mediated feedback model in which preTCR/CD3 complexes control the developmental transition from CD4-CD8- to CD4+CD8+thymocytes. PMID:8920871

  11. Serine residues in the LAT adaptor are essential for TCR-dependent signal transduction.

    PubMed

    Martínez-Florensa, Mario; García-Blesa, Antonio; Yélamos, José; Muñoz-Suano, Alba; Domínguez-Villar, Margarita; Valdor, Rut; Alonso, Antonio; García-Cózar, Francisco; Aparicio, Pedro; Malissen, Bernard; Aguado, Enrique

    2011-01-01

    The adaptor protein LAT has a prominent role in the transduction of intracellular signals elicited by the TCR/CD3 complex. Upon TCR engagement, LAT becomes tyrosine-phosphorylated and thereby, recruits to the membrane several proteins implicated in the activation of downstream signaling pathways. However, little is known about the role of other conserved motifs present in the LAT sequence. Here, we report that the adaptor LAT contains several conserved serine-based motifs, which are essential for proper signal transduction through the TCR. Mutation of these serine motifs in the human T cell line Jurkat prevents proper calcium influx, MAPK activation, and IL-2 production in response to TCR/CD3 stimulation. Moreover, this mutant form of LAT has a reduced ability to bind to PLC-γ1 and SLP-76, although phosphorylation of tyrosine residues 132, 171, and 191 is not decreased, raising a possible role for the serine-based motifs of LAT for the binding of important partners. The functional role of LAT serine-based motifs in signal transduction could be mediated by an effect on tyrosine phosphorylation, as their mutation significantly diminishes the phosphorylation of tyrosine residue 226. In addition, these serine motifs seem to have a regulatory role, given that upon their mutation, ZAP-70 shows enhanced phosphorylation. Therefore, the LAT serine-based motifs likely regulate signaling pathways that are essential for T cell physiology.

  12. Targeted loss of SHP1 in murine thymocytes dampens TCR signaling late in selection.

    PubMed

    Martinez, Ryan J; Morris, Anna B; Neeld, Dennis K; Evavold, Brian D

    2016-09-01

    SHP1 is a tyrosine phosphatase critical to proximal regulation of TCR signaling. Here, analysis of CD4-Cre SHP1(fl/fl) conditional knockout thymocytes using CD53, TCRβ, CD69, CD4, and CD8α expression demonstrates the importance of SHP1 in the survival of post selection (CD53(+) ), single-positive thymocytes. Using Ca(2+) flux to assess the intensity of TCR signaling demonstrated that SHP1 dampens the signal strength of these same mature, postselection thymocytes. Consistent with its dampening effect, TCR signal strength was also probed functionally using peptides that can mediate selection of the OT-I TCR, to reveal increased negative selection mediated by lower-affinity ligand in the absence of SHP1. Our data show that SHP1 is required for the survival of mature thymocytes and the generation of the functional T-cell repertoire, as its absence leads to a reduction in the numbers of CD4(+) and CD8(+) naïve T cells in the peripheral lymphoid compartments.

  13. Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation.

    PubMed

    Valdor, Rut; Mocholi, Enric; Botbol, Yair; Guerrero-Ros, Ignacio; Chandra, Dinesh; Koga, Hiroshi; Gravekamp, Claudia; Cuervo, Ana Maria; Macian, Fernando

    2014-11-01

    Chaperone-mediated autophagy (CMA) targets soluble proteins for lysosomal degradation. Here we found that CMA was activated in T cells in response to engagement of the T cell antigen receptor (TCR), which induced expression of the CMA-related lysosomal receptor LAMP-2A. In activated T cells, CMA targeted the ubiquitin ligase Itch and the calcineurin inhibitor RCAN1 for degradation to maintain activation-induced responses. Consequently, deletion of the gene encoding LAMP-2A in T cells caused deficient in vivo responses to immunization or infection with Listeria monocytogenes. Impaired CMA activity also occurred in T cells with age, which negatively affected their function. Restoration of LAMP-2A in T cells from old mice resulted in enhancement of activation-induced responses. Our findings define a role for CMA in regulating T cell activation through the targeted degradation of negative regulators of T cell activation.

  14. Visualization of the human CD4⁺ T-cell response in humanized HLA-DR4-expressing NOD/Shi-scid/γc(null) (NOG) mice by retrogenic expression of the human TCR gene.

    PubMed

    Takahashi, Takeshi; Katano, Ikumi; Ito, Ryoji; Ito, Mamoru

    2015-01-02

    The development of severe immunodeficient mouse strains containing various human genes, including cytokines or HLA, has enabled the reconstitution of functional human immune systems after transplantation of human hematopoietic stem cells (HSC). Accumulating evidence has suggested that HLA-restricted antigen-specific human T-cell responses can be generated in these humanized mice. To directly monitor immune responses of human CD4(+) T cells, we introduced β-lactoglobulin (BLG)-specific T cell receptor (TCR) genes derived from CD4(+) T-cell clones of cow-milk allergy patients into HSCs, and subsequently transplanted them into NOG-HLA-DR4 transgenic/I-Aβ deficient mice (NOG-DR4/I-A(o)). In the thymus, thymocytes with BLG-specific TCR preferentially differentiated into CD4(+)CD8(-) single-positive cells. Adoptive transfer of mature CD4(+) T cells expressing the TCR into recipient NOG-DR4/I-A(o) mice demonstrated that human CD4(+) T cells proliferated in response to antigenic stimulation and produced IFN-γ in vivo, suggesting that functional T-cell reactions (especially Th1-skewed responses) were induced in humanized mice.

  15. 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

  16. TCR Signal Strength Alters T-DC Activation and Interaction Times and Directs the Outcome of Differentiation.

    PubMed

    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.

  17. Dynamic Regulation of TCR–Microclusters and the Microsynapse for T Cell Activation

    PubMed Central

    Hashimoto-Tane, Akiko; Saito, Takashi

    2016-01-01

    The interaction between a T cell and an antigen-presenting cell is the initiating event in T cell-mediated adaptive immunity. The Immunological Synapse (IS) is formed at the interface between these two cell types, and is the site where antigen (Ag)-specific recognition and activation are induced through the T cell receptor (TCR). This occurs at the center of the IS, and cell adhesion is supported through integrins in the area surrounding the TCR. Recently, this model has been revised based on data indicating that the initial Ag-specific activation signal is triggered prior to IS formation at TCR–microclusters (MCs), sites where TCR, kinases and adaptors of TCR proximal downstream signaling molecules accumulate as an activation signaling cluster. TCR–MCs then move into the center of the cell–cell interface to generate the cSMAC. This translocation of TCR–MCs is mediated initially by the actin cytoskeleton and then by dynein-induced movement along microtubules. The translocation of TCR–MCs and cSMAC formation is induced upon strong TCR stimulation through the assembly of a TCR–dynein super complex with microtubules. The Ag-specific activation signal is induced at TCR–MCs, but the adhesion signal is now shown to be induced by generating a “microsynapse,” which is composed of a core of TCR–MCs and the surrounding adhesion ring of integrin and focal adhesion molecules. Since the microsynapse is critical for activation, particularly under weak TCR stimulation, this structure supports a weak TCR signal through a cell–cell adhesion signal. The microsynapse has a structure similar to the IS but on a micro-scale and regulates Ag-specific activation as well as cell–cell adhesion. We describe here the dynamic regulation of TCR–MCs, responsible for inducing Ag-specific activation signals, and the microsynapse, responsible for adhesion signals critical for cell–cell interactions, and their interrelationship. PMID:27446085

  18. Deep sequencing of the TCR-β repertoire of human forkhead box protein 3 (FoxP3)(+) and FoxP3(-) T cells suggests that they are completely distinct and non-overlapping.

    PubMed

    Golding, A; Darko, S; Wylie, W H; Douek, D C; Shevach, E M

    2017-04-01

    Maintenance of peripheral tolerance requires a balance between autoreactive conventional T cells (Tconv ) and thymically derived forkhead box protein 3 (FoxP3)(+) regulatory T cells (tTregs ). Considerable controversy exists regarding the similarities/differences in T cell receptor (TCR) repertoires expressed by Tconv and tTregs . We generated highly purified populations of human adult and cord blood Tconv and tTregs based on the differential expression of CD25 and CD127. The purity of the sorted populations was validated by intracellular staining for FoxP3 and Helios. We also purified an overlap group of CD4 T cells from adult donors to ensure that considerable numbers of shared clonotypes could be detected when present. We used deep sequencing of entire TCR-β CDR3 sequences to analyse the TCR repertoire of Tconv and tTregs . Our studies suggest that both neonatal and adult human Tconv and tTreg cells are, in fact, entirely distinct CD4 T cell lineages.

  19. Synovial Regulatory T Cells Occupy a Discrete TCR Niche in Human Arthritis and Require Local Signals To Stabilize FOXP3 Protein Expression

    PubMed Central

    Giannakopoulou, Eirini; Lom, Hannah; Wedderburn, Lucy R.

    2015-01-01

    Although there is great interest in harnessing the immunosuppressive potential of FOXP3+ regulatory T cells (Tregs) for treating autoimmunity, a sizeable knowledge gap exists regarding Treg fate in human disease. In juvenile idiopathic arthritis (JIA) patients, we have previously reported that atypical CD25+FOXP3− Treg-like cells uniquely populate the inflamed site. Intriguingly, their proportions relative to CD25+FOXP3+ Tregs associate with arthritis course, suggesting a role in disease. The ontogeny of these FOXP3− Treg-like cells is, however, unknown. In this study, we interrogated clonal relationships between CD4+ T cell subsets in JIA, using high-throughput TCR repertoire analysis. We reveal that FOXP3+ Tregs possess highly exclusive TCRβ usage from conventional T cells, in blood, and also at the inflamed site, where they are clonally expanded. Intriguingly, the repertoires of FOXP3+ Tregs in synovial fluid are highly overlapping with CD25+FOXP3− Treg-like cells, indicating fluctuations in FOXP3 expression in the inflamed joint. Furthermore, cultured synovial Tregs rapidly downregulated FOXP3 protein (but not mRNA), and this process was prevented by addition of synovial fluid from JIA patients, through an IL-6–independent mechanism. Our findings suggest that most Tregs arise from a separate lineage from conventional T cells, and that this repertoire divergence is largely maintained under chronic inflammatory conditions. We propose that subsequent Treg expansions at the inflamed site creates an environment that leads to competition for limited resources within the synovium, resulting in the destabilization of FOXP3 expression in some Tregs. PMID:26561546

  20. Downregulation of T cell receptor expression by CD8(+) lymphocytes in kidney allografts.

    PubMed Central

    Mannon, R B; Kotzin, B L; Nataraj, C; Ferri, K; Roper, E; Kurlander, R J; Coffman, T M

    1998-01-01

    Allospecific CD8(+) T lymphocytes are an important component of the cellular response in allograft rejection. These cells recognize and engage MHC class I antigens, leading to allospecific cytolytic responses and graft rejection. In mouse kidney allografts that survive to 3 wk after transplantation, we noted that the majority of CD8(+) cells do not express surface alpha/beta T cell receptor alpha/beta(TCR), gamma/deltaTCR, or CD3. However, these CD8(+)TCR- cells did express surface markers characteristic of T cells, including Thy1.2, CD2, and CD5. In addition, the CD8(+)TCR- cells expressed mRNA for TCR Vbeta gene families, and nearly half stained positive for cytoplasmic Vbeta8 protein, suggesting that they are T cells that have downregulated alpha/betaTCR protein expression from their cell surfaces. When these surface TCR- cells were isolated from kidney allografts by flow cytometry and cultured in the presence of either allogeneic or syngeneic stimulators, nearly 100% of cells reacquired normal levels of alpha/betaTCR expression with disproportionate usage of Vbeta8 chains. After recovery of their surface TCR expression, the CD8(+)TCR- population demonstrated strong alloreactivity in culture. These results suggest that the substantial number of CD8(+)TCR- cells found in long-term surviving mouse kidney allografts are alpha/beta-T cells that have downregulated their cell surface expression of TCR. While in other systems this phenotype may identify cells that have engaged antigen, our results indicate that loss of TCR expression by CD8(+) kidney graft-infiltrating cells may not depend on antigen engagement and that elements in the microenvironment of the kidney graft play a key role in this process. Factors that modulate expression of TCR by graft-infiltrating lymphocytes may have an important role in regulating rejection responses. PMID:9616223

  1. Grouper (Epinephelus coioides) TCR signaling pathway was involved in response against Cryptocaryon irritans infection.

    PubMed

    Li, Ze-Xiang; Li, Yan-Wei; Xu, Shun; Xu, Yang; Mo, Ze-Quan; Dan, Xue-Ming; Luo, Xiao-Chun

    2017-03-07

    T cell activation is a complicated process accompanying with the activation of T cell receptor (TCR) signaling pathway, which is not well described in teleost fish. The initiation of this pathway depends on the interaction of membrane TCR co-receptors (e.g. CD4/8, CD3 and CD45) and a series of cytoplasmic protein tyrosine kinases (e.g. Lck, Fyn and ZAP70). Cyptocaryon irritans is a ciliate pathogen of marine fish white spot disease causing huge economic lost in marine aquaculture. This parasite can infect fish gill and skin and is considered to be a good pathogen model for fish gill and skin mucosal immunity. Our previous studies showed the locally mucosal antibody response was important for fish defense against this parasite. While how TCR signaling pathway involved in T cell activation to help B cell activation in C. irritans infected fish is still not known. In the present study, we cloned a grouper TCR co-receptor gene EcCD3ε (537 bp) and its three kinase genes, including EcLck (1512 bp), EcFyn (1605 bp) and EcZAP70 (1893 bp). Homology analysis showed that they all shared the highest identity with corresponding genes from Takifugu rubripes (EcCD3ε 41%, EcLck 88%, EcFyn 98% and EcZAP70 93%), and their conserved motifs involved in the signaling transduction were analyzed. The tissue distribution analysis showed these four genes were high expressed in thymus, and it is interesting to find their comparative high expression in skin, gill and midgut mucosal immune tissues. In C. irritans infected grouper, the expression of three TCR co-receptors (EcCD4-1, EcCD3ε and EcCD45) and three kinases (EcLck, EcFyn and EcZAP70) was tested in skin, gill, head kidney and spleen at 0, 12 h, 24 h, 2 d, 3 d, 5 d and 7 d. All six genes were significantly up-regulated in skin at most tested time points, which indicate the possibility of skin local T cell activation to support the local antibody response. Compared to three TCR co-receptors, significantly up-regulation of three

  2. Posttranslational modification of gluten shapes TCR usage in celiac disease.

    PubMed

    Qiao, Shuo-Wang; Ráki, Melinda; Gunnarsen, Kristin S; Løset, Geir-Åge; Lundin, Knut E A; Sandlie, Inger; Sollid, Ludvig M

    2011-09-15

    Posttranslational modification of Ag is implicated in several autoimmune diseases. In celiac disease, a cereal gluten-induced enteropathy with several autoimmune features, T cell recognition of the gluten Ag is heavily dependent on the posttranslational conversion of Gln to Glu residues. Evidence suggests that the enhanced recognition of deamidated gluten peptides results from improved peptide binding to the MHC and TCR interaction with the peptide-MHC complex. In this study, we report that there is a biased usage of TCR Vβ6.7 chain among TCRs reactive to the immunodominant DQ2-α-II gliadin epitope. We isolated Vβ6.7 and DQ2-αII tetramer-positive CD4(+) T cells from peripheral blood of gluten-challenged celiac patients and sequenced the TCRs of a large number of single T cells. TCR sequence analysis revealed in vivo clonal expansion, convergent recombination, semipublic response, and the notable conservation of a non-germline-encoded Arg residue in the CDR3β loop. Functional testing of a prototype DQ2-α-II-reactive TCR by analysis of TCR transfectants and soluble single-chain TCRs indicate that the deamidated residue in the DQ2-α-II peptide poses constraints on the TCR structure in which the conserved Arg residue is a critical element. The findings have implications for understanding T cell responses to posttranslationally modified Ags.

  3. Disease etiology and diagnosis by TCR repertoire analysis goes viral.

    PubMed

    Attaf, Meriem; Sewell, Andrew K

    2016-11-01

    The importance of T-cell receptor (TCR) repertoire diversity is highlighted in murine models of immunodeficiency and in many human pathologies. However, the true extent of TCR diversity and how this diversity varies in health and disease is poorly understood. In a previous issue of the European Journal of Immunology, Lossius et al. [Eur. J. Immunol. 2014. 44: 3439-3452] dissected the composition of the TCR repertoire in the context of multiple sclerosis (MS) using high-throughput sequencing of TCR-β chains in cerebrospinal fluid samples and blood. The authors demonstrated that the TCR repertoire of the CSF was largely distinct from the blood and enriched in EBV-reactive CD8(+) T cells in MS patients. Studies of this kind have long been hindered by technical limitations and remain scarce in the literature. However, TCR sequencing methodologies are progressing apace and will undoubtedly shed light on the genetic basis of T-cell responses and the ontogeny of T-cell-mediated diseases, such as MS.

  4. Immunophenotypic Analysis of the TCR-Vβ Repertoire in 98 Persistent Expansions of CD3+/TCR-αβ+ Large Granular Lymphocytes

    PubMed Central

    Lima, Margarida; Almeida, Julia; Santos, Ana Helena; dos Anjos Teixeira, Maria; del Carmen Alguero, Maria; Queirós, Maria Luís; Balanzategui, Ana; Justiça, Benvindo; Gonzalez, Marcos; San Miguel, Jesús F.; Orfão, Alberto

    2001-01-01

    At present, a major challenge in the initial diagnosis of leukemia of large granular lymphocytes (LGLs) is to establish the clonal nature of the expanded population. In the present study we have analyzed by flow cytometry immunophenotyping the TCR-Vβ repertoire of 98 consecutive cases of persistent expansions of CD4+ or CD8+bright CD3+/TCR-αβ+ LGLs and compared the results with those obtained in molecular studies of TCR-β gene rearrangements. Fifty-eight cases were considered to be monoclonal in molecular studies whereas in the remaining 40 cases there was no evidence for monoclonality (11 cases were considered oligoclonal and 29 polyclonal). The TCR-Vβ repertoire was biased to the preferential use of one or more TCR-Vβ families in 96% of cases, a total of 124 TCR-Vβ expansions being diagnosed: one TCR-Vβ expansion in 71 cases and two or more TCR-Vβ expansions in 23 cases. The highest TCR-Vβ expansion observed in each case was higher among monoclonal (74 ± 19%) as compared to nonmonoclonal cases (24 ± 14%) (P = 0.001), as did the fraction of LGLs that exhibited a TCR-Vβ-restricted pattern (86 ± 16% and 42 ± 23%, respectively; P = 0.0001); by contrast, the proportion of cases displaying more than one TCR-Vβ expansion was higher in the latter group: 7% versus 48%, respectively (P = 0.001). Results obtained in oligoclonal cases were intermediate between those obtained in polyclonal and monoclonal cases and similar results were observed for CD4+ as for CD8+bright T-cell expansions. TCR-Vβ familiesexpressed in CD8+bright T-cell-LGL proliferations showed a pattern of distribution that mimics the frequency at which the individual TCR-Vβ families are represented in normal peripheral blood T cells. Assuming that a given proliferation of LGLs is monoclonal whenever there is an expansion of a given TCR-Vβ family of at least 40% of the total CD4+ or CD8+bright T-cell compartment, we were able to predict clonality with a sensitivity of 93% and a specificity

  5. Transcription factors and target genes of pre-TCR signaling.

    PubMed

    López-Rodríguez, Cristina; Aramburu, Jose; Berga-Bolaños, Rosa

    2015-06-01

    Almost 30 years ago pioneering work by the laboratories of Harald von Boehmer and Susumo Tonegawa provided the first indications that developing thymocytes could assemble a functional TCRβ chain-containing receptor complex, the pre-TCR, before TCRα expression. The discovery and study of the pre-TCR complex revealed paradigms of signaling pathways in control of cell survival and proliferation, and culminated in the recognition of the multifunctional nature of this receptor. As a receptor integrated in a dynamic developmental process, the pre-TCR must be viewed not only in the light of the biological outcomes it promotes, but also in context with those molecular processes that drive its expression in thymocytes. This review article focuses on transcription factors and target genes activated by the pre-TCR to drive its different outcomes.

  6. The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

    PubMed

    Weng, Xiufang; Liao, Chia-Min; Bagchi, Sreya; Cardell, Susanna L; Stein, Paul L; Wang, Chyung-Ru

    2014-12-01

    CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP.

  7. TCR engagement induces proline-rich tyrosine kinase-2 (Pyk2) translocation to the T cell-APC interface independently of Pyk2 activity and in an immunoreceptor tyrosine-based activation motif-mediated fashion.

    PubMed

    Sancho, David; Montoya, María C; Monjas, Alicia; Gordón-Alonso, Mónica; Katagiri, Takuya; Gil, Diana; Tejedor, Reyes; Alarcón, Balbino; Sánchez-Madrid, Francisco

    2002-07-01

    The relocation of kinases in T lymphocytes during their cognate interaction with APCs is essential for lymphocyte activation. We found that the proline-rich tyrosine kinase-2 (Pyk2) is rapidly translocated to the T cell-APC contact area upon T cell-specific recognition of superantigen-pulsed APCs. Stimulation with anti-CD3-coated latex microspheres was sufficient for Pyk2 reorientation, and the coengagement of CD28 boosted Pyk2 redistribution. Nevertheless, Pyk2 translocation did not result in its recruitment to lipid rafts. Two results support that Pyk2 translocation was independent of its kinase activity. First, Lck activity was required for TCR-induced Pyk2 translocation, but not for TCR-induced Pyk2 activation. Second, a kinase-dead Pyk2 mutant was equally translocated upon TCR triggering. In addition, Lck activity alone was insufficient to induce Pyk2 reorientation and activation, requiring the presence of at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Despite the dependence on functional Lck and on phosphorylated ITAM for Pyk2 translocation, the ITAM-binding tyrosine kinase zeta-associated protein 70 (ZAP-70) was not essential. All these data suggest that, by translocating to the vicinity of the immune synapse, Pyk2 could play an essential role in T cell activation and polarized secretion of cytokines.

  8. Analysis of TCR/CD3 Recycling at the Immune Synapse.

    PubMed

    Patrussi, Laura; Baldari, Cosima T

    2017-01-01

    Engagement of the T cell antigen receptor (TCR) by specific ligand bound to the major histocompatibility complex is the primary event that leads to the assembly of the immune synapse (IS). Central to this process is TCR clustering at the T cell-APC contact, which is achieved with the contribution of an endosomal pool that is delivered to the IS by polarized recycling. As the TCR recycling process has not been fully elucidated, we developed methods suitable to quantitate recycling to the plasma membrane of TCR/CD3 complexes that have been engaged at the cell surface and track their traffic through the intracellular vesicular compartments toward the IS.

  9. The humoral response in TCR alpha-/- mice. Can gammadelta-T cells support the humoral immune response?

    PubMed

    Lindroth, K; Troye-Blomberg, M; Singh, M; Dieli, F; Ivanyi, J; Fernández, C

    2002-03-01

    An optimal humoral response requires T-cell help; however, it has been questioned if this help comes exclusively from alphabeta-T cells or whether gammadelta-T cells also contribute. We have attempted to answer this question by studying the humoral response in T-cell receptor alpha-chain knockout (alpha-/-) mice, which lack the alphabetaT cell subset. Two model antigens were used to characterize the response: the thymus-independent (TI) antigen native dextran B512 (Dx), and the thymus-dependent (TD) antigen heat shock protein (HSP65) from Mycobacterium tuberculosis. When challenged with Dx, the alpha-/- mice elicited a strong antibody response and formed rudimentary germinal centres (GCs), a T-cell dependent reaction. In contrast, the humoral response to HSP65 was poor. However, alpha-/- mice became primed when challenged with HSP65, because when supplemented with wild-type thymocytes, the antigen-primed animals were able to mount a stronger response than the nonprimed ones when challenged with HSP65. A crucial step seems to be the collaboration between gammadeltaT cells and antigen presenting cells (APCs), as splenocytes from alpha-/- mice were able to respond to HSP65 in an environment containing primed-APCs. Based on these results, we propose a model for B-cell activation in the alpha-/- mice.

  10. Expression profiling of TCR-engineered T cells demonstrates overexpression of multiple inhibitory receptors in persisting lymphocytes

    PubMed Central

    Abate-Daga, Daniel; Hanada, Ken-ichi; Davis, Jeremy L.; Yang, James C.; Rosenberg, Steven A.

    2013-01-01

    Despite significant progress in the development of adoptive cell-transfer therapies (ACTs) using gene-engineered T cells, little is known about the fate of cells following infusion. To address that, we performed a comparative analysis of gene expression between T-cell receptor–engineered lymphocytes persisting in the circulation 1 month after administration and the product that was infused. We observed that 156 genes related to immune function were differentially expressed, including underexpression of stimulators of lymphocyte function and overexpression of inhibitory genes in postinfusion cells. Of genes overexpressed postinfusion, the product of programmed cell death 1 (PDCD1), coinhibitory receptor PD-1, was expressed at a higher percentage in postinfusion lymphocytes than in the infusion product. This was associated with a higher sensitivity to inhibition of cytokine production by interaction with its ligand PD-L1. Coinhibitory receptor CD160 was also overexpressed in persisting cells, and its expression was associated with decreased reactivity, which surprisingly was found to be ligand-independent. These results contribute to a deeper understanding of the properties of transgenic lymphocytes used to treat human malignancies and may provide a rationale for the development of combination therapies as a method to improve ACT. This trial was registered at www.clinicaltrials.gov as #NCT00509288, #NCT00923195, and #NCT01273181. PMID:23861247

  11. Macroautophagy regulates energy metabolism during effector T cell activation.

    PubMed

    Hubbard, Vanessa M; Valdor, Rut; Patel, Bindi; Singh, Rajat; Cuervo, Ana Maria; Macian, Fernando

    2010-12-15

    Macroautophagy is a highly conserved mechanism of lysosomal-mediated protein degradation that plays a key role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins, and regulating protein levels in response to extracellular signals. We have found that macroautophagy is induced after effector T cell activation. Engagement of the TCR and CD28 results in enhanced microtubule-associated protein 1 light chain 3 (LC3) processing, increased numbers of LC3-containing vesicles, and increased LC3 flux, indicating active autophagosome formation and clearance. The autophagosomes formed in stimulated T cells actively fuse with lysosomes to degrade their cargo. Using a conditional KO mouse model where Atg7, a critical gene for macroautophagy, is specifically deleted in T cells, we have found that macroautophagy-deficient effector Th cells have defective IL-2 and IFN-γ production and reduced proliferation after stimulation, with no significant increase in apoptosis. We have found that ATP generation is decreased when autophagy is blocked, and defects in activation-induced cytokine production are restored when an exogenous energy source is added to macroautophagy-deficient T cells. Furthermore, we present evidence showing that the nature of the cargo inside autophagic vesicles found in resting T cells differs from the cargo of autophagosomes in activated T cells, where mitochondria and other organelles are selectively excluded. These results suggest that macroautophagy is an actively regulated process in T cells that can be induced in response to TCR engagement to accommodate the bioenergetic requirements of activated T cells.

  12. Autophagy regulates T lymphocyte proliferation through selective degradation of the cell-cycle inhibitor CDKN1B/p27Kip1.

    PubMed

    Jia, Wei; He, Ming-Xiao; McLeod, Ian X; Guo, Jian; Ji, Dong; He, You-Wen

    2015-01-01

    The highly conserved cellular degradation pathway, macroautophagy, regulates the homeostasis of organelles and promotes the survival of T lymphocytes. Previous results indicate that Atg3-, Atg5-, or Pik3c3/Vps34-deficient T cells cannot proliferate efficiently. Here we demonstrate that the proliferation of Atg7-deficient T cells is defective. By using an adoptive transfer and Listeria monocytogenes (LM) mouse infection model, we found that the primary immune response against LM is intrinsically impaired in autophagy-deficient CD8(+) T cells because the cell population cannot expand after infection. Autophagy-deficient T cells fail to enter into S-phase after TCR stimulation. The major negative regulator of the cell cycle in T lymphocytes, CDKN1B, is accumulated in autophagy-deficient naïve T cells and CDKN1B cannot be degraded after TCR stimulation. Furthermore, our results indicate that genetic deletion of one allele of CDKN1B in autophagy-deficient T cells restores proliferative capability and the cells can enter into S-phase after TCR stimulation. Finally, we found that natural CDKN1B forms polymers and is physiologically associated with the autophagy receptor protein SQSTM1/p62 (sequestosome 1). Collectively, autophagy is required for maintaining the expression level of CDKN1B in naïve T cells and selectively degrades CDKN1B after TCR stimulation.

  13. Functional evidence for TCR-intrinsic specificity for MHCII.

    PubMed

    Parrish, Heather L; Deshpande, Neha R; Vasic, Jelena; Kuhns, Michael S

    2016-03-15

    How T cells become restricted to binding antigenic peptides within class I or class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) via clonotypic T-cell receptors (TCRs) remains debated. During development, if TCR-pMHC interactions exceed an affinity threshold, a signal is generated that positively selects the thymocyte to become a mature CD4(+) or CD8(+) T cell that can recognize foreign peptides within MHCII or MHCI, respectively. But whether TCRs possess an intrinsic, subthreshold specificity for MHC that facilitates sampling of the peptides within MHC during positive selection or T-cell activation is undefined. Here we asked if increasing the frequency of lymphocyte-specific protein tyrosine kinase (Lck)-associated CD4 molecules in T-cell hybridomas would allow for the detection of subthreshold TCR-MHC interactions. The reactivity of 10 distinct TCRs was assessed in response to selecting and nonselecting MHCII bearing cognate, null, or "shaved" peptides with alanine substitutions at known TCR contact residues: Three of the TCRs were selected on MHCII and have defined peptide specificity, two were selected on MHCI and have a known pMHC specificity, and five were generated in vitro without defined selecting or cognate pMHC. Our central finding is that IL-2 was made when each TCR interacted with selecting or nonselecting MHCII presenting shaved peptides. These responses were abrogated by anti-CD4 antibodies and mutagenesis of CD4. They were also inhibited by anti-MHC antibodies that block TCR-MHCII interactions. We interpret these data as functional evidence for TCR-intrinsic specificity for MHCII.

  14. Impaired functional responses in follicular lymphoma CD8(+)TIM-3(+) T lymphocytes following TCR engagement.

    PubMed

    Gravelle, Pauline; Do, Catherine; Franchet, Camille; Mueller, Sabina; Oberic, Lucie; Ysebaert, Loïc; Larocca, Luigi Maria; Hohaus, Stefan; Calmels, Marie-Noëlle; Frenois, François-Xavier; Kridel, Robert; Gascoyne, Randy D; Laurent, Guy; Brousset, Pierre; Valitutti, Salvatore; Laurent, Camille

    2016-01-01

    Upregulation of T cell immunoglobulin-3 (TIM-3) has been associated with negative regulation of the immune response in chronic infection and cancer, including lymphoma. Here, we investigated the possible correlation between TIM-3 expression by ex vivo cytotoxic T cells (CTL) from follicular lymphoma (FL) biopsies and their functional unresponsiveness that could limit the favorable impact of CTL on disease progression. We report a high percentage of CD8(+)TIM-3(+)T cells in lymph nodes of FL patients. When compared to their CD8(+)TIM-3(-) counterparts, CD8(+)TIM-3(+) T cells exhibited defective cytokine production following TCR engagement. Furthermore, CD8(+)TIM-3(+) T cells display ex vivo markers of lytic granule release and remain unresponsive to further TCR-induced activation of the lytic machinery. Although confocal microscopy showed that TIM-3 expression on CD8(+) T cells correlated with minor alterations of immunological synapse, a selective reduction of ERK signaling in CD8(+)TIM-3(+)T cells was observed by phospho-flow analysis. Finally, short relapse-free survival despite rituximab(R)-chemotherapy was observed in patients with high content of TIM-3(+) cells and a poor infiltrate of granzyme B(+) T cells in FL lymph nodes. Together, our data indicate that, besides selective TCR early signaling defects, TIM-3 expression correlates with unresponsiveness of ex vivo CD8(+) T cells in FL. They show that scores based on the combination of exhaustion and cytolytic markers in FL microenvironment might be instrumental to identify patients at early risk of relapses following R-chemotherapy.

  15. A sharp T-cell antigen receptor signaling threshold for T-cell proliferation

    PubMed Central

    Au-Yeung, Byron B.; Zikherman, Julie; Mueller, James L.; Ashouri, Judith F.; Matloubian, Mehrdad; Cheng, Debra A.; Chen, Yiling; Shokat, Kevan M.; Weiss, Arthur

    2014-01-01

    T-cell antigen receptor (TCR) signaling is essential for activation, proliferation, and effector function of T cells. Modulation of both intensity and duration of TCR signaling can regulate these events. However, it remains unclear how individual T cells integrate such signals over time to make critical cell-fate decisions. We have previously developed an engineered mutant allele of the critical T-cell kinase zeta-chain-associated protein kinase 70 kDa (Zap70) that is catalytically inhibited by a small molecule inhibitor, thereby blocking TCR signaling specifically and efficiently. We have also characterized a fluorescent reporter Nur77–eGFP transgenic mouse line in which T cells up-regulate GFP uniquely in response to TCR stimulation. The combination of these technologies unmasked a sharp TCR signaling threshold for commitment to cell division both in vitro and in vivo. Further, we demonstrate that this threshold is independent of both the magnitude of the TCR stimulus and Interleukin 2. Similarly, we identify a temporal threshold of TCR signaling that is required for commitment to proliferation, after which T cells are able to proliferate in a Zap70 kinase-independent manner. Taken together, our studies reveal a sharp threshold for the magnitude and duration of TCR signaling required for commitment of T cells to proliferation. These results have important implications for understanding T-cell responses to infection and optimizing strategies for immunomodulatory drug delivery. PMID:25136127

  16. Transcriptional regulation of kinases downstream of the T cell receptor: another immunomodulatory mechanism of glucocorticoids

    PubMed Central

    2014-01-01

    Background Glucocorticoids affect peripheral immune responses, including modulation of T-cell activation, differentiation, and apoptosis. The quantity and quality of T-cell receptor (TCR)-triggered intracellular signals modulate T-cell function. Thus, glucocorticoids may affect T cells by interfering with the TCR signaling cascade. The purpose of the study was to search for glucocorticoid-modulated kinases downstream of the TCR. Methods Gene modulation in lymphoid cells either treated with glucocorticoids or from glucocorticoid-treated mice was studied using a RNase protection assay, real-time PCR, and western blotting. The sensitivity of genetically modified thymocytes to glucocorticoid-induced apoptosis was studied by performing hypotonic propidium iodide staining and flow cytometry. The Student’s t-test was employed for statistical evaluation. Results We found that transcription of Itk, a non-receptor tyrosine kinase of the Tec family, was up-regulated in a mouse T-cell hybridoma by the synthetic glucocorticoid dexamethasone. In contrast, dexamethasone down-regulated the expression of Txk, a Tec kinase that functions redundantly with Itk, and Lck, the Src kinase immediately downstream of the TCR. We investigated the expression of Itk, Txk, and Lck in thymocytes and mature lymphocytes following in vitro and in vivo dexamethasone treatment at different time points and doses. Kinase expression was differentially modulated and followed distinct kinetics. Itk was up-regulated in all cell types and conditions tested. Txk was strongly up-regulated in mature lymphocytes but only weakly up-regulated or non-modulated in thymocytes in vitro or in vivo, respectively. Conversely, Lck was down-regulated in thymocytes, but not modulated or up-regulated in mature lymphocytes in the different experimental conditions. This complex behaviour correlates with the presence of both positive and negative glucocorticoid responsive elements (GRE and nGRE, respectively) in the Itk, Txk

  17. D120 and K152 within the PH Domain of T Cell Adapter SKAP55 Regulate Plasma Membrane Targeting of SKAP55 and LFA-1 Affinity Modulation in Human T Lymphocytes.

    PubMed

    Witte, Amelie; Meineke, Bernhard; Sticht, Jana; Philipsen, Lars; Kuropka, Benno; Müller, Andreas J; Freund, Christian; Schraven, Burkhart; Kliche, Stefanie

    2017-04-01

    The β2-integrin lymphocyte function-associated antigen 1 (LFA-1) is needed for the T cell receptor (TCR)-induced activation of LFA-1 to promote T cell adhesion and interaction with antigen-presenting cells (APCs). LFA-1-mediated cell-cell interactions are critical for proper T cell differentiation and proliferation. The Src kinase-associated phosphoprotein of 55 kDa (SKAP55) is a key regulator of TCR-mediated LFA-1 signaling (inside-out/outside-in signaling). To gain an understanding of how SKAP55 controls TCR-mediated LFA-1 activation, we assessed the functional role of its pleckstrin homology (PH) domain. We identified two critical amino acid residues within the PH domain of SKAP55, aspartic acid 120 (D120) and lysine 152 (K152). D120 facilitates the retention of SKAP55 in the cytoplasm of nonstimulated T cells, while K152 promotes SKAP55 membrane recruitment via actin binding upon TCR triggering. Importantly, the K152-dependent interaction of the PH domain with actin promotes the binding of talin to LFA-1, thus facilitating LFA-1 activation. These data suggest that K152 and D120 within the PH domain of SKAP55 regulate plasma membrane targeting and TCR-mediated activation of LFA-1.

  18. Age-related decrease in TCR repertoire diversity measured with deep and normalized sequence profiling.

    PubMed

    Britanova, Olga V; Putintseva, Ekaterina V; Shugay, Mikhail; Merzlyak, Ekaterina M; Turchaninova, Maria A; Staroverov, Dmitriy B; Bolotin, Dmitriy A; Lukyanov, Sergey; Bogdanova, Ekaterina A; Mamedov, Ilgar Z; Lebedev, Yuriy B; Chudakov, Dmitriy M

    2014-03-15

    The decrease of TCR diversity with aging has never been studied by direct methods. In this study, we combined high-throughput Illumina sequencing with unique cDNA molecular identifier technology to achieve deep and precisely normalized profiling of TCR β repertoires in 39 healthy donors aged 6-90 y. We demonstrate that TCR β diversity per 10(6) T cells decreases roughly linearly with age, with significant reduction already apparent by age 40. The percentage of naive T cells showed a strong correlation with measured TCR diversity and decreased linearly up to age 70. Remarkably, the oldest group (average age 82 y) was characterized by a higher percentage of naive CD4(+) T cells, lower abundance of expanded clones, and increased TCR diversity compared with the previous age group (average age 62 y), suggesting the influence of age selection and association of these three related parameters with longevity. Interestingly, cross-analysis of individual TCR β repertoires revealed a set >10,000 of the most representative public TCR β clonotypes, whose abundance among the top 100,000 clones correlated with TCR diversity and decreased with aging.

  19. The Adaptor Molecule SAP Regulates IFNγ and IL-4 Production in Vα14 Transgenic NKT cells via Effects on GATA-3 and T-bet Expression1

    PubMed Central

    Cen, Osman; Ueda, Aki; Guzman, Laura; Jain, Jimmy; Bassiri, Hamid; Nichols, Kim E.; Stein, Paul L.

    2008-01-01

    NKT cells comprise a rare regulatory T cell population of limited TCR diversity, with most cells utilizing a Vα14Jα18 TCR. These cells exhibit a critical dependence on the signaling adapter molecule SAP for their ontogeny, an aspect not seen in conventional αβ T cells. Prior studies demonstrate that SAP enhances TCR-induced activation of NF-kB in CD4+ T cells. Since NF-kB is required for NKT cell development, SAP might promote the ontogeny of this lineage by signaling to NF-kB. In this report, we demonstrate that forced expression of the NF-kB target gene, Bcl-xL, or IKKβ, a catalytic subunit of the IkB kinase complex essential for NF-kB activation, fails to restore NKT cell development in sap−/− mice, suggesting that SAP mediates NKT cell development independently of NF-kB. To examine the role of SAP in NKT cell function, we generated NKT cells in sap−/− mice by expressing a transgene encoding the Vα14Jα18 component of the invariant TCR. These cells bound α-GalCer loaded CD1d tetramers, but exhibited a very immature CD24+NK1.1- phenotype. While sap−/− tetramer-reactive cells proliferated in response to TCR activation, they did not produce appreciable levels of IL-4 or IFN-γ. The reduction in cytokine production correlated with the near absence of GATA-3 and T-bet, key transcription factors regulating cytokine expression and maturation of NKT cells. Ectopic expression of GATA-3 partially restored IL-4 production by the NKT cells. Collectively these data suggest that by promoting GATA3 and T-bet expression, SAP exerts control over NKT cell development and mature NKT cell cytokine production. PMID:19155483

  20. NCAM regulates cell motility.

    PubMed

    Prag, Søren; Lepekhin, Eugene A; Kolkova, Kateryna; Hartmann-Petersen, Rasmus; Kawa, Anna; Walmod, Peter S; Belman, Vadym; Gallagher, Helen C; Berezin, Vladimir; Bock, Elisabeth; Pedersen, Nina

    2002-01-15

    Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells independently of homophilic NCAM interactions. Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59(fyn) with which NCAM-140 interacts. Furthermore, we showed that the extracellular part of NCAM acted as a paracrine inhibitor of NCAM-negative cell locomotion through a heterophilic interaction with a cell-surface receptor. As we showed that the two N-terminal immunoglobulin modules of NCAM, which are known to bind to heparin, were responsible for this inhibition, we presume that this receptor is a heparan sulfate proteoglycan. A model for the inhibitory effect of NCAM is proposed, which involves competition between NCAM and extracellular components for the binding to membrane-associated heparan sulfate proteoglycan.

  1. Dissecting the two models of TCR structure-function relationships.

    PubMed

    Cohn, Melvin

    2016-08-01

    There are only two comprehensive models attempting to account for the TCR structure-function relationships, referred to as the Standard or Centric model (Model I) and the Tritope model (Model II). This essay is written to analyze comparatively the two formulations of restrictive reactivity, stressing in particular the logic of each. Model I is essentially built on an analogy between the TCR and the BCR. Given a TCR with only one combining site (paratope), restrictive recognition requires that its ligand be viewed as a composite structure between the peptide and restricting element. It is this relationship that entrains a set of correlates that makes Model I untenable. Model II is predicated on the postulate that the recognition of the allele-specific determinants expressed by MHC-encoded restricting elements (R) is germline encoded and selected, whereas the recognition of peptide (P) is somatically encoded and selected. These selective pressures must operate on definable structures and this, in turn, necessitates a multiply recognitive T cell antigen receptor (TCR) with independent anti-R and anti-P paratopes that function coherently to signal restrictive reactivity. The consequences of this "two repertoire" postulate give us a concept of TCR structure quite distinct from that at present generally accepted, as well as a surprising relationship between numbers of functional TCR V gene segments and allele-specific determinants in the species. In the end, both models must deal with the relationship between the epitope-paratope interaction(s) and the signals to the T cell necessary for its differentiation and function.

  2. T Cell Receptor Signaling in the Control of Regulatory T Cell Differentiation and Function

    PubMed Central

    Li, Ming O.; Rudensky, Alexander Y.

    2016-01-01

    Regulatory T cells (TReg cells), a specialized T cell lineage, have a pivotal function in the control of self-tolerance and inflammatory responses. Recent studies have revealed a discrete mode of TCR signaling that regulates Treg cell differentiation, maintenance and function and that impacts on gene expression, metabolism, cell adhesion and migration of these cells. Here, we discuss the emerging understanding of TCR-guided differentiation of Treg cells in the context of their function in health and disease. PMID:27026074

  3. Cell Proliferation, Cell Death, and Size Regulation

    DTIC Science & Technology

    1998-10-01

    Cell Death , and Size Regulation PRINCIPAL INVESTIGATOR: Nicholas E. Baker, Ph.D. CONTRACTING ORGANIZATION: Albert Einstein College of Medicine of Yeshiva...SUBTITLE 5. FUNDING NUMBERS Cell Proliferation, Cell Death , and Size Regulation DAMD17-97-1-7034 6. AUTHOR(S) Nicholas E. Baker, Ph.D. 7. PERFORMING...Contains unpublished data 5 CELL PROLIFERATION, CELL DEATH , AND SIZE REGULATION INTRODUCTION Cell proliferation and cell death come to attention through

  4. The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse.

    PubMed

    Finetti, Francesca; Patrussi, Laura; Galgano, Donatella; Cassioli, Chiara; Perinetti, Giuseppe; Pazour, Gregory J; Baldari, Cosima T

    2015-07-15

    IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11(+) endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR(+) endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis.

  5. The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

    PubMed Central

    Finetti, Francesca; Patrussi, Laura; Galgano, Donatella; Cassioli, Chiara; Perinetti, Giuseppe; Pazour, Gregory J.; Baldari, Cosima T.

    2015-01-01

    ABSTRACT IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11+ endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR+ endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis. PMID:26034069

  6. Stage and Tissue Specific Expression of Four TCR Subunits in Olive Flounder (Paralichthys olivaceus).

    PubMed

    Lee, Young Mee; Lee, Jeong-Ho; Noh, Jae Koo; Kim, Hyun Chul; Park, Choul-Ji; Park, Jong-Won; Hwang, In Joon; Kim, Sung Yeon

    2013-12-01

    TCR subunits are members of membrane-bound receptors which allow the fast and efficient elimination of the specific fish pathogens have regulated function in adaptive immunity. Sequence structure of TCR subunits have been reported for various teleosts, but the information of each TCR subunit functional characterization through expression analysis in fish was unknown. In this study, we examined the gene expression of TCR subunits in the early developmental stages and observed transcript levels in various tissues from healthy adult olive flounder by RT-PCR. The mRNA expression of alpha subunit was already detected in the previous hatching step. But the transcripts of another TCR subunit were not observed during embryo development and increased after hatching and maintained until metamorphosis at the same level. It was found that all TCR subunits mRNAs are commonly expressed in the immune-related organ such as spleen, kidney and gill, also weak expressed in fin and eye. TCR alpha and beta subunit were expressed in brain, whereas gamma and delta were not expressed same tissue. The sequence alignment analysis shows that there are more than 80% sequence homology between TCR subunits. Because it has a high similarity of amino acid sequence to expect similar in function, but expression analysis show that will have may functional diversity due to different time and place of expression.

  7. Evidence for a functional sidedness to the αβTCR

    PubMed Central

    Kuhns, Michael S.; Girvin, Andrew T.; Klein, Lawrence O.; Chen, Rebecca; Jensen, Kirk D.C.; Newell, Evan W.; Huppa, Johannes B.; Lillemeier, Björn F.; Huse, Morgan; Chien, Yueh-hsiu; Garcia, K. Christopher; Davis, Mark M.

    2010-01-01

    The T cell receptor (TCR) and associated CD3γε, δε, and ζζ signaling dimers allow T cells to discriminate between different antigens and respond accordingly, but our knowledge of how these parts fit and work together is incomplete. In this study, we provide additional evidence that the CD3 heterodimers congregate on one side of the TCR in both the αβ and γδTCR-CD3 complexes. We also report that the other side of the αβTCR mediates homotypic αβTCR interactions and signaling. Specifically, an erythropoietin receptor-based dimerization assay was used to show that, upon complex assembly, the CD3ε chains of two CD3 heterodimers are arranged side-by-side in both the αβ and γδTCR-CD3 complexes. This system was also used to show that αβTCRs can dimerize in the cell membrane and that mutating the unusual outer strands of the Cα domain impairs this dimerization. Finally, we present data showing that, for CD4 T cells, the mutations that impair αβTCR dimerization also alter ligand-induced calcium mobilization, TCR accumulation at the site of pMHC contact, and polarization toward the site of antigen contact. These data reveal a “functional-sidedness” to the αβTCR constant region, with dimerization occurring on the side of the TCR opposite from where the CD3 heterodimers are located. PMID:20202921

  8. MERTK as negative regulator of human T cell activation

    PubMed Central

    Cabezón, Raquel; Carrera-Silva, E. Antonio; Flórez-Grau, Georgina; Errasti, Andrea E.; Calderón-Gómez, Elisabeth; Lozano, Juan José; España, Carolina; Ricart, Elena; Panés, Julián; Rothlin, Carla Vanina; Benítez-Ribas, Daniel

    2015-01-01

    The aim of this study was to test the hypothesis whether MERTK, which is up-regulated in human DCs treated with immunosuppressive agents, is directly involved in modulating T cell activation. MERTK is a member of the TAM family and contributes to regulating innate immune response to ACs by inhibiting DC activation in animal models. However, whether MERTK interacts directly with T cells has not been addressed. Here, we show that MERTK is highly expressed on dex-induced human tol-DCs and participates in their tolerogenic effect. Neutralization of MERTK in allogenic MLR, as well as autologous DC–T cell cultures, leads to increased T cell proliferation and IFN-γ production. Additionally, we identify a previously unrecognized noncell-autonomous regulatory function of MERTK expressed on DCs. Mer-Fc protein, used to mimic MERTK on DCs, suppresses naïve and antigen-specific memory T cell activation. This mechanism is mediated by the neutralization of the MERTK ligand PROS1. We find that MERTK and PROS1 are expressed in human T cells upon TCR activation and drive an autocrine proproliferative mechanism. Collectively, these results suggest that MERTK on DCs controls T cell activation and expansion through the competition for PROS1 interaction with MERTK in the T cells. In conclusion, this report identified MERTK as a potent suppressor of T cell response. PMID:25624460

  9. Aurora-A shines on T cell activation through the regulation of Lck.

    PubMed

    Blas-Rus, Noelia; Bustos-Morán, Eugenio; Martín-Cófreces, Noa B; Sánchez-Madrid, Francisco

    2017-02-01

    Different protein kinases control signaling emanating from the T cell receptor (TCR) during antigen-specific T cell activation. Mitotic kinases, e.g. Aurora-A, have been widely studied in the context of mitosis due to their role during microtubule (MT) nucleation, becoming critical regulators of cell cycle progression. We have recently described a specific role for Aurora-A kinase in antigenic T cell activation. Blockade of Aurora-A in T cells severely disrupts the dynamics of MTs and CD3ζ-bearing signaling vesicles during T cell activation. Furthermore, Aurora-A deletion impairs the activation of signaling molecules downstream of the TCR. Targeting Aurora-A disturbs the activation of Lck, which is one of the first signals that drive T cell activation in an antigen-dependent manner. This work describes possible models of regulation of Lck by Aurora-A during T cell activation. We also discuss possible roles for Aurora-A in other systems similar to the IS, and its putative functions in cell polarization.

  10. Two common structural motifs for TCR recognition by staphylococcal enterotoxins

    PubMed Central

    Rödström, Karin E. J.; Regenthal, Paulina; Bahl, Christopher; Ford, Alex; Baker, David; Lindkvist-Petersson, Karin

    2016-01-01

    Superantigens are toxins produced by Staphylococcus aureus, called staphylococcal enterotoxins (abbreviated SEA to SEU). They can cross-link the T cell receptor (TCR) and major histocompatibility complex class II, triggering a massive T cell activation and hence disease. Due to high stability and toxicity, superantigens are potential agents of bioterrorism. Hence, antagonists may not only be useful in the treatment of disease but also serve as countermeasures to biological warfare. Of particular interest are inhibitors against SEA and SEB. SEA is the main cause of food poisoning, while SEB is a common toxin manufactured as a biological weapon. Here, we present the crystal structures of SEA in complex with TCR and SEE in complex with the same TCR, complemented with computational alanine-scanning mutagenesis of SEA, SEB, SEC3, SEE, and SEH. We have identified two common areas that contribute to the general TCR binding for these superantigens. This paves the way for design of single antagonists directed towards multiple toxins. PMID:27180909

  11. TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

    PubMed

    Tomkowicz, Brian; Walsh, Eileen; Cotty, Adam; Verona, Raluca; Sabins, Nina; Kaplan, Fred; Santulli-Marotto, Sandy; Chin, Chen-Ni; Mooney, Jill; Lingham, Russell B; Naso, Michael; McCabe, Timothy

    2015-01-01

    TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

  12. CD103 or LFA-1 engagement at the immune synapse between cytotoxic T cells and tumor cells promotes maturation and regulates T-cell effector functions.

    PubMed

    Franciszkiewicz, Katarzyna; Le Floc'h, Audrey; Boutet, Marie; Vergnon, Isabelle; Schmitt, Alain; Mami-Chouaib, Fathia

    2013-01-15

    T-cell adhesion/costimulatory molecules and their cognate receptors on target cells play a major role in T-cell receptor (TCR)-mediated activities. Here, we compared the involvement of CD103 and LFA-1, and their respective ligands, in the maturation of the cytotoxic immune synapse (cIS) and in the activation of CTL effector functions. Our results indicate that cytotoxicity toward cancer cells and, to a lesser extent, cytokine production by specific CTL require, together with TCR engagement, the interaction of either CD103 with E-cadherin or LFA-1 with ICAM-1. Flow-based adhesion assay showed that engagement of CD103 or LFA-1, together with TCR, enhances the strength of the T-cell/target cell interaction. Moreover, electron microscopic analyses showed that integrin-dependent mature cIS (mcIS) displays a cohesive ultrastructure, with tight membrane contacts separated by extensive clefts. In contrast, immature cIS (icIS), which is unable to trigger target cell lysis, is loose, with multiple protrusions in the effector cell membrane. Experiments using confocal microscopy revealed polarized cytokine release and degranulation at the mcIS associated with target cell killing, whereas icIS is characterized by failure of IFN-γ and granzyme B relocalization. Thus, interactive forces between CTL and epithelial tumor cells, mainly regulated by integrin engagement, correlate with maturity and the ultrastructure of the cIS and influence CTL effector functions. These results provide new insights into molecular mechanisms regulating antitumor CTL responses and may lead to the development of more efficient cancer immunotherapy strategies.

  13. Unpredicted phenotypes of two mutants of the TcR DMF5.

    PubMed

    Tadesse, Fitsum G; Mensali, Nadia; Fallang, Lars-Egil; Walseng, Even; Yang, Weiwen; Olweus, Johanna; Wälchli, Sébastien

    2015-10-01

    When a T-cell Receptor (TcR) interacts with its cognate peptide-MHC (pMHC), it triggers activation of a signaling cascade that results in the elicitation of a T cell effector function. Different models have been proposed to understand which parameters are needed to obtain an optimal activation of the signaling. It was speculated that improving the binding of a TcR could bring a stronger pMHC recognition, hence a stronger stimulation of the T cell. However, it was recently shown that an increase in affinity does not seem to be sufficient to guarantee improved functionality. A combination of factors is necessary to place the modified TcR in an optimal functional window. We here compared the binding parameters of two mutants of the melanoma antigen peptide MART-127-35 specific TcR DMF5. The first mutant was previously isolated by others in a screen for improved TcR. It was reported to have an increased CD8-independent activity. We confirmed these data and showed that the enhancement was neither due to change in half life (t1/2) nor Kd of the pMHC-TcR complex. The second mutant was designed based on a previous report claiming that a particular polymorphic residue in the TRAV12-2 chain was stabilizing the TcR. We created a DMF5 mutant for this residue and showed that, unexpectedly, this TcR had acquired a reduced overall activity although the TcR-pMHC complex was more stable when compared to the TcR wild type complex (increased t1/2). In addition, the soluble TcR form of this mutant bound target cells less efficiently. From this we concluded that kinetic parameters do not always predict the superior functionality of mutant TcRs.

  14. Activated PLC-γ1 is catalytically induced at LAT but activated PLC-γ1 is localized at both LAT- and TCR-containing complexes.

    PubMed

    Cruz-Orcutt, Noemi; Vacaflores, Aldo; Connolly, Sean F; Bunnell, Stephen C; Houtman, Jon C D

    2014-04-01

    Phospholipase C-γ1 (PLC-γ1) is a key regulator of T cell receptor (TCR)-induced signaling. Activation of the TCR enhances PLC-γ1 enzymatic function, resulting in calcium influx and the activation of PKC family members and RasGRP. The current model is that phosphorylation of LAT tyrosine 132 facilitates the recruitment of PLC-γ1, leading to its activation and function at the LAT complex. In this study, we examined the phosphorylation kinetics of LAT and PLC-γ1 and the cellular localization of activated PLC-γ1. We observed that commencement of the phosphorylation of LAT tyrosine 132 and PLC-γ1 tyrosine 783 occurred simultaneously, supporting the current model. However, once begun, PLC-γ1 activation occurred more rapidly than LAT tyrosine 132. The association of LAT and PLC-γ1 was more transient than the interaction of LAT and Grb2 and a pool of activated PLC-γ1 translocated away from LAT to cellular structures containing the TCR. These studies demonstrate that LAT and PLC-γ1 form transient interactions that catalyze the activation of PLC-γ1, but that activated PLC-γ1 resides in both LAT and TCR clusters. Together, this work highlights that our current model is incomplete and the activation and function of PLC-γ1 in T cells is highly complex.

  15. TCR triggering by pMHC ligands tethered on surfaces via poly(ethylene glycol) depends on polymer length.

    PubMed

    Ma, Zhengyu; LeBard, David N; Loverde, Sharon M; Sharp, Kim A; Klein, Michael L; Discher, Dennis E; Finkel, Terri H

    2014-01-01

    Antigen recognition by T cells relies on the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) at the interface between the T cell and the antigen presenting cell (APC). The pMHC-TCR interaction is two-dimensional (2D), in that both the ligand and receptor are membrane-anchored and their movement is limited to 2D diffusion. The 2D nature of the interaction is critical for the ability of pMHC ligands to trigger TCR. The exact properties of the 2D pMHC-TCR interaction that enable TCR triggering, however, are not fully understood. Here, we altered the 2D pMHC-TCR interaction by tethering pMHC ligands to a rigid plastic surface with flexible poly(ethylene glycol) (PEG) polymers of different lengths, thereby gradually increasing the ligands' range of motion in the third dimension. We found that pMHC ligands tethered by PEG linkers with long contour length were capable of activating T cells. Shorter PEG linkers, however, triggered TCR more efficiently. Molecular dynamics simulation suggested that shorter PEGs exhibit faster TCR binding on-rates and off-rates. Our findings indicate that TCR signaling can be triggered by surface-tethered pMHC ligands within a defined 3D range of motion, and that fast binding rates lead to higher TCR triggering efficiency. These observations are consistent with a model of TCR triggering that incorporates the dynamic interaction between T cell and antigen-presenting cell.

  16. Structure of the superantigen staphylococcal enterotoxin B in complex with TCR and peptide-MHC demonstrates absence of TCR-peptide contacts.

    PubMed

    Rödström, Karin E J; Elbing, Karin; Lindkvist-Petersson, Karin

    2014-08-15

    Superantigens are immune-stimulatory toxins produced by Staphylococcus aureus, which are able to interact with host immune receptors to induce a massive release of cytokines, causing toxic shock syndrome and possibly death. In this article, we present the x-ray structure of staphylococcal enterotoxin B (SEB) in complex with its receptors, the TCR and MHC class II, forming a ternary complex. The structure, in combination with functional analyses, clearly shows how SEB adopts a wedge-like position when binding to the β-chain of TCR, allowing for an interaction between the α-chain of TCR and MHC. Furthermore, the binding mode also circumvents contact between TCR and the peptide presented by MHC, which enables SEB to initiate a peptide-independent activation of T cells.

  17. Structural Model of the Extracellular Assembly of the TCR-CD3 Complex.

    PubMed

    Natarajan, Aswin; Nadarajah, Vidushan; Felsovalyi, Klara; Wang, Wenjuan; Jeyachandran, Vivian R; Wasson, Riley A; Cardozo, Timothy; Bracken, Clay; Krogsgaard, Michelle

    2016-03-29

    Antigen recognition of peptide-major histocompatibility complexes (pMHCs) by T cells, a key step in initiating adaptive immune responses, is performed by the T cell receptor (TCR) bound to CD3 heterodimers. However, the biophysical basis of the transmission of TCR-CD3 extracellular interaction into a productive intracellular signaling sequence remains incomplete. Here we used nuclear magnetic resonance (NMR) spectroscopy combined with mutational analysis and computational docking to derive a structural model of the extracellular TCR-CD3 assembly. In the inactivated state, CD3γε interacts with the helix 3 and helix 4-F strand regions of the TCR Cβ subunit, whereas CD3δε interacts with the F and C strand regions of the TCR Cα subunit in this model, placing the CD3 subunits on opposing sides of the TCR. This work identifies the molecular contacts between the TCR and CD3 subunits, identifying a physical basis for transmitting an activating signal through the complex.

  18. Th1 and Th17 cells regulate innate immune responses and bacterial clearance during central nervous system infection.

    PubMed

    Holley, Monica M; Kielian, Tammy

    2012-02-01

    Brain abscesses arise following parenchymal infection with pyogenic bacteria and are typified by inflammation and edema, which frequently results in a multitude of long-term health problems. The impact of adaptive immunity in shaping continued innate responses during late-stage brain abscess formation is not known but is important, because robust innate immunity is required for effective bacterial clearance. To address this issue, brain abscesses were induced in TCR αβ knockout (KO) mice, because CD4(+) and NKT cells represented the most numerous T cell infiltrates. TCR αβ KO mice exhibited impaired bacterial clearance during later stages of infection, which was associated with alterations in neutrophil and macrophage recruitment, as well as perturbations in cytokine/chemokine expression. Adoptive transfer of either Th1 or Th17 cells into TCR αβ KO mice restored bacterial burdens and innate immune cell infiltrates to levels detected in wild-type animals. Interestingly, adoptively transferred Th17 cells demonstrated plasticity within the CNS compartment and induced distinct cytokine secretion profiles in abscess-associated microglia and macrophages compared with Th1 transfer. Collectively, these studies identified an amplification loop for Th1 and Th17 cells in shaping established innate responses during CNS infection to maximize bacterial clearance and differentially regulate microglial and macrophage secretory profiles.

  19. Antigen receptor-regulated exocytosis in cytotoxic T lymphocytes

    PubMed Central

    1987-01-01

    We demonstrate here that T cell receptor for antigen (TCR)-triggered exocytosis in cytotoxic T lymphocytes (CTL) is not constitutive and is regulated through crosslinking of the TCR by antigen or monoclonal anti- TCR antibodies. Morphological and biochemical data using three different biochemical markers of granules and Percoll gradient fractionation analysis are presented, suggesting that TCR-triggered exocytosis is accompanied by the loss of granules from CTL and appearance of intragranular proteins and enzymatic activities in the incubation medium. The strict requirement for crosslinking of the TCR in exocytosis triggering could be bypassed by protein kinase C activators (phorbol esters or bryostatin I and II) acting in synergy with Ca2+ ionophores. It is shown that external Ca2+ is obligatory for both the TCR-triggered and for the PMA/A23187-triggered exocytosis, since Ca2+ chelators and divalent cations that compete with Ca2+ for A23187 can inhibit exocytosis of granules. These data suggest that Ca2+ from intracellular stores is not sufficient to support exocytosis in CTL. Ca2+ channel blockers and calmodulin antagonists significantly inhibited TCR-triggered exocytosis without affecting the basal level of secretion. The described results are consistent with a model in which exocytosis of granules in CTL is triggered by the crosslinking of TCR, transmembrane protein kinase C activation, and external Ca2+ translocation through CTL plasma membrane Ca2+ channels and modulation of activity of Ca2+, calmodulin-dependent enzymes, and cytoskeletal proteins. PMID:2442289

  20. Pre-TCR signaling and CD8 gene bivalent chromatin resolution during thymocyte development.

    PubMed

    Harker, Nicola; Garefalaki, Anna; Menzel, Ursula; Ktistaki, Eleni; Naito, Taku; Georgopoulos, Katia; Kioussis, Dimitris

    2011-06-01

    The CD8 gene is silent in CD4(-)CD8(-) double-negative thymocytes, expressed in CD4(+)CD8(+) double-positive cells, and silenced in cells committing to the CD4(+) single-positive (SP) lineage, remaining active in the CD8(+) SP lineage. In this study, we show that the chromatin of the CD8 locus is remodeled in C57BL/6 and B6/J Rag1(-/-) MOM double-negative thymocytes as indicated by DNaseI hypersensitivity and widespread bivalent chromatin marks. Pre-TCR signaling coincides with chromatin bivalency resolution into monovalent activating modifications in double-positive and CD8 SP cells. Shortly after commitment to CD4 SP cell lineage, monovalent repressive characteristics and chromatin inaccessibility are established. Differential binding of Ikaros, NuRD, and heterochromatin protein 1α on the locus during these processes may participate in the complex regulation of CD8.

  1. Cholesterol and sphingomyelin drive ligand-independent T-cell antigen receptor nanoclustering.

    PubMed

    Molnár, Eszter; Swamy, Mahima; Holzer, Martin; Beck-García, Katharina; Worch, Remigiusz; Thiele, Christoph; Guigas, Gernot; Boye, Kristian; Luescher, Immanuel F; Schwille, Petra; Schubert, Rolf; Schamel, Wolfgang W A

    2012-12-14

    The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRβ chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRβ chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.

  2. TCR usage, gene expression and function of two distinct FOXP3(+)Treg subsets within CD4(+)CD25(hi) T cells identified by expression of CD39 and CD45RO.

    PubMed

    Ye, Lingying; Goodall, Jane C; Zhang, Libin; Putintseva, Ekaterina V; Lam, Brian; Jiang, Lei; Liu, Wei; Yin, Jian; Lin, Li; Li, Ting; Wu, Xin; Yeo, Giles; Shugay, Mikhail; Chudakov, Dmitriy M; Gaston, Hill; Xu, Huji

    2016-03-01

    FOXP3+ regulatory T (Treg) cells are indispensable for immune homeostasis, but their study in humans is complicated by heterogeneity within Treg, the difficulty in purifying Tregs using surface marker expression (e.g. CD25) and the transient expression of FOXP3 by activated effector cells. Here, we report that expression of CD39 and CD45RO distinguishes three sub-populations within human CD4(+)CD25(hi) T cells. Initial phenotypic and functional analysis demonstrated that CD4(+)CD25(hi)CD39(+)CD45RO(+) cells had properties consistent with effector Treg, CD4(+)CD25(hi)CD39(-)CD45RO(-) cells were naïve Treg and CD4(+)CD25(hi)CD39(-)CD45RO(+) cells were predominantly non-Treg with effector T-cell function. Differences in these two newly identified Treg subsets were corroborated by studies of gene expression and TCR analysis. To apply this approach, we studied these two newly identified Treg subsets in ankylosing spondylitis, and showed impairment in both effector and naïve Treg. This work highlights the importance of discriminating Treg subsets to enable proper comparisons of immune regulatory capacity in healthy individuals and those with inflammatory disease.

  3. Tpl2 and ERK transduce antiproliferative T cell receptor signals and inhibit transformation of chronically stimulated T cells.

    PubMed

    Tsatsanis, Christos; Vaporidi, Katerina; Zacharioudaki, Vassiliki; Androulidaki, Ariadne; Sykulev, Yuri; Margioris, Andrew N; Tsichlis, Philip N

    2008-02-26

    The protein kinase encoded by the Tpl2 protooncogene plays an obligatory role in the transduction of Toll-like receptor and death receptor signals in macrophages, B cells, mouse embryo fibroblasts, and epithelial cells in culture and promotes inflammatory responses in animals. To address its role in T cell activation, we crossed the T cell receptor (TCR) transgene 2C, which recognizes class I MHC presented peptides, into the Tpl2(-/-) genetic background. Surprisingly, the TCR2C(tg/tg)/Tpl2(-/-) mice developed T cell lymphomas with a latency of 4-6 months. The tumor cells were consistently TCR2C(+)CD8(+)CD4(-), suggesting that they were derived either from chronically stimulated mature T cells or from immature single positive (ISP) cells. Further studies showed that the population of CD8(+) ISP cells was not expanded in the thymus of TCR2C(tg/tg)/Tpl2(-/-) mice, making the latter hypothesis unlikely. Mature peripheral T cells of Tpl2(-/-) mice were defective in ERK activation and exhibited enhanced proliferation after TCR stimulation. The same cells were defective in the induction of CTLA4, a negative regulator of the T cell response, which is induced by TCR signals via ERK. These findings suggest that Tpl2 functions normally in a feedback loop that switches off the T cell response to TCR stimulation. As a result, Tpl2, a potent oncogene, functions as a tumor suppressor gene in chronically stimulated T cells.

  4. Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

    PubMed Central

    Ibiza, Sales; Pérez-Rodríguez, Andrea; Ortega, Ángel; Martínez-Ruiz, Antonio; Barreiro, Olga; García-Domínguez, Carlota A.; Víctor, Víctor M.; Esplugues, Juan V.; Rojas, José M.; Sánchez-Madrid, Francisco; Serrador, Juan M.

    2008-01-01

    Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys118, suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys118 contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments. PMID:18641128

  5. The kinases MEKK2 and MEKK3 regulate transforming growth factor-β-mediated helper T cell differentiation.

    PubMed

    Chang, Xing; Liu, Fang; Wang, Xiaofang; Lin, Aiping; Zhao, Hongyu; Su, Bing

    2011-02-25

    Mitogen-activated protein kinases (MAPKs) are key mediators of the T cell receptor (TCR) signals but their roles in T helper (Th) cell differentiation are unclear. Here we showed that the MAPK kinase kinases MEKK2 (encoded by Map3k2) and MEKK3 (encoded by Map3k3) negatively regulated transforming growth factor-β (TGF-β)-mediated Th cell differentiation. Map3k2(-/-)Map3k3(Lck-Cre/-) mice showed an abnormal accumulation of regulatory T (Treg) and Th17 cells in the periphery, consistent with Map3k2(-/-)Map3k3(Lck-Cre/-) naive CD4(+) T cells' differentiation into Treg and Th17 cells with a higher frequency than wild-type (WT) cells after TGF-β stimulation in vitro. In addition, Map3k2(-/-)Map3k3(Lck-Cre/-) mice developed more severe experimental autoimmune encephalomyelitis. Map3k2(-/-)Map3k3(Lck-Cre/-) T cells exhibited impaired phosphorylation of SMAD2 and SMAD3 proteins at their linker regions, which negatively regulated the TGF-β responses in T cells. Thus, the crosstalk between TCR-induced MAPK and the TGF-β signaling pathways is important in regulating Th cell differentiation.

  6. Regulators of Glucose Metabolism in CD4(+) and CD8(+) T Cells.

    PubMed

    Palmer, Clovis S; Hussain, Tabinda; Duette, Gabriel; Weller, Thomas J; Ostrowski, Matias; Sada-Ovalle, Isabel; Crowe, Suzanne M

    2016-11-01

    Much like cancer cells, activated T cells undergo various metabolic changes that allow them to grow and proliferate rapidly. By adopting aerobic glycolysis upon activation, T cells effectively prioritize efficiency in biosynthesis over energy generation. There are distinct differences in the way CD4(+) and CD8(+) T cells process activation signals. CD8(+) effector T cells are less dependent on Glut1 and oxygen levels compared to their CD4(+) counterparts. Similarly the downstream signaling by TCR also differs in both effector T cell types. Recent studies have explored PI3K/Akt, mTORC, HIF1α, p70S6K and Bcl-6 signaling in depth providing definition of the crucial roles of these regulators in glucose metabolism. These new insights may allow improved therapeutic manipulation against inflammatory conditions that are associated with dysfunctional T-cell metabolism such as autoimmune disorders, metabolic syndrome, HIV, and cancers.

  7. Increased Peptide Contacts Govern High Affinity Binding of a Modified TCR Whilst Maintaining a Native pMHC Docking Mode.

    PubMed

    Cole, David K; Sami, Malkit; Scott, Daniel R; Rizkallah, Pierre J; Borbulevych, Oleg Y; Todorov, Penio T; Moysey, Ruth K; Jakobsen, Bent K; Boulter, Jonathan M; Baker, Brian M; Yi Li

    2013-01-01

    Natural T cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A(∗)0201) complexed with human T cell lymphotropic virus type 111-19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3β loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134 TCR made increased contacts with the Tax peptide compared with the A6wt/A2-Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134 TCR CDR3β loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies.

  8. Distinctive properties of identical twins' TCR repertoires revealed by high-throughput sequencing.

    PubMed

    Zvyagin, Ivan V; Pogorelyy, Mikhail V; Ivanova, Marina E; Komech, Ekaterina A; Shugay, Mikhail; Bolotin, Dmitry A; Shelenkov, Andrey A; Kurnosov, Alexey A; Staroverov, Dmitriy B; Chudakov, Dmitriy M; Lebedev, Yuri B; Mamedov, Ilgar Z

    2014-04-22

    Adaptive immunity in humans is provided by hypervariable Ig-like molecules on the surface of B and T cells. The final set of these molecules in each organism is formed under the influence of two forces: individual genetic traits and the environment, which includes the diverse spectra of alien and self-antigens. Here we assess the impact of individual genetic factors on the formation of the adaptive immunity by analyzing the T-cell receptor (TCR) repertoires of three pairs of monozygous twins by next-generation sequencing. Surprisingly, we found that an overlap between the TCR repertoires of monozygous twins is similar to an overlap between the TCR repertoires of nonrelated individuals. However, the number of identical complementary determining region 3 sequences in two individuals is significantly increased for twin pairs in the fraction of highly abundant TCR molecules, which is enriched by the antigen-experienced T cells. We found that the initial recruitment of particular TCR V genes for recombination and subsequent selection in the thymus is strictly determined by individual genetic factors. J genes of TCRs are selected randomly for recombination; however, the subsequent selection in the thymus gives preference to some α but not β J segments. These findings provide a deeper insight into the mechanism of TCR repertoire generation.

  9. Identification of peptide-specific TCR genes by in vitro peptide stimulation and CDR3 length polymorphism analysis.

    PubMed

    Shao, Hongwei; Lin, Yanmei; Wang, Teng; Ou, Yusheng; Shen, Han; Tao, Changli; Wu, Fenglin; Zhang, Wenfeng; Bo, Huaben; Wang, Hui; Huang, Shulin

    2015-07-10

    Identification of TCR genes specific for tumor-associated antigens (TAAs) is necessary for TCR gene modification of T cells, which is applied in anti-tumor adoptive T cell therapy (ACT). The usual identification methods are based on isolating single peptide-responding T cells and cloning the TCR gene by in vitro expansion or by single-cell RT-PCR. However, the long and exacting in vitro culture period and demanding operational requirements restrict the application of these methods. Immunoscope is an effective tool that profiles a repertoire of TCRs and identifies significantly expanded clones through CDR3 length analysis. In this study, a survivin-derived mutant peptide optimized for HLA-A2 binding was selected to load DCs and activate T cells. The monoclonal expansion of TCRA and TCRB genes was separately identified by Immunoscope analysis and following sequence identification, the properly paired TCR genes were transferred into T cells. Peptide recognition and cytotoxicity assays indicated that TCR-modified PBMCs could respond to both the mutant and wild type peptides and lyse target cells. These results show that combining Immunoscope with in vitro peptide stimulation provides an alternative and superior method for identifying specific TCR genes, which represents a significant advance for the application of TCR gene-modified T cells.

  10. Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif

    SciTech Connect

    Carlow, D.A.; Teh, H.S.; Marth, J.

    1995-02-15

    In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4{sup +}8{sup +} thymocytes upon TCR cross-linking. In contrast, expression of Tgtp in peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of {approx}50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation. 51 refs., 6 figs.

  11. Negative regulators of cell proliferation

    NASA Technical Reports Server (NTRS)

    Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Cell proliferation is governed by the influence of both mitogens and inhibitors. Although cell contact has long been thought to play a fundamental role in cell cycling regulation, and negative regulators have long been suspected to exist, their isolation and purification has been complicated by a variety of technical difficulties. Nevertheless, over recent years an ever-expanding list of putative negative regulators have emerged. In many cases, their biological inhibitory activities are consistent with density-dependent growth inhibition. Most likely their interactions with mitogenic agents, at an intracellular level, are responsible for either mitotic arrest or continued cell cycling. A review of naturally occurring cell growth inhibitors is presented with an emphasis on those factors shown to be residents of the cell surface membrane. Particular attention is focused on a cell surface sialoglycopeptide, isolated from intact bovine cerebral cortex cells, which has been shown to inhibit the proliferation of an unusually wide range of target cells. The glycopeptide arrest cells obtained from diverse species, both fibroblasts and epithelial cells, and a broad variety of transformed cells. Signal transduction events and a limited spectrum of cells that are refractory to the sialoglycopeptide have provided insight into the molecular events mediated by this cell surface inhibitor.

  12. Superantigen involvement and susceptibility factors in Kawasaki disease: profiles of TCR Vβ2+ T cells and HLA-DRB1, TNF-α and ITPKC genes among Filipino patients.

    PubMed

    Natividad, Magdalena F; Torres-Villanueva, Celia Aurora T; Saloma, Cynthia P

    2013-01-01

    Superantigens and genetic factors may play roles in the etiology and susceptibility to Kawasaki disease (KD). To investigate these roles, percentages of TCR-Vβ2+ T cells were compared by flow cytometry using anti-Vβ2 monoclonal antibodies and genotyping was done on HLA-DRB1 exon 2, the -308 site of the TNF-α promoter region, and ITPKC SNP rs28493229 by polymerase chain reaction followed by direct sequencing. There were higher percentages of Vβ2+ T-cells in KD patients (9.5 ± 2.15%) compared to healthy controls (7.25 ± 1.48%) (P<0.05, Student's t-test, n=6-8/group). However, no polymorphisms were observed in exon 2 of HLA-DRB1 and in the -308 region of the TNF-α promoter. The ITPKC SNP rs28493229 G/C polymorphism was observed in 1 KD patient and 4 healthy controls. This study suggests that KD etiology may be associated with a superantigen and that HLA-DRB1 exon2, TNF-α -308 region and ITPKC SNP rs28493229 may not be associated with KD. This is the first study investigating Vβ2+ T cells and candidate genes involvement among Filipino KD patients.

  13. Superantigen involvement and susceptibility factors in Kawasaki disease: profiles of TCR Vβ2+ T cells and HLA-DRB1, TNF-α and ITPKC genes among Filipino patients

    PubMed Central

    Natividad, Magdalena F; Torres-Villanueva, Celia Aurora T; Saloma, Cynthia P

    2013-01-01

    Superantigens and genetic factors may play roles in the etiology and susceptibility to Kawasaki disease (KD). To investigate these roles, percentages of TCR-Vβ2+ T cells were compared by flow cytometry using anti-Vβ2 monoclonal antibodies and genotyping was done on HLA-DRB1 exon 2, the -308 site of the TNF-α promoter region, and ITPKC SNP rs28493229 by polymerase chain reaction followed by direct sequencing. There were higher percentages of Vβ2+ T-cells in KD patients (9.5 ± 2.15%) compared to healthy controls (7.25 ± 1.48%) (P<0.05, Student’s t-test, n=6-8/group). However, no polymorphisms were observed in exon 2 of HLA-DRB1 and in the -308 region of the TNF-α promoter. The ITPKC SNP rs28493229 G/C polymorphism was observed in 1 KD patient and 4 healthy controls. This study suggests that KD etiology may be associated with a superantigen and that HLA-DRB1 exon2, TNF-α -308 region and ITPKC SNP rs28493229 may not be associated with KD. This is the first study investigating Vβ2+ T cells and candidate genes involvement among Filipino KD patients. PMID:23565324

  14. TCR Triggering Induces the Formation of Lck-RACK1-Actinin-1 Multiprotein Network Affecting Lck Redistribution.

    PubMed

    Ballek, Ondřej; Valečka, Jan; Dobešová, Martina; Broučková, Adéla; Manning, Jasper; Řehulka, Pavel; Stulík, Jiří; Filipp, Dominik

    2016-01-01

    The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor (TCR) triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, receptor for activated C kinase 1 (RACK1), was chosen as a viable option, and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant, and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1-Lck complexes were detectable in primary CD4(+) T-cells with their maximum levels peaking 10 s after TCR-CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394(Lck), which co-purifies with high molecular weight cellular fractions. The formation of RACK1-Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1-pY394(Lck)-α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4(+) T-cells with nocodazole, which disrupts the microtubular network, also blocked the

  15. Self Regulating Fiber Fuel Cell

    DTIC Science & Technology

    2010-08-16

    energy numbers are 2.3X and 5.7X the theoretical values for lithium thionyl chloride respectively (1100 Whr/liter and 590 Whr/kg), which has the...REPORT Self Regulating Fiber Fuel Cell 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: Advances in lithium primary battery technology, which serves as the...Prescribed by ANSI Std. Z39.18 - 16-Aug-2010 Self Regulating Fiber Fuel Cell Report Title ABSTRACT Advances in lithium primary battery technology

  16. A T-cell specific transcriptional enhancer element 3 prime of C sub. alpha. in the human T-cell receptor. alpha. locus

    SciTech Connect

    Ho, Icheng; Yang, Lihsuan; Morle, G.; Leiden, J.M. )

    1989-09-01

    A transcriptional enhancer element has been identified 4.5 kilobases 3{prime} of C{sub {alpha}} (constant region {alpha} chain) in the human T-cell receptor (TCR) {alpha}-chain locus. This enhancer is active on both a TCR V{sub {alpha}} (variable region {alpha} chain) promoter and the minimal simian virus 40 promoter in TCR {alpha}/{beta} Jurkat and EL4 cells but is inactive on a V{sub {alpha}} promoter TCR {gamma}/{delta} PEER and Molt-13 cells, clone 13 B cells, and HeLa fibroblasts. The enhancer has been localized to a 116-base-pair BstXI/Dra I restriction enzyme fragment, which lacks immunoglobulin octamer and {kappa}B enhancer motifs but does contain a consensus cAMP-response element (CRE). DNase I footprint analyses demonstrated that the minimal enhancer contains two binding sites for Jurkat nuclear proteins. One of these sites corresponds to the CRE, while the other does not correspond to a known transcriptional enhancer motif. These data support a model in which TCR {alpha} gene transcription is regulated by a unique set of cis-acting sequences and trans-acting factors, which are differentially active in cells of the TCR {alpha}/{beta} lineage. In addition, the TCR {alpha} enhancer may play a role in activating oncogene expression in T-lymphoblastoid tumors that have previously been shown to display chromosomal translocations into the human TCR {alpha} locus.

  17. αβ T cell receptor germline CDR regions moderate contact with MHC ligands and regulate peptide cross-reactivity

    PubMed Central

    Attaf, Meriem; Holland, Stephan J.; Bartok, Istvan; Dyson, Julian

    2016-01-01

    αβ T cells respond to peptide epitopes presented by major histocompatibility complex (MHC) molecules. The role of T cell receptor (TCR) germline complementarity determining regions (CDR1 and 2) in MHC restriction is not well understood. Here, we examine T cell development, MHC restriction and antigen recognition where germline CDR loop structure has been modified by multiple glycine/alanine substitutions. Surprisingly, loss of germline structure increases TCR engagement with MHC ligands leading to excessive loss of immature thymocytes. MHC restriction is, however, strictly maintained. The peripheral T cell repertoire is affected similarly, exhibiting elevated cross-reactivity to foreign peptides. Our findings are consistent with germline TCR structure optimising T cell cross-reactivity and immunity by moderating engagement with MHC ligands. This strategy may operate alongside co-receptor imposed MHC restriction, freeing germline TCR structure to adopt this novel role in the TCR-MHC interface. PMID:27775030

  18. αβ T cell receptor germline CDR regions moderate contact with MHC ligands and regulate peptide cross-reactivity.

    PubMed

    Attaf, Meriem; Holland, Stephan J; Bartok, Istvan; Dyson, Julian

    2016-10-24

    αβ T cells respond to peptide epitopes presented by major histocompatibility complex (MHC) molecules. The role of T cell receptor (TCR) germline complementarity determining regions (CDR1 and 2) in MHC restriction is not well understood. Here, we examine T cell development, MHC restriction and antigen recognition where germline CDR loop structure has been modified by multiple glycine/alanine substitutions. Surprisingly, loss of germline structure increases TCR engagement with MHC ligands leading to excessive loss of immature thymocytes. MHC restriction is, however, strictly maintained. The peripheral T cell repertoire is affected similarly, exhibiting elevated cross-reactivity to foreign peptides. Our findings are consistent with germline TCR structure optimising T cell cross-reactivity and immunity by moderating engagement with MHC ligands. This strategy may operate alongside co-receptor imposed MHC restriction, freeing germline TCR structure to adopt this novel role in the TCR-MHC interface.

  19. NKG2D receptor regulates human effector T-cell cytokine production

    PubMed Central

    Barber, Amorette

    2011-01-01

    Although innate immune signals shape the activation of naive T cells, it is unclear how innate signals influence effector T-cell function. This study determined the effects of stimulating the NKG2D receptor in conjunction with the TCR on human effector CD8+ T cells. Stimulation of CD8+ T cells through CD3 and NKG2D simultaneously or through a chimeric NKG2D receptor, which consists of NKG2D fused to the intracellular region of CD3ζ, activated β-catenin and increased expression of β-catenin–induced genes, whereas T cells stimulated through the TCR or a combination of the TCR and CD28 did not. Activation by TCR and NKG2D prevented expression and production of anti-inflammatory cytokines IL-10, IL-9, IL-13, and VEGF-α in a β-catenin– and PPARγ- dependent manner. NKG2D stimulation also modulated the cytokine secretion of T cells activated simultaneously through CD3 and CD28. These data indicate that activating CD8+ T cells through the NKG2D receptor along with the TCR modulates signal transduction and the production of anti-inflammatory cytokines. Thus, human effector T cells alter their function depending on which innate receptors are engaged in conjunction with the TCR complex. PMID:21518928

  20. Relating TCR-peptide-MHC affinity to immunogenicity for the design of tumor vaccines

    PubMed Central

    McMahan, Rachel H.; McWilliams, Jennifer A.; Jordan, Kimberly R.; Dow, Steven W.; Wilson, Darcy B.; Slansky, Jill E.

    2006-01-01

    One approach to enhancing the T cell response to tumors is vaccination with mimotopes, mimics of tumor epitopes. While mimotopes can stimulate proliferation of T cells that recognize tumor-associated antigens (TAAs), this expansion does not always correlate with control of tumor growth. We hypothesized that vaccination with mimotopes of optimal affinity in this interaction will improve antitumor immunity. Using a combinatorial peptide library and a cytotoxic T lymphocyte clone that recognizes a TAA, we identified a panel of mimotopes that, when complexed with MHC, bound the TAA-specific TCR with a range of affinities. As expected, in vitro assays showed that the affinity of the TCR-peptide-MHC (TCR-pMHC) interaction correlated with activity of the T cell clone. However, only vaccination with mimotopes in the intermediate-affinity range elicited functional T cells and provided protection against tumor growth in vivo. Vaccination with mimotopes with the highest-affinity TCR-pMHC interactions elicited TAA-specific T cells to the tumor, but did not control tumor growth at any of the peptide concentrations tested. Further analysis of these T cells showed functional defects in response to the TAA. Thus, stimulation of an antitumor response by mimotopes may be optimal with peptides that increase but do not maximize the affinity of the TCR-pMHC interaction. PMID:16932807

  1. Engineered cytotoxic T lymphocytes with AFP-specific TCR gene for adoptive immunotherapy in hepatocellular carcinoma.

    PubMed

    Sun, Longhao; Guo, Hao; Jiang, Ruoyu; Lu, Li; Liu, Tong; He, Xianghui

    2016-01-01

    Alpha-fetoprotein (AFP) is overexpressed in hepatocellular carcinoma (HCC) and could serve as a tumor-associated antigen (TAA) and potential target for adoptive immunotherapy. However, low frequency and severe functional impairment of AFP-specific T cells in vivo hamper adoptive infusion. TAA-specific T cell receptor (TCR) gene transfer could be an efficient and reliable alternation to generate AFP-specific cytotoxic T lymphocytes (CTLs). Autologous dendritic cells (DC) pulsed with AFP158-166 peptides were used to stimulate AFP-specific CTLs. TCR α/β chain genes of AFP-specific CTLs were cloned and linked by 2A peptide to form full-length TCR coding sequence synthesized into a lentiviral vector. Nonspecific activated T cells were engineered by lentivirus infection. Transgenetic CTLs were evaluated for transfection efficiency, expression of AFP158-166-specific TCR, interferon (IFN)-γ secretion, and specific cytotoxicity toward AFP+ HCC cells in vitro and in vivo. Flow cytometry revealed the AFP158-166-MHC-Pentamer positive transgenetic CTLs was 9.86 %. The number of IFN-γ secretion T cells and the specific cytotoxicity toward HpeG2 in vitro and in tumor-bearing NOD/SCID mice were significantly raised in transgenetic CTLs than that of AFP158-166-specific CTLs obtained by peptide-pulsed DCs or control group. TCR gene transfer is a promising strategy to generate AFP158-166-specific CTLs for the treatment of HCC.

  2. SNX17 Affects T Cell Activation by Regulating T Cell Receptor and Integrin Recycling

    PubMed Central

    Osborne, Douglas G.; Piotrowski, Joshua T.; Dick, Christopher J.; Zhang, Jin-San; Billadeau, Daniel D.

    2015-01-01

    A key component in T cell activation is the endosomal recycling of receptors to the cell surface, thereby allowing continual integration of signaling and antigen recognition. One protein potentially involved in T cell receptor transport is sorting nexin 17 (SNX17). SNX proteins have been found to bind proteins involved in T cell activation, but specifically the role of SNX17 in receptor recycling and T cell activation is unknown. Using immunofluorescence, we find that SNX17 co-localizes with TCR and localizes to the immune synapse in T-APC conjugates. Significantly, knockdown of the SNX17 resulted in fewer T-APC conjugates, lower CD69, TCR, and LFA-1 surface expression, as well as lower overall TCR recycling compared to control T cells. Lastly, we identified the FERM-domain of SNX17 as being responsible in the binding and trafficking of TCR and LFA-1 to the cell surface. These data suggest that SNX17 plays a role in the maintenance of normal surface levels of activating receptors and integrins to permit optimum T cell activation at the immune synapse. PMID:25825439

  3. Nuclear transfer nTreg model reveals fate-determining TCR-β and novel peripheral nTreg precursors.

    PubMed

    Ku, Manching; Chang, Shih-En; Hernandez, Julio; Abadejos, Justin R; Sabouri-Ghomi, Mohsen; Muenchmeier, Niklas J; Schwarz, Anna; Valencia, Anna M; Kirak, Oktay

    2016-04-19

    To study the development and function of "natural-arising" T regulatory (nTreg) cells, we developed a novel nTreg model on pure nonobese diabetic background using epigenetic reprogramming via somatic cell nuclear transfer. On RAG1-deficient background, we found that monoclonal FoxP3(+)CD4(+)Treg cells developed in the thymus in the absence of other T cells. Adoptive transfer experiments revealed that the thymic niche is not a limiting factor in nTreg development. In addition, we showed that the T-cell receptor (TCR) β-chain of our nTreg model was not only sufficient to bias T-cell development toward the CD4 lineage, but we also demonstrated that this TCR β-chain was able to provide stronger TCR signals. This TCR-β-driven mechanism would thus unify former per se contradicting hypotheses of TCR-dependent and -independent nTreg development. Strikingly, peripheral FoxP3(-)CD4(+)T cells expressing the same TCR as this somatic cell nuclear transfer nTreg model had a reduced capability to differentiate into Th1 cells but were poised to differentiate better into induced nTreg cells, both in vitro and in vivo, representing a novel peripheral precursor subset of nTreg cells to which we refer to as pre-nTreg cells.

  4. Epigenetic Regulation of Myeloid Cells

    PubMed Central

    IVASHKIV, LIONEL B.; PARK, SUNG HO

    2017-01-01

    Epigenetic regulation in myeloid cells is crucial for cell differentiation and activation in response to developmental and environmental cues. Epigenetic control involves posttranslational modification of DNA or chromatin, and is also coupled to upstream signaling pathways and transcription factors. In this review, we summarize key epigenetic events and how dynamics in the epigenetic landscape of myeloid cells shape the development, immune activation, and innate immune memory. PMID:27337441

  5. CD5-mediated inhibition of TCR signaling proceeds normally in the absence of SHP-1

    PubMed Central

    DONG, BAOXIA; SOMANI, ALLY-KHAN; LOVE, PAUL E.; ZHENG, XUAN; CHEN, XIEQUN; ZHANG, JINYI

    2016-01-01

    The CD5 transmembrane glycoprotein functions as a co-receptor in the signaling pathway linking T-cell antigen receptor (TCR) engagement to activation and differentiation. Although CD5 effects on TCR signaling have been shown to be primarily inhibitory, the underlying mechanisms remain unclear. In view of recent data revealing the ability of CD5 to associate with the SHP-1 tyrosine phosphatase, a protein that also downregulates TCR signaling, we examined the role of SHP-1 in modulating CD5 function using thymocytes from SHP-1-deficient viable motheaten (mev) mice. The results revealed the association of SHP-1 with CD5 to be markedly increased following TCR stimulation and indicated that this interaction was enhanced by and was dependent on CD5 tyrosine phosphorylation. However, there was no difference of the tyrosine phosphorylation status of CD5 between resting and TCR-stimulated cells in SHP-1-deficient compared to wild-type thymocytes. Lack of SHP-1 activity did not affect the levels of CD5 surface expression, CD5 co-immunoprecipitable tyrosine phosphatase activity and intracellular calcium increase following co-crosslinking of the TCR and CD5. Similarly, an analysis of T-cell thymocyte populations in mev mice expressing an H-Y transgene as well as a construct mediating T-cell restricted CD5 overexpression, revealed that the reduction in the positive selection conferred by CD5 overexpression was unaffected by SHP-1 deficiency. CD5 is not a SHP-1 substrate and SHP-1 is not required for and possibly not involved in the CD5-mediated modulation of TCR signaling. PMID:27221212

  6. Rapid Evolution of the CD8+ TCR Repertoire in Neonatal Mice.

    PubMed

    Carey, Alison J; Gracias, Donald T; Thayer, Jillian L; Boesteanu, Alina C; Kumova, Ogan K; Mueller, Yvonne M; Hope, Jennifer L; Fraietta, Joseph A; van Zessen, David B H; Katsikis, Peter D

    2016-03-15

    Currently, there is little consensus regarding the most appropriate animal model to study acute infection and the virus-specific CD8(+) T cell (CTL) responses in neonates. TCRβ high-throughput sequencing in naive CTL of differently aged neonatal mice was performed, which demonstrated differential Vβ family gene usage. Using an acute influenza infection model, we examined the TCR repertoire of the CTL response in neonatal and adult mice infected with influenza type A virus. Three-day-old mice mounted a greatly reduced primary NP(366-374)-specific CTL response when compared with 7-d-old and adult mice, whereas secondary CTL responses were normal. Analysis of NP(366-374)-specific CTL TCR repertoire revealed different Vβ gene usage and greatly reduced public clonotypes in 3-d-old neonates. This could underlie the impaired CTL response in these neonates. To directly test this, we examined whether controlling the TCR would restore neonatal CTL responses. We performed adoptive transfers of both nontransgenic and TCR-transgenic OVA(257-264)-specific (OT-I) CD8(+) T cells into influenza-infected hosts, which revealed that naive neonatal and adult OT-I cells expand equally well in neonatal and adult hosts. In contrast, nontransgenic neonatal CD8(+) T cells when transferred into adults failed to expand. We further demonstrate that differences in TCR avidity may contribute to decreased expansion of the endogenous neonatal CTL. These studies highlight the rapid evolution of the neonatal TCR repertoire during the first week of life and show that impaired neonatal CTL immunity results from an immature TCR repertoire, rather than intrinsic signaling defects or a suppressive environment.

  7. CD5-mediated inhibition of TCR signaling proceeds normally in the absence of SHP-1.

    PubMed

    Dong, Baoxia; Somani, Ally-Khan; Love, Paul E; Zheng, Xuan; Chen, Xiequn; Zhang, Jinyi

    2016-07-01

    The CD5 transmembrane glycoprotein functions as a co-receptor in the signaling pathway linking T-cell antigen receptor (TCR) engagement to activation and differentiation. Although CD5 effects on TCR signaling have been shown to be primarily inhibitory, the underlying mechanisms remain unclear. In view of recent data revealing the ability of CD5 to associate with the SHP-1 tyrosine phosphatase, a protein that also downregulates TCR signaling, we examined the role of SHP-1 in modulating CD5 function using thymocytes from SHP-1‑deficient viable motheaten (mev) mice. The results revealed the association of SHP-1 with CD5 to be markedly increased following TCR stimulation and indicated that this interaction was enhanced by and was dependent on CD5 tyrosine phosphorylation. However, there was no difference of the tyrosine phosphorylation status of CD5 between resting and TCR-stimulated cells in SHP-1‑deficient compared to wild-type thymocytes. Lack of SHP-1 activity did not affect the levels of CD5 surface expression, CD5 co-immunoprecipitable tyrosine phosphatase activity and intracellular calcium increase following co-crosslinking of the TCR and CD5. Similarly, an analysis of T‑cell thymocyte populations in mev mice expressing an H-Y transgene as well as a construct mediating T‑cell restricted CD5 overexpression, revealed that the reduction in the positive selection conferred by CD5 overexpression was unaffected by SHP-1 deficiency. CD5 is not a SHP-1 substrate and SHP-1 is not required for and possibly not involved in the CD5-mediated modulation of TCR signaling.

  8. Clonal CD8+ TCR-Vbeta expanded populations with effector memory phenotype in Churg Strauss syndrome.

    PubMed

    Guida, Giuseppe; Vallario, Antonella; Stella, Stefania; Boita, Monica; Circosta, Paola; Mariani, Sara; Prato, Giuseppina; Heffler, Enrico; Bergia, Roberta; Sottile, Antonino; Rolla, Giovanni; Cignetti, Alessandro

    2008-07-01

    Churg Strauss Syndrome (CSS) is a systemic vasculitis in which oligoclonal T cell expansions might be involved in the pathogenesis. Combined analysis of TCR-Vbeta expression profile by flow cytometry and of TCR gene rearrangement by heteroduplex PCR was used to detect and characterize T cell expansions in 8 CSS patients, 10 asthmatics and 42 healthy subjects. In all CSS patients one or two Vbeta families were expanded among CD8+ cells, with an effector memory phenotype apt to populate tissues and inflammatory sites. Heteroduplex PCR showed the presence of one or more clonal TCR rearrangements, which reveals monoclonal or oligoclonal T cells subpopulations. After purification with a Vbeta specific monoclonal antibody, each CD8+/Vbeta+ expanded family showed a single TCR rearrangement, clearly suggestive of monoclonality. All CD8+ expansions were detectable throughout the disease course. TCR-Vbeta expanded or deleted populations were not observed in asthmatic patients. Clonal CD8+/Vbeta+ T cell expansions might be useful as a disease marker.

  9. Intraflagellar transport is required for polarized recycling of the TCR/CD3 complex to the immune synapse.

    PubMed

    Finetti, Francesca; Paccani, Silvia Rossi; Riparbelli, Maria Giovanna; Giacomello, Emiliana; Perinetti, Giuseppe; Pazour, Gregory J; Rosenbaum, Joel L; Baldari, Cosima T

    2009-11-01

    Most eukaryotic cells have a primary cilium which functions as a sensory organelle. Cilia are assembled by intraflagellar transport (IFT), a process mediated by multimeric IFT particles and molecular motors. Here we show that lymphoid and myeloid cells, which lack primary cilia, express IFT proteins. IFT20, an IFT component essential for ciliary assembly, was found to colocalize with both the microtubule organizing centre (MTOC) and Golgi and post-Golgi compartments in T-lymphocytes. In antigen-specific conjugates, IFT20 translocated to the immune synapse. IFT20 knockdown resulted in impaired T-cell receptor/CD3 (TCR/CD3) clustering and signalling at the immune synapse, due to defective polarized recycling. Moreover, IFT20 was required for the inducible assembly of a complex with other IFT components (IFT57 and IFT88) and the TCR. The results identify IFT20 as a new regulator of immune synapse assembly in T cells and provide the first evidence to implicate IFT in membrane trafficking in cells lacking primary cilia, thereby introducing a new perspective on IFT function beyond its role in ciliogenesis.

  10. Fixed expression of single influenza virus-specific TCR chains demonstrates the capacity for TCR α- and β-chain diversity in the face of peptide-MHC class I specificity.

    PubMed

    Clemens, E Bridie; Doherty, Peter C; La Gruta, Nicole L; Turner, Stephen J

    2015-02-01

    The characteristics of the TCR repertoire expressed by epitope-specific CD8(+) T cells can be an important determinant of the quality of immune protection against virus infection. Most studies of epitope-specific TCR repertoires focus solely on an analysis of TCR β-chains, rather than the combined TCRαβ heterodimers that confer specificity. Hence, the importance of complementary α- and β-chain pairing in determining TCR specificity and T cell function is not well understood. Our earlier study of influenza-specific TCR repertoires in a C57BL/6J mouse model described a structural basis for preferred TCRαβ pairing that determined exquisite specificity for the D(b)PA224 epitope from influenza A virus. We have now extended this analysis using retrogenic mice engineered to express single TCR α- or β-chains specific for the D(b)NP366 or D(b)PA224 epitopes derived from influenza A virus. We found that particular TCRαβ combinations were selected for recognition of these epitopes following infection, indicating that pairing of certain α- and β-chain sequences is key for determining TCR specificity. Furthermore, we demonstrated that some TCRαβ heterodimers were preferentially expanded from the naive repertoire in response to virus infection, suggesting that appropriate αβ pairing confers optimal T cell responsiveness to Ag.

  11. The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-C{beta} antibody

    SciTech Connect

    Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji; Jin, Aishun; Kishi, Hiroyuki; Muraguchi, Atsushi

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V

  12. Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions

    PubMed Central

    Swee, Lee Kim; Tan, Zhen Wei; Sanecka, Anna; Yoshida, Nagisa; Patel, Harshil; Grotenbreg, Gijsbert; Ploegh, Hidde L.

    2016-01-01

    T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity—hence reactivity to self—and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 Ld-restricted epitope, derived from the Rop7 protein of Toxoplasma gondii. We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro. Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds. PMID:27881740

  13. Accurate detection of the tumor clone in peripheral T-cell lymphoma biopsies by flow cytometric analysis of TCR-Vβ repertoire.

    PubMed

    Salameire, Dimitri; Solly, Françoise; Fabre, Blandine; Lefebvre, Christine; Chauvet, Martine; Gressin, Rémy; Corront, Bernadette; Ciapa, Agnès; Pernollet, Martine; Plumas, Joël; Macintyre, Elizabeth; Callanan, Mary B; Leroux, Dominique; Jacob, Marie-Christine

    2012-09-01

    Multiparametric flow cytometry has proven to be a powerful method for detection and immunophenotypic characterization of clonal subsets, particularly in lymphoproliferative disorders of the B-cell lineage. Although in theory promising, this approach has not been comparably fulfilled in mature T-cell malignancies. Specifically, the T-cell receptor-Vβ repertoire analysis in blood can provide strong evidence of clonality, particularly when a single expanded Vß family is detected. The purpose of this study was to determine the relevance of this approach when applied to biopsies, at the site of tumor involvement. To this end, 30 peripheral T-cell lymphoma and 94 control biopsies were prospectively studied. Vβ expansions were commonly detected within CD4+ or CD8+ T cells (97% of peripheral T-cell lymphoma and 54% of non-peripheral T-cell lymphoma cases); thus, not differentiating malignant from reactive processes. Interestingly, we demonstrated that using a standardized evaluation, the detection of a high Vβ expansion was closely associated with diagnosis of peripheral T-cell lymphoma, with remarkable specificity (98%) and sensitivity (90%). This approach also identified eight cases of peripheral T-cell lymphoma that were not detectable by other forms of immunophenotyping. Moreover, focusing Vβ expression analysis to T-cell subsets with aberrant immunophenotypes, we demonstrated that the T-cell clone might be heterogeneous with regard to surface CD7 or CD10 expression (4/11 cases), providing indication on 'phenotypic plasticity'. Finally, among the wide variety of Vβ families, the occurrence of a Vβ17 expansion in five cases was striking. To our knowledge, this is the first report demonstrating the power of T-cell receptor-Vβ repertoire analysis by flow cytometry in biopsies as a basis for peripheral T-cell lymphoma diagnosis and precise T-cell clone identification and characterization.

  14. Differential T cell receptor-mediated signaling in naive and memory CD4 T cells.

    PubMed

    Farber, D L; Acuto, O; Bottomly, K

    1997-08-01

    Naive and memory CD4 T cells differ in cell surface phenotype, function, activation requirements, and modes of regulation. To investigate the molecular bases for the dichotomies between naive and memory CD4 T cells and to understand how the T cell receptor (TCR) directs diverse functional outcomes, we investigated proximal signaling events triggered through the TCR/CD3 complex in naive and memory CD4 T cell subsets isolated on the basis of CD45 isoform expression. Naive CD4 T cells signal through TCR/CD3 similar to unseparated CD4 T cells, producing multiple tyrosine-phosphorylated protein species overall and phosphorylating the T cell-specific ZAP-70 tyrosine kinase which is recruited to the CD3zeta subunit of the TCR. Memory CD4 T cells, however, exhibit a unique pattern of signaling through TCR/CD3. Following stimulation through TCR/CD3, memory CD4 T cells produce fewer species of tyrosine-phosphorylated substrates and fail to phosphorylate ZAP-70, yet unphosphorylated ZAP-70 can associate with the TCR/CD3 complex. Moreover, a 26/28-kDa phosphorylated doublet is associated with CD3zeta in resting and activated memory but not in naive CD4 T cells. Despite these differences in the phosphorylation of ZAP-70 and CD3-associated proteins, the ZAP-70-related kinase, p72syk, exhibits similar phosphorylation in naive and memory T cell subsets, suggesting that this kinase could function in place of ZAP-70 in memory CD4 T cells. These results indicate that proximal signals are differentially coupled to the TCR in naive versus memory CD4 T cells, potentially leading to distinct downstream signaling events and ultimately to the diverse functions elicited by these two CD4 T cell subsets.

  15. Cell Therapy Regulation in Taiwan.

    PubMed

    Chen, Yuan-Chuan; Cheng, Hwei-Fang; Yeh, Ming-Kung

    2017-03-13

    Cell therapy is not only a novel medical practice but also a medicinal product [cell therapy product (CTP)]. More and more CTPs are being approved for marketing globally because of the rapid development of biomedicine in cell culture, preservation, and preparation. However, regulation is the most important criterion for the development of CTPs. Regulations must be flexible to expedite the process of marketing for new CTPs. Recently, the Taiwan Food and Drug Administration (TFDA) updated the related regulations such as regulation of development, current regulatory framework and process, and the application and evaluation processes. When the quality of CTPs has been improved significantly, their safety and efficacy are further ensured. The treatment protocol, a new design for adaptive licensing to current clinical practice, is a rapid process for patients with life-threatening diseases or serious conditions for which there are no suitable drugs, medical devices, or other therapeutic methods available. The hospital can submit the treatment protocol to apply for cell therapy as a medical practice, which may result in easier and faster cell therapy development, and personalized treatment for individual patients will evolve quickly.

  16. Helper T cells down-regulate CD4 expression upon chronic stimulation giving rise to double-negative T cells.

    PubMed

    Grishkan, Inna V; Ntranos, Achilles; Calabresi, Peter A; Gocke, Anne R

    2013-01-01

    Double-negative T (DNT) cells are αβTCR(+)CD3(+)CD4(-)CD8(-)NK1.1(-) cells that constitute a small but significant proportion of the αβTCR(+) T cells. Their developmental pathway and pathological significance remain unclear. In the present study, we utilized chronic in vitro stimulation of CD4(+) T cells to mimic immune hyper-activation of autoimmune lymphoproliferative syndrome and systemic lupus erythematosus, conditions characterized by DNT cells accumulation. After approximately 4-5 rounds of stimulation, the CD3(+)CD4(-) population became apparent. These cells did not express CD8, NK1.1, γδTCR, or B220, exhibited a highly proliferative effector phenotype, and were dependent on T cell receptor (TCR) stimulation for survival. Moreover, CD3(+)CD4(-) cells expressed MHC class II-restricted αβTCR, indicative of their origin from a CD4(+) T cell population. The results presented herein illustrate a novel method of DNT cell generation in vitro and suggest that immune hyper-activation could also be implicated in the genesis of the disease-associated DNT cells in vivo.

  17. 76 FR 35739 - Foreign Assets Control Regulations; Transaction Control Regulations (Regulations Prohibiting...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-20

    ... CFR part 500 (the ``FACR''), and the Transaction Control Regulations, 31 CFR part 505 (the ``TCR... FACR and TCR immediately prior to the issuance of Proclamation 8271 were those that related to North... TCR from 31 CFR chapter V. Public Participation Because the Regulations involve a foreign...

  18. Computational spatiotemporal analysis identifies WAVE2 and Cofilin as joint regulators of costimulation-mediated T cell actin dynamics

    PubMed Central

    Roybal, Kole T.; Buck, Taráz E.; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J.; Ambler, Rachel; Tunbridge, Helen M.; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F.

    2016-01-01

    Fluorescence microscopy is one of the most important tools in cell biology research and it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells; however, given extensive cell-to-cell variation, methods do not currently exist to assemble these data into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. Here, we have developed one such method and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28 and we have determined how CD28 modulates actin dynamics. We imaged actin and eight core actin regulators under conditions where CD28 in the context of a strong TCR signal was engaged or blocked to yield over a thousand movies. Our computational analysis identified diminished recruitment of the activator of actin nucleation WAVE2 and the actin severing protein cofilin to F-actin as the dominant difference upon costimulation blockade. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics upon costimulation blockade. Thus we have developed and validated an approach to quantify protein distributions in time and space for analysis of complex regulatory systems. PMID:27095595

  19. Unraveling a Hotspot for TCR Recognition on HLA-A2: Evidence Against the Existence of Peptide-independent TCR Binding Determinants

    SciTech Connect

    Gagnon, Susan J.; Borbulevych, Oleg Y.; Davis-Harrison, Rebecca L.; Baxter, Tiffany K.; Clemens, John R.; Armstrong, Kathryn M.; Turner, Richard V.; Damirjian, Marale; Biddison, William E.; Baker, Brian M.

    2010-07-19

    T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 {alpha}1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.

  20. Changes of TCR repertoire diversity in colorectal cancer after Erbitux (cetuximab) in combination with chemotherapy.

    PubMed

    Luo, Wei; He, Wen-Ting; Wen, Qian; Chen, Shu; Wu, Jing; Chen, Xiang-Ping; Ma, Li

    2014-01-01

    We have previous found a positive correlation between post-therapy TCR repertoire normalization and remission of colorectal cancer (CRC) patients following fluorouracil, leucovorin, and irinotecan (FOLFIRI) plus bevacizumab or Rh-endostatin therapy. To further define the TCR repertoire diversity changes following treatment in CRC patients, and confirm its potential prognostic value, the present study extended the sample size of follow-up and used an alternative therapy regime to investigate changes of TCR repertoires following Erbitux plus FOLFIRI therapy. Inclusion and exclusion criteria have been established to screen out 26 patients to receive Erbitux plus FOLFIRI therapy. Efficacy and toxicity assessment have been made for them after 3 months' treatment as well as the TCR repertoire diversity has been determined. A CDR3 complex scoring system was used to quantify the diversity of TCR repertoire. The results showing that the diversity of CD4(+) T cells in PR group was significantly higher than that of SD and PD groups, and the difference was enlargement after treatment. The diversity of CD8(+) T cells in PR group has no difference before and after treatment, but significant decrease in SD and PD group after treatment. In conclusion, analysis the diversity of T cell repertoire has an important prognosis value for CRC patients.

  1. Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

    PubMed Central

    Jahan, Akhee S.; Lestra, Maxime; Swee, Lee Kim; Fan, Ying; Lamers, Mart M.; Tafesse, Fikadu G.; Theile, Christopher S.; Spooner, Eric; Bruzzone, Roberto; Ploegh, Hidde L.; Sanyal, Sumana

    2016-01-01

    Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12−/− Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12−/− cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades. PMID:26811477

  2. RIPK1 and RIPK3: critical regulators of inflammation and cell death.

    PubMed

    Newton, Kim

    2015-06-01

    RIPK1 and RIPK3 (receptor-interacting serine/threonine protein kinases 1/3) interact by virtue of their RIP homotypic interaction motifs to mediate a form of cell death called necroptosis, although mice lacking these kinases have very different phenotypes. RIPK1-deficient mice die soon after birth, whereas RIPK3-deficient mice are healthy. Necroptosis involves cell rupture and is triggered by tumor necrosis factor (TNF), Toll-like receptors (TLRs), or the T cell receptor (TCR) when pro-apoptotic caspase-8 is inhibited. Various mouse models of disease are ameliorated by RIPK3 deficiency, suggesting that necroptosis contributes to pathology. Genetic rescue experiments now reveal why RIPK3-deficient are viable but RIPK1-deficient mice are not. These and other experiments indicate unexpected complexity in the regulation of both apoptosis and necroptosis by RIPK1 and RIPK3.

  3. Human αβ and γδ Thymocyte Development: TCR Gene Rearrangements, Intracellular TCRβ Expression and γδ Developmental Potential – Differences between Men and Mice1,2

    PubMed Central

    Joachims, Michelle L.; Chain, Jennifer L.; Hooker, Scott W.; Knott-Craig, Christopher J.; Thompson, Linda F.

    2006-01-01

    To evaluate the role of the TCR in the αβ/γδ lineage choice during human thymocyte development, molecular analyses of the TCRβ locus in γδ cells and the TCRγ and δ loci in αβ cells were undertaken. TCRβ variable gene segments remained largely in germline configuration in γδ cells, indicating that commitment to the γδ lineage occurred prior to complete TCRβ rearrangements in most cases. The few TCRβ rearrangements detected were primarily out-of-frame, suggesting that productive TCRβ rearrangements diverted cells away from the γδ lineage. In contrast, in αβ cells, the TCRγ locus was almost completely rearranged with a random productivity profile; the TCRδ locus contained primarily non-productive rearrangements. Productive γ rearrangements were, however, depleted compared to pre-selected cells. Productive TCRγ and δ rearrangements rarely occurred in the same cell, suggesting that αβ cells developed from cells unable to produce a functional γδ TCR. Intracellular TCRβ expression correlated with the up regulation of CD4 and concomitant down regulation of CD34, and plateaued at the early double positive stage. Surprisingly, however, some early double positive thymocytes retained γδ potential in culture. We present a model for human thymopoiesis which includes γδ development as a default pathway, an instructional role for the TCR in the αβ/γδ lineage choice, and a prolonged developmental window for β-selection and γδ lineage commitment. Aspects that differ from the mouse are the status of TCR gene rearrangements at the non-expressed loci, the timing of β-selection, and maintenance of γδ potential through the early double positive stage of development. PMID:16424183

  4. TCR Signaling Emerges from the Sum of Many Parts

    PubMed Central

    Kuhns, Michael S.; Davis, Mark M.

    2012-01-01

    “How does T cell receptor signaling begin?” Answering this question requires an understanding of how the parts of the molecular machinery that mediates this process fit and work together. Ultimately this molecular architecture must (i) trigger the relay of information from the TCR-pMHC interface to the signaling substrates of the CD3 molecules and (ii) bring the kinases that modify these substrates in close proximity to interact, initiate, and sustain signaling. In this contribution we will discuss advances of the last decade that have increased our understanding of the complex machinery and interactions that underlie this type of signaling. PMID:22737151

  5. Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

    PubMed

    Supper, Verena; Schiller, Herbert B; Paster, Wolfgang; Forster, Florian; Boulègue, Cyril; Mitulovic, Goran; Leksa, Vladimir; Ohradanova-Repic, Anna; Machacek, Christian; Schatzlmaier, Philipp; Zlabinger, Gerhard J; Stockinger, Hannes

    2016-02-01

    The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression.

  6. Regulators of Tfh Cell Differentiation

    PubMed Central

    Jogdand, Gajendra M.; Mohanty, Suchitra; Devadas, Satish

    2016-01-01

    The follicular helper T (Tfh) cells help is critical for activation of B cells, antibody class switching, and germinal center (GC) formation. The Tfh cells are characterized by the expression of CXC chemokine receptor 5 (CXCR5), ICOS, programed death 1 (PD-1), B cell lymphoma 6 (BCL-6), and IL-21. They are involved in clearing infections and are adversely linked with autoimmune diseases and also have a role in viral replication as well as clearance. On the one hand, Tfh cells are generated from naive CD4+ T cells with sequential steps involving cytokine signaling (IL-21, IL-6, IL-12, activin A), migration, and positioning in the GC by CXCR5, surface receptors (ICOS/ICOSL, signaling lymphocyte activation molecule-associated protein/signaling lymphocyte activation molecule) as well as transcription factor (BCL-6, c-Maf, and signal transducer and activator of transcription 3) signaling and repressor miR155. On the other hand, Tfh generation is negatively regulated at specific steps of Tfh generation by specific cytokine (IL-2, IL-7), surface receptor (PD-1, CTLA-4), transcription factors B lymphocyte maturation protein 1, signal transducer and activator of transcription 5, T-bet, KLF-2 signaling, and repressor miR 146a. Interestingly, miR-17–92 and FOXO1 act as a positive as well as a negative regulator of Tfh differentiation depending on the time of expression and disease specificity. Tfh cells are also generated from the conversion of other effector T cells as exemplified by Th1 cells converting into Tfh during viral infection. The mechanistic details of effector T cells conversion into Tfh are yet to be clear. To manipulate Tfh cells for therapeutic implication and or for effective vaccination strategies, it is important to know positive and negative regulators of Tfh generation. Hence, in this review, we have highlighted and interlinked molecular signaling from cytokines, surface receptors, transcription factors, ubiquitin ligase, and microRNA as positive and

  7. Loss of protein kinase C theta, Bcl10, or Malt1 selectively impairs proliferation and NF-kappa B activation in the CD4+ T cell subset.

    PubMed

    Kingeter, Lara M; Schaefer, Brian C

    2008-11-01

    The cytosolic proteins protein kinase Ctheta (PKCtheta), Bcl10, and Malt1 play critical roles in TCR signaling to the transcription factor NF-kappaB. Our data confirm that CD4(+) T cells from PKCtheta, Bcl10, and Malt1 knockout mice show severe impairment of proliferation in response to TCR stimulation. Unexpectedly, we find that knockout CD8(+) T cells proliferate to a similar extent as wild-type cells in response to strong TCR signals, although a survival defect prevents their accumulation. Both CD4(+) and CD8(+) knockout T cells express activation markers, including CD25, following TCR stimulation. Addition of exogenous IL-2 rescues survival of knockout CD4(+) and CD8(+) T cells, but fails to overcome the proliferation defect of CD4(+) T cells. CD4(+) T cells from knockout mice are extremely deficient in TCR-induced NF-kappaB activation, whereas NF-kappaB activation is only partially impaired in CD8(+) T cells. Overall, our results suggest that defects in TCR signaling through PKCtheta, Bcl10, and Malt1 predominantly impair NF-kappaB activation and downstream functional responses of CD4(+) T cells. In contrast, CD8(+) T cells maintain substantial NF-kappaB signaling, implying the existence of a significant TCR-regulated NF-kappaB activation pathway in CD8(+) T cells that is independent of PKCtheta, Bcl10, and Malt1.

  8. A Carma1/MALT1-dependent, Bcl10-independent, pathway regulates antigen receptor-mediated mTOR signaling in T cells

    PubMed Central

    Hamilton, Kristia S.; Phong, Binh; Corey, Catherine; Cheng, Jing; Gorentla, Balachandra; Zhong, Xiaoping; Shiva, Sruti; Kane, Lawrence P.

    2015-01-01

    Signaling to the mechanistic target of rapamycin (mTOR) regulates diverse cellular processes, including protein translation, cellular proliferation, metabolism, and autophagy. These effects are mediated in part by the mTOR targets S6 kinase (S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). Most models place Akt upstream of the best-studied mTOR complex, mTORC1; however, studies have called into question whether Akt is necessary for this pathway, at least in T cells. We found that the adaptor protein Carma1 [caspase recruitment domain (CARD)-containing membrane-associated protein 1 (Carma1)] and at least one of its associated proteins, the paracaspase MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), were required for optimal activation of mTOR in T cells in response to stimulation of the T cell receptor (TCR) and the coreceptor CD28. However, another common binding partner of Carma1 and MALT1, Bcl10, was not required for TCR-dependent activation of the mTOR pathway. Consistent with these findings, MALT1 activity was required for the proliferation of CD4+ T cells, but not early TCR-dependent activation events. Also consistent with an effect on mTOR, MALT1 activity was required for the increased metabolic flux in activated CD4+ T cells. Together, our data suggest that Carma1 and MALT1 play previously unappreciated roles in the activation of mTOR signaling in T cells after engagement of the TCR. PMID:24917592

  9. The dynamics of T-cell receptor repertoire diversity following thymus transplantation for DiGeorge anomaly.

    PubMed

    Ciupe, Stanca M; Devlin, Blythe H; Markert, M Louise; Kepler, Thomas B

    2009-06-01

    T cell populations are regulated both by signals specific to the T-cell receptor (TCR) and by signals and resources, such as cytokines and space, that act independently of TCR specificity. Although it has been demonstrated that disruption of either of these pathways has a profound effect on T-cell development, we do not yet have an understanding of the dynamical interactions of these pathways in their joint shaping of the T cell repertoire. Complete DiGeorge Anomaly is a developmental abnormality that results in the failure of the thymus to develop, absence of T cells, and profound immune deficiency. After receiving thymic tissue grafts, patients suffering from DiGeorge anomaly develop T cells derived from their own precursors but matured in the donor tissue. We followed three DiGeorge patients after thymus transplantation to utilize the remarkable opportunity these subjects provide to elucidate human T-cell developmental regulation. Our goal is the determination of the respective roles of TCR-specific vs. TCR-nonspecific regulatory signals in the growth of these emerging T-cell populations. During the course of the study, we measured peripheral blood T-cell concentrations, TCRbeta V gene-segment usage and CDR3-length spectratypes over two years or more for each of the subjects. We find, through statistical analysis based on a novel stochastic population-dynamic T-cell model, that the carrying capacity corresponding to TCR-specific resources is approximately 1000-fold larger than that of TCR-nonspecific resources, implying that the size of the peripheral T-cell pool at steady state is determined almost entirely by TCR-nonspecific mechanisms. Nevertheless, the diversity of the TCR repertoire depends crucially on TCR-specific regulation. The estimated strength of this TCR-specific regulation is sufficient to ensure rapid establishment of TCR repertoire diversity in the early phase of T cell population growth, and to maintain TCR repertoire diversity in the face of

  10. Positive and negative regulation of T-cell activation through kinases and phosphatases.

    PubMed Central

    Mustelin, Tomas; Taskén, Kjetil

    2003-01-01

    The sequence of events in T-cell antigen receptor (TCR) signalling leading to T-cell activation involves regulation of a number of protein tyrosine kinases (PTKs) and the phosphorylation status of many of their substrates. Proximal signalling pathways involve PTKs of the Src, Syk, Csk and Tec families, adapter proteins and effector enzymes in a highly organized tyrosine-phosphorylation cascade. In intact cells, tyrosine phosphorylation is rapidly reversible and generally of a very low stoichiometry even under induced conditions due to the fact that the enzymes removing phosphate from tyrosine-phosphorylated substrates, the protein tyrosine phosphatases (PTPases), have a capacity that is several orders of magnitude higher than that of the PTKs. It follows that a relatively minor change in the PTK/PTPase balance can have a major impact on net tyrosine phosphorylation and thereby on activation and proliferation of T-cells. This review focuses on the involvement of PTKs and PTPases in positive and negative regulation of T-cell activation, the emerging theme of reciprocal regulation of each type of enzyme by the other, as well as regulation of phosphotyrosine turnover by Ser/Thr phosphorylation and regulation of localization of signal components. PMID:12485116

  11. Characteristics of the TCR Vβ repertoire in imatinib-resistant chronic myeloid leukemia patients with ABL mutations.

    PubMed

    Xu, Ling; Lu, YuHong; Lai, Jing; Yu, Wei; Zhang, YiKai; Jin, ZhenYi; Xu, Yan; Chen, Jie; Zha, XianFeng; Chen, ShaoHua; Yang, LiJian; Li, YangQiu

    2015-12-01

    Diversity in the T cell receptor (TCR) repertoire provides a miniature defense ability for the T cell immune system that may be related to tumor initiation and progression. Understanding the T cell immune status of leukemia patients is critical for establishing specific immunotherapies. Previous studies have reported abnormal TCR repertoires and clonally expanded TCR Vβ T cells in chronic myeloid leukemia in chronic phase (CP-CML). In this study, we investigated the distribution and clonality of the TCR Vβ repertoire in 4 cases with imatinib-resistant CML in blast crisis (BC-CML) with abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase domain mutations (KDMs). Examination of TCR V expression and clonality was performed by reverse transcription-polymerase chain reaction (RT-PCR) and GeneScan analysis. Significantly skewed TCR Vβ repertoires were observed in BC-CML patients with different KDMs, and 4 to 8 oligoclonally expanded TCR Vβ subfamilies could be identified in each sample. Intriguingly, a relatively highly expanded Vβ9 clone with the same length as complementarity- determining region 3 (CDR3) (139 bp) was found in all three CML patients in lymphoid blast crisis (LBC-CML) who had different KDMs, but the clone was not detected in the only CML patient in myeloid blast crisis (MBC-CML). In conclusion, restricted TCR Vβ repertoire expression and decreased clone complexity was a general phenomenon observed in the BC-CML patients with different KDMs, indicating the T-cell immunodeficiency of these patients. In addition, clonally expanded Vβ9 T cell clones may indicate a specific immune response to leukemia-associated antigens in LBC-CML patients.

  12. Evolution and function of the TCR Vgamma9 chain repertoire: It's good to be public.

    PubMed

    Pauza, C David; Cairo, Cristiana

    2015-07-01

    Lymphocytes expressing a T cell receptor (TCR) composed of Vgamma9 and Vdelta2 chains represent a minor fraction of human thymocytes. Extrathymic selection throughout post-natal life causes the proportion of cells with a Vgamma9-JP rearrangement to increase and elevates the capacity for responding to non-peptidic phosphoantigens. Extrathymic selection is so powerful that phosphoantigen-reactive cells comprise about 1 in 40 circulating memory T cells in healthy adults and the subset expands rapidly upon infection or in response to malignancy. Skewing of the gamma delta TCR repertoire is accompanied by selection for public gamma chain sequences such that many unrelated individuals overlap extensive in their circulating repertoire. This type of selection implies the presence of a monomorphic antigen-presenting molecule that is an object of current research but remains incompletely defined. While selection on a monomorphic presenting molecule may seem unusual, similar mechanisms shape the alpha beta T cell repertoire including the extreme examples of NKT or mucosal-associated invariant T cells (MAIT) and the less dramatic amplification of public Vbeta chain rearrangements driven by individual MHC molecules and associated with resistance to viral pathogens. Selecting and amplifying public T cell receptors whether alpha beta or gamma delta, are important steps in developing an anticipatory TCR repertoire. Cell clones expressing public TCR can accelerate the kinetics of response to pathogens and impact host survival.

  13. Znhit1 causes cell cycle arrest and down-regulates CDK6 expression

    SciTech Connect

    Yang, Zhengmin; Cao, Yonghao; Zhu, Xiaoyan; Huang, Ying; Ding, Yuqiang; Liu, Xiaolong

    2009-08-14

    Cyclin-dependent kinase 6 (CDK6) is the key element of the D-type cyclin holoenzymes which has been found to function in the regulation of G1-phase of the cell cycle and is presumed to play important roles in T cell function. In this study, Znhit1, a member of a new zinc finger protein family defined by a conserved Zf-HIT domain, induced arrest in the G1-phase of the cell cycle in NIH/3T3 cells. Of the G1 cell cycle factors examined, the expression of CDK6 was found to be strongly down-regulated by Znhit1 via transcriptional repression. This effect may have correlations with the decreased acetylation level of histone H4 in the CDK6 promoter region. In addition, considering that CDK6 expression predominates in T cells, the negative regulatory role of Znhit1 in TCR-induced T cell proliferation was validated using transgenic mice. These findings identified Znhit1 as a CDK6 regulator that plays an important role in cell proliferation.

  14. Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

    PubMed Central

    Axmann, Markus; Schütz, Gerhard J.; Huppa, Johannes B.

    2015-01-01

    T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (µM range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on Förster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment. PMID:26555227

  15. Transglutaminase Regulation of Cell Function

    PubMed Central

    Kaartinen, Mari T.; Nurminskaya, Maria; Belkin, Alexey M.; Colak, Gozde; Johnson, Gail V. W.; Mehta, Kapil

    2014-01-01

    Transglutaminases (TGs) are multifunctional proteins having enzymatic and scaffolding functions that participate in regulation of cell fate in a wide range of cellular systems and are implicated to have roles in development of disease. This review highlights the mechanism of action of these proteins with respect to their structure, impact on cell differentiation and survival, role in cancer development and progression, and function in signal transduction. We also discuss the mechanisms whereby TG level is controlled and how TGs control downstream targets. The studies described herein begin to clarify the physiological roles of TGs in both normal biology and disease states. PMID:24692352

  16. V(D)J recombination and allelic exclusion of a TCR {beta}-chain minilocus occurs in the absence of a functional promoter

    SciTech Connect

    Alvarez, J.D.; Anderson, S.J.; Loh, D.Y.

    1995-08-01

    Transcriptional activation of rearranging Ag receptor gene segments has been hypothesized to regulate their accessibility to V(D)J recombination. We analyzed the role of a functional promoter in the rearrangement of the murine TCR {beta}-chain locus using two transgenic minilocus constructs. These miniloci each contain an unrearranged V{beta}8.3 gene. One has a wild-type V{beta}8.3 gene, but the other has a V{beta}8.3 gene with a promoter mutation that was previously shown to abrogate transcription in tissue culture. FACS analysis of thymus and lymph node cells from transgenic mouse lines showed that only the lines with the wild-type V{beta}8.3 gene promoter express an 8.3 TCR {beta}-chain. Consistent with the protein expression data, V{beta}8.3 gene transcripts were found only in the transgenic lines with the wild-type promoter. Using a quantitative PCR-based assay, it was shown that both types of transgenic lines recombine the V{beta}8.3 gene at similar levels. Rearrangement of the transgenes was normal with respect to thymic development and junctional reading frame. Interestingly, both types of miniloci also underwent allelic exclusion in that recombination was blocked by the expression of a rearranged TCR {beta}-chain transgene. We conclude that a functional V{beta} gene promoter is not necessary for proper V(D)J recombination to occur.

  17. Accelerated Loss of TCR Repertoire Diversity in Common Variable Immunodeficiency

    PubMed Central

    Wong, Gabriel K.; Millar, David; Penny, Sarah; Heather, James M.; Mistry, Punam; Buettner, Nico; Bryon, Jane; Huissoon, Aarnoud P.

    2016-01-01

    Although common variable immunodeficiency (CVID) has long been considered as a group of primary Ab deficiencies, growing experimental data now suggest a global disruption of the entire adaptive immune response in a segment of patients. Oligoclonality of the TCR repertoire was previously demonstrated; however, the manner in which it relates to other B cell and T cell findings reported in CVID remains unclear. Using a combination approach of high-throughput TCRβ sequencing and multiparametric flow cytometry, we compared the TCR repertoire diversity between various subgroups of CVID patients according to their B cell immunophenotypes. Our data suggest that the reduction in repertoire diversity is predominantly restricted to those patients with severely reduced class-switched memory B cells and an elevated level of CD21lo B cells (Freiburg 1a), and may be driven by a reduced number of naive T cells unmasking underlying memory clonality. Moreover, our data indicate that this loss in repertoire diversity progresses with advancing age far exceeding the expected physiological rate. Radiological evidence supports the loss in thymic volume, correlating with the decrease in repertoire diversity. Evidence now suggests that primary thymic failure along with other well-described B cell abnormalities play an important role in the pathophysiology in Freiburg group 1a patients. Clinically, our findings emphasize the integration of combined B and T cell testing to identify those patients at the greatest risk for infection. Future work should focus on investigating the link between thymic failure and the severe reduction in class-switched memory B cells, while gathering longitudinal laboratory data to examine the progressive nature of the disease. PMID:27481850

  18. TCR Triggering Induces the Formation of Lck–RACK1–Actinin-1 Multiprotein Network Affecting Lck Redistribution

    PubMed Central

    Ballek, Ondřej; Valečka, Jan; Dobešová, Martina; Broučková, Adéla; Manning, Jasper; Řehulka, Pavel; Stulík, Jiří; Filipp, Dominik

    2016-01-01

    The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor (TCR) triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, receptor for activated C kinase 1 (RACK1), was chosen as a viable option, and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant, and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1–Lck complexes were detectable in primary CD4+ T-cells with their maximum levels peaking 10 s after TCR–CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394Lck, which co-purifies with high molecular weight cellular fractions. The formation of RACK1–Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1–pY394Lck–α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4+ T-cells with nocodazole, which disrupts the microtubular network, also blocked the

  19. Constitutively active Lck kinase in T cells drives antigen receptor signal transduction.

    PubMed

    Nika, Konstantina; Soldani, Cristiana; Salek, Mogjiborahman; Paster, Wolfgang; Gray, Adrian; Etzensperger, Ruth; Fugger, Lars; Polzella, Paolo; Cerundolo, Vincenzo; Dushek, Omer; Höfer, Thomas; Viola, Antonella; Acuto, Oreste

    2010-06-25

    T cell antigen receptor (TCR) and coreceptor ligation is thought to initiate signal transduction by inducing activation of the kinase Lck. Here we showed that catalytically active Lck was present in unstimulated naive T cells and thymocytes and was readily detectable in these cells in lymphoid organs. In naive T cells up to approximately 40% of total Lck was constitutively activated, part of which was also phosphorylated on the C-terminal inhibitory site. Formation of activated Lck was independent of TCR and coreceptors but required Lck catalytic activity and its maintenance relied on monitoring by the HSP90-CDC37 chaperone complex to avoid degradation. The amount of activated Lck did not change after TCR and coreceptor engagement; however it determined the extent of TCR-zeta phosphorylation. Our findings suggest a dynamic regulation of Lck activity that can be promptly utilized to initiate T cell activation and have implications for signaling by other immune receptors.

  20. Stimulation through CD50 preferentially induces apoptosis of TCR1+ human peripheral blood lymphocytes.

    PubMed

    López-Briones, S; Portales-Pérez, D P; Baranda, L; de la Fuente, H; Rosenstein, Y; González-Amaro, R

    1998-01-01

    Apoptosis has an important role in several key immunological phenomena such as regulation of the immune response, and deletion of auto-reactive cells. This phenomenon is induced following the interaction of several cell membrane receptors with their respective ligands or after cell activation. We have studied the possible effect of signaling through CD50/ICAM-3 and CD69/AIM on apoptosis of peripheral blood lymphocytes. Apoptosis was assessed by both flow cytometry analysis (content of cell DNA and binding to annexin V), and detection of DNA fragmentation by agarose gel electrophoresis. We found that a stimulatory anti-CD50 mAb was able to induce a small but significant degree of apoptosis in resting peripheral blood mononuclear cells from most donors; this effect was dose-dependent and was evident as early as at 12 h, with a maximal induction at 48 h. Studies with T and non-T cells showed that only the former cell population was sensitive to the induction of apoptosis through CD50. Further experiments revealed that the anti-ICAM-3 mAb preferentially induced apoptosis of TCR gamma delta-bearing cells. In addition, we found a significant increase in Cai2+ in PBMC stimulated with an anti-CD50 mAb, suggesting the involvement of this signaling pathway in the induction of apoptosis through this adhesion receptor. In contrast, under our experimental conditions, stimulation through CD69 did not have any effect on the induction of apoptosis on either cultured T lymphoblasts or PMA-stimulated PBMC. Our findings suggest that the interaction of CD50 with its natural ligand LFA-1 results in the induction of apoptosis in a significant fraction of resting PBMC. This phenomenon may be involved in immune regulation, lymphocyte turnover and peripheral deletion of auto-reactive cells.

  1. Genome regulation in mammalian cells.

    PubMed

    Puck, T T; Krystosek, A; Chan, D C

    1990-05-01

    A theory is presented proposing that genetic regulation in mammalian cells is at least a two-tiered effect; that one level of regulation involves the transition between gene exposure and sequestration; that normal differentiation requires a different spectrum of genes to be exposed in each separate state of differentiation; that the fiber systems of the cell cytoskeleton and the nuclear matrix together control the degree of gene exposure; that specific phosphorylation of these elements causes them to assume a different organizational network and to impose a different pattern of sequestration and exposure on the elements of the genome; that the varied gene phosphorylation mechanisms in the cell are integrated in this function; that attachment of this network system to specific parts of the chromosomes brings about sequestration or exposure of the genes in their neighborhood in a fashion similar to that observed when microtubule elements attach through the kinetochore to the centromeric DNA; that one function of repetitive sequences is to serve as elements for the final attachment of this fibrous network to the specific chromosomal loci; and that at least an important part of the calcium manifestation as a metabolic trigger of different differentiation states involves its acting as a binding agent to centers of electronegativity, in particular proteins and especially phosphorylated groups, so as to change the conformation of the fiber network that ultimately controls gene exposure in the mammalian cell. It would appear essential to determine what abnormal gene exposures and sequestrations are characteristic of each type of cancer; which agonists, if any, will bring about reverse transformation; and whether these considerations can be used in therapy.

  2. CD3ε recruits Numb to promote TCR degradation.

    PubMed

    Martin-Blanco, Nadia; Jiménez Teja, Daniel; Bretones, Gabriel; Borroto, Aldo; Caraballo, Michael; Screpanti, Isabella; León, Javier; Alarcón, Balbino; Canelles, Matilde

    2016-03-01

    Modulation of TCR signaling upon ligand binding is achieved by changes in the equilibrium between TCR degradation, recycling and synthesis; surprisingly, the molecular mechanism of such an important process is not fully understood. Here, we describe the role of a new player in the mediation of TCR degradation: the endocytic adaptor Numb. Our data show that Numb inhibition leads to abnormal intracellular distribution and defective TCR degradation in mature T lymphocytes. In addition, we find that Numb simultaneously binds to both Cbl and a site within CD3ε that overlaps with the Nck binding site. As a result, Cbl couples specifically to the CD3ε chain to mediate TCR degradation. The present study unveils a novel role of Numb that lies at the heart of TCR signaling initiation and termination.

  3. The FRK/RAK-SHB signaling cascade: a versatile signal-transduction pathway that regulates cell survival, differentiation and proliferation.

    PubMed

    Annerén, Cecilia; Lindholm, Cecilia K; Kriz, Vitezslav; Welsh, Michael

    2003-06-01

    Recent experiments have unravelled novel signal transduction pathways that involve the SRC homology 2 (SH2) domain adapter protein SHB. SHB is ubiquitously expressed and contains proline rich motifs, a phosphotyrosine binding (PTB) domain, tyrosine phosphorylation sites and an SH2 domain and serves a role in generating signaling complexes in response to tyrosine kinase activation. SHB mediates certain responses in platelet-derived growth factor (PDGF) receptor-, fibroblast growth factor (FGF) receptor-, neural growth factor (NGF) receptor TRKA-, T cell receptor-, interleukin-2 (IL-2) receptor- and focal adhesion kinase- (FAK) signaling. Upstream of SHB in some cells lies the SRC-like FYN-Related Kinase FRK/RAK (also named BSK/IYK or GTK). FRK/RAK and SHB exert similar effects when overexpressed in rat phaeochromocytoma (PC12) and beta-cells, where they both induce PC12 cell differentiation and beta-cell proliferation. Furthermore, beta-cell apoptosis is augmented by these proteins under conditions that cause beta-cell degeneration. The FRK/RAK-SHB responses involve FAK and insulin receptor substrates (IRS) -1 and -2. Besides regulating apoptosis, proliferation and differentiation, SHB is also a component of the T cell receptor (TCR) signaling response. In Jurkat T cells, SHB links several signaling components with the TCR and is thus required for IL-2 production. In endothelial cells, SHB both promotes apoptosis under conditions that are anti-angiogenic, but is also required for proper mitogenicity, spreading and tubular morphogenesis. In embryonic stem cells, dominant-negative SHB (R522K) prevents early cavitation of embryoid bodies and reduces differentiation to cells expressing albumin, amylase, insulin and glucagon, suggesting a role of SHB in development. In summary, SHB is a versatile signal transduction molecule that produces diverse biological responses in different cell types under various conditions. SHB operates downstream of GTK in cells that express

  4. K33-linked polyubiquitination of Zap70 by Nrdp1 controls CD8(+) T cell activation.

    PubMed

    Yang, Mingjin; Chen, Taoyong; Li, Xuelian; Yu, Zhou; Tang, Songqing; Wang, Chen; Gu, Yan; Liu, Yanfang; Xu, Sheng; Li, Weihua; Zhang, Xuemin; Wang, Jianli; Cao, Xuetao

    2015-12-01

    The key molecular mechanisms that control signaling via T cell antigen receptors (TCRs) remain to be fully elucidated. Here we found that Nrdp1, a ring finger-type E3 ligase, mediated Lys33 (K33)-linked polyubiquitination of the signaling kinase Zap70 and promoted the dephosphorylation of Zap70 by the acidic phosphatase-like proteins Sts1 and Sts2 and thereby terminated early TCR signaling in CD8(+) T cells. Nrdp1 deficiency significantly promoted the activation of naive CD8(+) T cells but not that of naive CD4(+) T cells after engagement of the TCR. Nrdp1 interacted with Zap70 and with Sts1 and Sts2 and connected K33 linkage of Zap70 to Sts1- and Sts2-mediated dephosphorylation. Our study suggests that Nrdp1 terminates early TCR signaling by inactivating Zap70 and provides new mechanistic insights into the non-proteolytic regulation of TCR signaling by E3 ligases.

  5. Preferential Use of Public TCR during Autoimmune Encephalomyelitis.

    PubMed

    Zhao, Yunqian; Nguyen, Phuong; Ma, Jing; Wu, Tianhua; Jones, Lindsay L; Pei, Deqing; Cheng, Cheng; Geiger, Terrence L

    2016-06-15

    How the TCR repertoire, in concert with risk-associated MHC, imposes susceptibility for autoimmune diseases is incompletely resolved. Due largely to recombinatorial biases, a small fraction of TCRα or β-chains are shared by most individuals, or public. If public TCR chains modulate a TCRαβ heterodimer's likelihood of productively engaging autoantigen, because they are pervasive and often high frequency, they could also broadly influence disease risk and progression. Prior data, using low-resolution techniques, have identified the heavy use of select public TCR in some autoimmune models. In this study, we assess public repertoire representation in mice with experimental autoimmune encephalomyelitis at high resolution. Saturation sequencing was used to identify >18 × 10(6) TCRβ sequences from the CNSs, periphery, and thymi of mice at different stages of autoimmune encephalomyelitis and healthy controls. Analyses indicated the prominent representation of a highly diverse public TCRβ repertoire in the disease response. Preferential formation of public TCR implicated in autoimmunity was identified in preselection thymocytes, and, consistently, public, disease-associated TCRβ were observed to be commonly oligoclonal. Increased TCR sharing and a focusing of the public TCR response was seen with disease progression. Critically, comparisons of peripheral and CNS repertoires and repertoires from preimmune and diseased mice demonstrated that public TCR were preferentially deployed relative to nonshared, or private, sequences. Our findings implicate public TCR in skewing repertoire response during autoimmunity and suggest that subsets of public TCR sequences may serve as disease-specific biomarkers or influence disease susceptibility or progression.

  6. Regulation of vesicular traffic at the T cell immune synapse: lessons from the primary cilium.

    PubMed

    Finetti, Francesca; Onnis, Anna; Baldari, Cosima T

    2015-03-01

    The signals that orchestrate the process of T cell activation are coordinated at the specialized interface that forms upon contact with an antigen presenting cell displaying a specific MHC-associated peptide ligand, known as the immune synapse. The central role of vesicular traffic in the assembly of the immune synapse has emerged only in recent years with the finding that sustained T-cell receptor (TCR) signaling involves delivery of TCR/CD3 complexes from an intracellular pool associated with recycling endosomes. A number of receptors as well as membrane-associated signaling mediators have since been demonstrated to exploit this process to localize to the immune synapse. Here, we will review our current understanding of the mechanisms responsible for TCR recycling, with a focus on the intraflagellar transport system, a multimolecular complex that is responsible for the assembly and function of the primary cilium which we have recently implicated in polarized endosome recycling to the immune synapse.

  7. The protein tyrosine phosphatase PTPN22 controls forkhead box protein 3 T regulatory cell induction but is dispensable for T helper type 1 cell polarization

    PubMed Central

    Fousteri, G; Jofra, T; Debernardis, I; Stanford, S M; Laurenzi, A; Bottini, N; Battaglia, M

    2014-01-01

    Protein tyrosine phosphatases (PTPs) regulate T cell receptor (TCR) signalling and thus have a role in T cell differentiation. Here we tested whether the autoimmune predisposing gene PTPN22 encoding for a PTP that inhibits TCR signalling affects the generation of forkhead box protein 3 (FoxP3)+ T regulatory (Treg) cells and T helper type 1 (Th1) cells. Murine CD4+ T cells isolated from Ptpn22 knock-out (Ptpn22KO) mice cultured in Treg cell polarizing conditions showed increased sensitivity to TCR activation compared to wild-type (WT) cells, and subsequently reduced FoxP3 expression at optimal-to-high levels of activation. However, at lower levels of TCR activation, Ptpn22KO CD4+ T cells showed enhanced expression of FoxP3. Similar experiments in humans revealed that at optimal levels of TCR activation PTPN22 knock-down by specific oligonucleotides compromises the differentiation of naive CD4+ T cells into Treg cells. Notably, in vivo Treg cell conversion experiments in mice showed delayed kinetic but overall increased frequency and number of Treg cells in the absence of Ptpn22. In contrast, the in vitro and in vivo generation of Th1 cells was comparable between WT and Ptpn22KO mice, thus suggesting PTPN22 as a FoxP3-specific regulating factor. Together, these results propose PTPN22 as a key factor in setting the proper threshold for FoxP3+ Treg cell differentiation. PMID:24905474

  8. Genetic and epigenetic determinants mediate proneness of oncogene breakpoint sites for involvement in TCR translocations.

    PubMed

    Larmonie, N S D; van der Spek, A; Bogers, A J J C; van Dongen, J J M; Langerak, A W

    2014-03-01

    T-cell receptor (TCR) translocations are a genetic hallmark of T-cell acute lymphoblastic leukemia and lead to juxtaposition of oncogene and TCR loci. Oncogene loci become involved in translocations because they are accessible to the V(D)J recombination machinery. Such accessibility is predicted at cryptic recombination signal sequence (cRSS) sites ('Type 1') as well as other sites that are subject to DNA double-strand breaks (DSBs) ('Type 2') during early stages of thymocyte development. As chromatin accessibility markers have not been analyzed in the context of TCR-associated translocations, various genetic and epigenetic determinants of LMO2, TAL1 and TLX1 translocation breakpoint (BP) sites and BP cluster regions (BCRs) were examined in human thymocytes to establish DSB proneness and heterogeneity of BP site involvement in TCR translocations. Our data show that DSBs in BCRs are primarily induced in the presence of a genetic element of sequence vulnerability (cRSSs, transposable elements), whereas breaks at single BP sites lacking such elements are more likely induced by chance or perhaps because of patient-specific genetic vulnerability. Vulnerability to obtain DSBs is increased by features that determine chromatin organization, such as methylation status and nucleosome occupancy, although at different levels at different BP sites.

  9. Stem cell regulation: Implications when differentiated cells regulate symmetric stem cell division.

    PubMed

    Høyem, Marte Rørvik; Måløy, Frode; Jakobsen, Per; Brandsdal, Bjørn Olav

    2015-09-07

    We use a mathematical model to show that if symmetric stem cell division is regulated by differentiated cells, then changes in the population dynamics of the differentiated cells can lead to changes in the population dynamics of the stem cells. More precisely, the relative fitness of the stem cells can be affected by modifying the death rate of the differentiated cells. This result is interesting because stem cells are less sensitive than differentiated cells to environmental factors, such as medical therapy. Our result implies that stem cells can be manipulated indirectly by medical treatments that target the differentiated cells.

  10. A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor gene expression

    NASA Astrophysics Data System (ADS)

    Falvo, James V.; Brinkman, Brigitta M. N.; Tsytsykova, Alla V.; Tsai, Eunice Y.; Yao, Tso-Pang; Kung, Andrew L.; Goldfeld, Anne E.

    2000-04-01

    The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor (TNF-) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF- gene induction by TCR activation is inhibited, whereas virus induction of the TNF- gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-α gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

  11. TCR backscattering characterization for microwave remote sensing

    NASA Astrophysics Data System (ADS)

    Riccio, Giovanni; Gennarelli, Claudio

    2014-05-01

    A Trihedral Corner Reflector (TCR) is formed by three mutually orthogonal metal plates of various shapes and is a very important scattering structure since it exhibits a high monostatic Radar Cross Section (RCS) over a wide angular range. Moreover it is a handy passive device with low manufacturing costs and robust geometric construction, the maintenance of its efficiency is not difficult and expensive, and it can be used in all weather conditions (i.e., fog, rain, smoke, and dusty environment). These characteristics make it suitable as reference target and radar enhancement device for satellite- and ground-based microwave remote sensing techniques. For instance, TCRs have been recently employed to improve the signal-to-noise ratio of the backscattered signal in the case of urban ground deformation monitoring [1] and dynamic survey of civil infrastructures without natural corners as the Musmeci bridge in Basilicata, Italy [2]. The region of interest for the calculation of TCR's monostatic RCS is here confined to the first quadrant containing the boresight direction. The backscattering term is presented in closed form by evaluating the far-field scattering integral involving the contributions related to the direct illumination and the internal bouncing mechanisms. The Geometrical Optics (GO) laws allow one to determine the field incident on each TCR plate and the patch (integration domain) illuminated by it, thus enabling the use of a Physical Optics (PO) approximation for the corresponding surface current densities to consider for integration on each patch. Accordingly, five contributions are associated to each TCR plate: one contribution is due to the direct illumination of the whole internal surface; two contributions originate by the impinging rays that are simply reflected by the other two internal surfaces; and two contributions are related to the impinging rays that undergo two internal reflections. It is useful to note that the six contributions due to the

  12. MMTV superantigens coerce an unconventional topology between the TCR and MHC class II.

    PubMed

    Fortin, Jean-Simon; Genève, Laetitia; Gauthier, Catherine; Shoukry, Naglaa H; Azar, Georges A; Younes, Souheil; Yassine-Diab, Bader; Sékaly, Rafick-Pierre; Fremont, Daved H; Thibodeau, Jacques

    2014-02-15

    Mouse mammary tumor virus superantigens (vSAGs) are notorious for defying structural characterization, and a consensus has yet to be reached regarding their ability to bridge the TCR to MHC class II (MHCII). In this study, we determined the topology of the T cell signaling complex by examining the respective relation of vSAG7 with the MHCII molecule, MHCII-associated peptide, and TCR. We used covalently linked peptide/MHCII complexes to demonstrate that vSAG presentation is tolerant to variation in the protruding side chains of the peptide, but can be sensitive to the nature of the protruding N-terminal extension. An original approach in which vSAG was covalently linked to either MHCII chain confirmed that vSAG binds outside the peptide binding groove. Also, whereas the C-terminal vSAG segment binds to the MHCII α-chain in a conformation-sensitive manner, the membrane-proximal N-terminal domain binds the β-chain. Because both moieties of the mature vSAG remain noncovalently associated after processing, our results suggest that vSAG crosslinks MHCII molecules. Comparing different T cell hybridomas, we identified key residues on the MHCII α-chain that are differentially recognized by the CDR3β when engaged by vSAG. Finally, we show that the highly conserved tyrosine residue found in the vSAg TGXY motif is required for T cell activation. Our results reveal a novel SAG/MHCII/TCR architecture in which vSAGs coerce a near-canonical docking between MHCII and TCR that allows eschewing of traditional CDR3 binding with the associated peptide in favor of MHCII α-chain binding. Our findings highlight the plasticity of the TCR CDRs.

  13. β-arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the immunological synapse by a PKC-mediated mechanism

    PubMed Central

    Fernández-Arenas, Elena; Calleja, Enrique; Martínez-Martín, Nadia; Gharbi, Severine I; Navajas, Rosana; García-Medel, Noel; Penela, Petronila; Alcamí, Antonio; Mayor, Federico; Albar, Juan P; Alarcón, Balbino

    2014-01-01

    T-cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen-presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β-arrestin-1 as a ligand of non-phosphorylated resting TCRs. Using dominant-negative and knockdown approaches we demonstrate that β-arrestin-1 is required for the internalization and downregulation of non-engaged bystander TCRs. Furthermore, TCR triggering provokes the β-arrestin-1-mediated downregulation of the G-protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β-arrestin-1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR-triggered PKC-mediated phosphorylation of β-arrestin-1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal. PMID:24502978

  14. T cell activation.

    PubMed

    Smith-Garvin, Jennifer E; Koretzky, Gary A; Jordan, Martha S

    2009-01-01

    This year marks the 25th anniversary of the first Annual Review of Immunology article to describe features of the T cell antigen receptor (TCR). In celebration of this anniversary, we begin with a brief introduction outlining the chronology of the earliest studies that established the basic paradigm for how the engaged TCR transduces its signals. This review continues with a description of the current state of our understanding of TCR signaling, as well as a summary of recent findings examining other key aspects of T cell activation, including cross talk between the TCR and integrins, the role of costimulatory molecules, and how signals may negatively regulate T cell function.Acronyms and DefinitionsAdapter protein: cellular protein that functions to bridge molecular interactions via characteristic domains able to mediate protein/protein or protein/lipid interactions Costimulation: signals delivered to T cells by cell surface receptors other than the TCR itself that potentiate T cell activation cSMAC: central supramolecular activation cluster Immunoreceptor tyrosine-based activation motif (ITAM): a short peptide sequence in the cytoplasmic tails of key surface receptors on hematopoietic cells that is characterized by tyrosine residues that are phosphorylated by Src family PTKs, enabling the ITAM to recruit activated Syk family kinases Inside-out signaling: signals initiated by engagement of immunoreceptors that lead to conformational changes and clustering of integrins, thereby increasing the affinity and avidity of the integrins for their ligands NFAT: nuclear factor of activated T cells PI3K: phosphoinositide 3-kinase PKC: protein kinase C PLC: phospholipase C pMHC: peptide major histocompatibility complex (MHC) complex pSMAC: peripheral supramolecular activation cluster PTK: protein tyrosine kinase Signal transduction: biochemical events linking surface receptor engagement to cellular responses TCR: T cell antigen receptor

  15. IL-15 regulates memory CD8+ T cell O-glycan synthesis and affects trafficking

    PubMed Central

    Nolz, Jeffrey C.; Harty, John T.

    2014-01-01

    Memory and naive CD8+ T cells exhibit distinct trafficking patterns. Specifically, memory but not naive CD8+ T cells are recruited to inflamed tissues in an antigen-independent manner. However, the molecular mechanisms that regulate memory CD8+ T cell trafficking are largely unknown. Here, using murine models of infection and T cell transfer, we found that memory but not naive CD8+ T cells dynamically regulate expression of core 2 O-glycans, which interact with P- and E-selectins to modulate trafficking to inflamed tissues. Following infection, antigen-specific effector CD8+ T cells strongly expressed core 2 O-glycans, but this glycosylation pattern was lost by most memory CD8+ T cells. After unrelated infection or inflammatory challenge, memory CD8+ T cells synthesized core 2 O-glycans independently of antigen restimulation. The presence of core 2 O-glycans subsequently directed these cells to inflamed tissue. Memory and naive CD8+ T cells exhibited the opposite pattern of epigenetic modifications at the Gcnt1 locus, which encodes the enzyme that initiates core 2 O-glycan synthesis. The open chromatin configuration in memory CD8+ T cells permitted de novo generation of core 2 O-glycans in a TCR-independent, but IL-15–dependent, manner. Thus, IL-15 stimulation promotes antigen-experienced memory CD8+ T cells to generate core 2 O-glycans, which subsequently localize them to inflamed tissues. These findings suggest that CD8+ memory T cell trafficking potentially can be manipulated to improve host defense and immunotherapy. PMID:24509081

  16. GvL effects in T-prolymphocytic leukemia: evidence from MRD kinetics and TCR repertoire analyses.

    PubMed

    Sellner, L; Brüggemann, M; Schlitt, M; Knecht, H; Herrmann, D; Reigl, T; Krejci, A; Bystry, V; Darzentas, N; Rieger, M; Dietrich, S; Luft, T; Ho, A D; Kneba, M; Dreger, P

    2016-12-12

    Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.Bone Marrow Transplantation advance online publication, 12 December 2016; doi:10.1038/bmt.2016.305.

  17. Substrate rigidity regulates human T cell activation and proliferation.

    PubMed

    O'Connor, Roddy S; Hao, Xueli; Shen, Keyue; Bashour, Keenan; Akimova, Tatiana; Hancock, Wayne W; Kam, Lance C; Milone, Michael C

    2012-08-01

    Adoptive immunotherapy using cultured T cells holds promise for the treatment of cancer and infectious disease. Ligands immobilized on surfaces fabricated from hard materials such as polystyrene plastic are commonly employed for T cell culture. The mechanical properties of a culture surface can influence the adhesion, proliferation, and differentiation of stem cells and fibroblasts. We therefore explored the impact of culture substrate stiffness on the ex vivo activation and expansion of human T cells. We describe a simple system for the stimulation of the TCR/CD3 complex and the CD28 receptor using substrates with variable rigidity manufactured from poly(dimethylsiloxane), a biocompatible silicone elastomer. We show that softer (Young's Modulus [E] < 100 kPa) substrates stimulate an average 4-fold greater IL-2 production and ex vivo proliferation of human CD4(+) and CD8(+) T cells compared with stiffer substrates (E > 2 MPa). Mixed peripheral blood T cells cultured on the stiffer substrates also demonstrate a trend (nonsignificant) toward a greater proportion of CD62L(neg), effector-differentiated CD4(+) and CD8(+) T cells. Naive CD4(+) T cells expanded on softer substrates yield an average 3-fold greater proportion of IFN-γ-producing Th1-like cells. These results reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation, and Th differentiation. Substrate rigidity should therefore be a consideration in the development of T cell culture systems as well as when interpreting results of T cell activation based upon solid-phase immobilization of TCR/CD3 and CD28 ligands.

  18. Substrate rigidity regulates human T cell activation and proliferation1

    PubMed Central

    O’Connor, Roddy S.; Hao, Xueli; Shen, Keyue; Bashour, Keenan; Akimova, Tatiana; Hancock, Wayne W.; Kam, Lance; Milone, Michael C.

    2012-01-01

    Adoptive immunotherapy using cultured T cells holds promise for the treatment of cancer and infectious disease. Ligands immobilized on surfaces fabricated from hard materials such as polystyrene plastic are commonly employed for T cell culture. The mechanical properties of a culture surface can influence the adhesion, proliferation, and differentiation of stem cells and fibroblasts. We therefore explored the impact of culture substrate stiffness on the ex vivo activation and expansion of human T cells. We describe a simple system for the stimulation of the TCR/CD3 complex and the CD28 receptor using substrates with variable rigidity manufactured from poly(dimethylsiloxane) (PDMS), a biocompatible silicone elastomer. We show that softer (Young’s Modulus [E] < 100 kPa) substrates stimulate an average 4-fold greater IL-2 production and ex vivo proliferation of human CD4+ and CD8+ T cells compared with stiffer substrates (E >2 MPa). Mixed peripheral blood T cells cultured on the stiffer substrates also demonstrate a trend (non-significant) towards a greater proportion of CD62Lneg, effector-differentiated CD4+ and CD8+ T cells. Naïve CD4+ T cells expanded on softer substrates yield an average 3-fold greater proportion of IFN-γ producing TH1-like cells. These results reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation and TH differentiation. Substrate rigidity should therefore be a consideration in the development of T cell culture systems as well as when interpreting results of T cell activation based upon solid-phase immobilization of TCR/CD3 and CD28 ligands. PMID:22732590

  19. Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation

    PubMed Central

    D’Souza, Anthony D.; Parikh, Neal; Kaech, Susan M.; Shadel, Gerald S.

    2009-01-01

    The quantity and activity of mitochondria vary dramatically in tissues and are modulated in response to changing cellular energy demands and environmental factors. The amount of mitochondrial DNA (mtDNA), which encodes essential subunits of the oxidative phosphorylation complexes required for cellular ATP production, is also tightly regulated, but by largely unknown mechanisms. Using murine T cells as a model system, we have addressed how specific signaling pathways influence mitochondrial biogenesis and mtDNA levels. T cell receptor (TCR) activation results in a large increase in mitochondrial mass and membrane potential and a corresponding increase of mtDNA copy number, indicating the vital role for mitochondrial function for the growth and proliferation of these cells. Independent activation of protein kinase C (via PMA) or calcium-related pathways (via ionomycin) had differential and sub-maximal effects on these mitochondrial parameters, as did activation of naïve T cells with proliferative cytokines. Thus, the robust mitochondrial biogenesis response observed upon TCR activation requires synergy of multiple downstream signaling pathways. One such pathway involves AMP-activated protein kinase (AMPK), which we show has an unprecedented role in negatively regulating mitochondrial biogenesis that is mammalian target of rapamycin (mTOR)-dependent. That is, inhibition of AMPK after TCR signaling commences results in excessive, but uncoordinated mitochondrial proliferation. We propose that mitochondrial biogenesis is not under control of a master regulatory circuit, but rather requires the convergence of multiple signaling pathways with distinct downstream consequences on the organelle’s structure, composition, and function. PMID:17890163

  20. Metaboloepigenetic Regulation of Pluripotent Stem Cells

    PubMed Central

    Harvey, Alexandra J.; Gardner, David K.

    2016-01-01

    The differentiation of pluripotent stem cells is associated with extensive changes in metabolism, as well as widespread remodeling of the epigenetic landscape. Epigenetic regulation is essential for the modulation of differentiation, being responsible for cell type specific gene expression patterns through the modification of DNA and histones, thereby establishing cell identity. Each cell type has its own idiosyncratic pattern regarding the use of specific metabolic pathways. Rather than simply being perceived as a means of generating ATP and building blocks for cell growth and division, cellular metabolism can directly influence cellular regulation and the epigenome. Consequently, the significance of nutrients and metabolites as regulators of differentiation is central to understanding how cells interact with their immediate environment. This review serves to integrate studies on pluripotent stem cell metabolism, and the regulation of DNA methylation and acetylation and identifies areas in which current knowledge is limited. PMID:26839556

  1. GeneScanning analysis of Ig/TCR gene rearrangements to detect clonality in canine lymphomas.

    PubMed

    Gentilini, Fabio; Calzolari, Claudia; Turba, Maria E; Bettini, Giuliano; Famigli-Bergamini, Paolo

    2009-01-15

    The diagnosis of canine lymphoma is achieved using morphological and immunological methods. In a certain percentage of cases, difficulties in making a definitive diagnosis of lymphoproliferative disorders may occur despite extensive immunophenotyping. Therefore, additional diagnostics, such as molecular assessment of Ig/TCR gene rearrangements clonality, may confirm the final diagnosis. Polyacrylamide gel electrophoresis and heteroduplex analysis have already been proven to be suitable for detecting clonality but are cumbersome and labor-intensive. In the present study, GeneScanning analysis of PCR products originating from different primer sets targeting different regions of Ig and TCR was validated in improving sensitivity as well as in reducing the turnaround time of gene rearrangement assays. GeneScanning exploits 5' fluorescently labelled primers for the automated and fast analysis of PCR products either as singleplex or multiplex runs. Initially, the assay was set up using DNA purified from normal tissues (n=6), hyperplastic/reactive tissues (n=10) and a small set of immunophenotyped lymphoma samples (n=12). The optimized methods were then used in a large set of 96 canine lymphoma samples. Normal and hyperplastic/reactive lymphoid tissues showed typically polyclonal or, occasionally, oligoclonal PCR products. Lymphoma samples showed monoclonal peaks arranged as a single or, occasionally, a double narrow base peak sometimes embedded in a polyclonal background. In all immunophenotyped cases, an Ig or TCR clonal finding corresponded to B- and T-cell lymphomas, respectively. Overall, 94/96 (97.9%) samples showed clonal Ig/TCR clonal rearrangements among which clonal Ig was found in 61/96 (63.5%) of samples and clonal TCR in 33/35 Ig negative samples (34.4% of all cases). In one out of ten randomly chosen cases, both Ig and TCR clonal gene rearrangements were found. Among the factors affecting assay accuracy, DNA quality has been shown to be critical and the

  2. Regulation of Murine Natural Killer Cell Development

    PubMed Central

    Goh, Wilford; Huntington, Nicholas D.

    2017-01-01

    Natural killer (NK) cells are effector lymphocytes of the innate immune system that are known for their ability to kill transformed and virus-infected cells. NK cells originate from hematopoietic stem cells in the bone marrow, and studies on mouse models have revealed that NK cell development is a complex, yet tightly regulated process, which is dependent on both intrinsic and extrinsic factors. The development of NK cells can be broadly categorized into two phases: lineage commitment and maturation. Efforts to better define the developmental framework of NK cells have led to the identification of several murine NK progenitor populations and mature NK cell subsets, each defined by a varied set of cell surface markers. Nevertheless, the relationship between some of these NK cell subsets remains to be determined. The classical approach to studying both NK cell development and function is to identify the transcription factors involved and elucidate the mechanistic action of each transcription factor. In this regard, recent studies have provided further insight into the mechanisms by which transcription factors, such as ID2, FOXO1, Kruppel-like factor 2, and GATA-binding protein 3 regulate various aspects of NK cell biology. It is also becoming evident that the biology of NK cells is not only transcriptionally regulated but also determined by epigenetic alterations and posttranscriptional regulation of gene expression by microRNAs. This review summarizes recent progress made in NK development, focusing primarily on transcriptional regulators and their mechanistic actions. PMID:28261203

  3. In vitro membrane reconstitution of the T cell receptor proximal signaling network

    PubMed Central

    Hui, Enfu; Vale, Ronald D.

    2014-01-01

    T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45, and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over a ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that coclustering TCR-Lck or detaching Csk from the membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs. PMID:24463463

  4. STIM1 controls T cell-mediated immune regulation and inflammation in chronic infection.

    PubMed

    Desvignes, Ludovic; Weidinger, Carl; Shaw, Patrick; Vaeth, Martin; Ribierre, Theo; Liu, Menghan; Fergus, Tawania; Kozhaya, Lina; McVoy, Lauren; Unutmaz, Derya; Ernst, Joel D; Feske, Stefan

    2015-06-01

    Chronic infections induce a complex immune response that controls pathogen replication, but also causes pathology due to sustained inflammation. Ca2+ influx mediates T cell function and immunity to infection, and patients with inherited mutations in the gene encoding the Ca2+ channel ORAI1 or its activator stromal interaction molecule 1 (STIM1) are immunodeficient and prone to chronic infection by various pathogens, including Mycobacterium tuberculosis (Mtb). Here, we demonstrate that STIM1 is required for T cell-mediated immune regulation during chronic Mtb infection. Compared with WT animals, mice with T cell-specific Stim1 deletion died prematurely during the chronic phase of infection and had increased bacterial burdens and severe pulmonary inflammation, with increased myeloid and lymphoid cell infiltration. Although STIM1-deficient T cells exhibited markedly reduced IFN-γ production during the early phase of Mtb infection, bacterial growth was not immediately exacerbated. During the chronic phase, however, STIM1-deficient T cells displayed enhanced IFN-γ production in response to elevated levels of IL-12 and IL-18. The lack of STIM1 in T cells was associated with impaired activation-induced cell death upon repeated TCR engagement and pulmonary lymphocytosis and hyperinflammation in Mtb-infected mice. Chronically Mtb-infected, STIM1-deficient mice had reduced levels of inducible regulatory T cells (iTregs) due to a T cell-intrinsic requirement for STIM1 in iTreg differentiation and excessive production of IFN-γ and IL-12, which suppress iTreg differentiation and maintenance. Thus, STIM1 controls multiple aspects of T cell-mediated immune regulation to limit injurious inflammation during chronic infection.

  5. Arrested rearrangement of TCR V[beta] genes in thymocytes from children with x-linked severe combined immunodeficiency disease

    SciTech Connect

    Sleasman, J.W.; Harville, T.O.; White, G.B.; Barrett, D.J. ); George, J.F. ); Goodenow, M.M. Univ. of Alabama, Birmingham, AL )

    1994-07-01

    Human X-linked severe combined immunodeficiency disease (SCID) is an immunodeficiency disorder in which T cell development is arrested in the thymic cortex. B lymphocytes in children with X-linked SCID seem to differentiate normally. X-linked SCID is associated with a mutation in the gene that encodes the IL-2R [gamma]-chain. Because TCR-[beta] gene recombination is a pivotal initial event in T lymphocyte onteogeny within the thymus, the authors hypothesized that a failure to express normal IL-2R[gamma] could lead to impaired TCR-[beta] gene recombination in early thymic development. PCR was used to determine the status of TCR-[beta] gene-segment rearrangements in thymic DNA that had been obtained from children with X-linked SCID. The initial step in TCR-[beta] gene rearrangement, that of D[beta] to J[beta] recombination, was readily detected in all thymus samples from children with X-linked SCID; in contrast, V[beta] to DJ[beta] gene rearrangements were undetectable in the same samples. Both D[beta] to J[beta] and V[beta] to DJ[beta] TCR genes were rearranged in the thymic tissues obtained from immunologically normal children. The authors conclude that TCR[beta]-chain gene rearrangement is arrested in children with X-linked SCID. The results suggest a causative relationship between the failure of TCR [beta]-chain gene arrangements to proceed beyond DJ[beta] rearrangements and the production of a nonfunctional IL-2R [gamma]-chain. 45 refs., 3 figs.

  6. Materials as stem cell regulators

    PubMed Central

    Murphy, William L.; McDevitt, Todd C.; Engler, Adam J.

    2014-01-01

    The stem cell/material interface is a complex, dynamic microenvironment in which the cell and the material cooperatively dictate one another's fate: the cell by remodelling its surroundings, and the material through its inherent properties (such as adhesivity, stiffness, nanostructure or degradability). Stem cells in contact with materials are able to sense their properties, integrate cues via signal propagation and ultimately translate parallel signalling information into cell fate decisions. However, discovering the mechanisms by which stem cells respond to inherent material characteristics is challenging because of the highly complex, multicomponent signalling milieu present in the stem cell environment. In this Review, we discuss recent evidence that shows that inherent material properties may be engineered to dictate stem cell fate decisions, and overview a subset of the operative signal transduction mechanisms that have begun to emerge. Further developments in stem cell engineering and mechanotransduction are poised to have substantial implications for stem cell biology and regenerative medicine. PMID:24845994

  7. Materials as stem cell regulators

    NASA Astrophysics Data System (ADS)

    Murphy, William L.; McDevitt, Todd C.; Engler, Adam J.

    2014-06-01

    The stem cell/material interface is a complex, dynamic microenvironment in which the cell and the material cooperatively dictate one another's fate: the cell by remodelling its surroundings, and the material through its inherent properties (such as adhesivity, stiffness, nanostructure or degradability). Stem cells in contact with materials are able to sense their properties, integrate cues via signal propagation and ultimately translate parallel signalling information into cell fate decisions. However, discovering the mechanisms by which stem cells respond to inherent material characteristics is challenging because of the highly complex, multicomponent signalling milieu present in the stem cell environment. In this Review, we discuss recent evidence that shows that inherent material properties may be engineered to dictate stem cell fate decisions, and overview a subset of the operative signal transduction mechanisms that have begun to emerge. Further developments in stem cell engineering and mechanotransduction are poised to have substantial implications for stem cell biology and regenerative medicine.

  8. RIAM Regulates the Cytoskeletal Distribution and Activation of PLC-γ1 in T cells

    PubMed Central

    Patsoukis, Nikolaos; Lafuente, Esther M.; Meraner, Paul; Kim, Jin sub; Dombkowski, David; Li, Lequn; Boussiotis, Vassiliki A.

    2010-01-01

    Rap1-GTP-interacting adaptor molecule (RIAM), an adaptor molecule of the Mig-10/RIAM/Lamellipodin (MRL) family, plays a critical role in actin reorganization and inside-out activation of integrins in lymphocytes and platelets. We investigated the role of RIAM in T cell receptor (TCR)-mediated signaling. Elimination of endogenous RIAM by short hairpin RNA (shRNA) resulted in impaired generation of inositol 1,4,5-trisphosphate (IP3) and mobilization of intracellular Ca2+, whereas phosphorylation of ζ chain–associated protein kinase of 70 kD (ZAP-70) and formation of the linker of activated T cells (LAT) signalosome were unaffected. Knockdown of RIAM also resulted in defective nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT) and activation of Ras guanine nucleotide-releasing protein 1 (RasGRP)1, which led to the diminished transcription of Il2. These events were associated with the impaired translocation of phosphorylated phospholipase C γ1 (PLC-γ1) to the actin cytoskeleton, which was required for the recruitment of PLC-γ1 to the immediate proximity of its substrate phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5)P2], and were reversed by reconstitution of cells with RIAM. Thus, by regulating the activation of PLC-γ1, RIAM has a central role in the activation of T cells and the transcription of target genes. PMID:19952372

  9. Natural killer cell regulation - beyond the receptors

    PubMed Central

    Urlaub, Doris; Fasbender, Frank; Claus, Maren

    2014-01-01

    Natural killer (NK) cells are lymphocytes that are important for early and effective immune responses against infections and cancer. In the last 40 years, many receptors, their corresponding ligands and signaling pathways that regulate NK cell functions have been identified. However, we now know that additional processes, such as NK cell education, differentiation and also the formation of NK cell memory, have a great impact on the reactivity of these cells. Here, we summarize the current knowledge about these modulatory processes. PMID:25374665

  10. Evolutionarily conserved amino acids in TCR V regions and MHC control their interaction

    PubMed Central

    Marrack, Philippa; Scott-Browne, James P.; Dai, Shaodong; Gapin, Laurent; Kappler, John W.

    2011-01-01

    Alpha/beta T cell receptors (TCRs) react with major histocompatibility complex proteins (MHC) plus peptides, a poorly understood phenomenon, probably because thymocytes bearing TCRs that manifest MHC-reactivity too well are lost by negative selection. Only TCRs with attenuated ability to react with MHC appear on mature T cells. Also, the interaction sites between TCRs and MHC may be inherently flexible and hence difficult to spot. Contacts between TCRs and MHC in the solved structures of their complexes were reevaluated with these points in mind. The results show that frequently used amino acids in TCR CDR1 and CDR2 regions are often used to bind MHC, in areas around small amino acids on the surfaces of MHC α helices that form a cup, allowing somewhat flexible binding of the TCRs. The TCR amino acids involved are specific to families of V regions and partially different rules govern recognition of MHC1 versus MHCII. PMID:18304006

  11. Cell competition in vertebrate organ size regulation.

    PubMed

    Penzo-Méndez, Alfredo I; Stanger, Ben Z

    2014-01-01

    The study of animal organ size determination has provided evidence of the existence of organ-intrinsic mechanisms that 'sense' and adjust organ growth. Cell competition, a form of cell interaction that equalizes cell population growth, has been proposed to play a role in organ size regulation. Cell competition involves a cell-context dependent response triggered by perceived differences in cell growth and/or proliferation rates, resulting in apoptosis in growth-disadvantaged cells and compensatory expansion of the more 'fit' cells. The mechanisms that allow cells to compare growth are not yet understood, but a number of genes and pathways have been implicated in cell competition. These include Myc, the members of the Hippo, JAK/STAT and WNT signaling pathways, and the Dlg/Lgl/Scrib and the Crb/Std/PatJ membrane protein complexes. Cell competition was initially characterized in the Drosophila imaginal disc, but several recent studies have shown that cell competition occurs in mouse embryonic stem cells and in the embryonic epiblast, where it plays a role in the regulation of early embryo size. In addition, competition-like behavior has been described in the adult mouse liver and the hematopoietic stem cell compartment. These data indicate that cell competition plays a more universal role in organ size regulation. In addition, as some authors have suggested that similar types of competitive behavior may operate in during tumorigenesis, there may be additional practical reasons for understanding this fundamental process of intercellular communication.

  12. Cell Cycle Regulation by Checkpoints

    PubMed Central

    Barnum, Kevin J.; O’Connell, Matthew J.

    2016-01-01

    Cell cycle checkpoints are surveillance mechanisms that monitor the order, integrity, and fidelity of the major events of the cell cycle. These include growth to the appropriate cell size, the replication and integrity of the chromosomes, and their accurate segregation at mitosis. Many of these mechanisms are ancient in origin and highly conserved, and hence have been heavily informed by studies in simple organisms such as the yeasts. Others have evolved in higher organisms, and control alternative cell fates with significant impact on tumor suppression. Here, we consider these different checkpoint pathways and the consequences of their dysfunction on cell fate. PMID:24906307

  13. Cell cycle regulation by checkpoints.

    PubMed

    Barnum, Kevin J; O'Connell, Matthew J

    2014-01-01

    Cell cycle checkpoints are surveillance mechanisms that monitor the order, integrity, and fidelity of the major events of the cell cycle. These include growth to the appropriate cell size, the replication and integrity of the chromosomes, and their accurate segregation at mitosis. Many of these mechanisms are ancient in origin and highly conserved, and hence have been heavily informed by studies in simple organisms such as the yeasts. Others have evolved in higher organisms, and control alternative cell fates with significant impact on tumor suppression. Here, we consider these different checkpoint pathways and the consequences of their dysfunction on cell fate.

  14. Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

    PubMed Central

    2015-01-01

    Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy. PMID:24564881

  15. How structural adaptability exists alongside HLA-A2 bias in the human αβ TCR repertoire

    PubMed Central

    Blevins, Sydney J.; Pierce, Brian G.; Singh, Nishant K.; Riley, Timothy P.; Wang, Yuan; Spear, Timothy T.; Nishimura, Michael I.; Weng, Zhiping; Baker, Brian M.

    2016-01-01

    How T-cell receptors (TCRs) can be intrinsically biased toward MHC proteins while simultaneously display the structural adaptability required to engage diverse ligands remains a controversial puzzle. We addressed this by examining αβ TCR sequences and structures for evidence of physicochemical compatibility with MHC proteins. We found that human TCRs are enriched in the capacity to engage a polymorphic, positively charged “hot-spot” region that is almost exclusive to the α1-helix of the common human class I MHC protein, HLA-A*0201 (HLA-A2). TCR binding necessitates hot-spot burial, yielding high energetic penalties that must be offset via complementary electrostatic interactions. Enrichment of negative charges in TCR binding loops, particularly the germ-line loops encoded by the TCR Vα and Vβ genes, provides this capacity and is correlated with restricted positioning of TCRs over HLA-A2. Notably, this enrichment is absent from antibody genes. The data suggest a built-in TCR compatibility with HLA-A2 that biases receptors toward, but does not compel, particular binding modes. Our findings provide an instructional example for how structurally pliant MHC biases can be encoded within TCRs. PMID:26884163

  16. Indoctrinating T cells to attack pathogens through homeschooling

    PubMed Central

    Parello, Caitlin S.; Huseby, Eric S.

    2015-01-01

    Adaptive immunity is predicated on the ability of the T cell repertoire to have pre-existing specificity for the universe of potential pathogens. Recent findings suggest that TCR-self-pMHC interactions limit autoimmune responses while enhancing T cell response to foreign antigens. We review these findings here, placing them in context of the current understanding of how TCR-self-pMHC interactions regulate T cell activation thresholds, and suggest that TCR-self-pMHC interactions increase the efficiency of the T cell repertoire by giving a competitive advantage to peptide cross-reactive T cells. We propose that self-reactivity and peptide-cross-reactivity are controlled by particular CDR3 sequence motifs, which would allow thymic selection to contribute to solving the feat of broad pathogen-specificity by exporting T cells that are pre-screened by positive and negative selection for the ability to be ‘moderately’ peptide cross-reactive. PMID:25979654

  17. Dopamine receptors D3 and D5 regulate CD4(+)T-cell activation and differentiation by modulating ERK activation and cAMP production.

    PubMed

    Franz, Dafne; Contreras, Francisco; González, Hugo; Prado, Carolina; Elgueta, Daniela; Figueroa, Claudio; Pacheco, Rodrigo

    2015-07-15

    Dopamine receptors have been described in T-cells, however their signalling pathways coupled remain unknown. Since cAMP and ERKs play key roles regulating T-cell physiology, we aim to determine whether cAMP and ERK1/2-phosphorylation are modulated by dopamine receptor 3 (D3R) and D5R, and how this modulation affects CD4(+) T-cell activation and differentiation. Our pharmacologic and genetic evidence shows that D3R-stimulation reduced cAMP levels and ERK2-phosphorylation, consequently increasing CD4(+) T-cell activation and Th1-differentiation, respectively. Moreover, D5R expression reinforced TCR-triggered ERK1/2-phosphorylation and T-cell activation. In conclusion, these findings demonstrate how D3R and D5R modulate key signalling pathways affecting CD4(+) T-cell activation and Th1-differentiation.

  18. Regulation of Ras Exchange Factors and Cellular Localization of Ras Activation by Lipid Messengers in T Cells

    PubMed Central

    Jun, Jesse E.; Rubio, Ignacio; Roose, Jeroen P.

    2013-01-01

    The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs) catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and Son of Sevenless (SOS)-family GEFs. Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood. One large group of biomolecules critically involved in the control of RasGEFs functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR) stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells. PMID:24027568

  19. Decreased ER-associated degradation of α-TCR induced by Grp78 depletion with the SubAB cytotoxin

    PubMed Central

    Lass, Agnieszka; Kujawa, Marek; McConnell, Elizabeth; Paton, Adrienne W.; Paton, James C.; Wójcik, Cezary

    2008-01-01

    HeLa cells stably expressing the α chain of T-cell receptor (αTCR), a model substrate of ERAD (ER-associated degradation), were used to analyze the effects of BiP/Grp78 depletion by the SubAB cytotoxin. SubAB induced XBP1 splicing, followed by JNK phosphorylation, eIF2α phosphorylation, upregulation of ATF3/4 and partial ATF6 cleavage. Other markers of ER stress, including elements of ER-associated degradation (ERAD) pathway, as well as markers of cytoplasmic stress, were not induced. SubAB treatment decreased absolute levels of αTCR, which was caused by inhibition of protein synthesis. At the same time, the half-life of αTCR was extended almost fourfold from 70 min to 210 min, suggesting that BiP normally facilitates ERAD. Depletion ofp97/VCP partially rescued SubAB-induced depletion of αTCR, confirming the role of VCP in ERAD of αTCR. It therefore appears that ERAD of αTCR is driven by at least two different ATP-ase systems located at two sides of the ER membrane, BiP located on the lumenal side, while p97/VCP on the cytoplasmic side. While SubAB altered cell morphology by inducing cytoplasm vacuolization and accumulation of lipid droplets, caspase activation was partial and subsided after prolonged incubation. Expression of CHOP/GADD153 occurred only after prolonged incubation and was not associated with apoptosis. PMID:18611445

  20. SIT and TRIM determine T cell fate in the thymus.

    PubMed

    Koelsch, Uwe; Schraven, Burkhart; Simeoni, Luca

    2008-11-01

    Thymic selection is a tightly regulated developmental process essential for establishing central tolerance. The intensity of TCR-mediated signaling is a key factor for determining cell fate in the thymus. It is widely accepted that low-intensity signals result in positive selection, whereas high-intensity signals induce negative selection. Transmembrane adaptor proteins have been demonstrated to be important regulators of T cell activation. However, little is known about their role during T cell development. Herein, we show that SIT (SHP2 Src homology domain containing tyrosine phosphatase 2-interacting transmembrane adaptor protein) and TRIM (TCR-interacting molecule), two structurally related transmembrane adaptors, cooperatively regulate TCR signaling potential, thereby influencing the outcome of thymic selection. Indeed, loss of both SIT and TRIM resulted in the up-regulation of CD5, CD69, and TCRbeta, strong MAPK activation, and, consequently, enhanced positive selection. Moreover, by crossing SIT/TRIM double-deficient mice onto transgenic mice bearing TCRs with different avidity/affinity, we found profound alterations in T cell development. Indeed, in female HY TCR transgenic mice, positive selection was completely converted into negative selection resulting in small thymi devoided of double-positive thymocytes. More strikingly, in a nonselecting background, SIT/TRIM double-deficient single-positive T cells developed, were functional, and populated the periphery. In summary, we demonstrated that SIT and TRIM regulate cell fate of developing thymocytes, thus identifying them as essential regulators of central tolerance.

  1. Small GTPases as regulators of cell division.

    PubMed

    Militello, Rodrigo; Colombo, María I

    2013-09-01

    The superfamily of small GTPases serves as a signal transducer to regulate a diverse array of cellular functions. The members of this superfamily are structurally and functionally classified into at least 5 groups (Ras, Rho/Rac, Rab, Arf, and Ran) and they are involved in the control of cell proliferation and differentiation, regulation of the actin cytoskeleton, membrane trafficking, and nuclear transport. It is widely reported that members of the Rab family participate in the control of intracellular membrane trafficking through the interaction with specific effector molecules. However, many Rabs and other small GTPases have also been shown to function in cell division. In this review, we discuss current knowledge about Rab proteins regulating different stages of the cell cycle, such as the congregation and segregation of chromosomes (during metaphase) and the final stage of cell division known as cytokinesis, in which a cell is cleaved originating 2 daughter cells.

  2. Tumor cell "dead or alive": caspase and survivin regulate cell death, cell cycle and cell survival.

    PubMed

    Suzuki, A; Shiraki, K

    2001-04-01

    Cell death and cell cycle progression are two sides of the same coin, and these two different phenomenons are regulated moderately to maintain the cellular homeostasis. Tumor is one of the disease states produced as a result of the disintegrated regulation and is characterized as cells showing an irreversible progression of cell cycle and a resistance to cell death signaling. Several investigations have been performed for the understanding of cell death or cell cycle, and cell death research has remarkably progressed in these 10 years. Caspase is a nomenclature referring to ICE/CED-3 cysteine proteinase family and plays a central role during cell death. Recently, several investigations raised some possible hypotheses that caspase is also involved in cell cycle regulation. In this issue, therefore, we review the molecular basis of cell death and cell cycle regulated by caspase in tumor, especially hepatocellular carcinoma cells.

  3. Conformational Melding Permits a Conserved Binding Geometry in TCR Recognition of Foreign and Self Molecular Mimics

    SciTech Connect

    Borbulevych, Oleg Y.; Piepenbrink, Kurt H.; Baker, Brian M.

    2012-03-16

    Molecular mimicry between foreign and self Ags is a mechanism of TCR cross-reactivity and is thought to contribute to the development of autoimmunity. The {alpha}{beta} TCR A6 recognizes the foreign Ag Tax from the human T cell leukemia virus-1 when presented by the class I MHC HLA-A2. In a possible link with the autoimmune disease human T cell leukemia virus-1-associated myelopathy/tropical spastic paraparesis, A6 also recognizes a self peptide from the neuronal protein HuD in the context of HLA-A2. We found in our study that the complexes of the HuD and Tax epitopes with HLA-A2 are close but imperfect structural mimics and that in contrast with other recent structures of TCRs with self Ags, A6 engages the HuD Ag with the same traditional binding mode used to engage Tax. Although peptide and MHC conformational changes are needed for recognition of HuD but not Tax and the difference of a single hydroxyl triggers an altered TCR loop conformation, TCR affinity toward HuD is still within the range believed to result in negative selection. Probing further, we found that the HuD-HLA-A2 complex is only weakly stable. Overall, these findings help clarify how molecular mimicry can drive self/nonself cross-reactivity and illustrate how low peptide-MHC stability can permit the survival of T cells expressing self-reactive TCRs that nonetheless bind with a traditional binding mode.

  4. Allograft rejection mediated by memory T cells is resistant to regulation.

    PubMed

    Yang, Jaeseok; Brook, Matthew O; Carvalho-Gaspar, Manuela; Zhang, Jidong; Ramon, Hilda E; Sayegh, Mohamed H; Wood, Kathryn J; Turka, Laurence A; Jones, Nick D

    2007-12-11

    Alloreactive memory T cells may be refractory to many of the tolerance-inducing strategies that are effective against naive T cells and thus present a significant barrier to long-term allograft survival. Because CD4(+)CD25(+) regulatory T cells (Tregs) are critical elements of many approaches to successful induction/maintenance of transplantation tolerance, we used MHC class I and II alloreactive TCR-transgenic models to explore the ability of antigen-specific Tregs to control antigen-specific memory T cell responses. Upon coadoptive transfer into RAG-1(-/-) mice, we found that Tregs effectively suppressed the ability of naive T cells to reject skin grafts, but neither antigen-unprimed nor antigen-primed Tregs suppressed rejection by memory T cells. Interestingly, different mechanisms appeared to be active in the ability of Tregs to control naive T cell-mediated graft rejection in the class II versus class I alloreactive models. In the former case, we observed decreased early expansion of effector cells in lymphoid tissue. In contrast, in the class I model, an effect of Tregs on early proliferation and expansion was not observed. However, at a late time point, significant differences in cell numbers were seen, suggesting effects on responding T cell survival. Overall, these data indicate that the relative resistance of both CD4(+) and CD8(+) alloreactive memory T cells to regulation may mediate resistance to tolerance induction seen in hosts with preexisting alloantigen-specific immunity and further indicate the multiplicity of mechanisms by which Tregs may control alloimmune responses in vivo.

  5. Discrete TCR repertoires and CDR3 features distinguish effector and Foxp3+ regulatory T lymphocytes in MOG-EAE

    PubMed Central

    Nguyen, Phuong; Liu, Wei; Ma, Jing; Manirarora, Jean N.; Liu, Xin; Cheng, Cheng; Geiger, Terrence L.

    2010-01-01

    Regulatory T lymphocytes (Treg) expressing the Forkhead Box Transcription Factor 3 (Foxp3) are critical modulators of autoimmunity. Foxp3+ Treg may develop in the thymus as a population distinct from conventional Foxp3− αβ T cells (Tconv). Alternatively, plasticity in Foxp3 expression may allow for the interconversion of mature Treg and Tconv. We examined >160,000 TCR sequences from Foxp3+ or Foxp3− populations in the spleens or CNS of wild type mice with experimental allergic encephalomyelitis (EAE) to determine their relatedness and identify distinguishing TCR features. Our results indicate that the CNS infiltrating Treg and Tconv arise predominantly from distinct sources. The repertoires of CNS Treg or Tconv TCR showed limited overlap with heterologous populations in either the CNS or spleen, indicating that they are largely unrelated. Indeed, Treg and Tconv TCR in the CNS were significantly less related than those populations in the spleen. In contrast, CNS Treg and Tconv repertoires strongly intersected those of the homologous cell type in the spleen. High frequency sequences more likely to be disease associated showed similar results, and some public TCR demonstrated Treg or Tconv-specific motifs. Different charge characteristics and amino acid use preferences were identified in the CDR3β of Treg and Tconv infiltrating the CNS, further indicating that their repertoires are qualitatively distinct. Therefore discrete populations of Treg and Tconv that do not substantially interconvert respond during EAE. Differences in sequence and physical characteristics distinguish Treg and Tconv TCR and imply dissimilar antigen recognition properties. PMID:20810983

  6. Regulation of germ cell function by SUMOylation

    PubMed Central

    Rodriguez, Amanda; Pangas, Stephanie A.

    2015-01-01

    Oogenesis and spermatogenesis are tightly regulated complex processes that are critical for fertility function. Germ cells undergo meiosis to generate haploid cells necessary for reproduction. Errors in meiosis, including the generation of chromosomal abnormalities, can result in reproductive defects and infertility. Meiotic proteins are regulated by post-translational modifications including SUMOylation, the covalent attachment of small ubiquitin-like modifier (SUMO) proteins. Here, we review the role of SUMO proteins in controlling germ cell development and maturation based on recent findings from mouse models. Several studies have characterized the localization of SUMO proteins in male and female germ cells. However, a deeper understanding of how SUMOylation regulates proteins with essential roles in oogenesis and spermatogenesis will provide useful insight into the underlying mechanisms of germ cell development and fertility. PMID:26374733

  7. Cartilage stem cells: regulation of differentiation.

    PubMed

    Solursh, M

    1989-01-01

    The developing limb bud is a useful source of cartilage stem cells for studies on the regulation of chondrogenesis. In high density cultures these cells can progress through all stages of chondrogenesis to produce mineralized hypertrophic cartilage. If the cells are maintained in a spherical shape, single stem cells can progress through a similar sequence. The actin cytoskeleton is implicated in the regulation of chondrogenesis since conditions that favor its disruption promote chondrogenesis and conditions that favor actin assembly inhibit chondrogenesis. Since a number of extracellular matrix receptors mediate effects of the extracellular matrix on cytoskeletal organization and some of these receptors are developmentally regulated, it is proposed that matrix receptor expression plays a central role in the divergence of connective tissue cells during development.

  8. Cell Size Regulation in Bacteria

    NASA Astrophysics Data System (ADS)

    Amir, Ariel

    2014-05-01

    Various bacteria such as the canonical gram negative Escherichia coli or the well-studied gram positive Bacillus subtilis divide symmetrically after they approximately double their volume. Their size at division is not constant, but is typically distributed over a narrow range. Here, we propose an analytically tractable model for cell size control, and calculate the cell size and interdivision time distributions, as well as the correlations between these variables. We suggest ways of extracting the model parameters from experimental data, and show that existing data for E. coli supports partial size control, and a particular explanation: a cell attempts to add a constant volume from the time of initiation of DNA replication to the next initiation event. This hypothesis accounts for the experimentally observed correlations between mother and daughter cells as well as the exponential dependence of size on growth rate.

  9. Protein-Protein Interaction Investigated by Steered Molecular Dynamics: The TCR-pMHC Complex

    PubMed Central

    Cuendet, Michel A.; Michielin, Olivier

    2008-01-01

    We present a novel steered molecular dynamics scheme to induce the dissociation of large protein-protein complexes. We apply this scheme to study the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) presenting a peptide (p). Two TCR-pMHC complexes are considered, which only differ by the mutation of a single amino acid on the peptide; one is a strong agonist that produces T cell activation in vivo, while the other is an antagonist. We investigate the interaction mechanism from a large number of unbinding trajectories by analyzing van der Waals and electrostatic interactions and by computing energy changes in proteins and solvent. In addition, dissociation potentials of mean force are calculated with the Jarzynski identity, using an averaging method developed for our steering scheme. We analyze the convergence of the Jarzynski exponential average, which is hampered by the large amount of dissipative work involved and the complexity of the system. The resulting dissociation free energies largely underestimate experimental values, but the simulations are able to clearly differentiate between wild-type and mutated TCR-pMHC and give insights into the dissociation mechanism. PMID:18621828

  10. Tcra enhancer activation by inducible transcription factors downstream of pre-TCR signaling.

    PubMed

    del Blanco, Beatriz; García-Mariscal, Alberto; Wiest, David L; Hernández-Munain, Cristina

    2012-04-01

    The Tcra enhancer (Eα) is essential for pre-TCR-mediated activation of germline transcription and V(D)J recombination. Eα is considered an archetypical enhanceosome that acts through the functional synergy and cooperative binding of multiple transcription factors. Based on dimethylsulfate genomic footprinting experiments, there has been a long-standing paradox regarding Eα activation in the absence of differences in enhancer occupancy. Our data provide the molecular mechanism of Eα activation and an explanation of this paradox. We found that germline transcriptional activation of Tcra is dependent on constant phospholipase Cγ, as well as calcineurin- and MAPK/ERK-mediated signaling, indicating that inducible transcription factors are crucially involved. NFAT, AP-1, and early growth response factor 1, together with CREB-binding protein/p300 coactivators, bind to Eα as part of an active enhanceosome assembled during pre-TCR signaling. We favor a scenario in which the binding of lymphoid-restricted and constitutive transcription factors to Eα prior to its activation forms a regulatory scaffold to recruit factors induced by pre-TCR signaling. Thus, the combinatorial assembly of tissue- and signal-specific transcription factors dictates the Eα function. This mechanism for enhancer activation may represent a general paradigm in tissue-restricted and stimulus-responsive gene regulation.

  11. Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αβ surface TCR expression

    PubMed Central

    2013-01-01

    Background The T cell antigen receptors (TCR) of αβ and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αβ or γδ TCR chains incorporate a CD3δε dimer, then a CD3γε dimer and finally a ζζ homodimer, resulting in TCR complexes with the same CD3 dimer stoichiometry. Partial reduction in the expression of the highly homologous CD3γ and CD3δ proteins would thus be expected to have a similar impact in the assembly and surface expression of both TCR isotypes. To test this hypothesis, we compared the surface TCR expression of primary αβ and γδ T cells from healthy donors carrying a single null or leaky mutation in CD3G (γ+/−) or CD3D (δ+/−, δ+/leaky) with that of normal controls. Results Although the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αβ T lymphocytes in CD3γ+/− individuals, whereas CD3δ+/− and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages. Therefore, surface γδ TCR expression was more dependent on available CD3γ than surface αβ TCR expression. Conclusions The results support the existence of differential structural constraints in the two human TCR isotypes regarding the incorporation of CD3γε and CD3δε dimers, as revealed by their discordant surface expression behaviour when confronted with reduced amounts of CD3γ, but not of the homologous CD3δ chain. A modified version of the prevailing TCR assembly model is proposed to accommodate these new data. PMID:23336327

  12. Epidermal stem cells and their epigenetic regulation.

    PubMed

    Shen, Qi; Jin, Hongchuan; Wang, Xian

    2013-08-30

    Stem cells play an essential role in embryonic development, cell differentiation and tissue regeneration. Tissue homeostasis in adults is maintained by adult stem cells resident in the niches of different tissues. As one kind of adult stem cell, epidermal stem cells have the potential to generate diversified types of progeny cells in the skin. Although its biology is still largely unclarified, epidermal stem cells are widely used in stem cell research and regenerative medicine given its easy accessibility and pluripotency. Despite the same genome, cells within an organism have different fates due to the epigenetic regulation of gene expression. In this review, we will briefly discuss the current understanding of epigenetic modulation in epidermal stem cells.

  13. Cell cycle regulation and regeneration.

    PubMed

    Heber-Katz, Ellen; Zhang, Yong; Bedelbaeva, Khamila; Song, Fengyu; Chen, Xiaoping; Stocum, David L

    2013-01-01

    Regeneration of ear punch holes in the MRL mouse and amputated limbs of the axolotl show a number of similarities. A large proportion of the fibroblasts of the uninjured MRL mouse ear are arrested in G2 of the cell cycle, and enter nerve-dependent mitosis after injury to form a ring-shaped blastema that regenerates the ear tissue. Multiple cell types contribute to the establishment of the regeneration blastema of the urodele limb by dedifferentiation, and there is substantial reason to believe that the cells of this early blastema are also arrested in G2, and enter mitosis under the influence of nerve-dependent factors supplied by the apical epidermal cap. Molecular analysis reveals other parallels, such as; (1) the upregulation of Evi5, a centrosomal protein that prevents mitosis by stabilizing Emi1, a protein that inhibits the degradation of cyclins by the anaphase promoting complex and (2) the expression of sodium channels by the epidermis. A central feature in the entry into the cell cycle by MRL ear fibroblasts is a natural downregulation of p21, and knockout of p21 in wild-type mice confers regenerative capacity on non-regenerating ear tissue. Whether the same is true for entry into the cell cycle in regenerating urodele limbs is presently unknown.

  14. Cell Proliferation, Cell Death, and Size Regulation

    DTIC Science & Technology

    2000-10-01

    predicted to encode a novel 582 amino acid protein, perhaps interacting with molybdopterin. It is possible that the pie gene encodes a novel enzyme protecting against cell death during growth and development.

  15. Circulating regulatory anti–T cell receptor antibodies in patients with myasthenia gravis

    PubMed Central

    Jambou, Florence; Zhang, Wei; Menestrier, Monique; Klingel-Schmitt, Isabelle; Michel, Olivier; Caillat-Zucman, Sophie; Aissaoui, Abderrahim; Landemarre, Ludovic; Berrih-Aknin, Sonia; Cohen-Kaminsky, Sylvia

    2003-01-01

    Serum anti–T cell receptor (TCR) Ab’s are involved in immune regulation directed against pathogenic T cells in experimental models of autoimmune diseases. Our identification of a dominant T cell population expressing the Vβ5.1 TCR gene (TCRBV5-1), which is responsible for the production of pathogenic anti-acetylcholine receptor (AChR) autoantibodies in HLA-DR3 patients with early-onset myasthenia gravis (EOMG), prompted us to explore the occurrence, reactivity, and regulatory role of anti-TCR Ab’s in EOMG patients and disease controls with clearly defined other autoantibodies. In the absence of prior vaccination against the TCR, EOMG patients had elevated anti-Vβ5.1 Ab’s of the IgG class. This increase was restricted largely to EOMG cases with HLA-DR3 and with less severe disease, and it predicted clinical improvement in follow-up studies. EOMG patient sera containing anti-TCR Ab’s bound specifically the native TCR on intact Vβ5.1-expressing cells and specifically inhibited the proliferation and IFN-γ production of purified Vβ5.1-expressing cells to alloantigens in mixed lymphocyte reaction and the proliferation of a Vβ5.1-expressing T cell clone to an AChR peptide, indicating a regulatory function for these Ab’s. This evidence of spontaneously active anti-Vβ5.1 Ab’s in EOMG patients suggests dynamic protective immune regulation directed against the excess of pathogenic Vβ5.1-expressing T cells. Though not sufficient to prevent a chronic, exacerbated autoimmune process, it might be boosted using a TCR peptide as vaccine. PMID:12865414

  16. Collecting Duct Intercalated Cell Function and Regulation

    PubMed Central

    Roy, Ankita; Al-bataineh, Mohammad M.

    2015-01-01

    Intercalated cells are kidney tubule epithelial cells with important roles in the regulation of acid-base homeostasis. However, in recent years the understanding of the function of the intercalated cell has become greatly enhanced and has shaped a new model for how the distal segments of the kidney tubule integrate salt and water reabsorption, potassium homeostasis, and acid-base status. These cells appear in the late distal convoluted tubule or in the connecting segment, depending on the species. They are most abundant in the collecting duct, where they can be detected all the way from the cortex to the initial part of the inner medulla. Intercalated cells are interspersed among the more numerous segment-specific principal cells. There are three types of intercalated cells, each having distinct structures and expressing different ensembles of transport proteins that translate into very different functions in the processing of the urine. This review includes recent findings on how intercalated cells regulate their intracellular milieu and contribute to acid-base regulation and sodium, chloride, and potassium homeostasis, thus highlighting their potential role as targets for the treatment of hypertension. Their novel regulation by paracrine signals in the collecting duct is also discussed. Finally, this article addresses their role as part of the innate immune system of the kidney tubule. PMID:25632105

  17. Cell Shape Dependent Regulation of Nuclear Morphology

    PubMed Central

    Chen, Bo; Co, Carlos; Ho, Chia-Chi

    2015-01-01

    Recent studies suggest that actin filaments are essential in how a cell controls its nuclear shape. However, little is known about the relative importance of membrane tension in determining nuclear morphology. In this study, we used adhesive micropatterned substrates to alter the cellular geometry (aspect ratio, size, and shape) that allowed direct membrane tension or without membrane lateral contact with the nucleus and investigate nuclear shape remodeling and orientation on a series of rectangular shapes. Here we showed that at low cell aspect ratios the orientation of the nucleus was regulated by actin filaments while cells with high aspect ratios can maintain nuclear shape and orientation even when actin polymerization was blocked. A model adenocarcinoma cell showed similar behavior in the regulation of nuclear shape in response to changes in cell shape but actin filaments were essential in maintaining cell shape. Our results highlight the two distinct mechanisms to regulate nuclear shape through cell shape control and the difference between fibroblasts and a model cancerous cell in cell adhesion and cell shape control. PMID:26210179

  18. Regulation of germ line stem cell homeostasis

    PubMed Central

    Garcia, T.X.; Hofmann, M.C.

    2015-01-01

    Mammalian spermatogenesis is a complex process in which spermatogonial stem cells of the testis (SSCs) develop to ultimately form spermatozoa. In the seminiferous epithelium, SSCs self-renew to maintain the pool of stem cells throughout life, or they differentiate to generate a large number of germ cells. A balance between SSC self-renewal and differentiation is therefore essential to maintain normal spermatogenesis and fertility. Stem cell homeostasis is tightly regulated by signals from the surrounding microenvironment, or SSC niche. By physically supporting the SSCs and providing them with these extrinsic molecules, the Sertoli cell is the main component of the niche. Earlier studies have demonstrated that GDNF and CYP26B1, produced by Sertoli cells, are crucial for self-renewal of the SSC pool and maintenance of the undifferentiated state. Down-regulating the production of these molecules is therefore equally important to allow germ cell differentiation. We propose that NOTCH signaling in Sertoli cells is a crucial regulator of germ cell fate by counteracting these stimulatory factors to maintain stem cell homeostasis. Dysregulation of this essential niche component can lead by itself to sterility or facilitate testicular cancer development.

  19. Regulation of cell-cell fusion by nanotopography

    NASA Astrophysics Data System (ADS)

    Padmanabhan, Jagannath; Augelli, Michael J.; Cheung, Bettina; Kinser, Emily R.; Cleary, Barnett; Kumar, Priyanka; Wang, Renhao; Sawyer, Andrew J.; Li, Rui; Schwarz, Udo D.; Schroers, Jan; Kyriakides, Themis R.

    2016-09-01

    Cell-cell fusion is fundamental to a multitude of biological processes ranging from cell differentiation and embryogenesis to cancer metastasis and biomaterial-tissue interactions. Fusogenic cells are exposed to biochemical and biophysical factors, which could potentially alter cell behavior. While biochemical inducers of fusion such as cytokines and kinases have been identified, little is known about the biophysical regulation of cell-cell fusion. Here, we designed experiments to examine cell-cell fusion using bulk metallic glass (BMG) nanorod arrays with varying biophysical cues, i.e. nanotopography and stiffness. Through independent variation of stiffness and topography, we found that nanotopography constitutes the primary biophysical cue that can override biochemical signals to attenuate fusion. Specifically, nanotopography restricts cytoskeletal remodeling-associated signaling, which leads to reduced fusion. This finding expands our fundamental understanding of the nanoscale biophysical regulation of cell fusion and can be exploited in biomaterials design to induce desirable biomaterial-tissue interactions.

  20. Regulation of cell-cell fusion by nanotopography

    PubMed Central

    Padmanabhan, Jagannath; Augelli, Michael J.; Cheung, Bettina; Kinser, Emily R.; Cleary, Barnett; Kumar, Priyanka; Wang, Renhao; Sawyer, Andrew J.; Li, Rui; Schwarz, Udo D.; Schroers, Jan; Kyriakides, Themis R.

    2016-01-01

    Cell-cell fusion is fundamental to a multitude of biological processes ranging from cell differentiation and embryogenesis to cancer metastasis and biomaterial-tissue interactions. Fusogenic cells are exposed to biochemical and biophysical factors, which could potentially alter cell behavior. While biochemical inducers of fusion such as cytokines and kinases have been identified, little is known about the biophysical regulation of cell-cell fusion. Here, we designed experiments to examine cell-cell fusion using bulk metallic glass (BMG) nanorod arrays with varying biophysical cues, i.e. nanotopography and stiffness. Through independent variation of stiffness and topography, we found that nanotopography constitutes the primary biophysical cue that can override biochemical signals to attenuate fusion. Specifically, nanotopography restricts cytoskeletal remodeling-associated signaling, which leads to reduced fusion. This finding expands our fundamental understanding of the nanoscale biophysical regulation of cell fusion and can be exploited in biomaterials design to induce desirable biomaterial-tissue interactions. PMID:27615159

  1. Ion channels regulating mast cell biology.

    PubMed

    Ashmole, I; Bradding, P

    2013-05-01

    Mast cells play a central role in the pathophysiology of asthma and related allergic conditions. Mast cell activation leads to the degranulation of preformed mediators such as histamine and the secretion of newly synthesised proinflammatory mediators such as leukotrienes and cytokines. Excess release of these mediators contributes to allergic disease states. An influx of extracellular Ca2+ is essential for mast cell mediator release. From the Ca2+ channels that mediate this influx, to the K+ , Cl- and transient receptor potential channels that set the cell membrane potential and regulate Ca2+ influx, ion channels play a critical role in mast cell biology. In this review we provide an overview of our current knowledge of ion channel expression and function in mast cells with an emphasis on how channels interact to regulate Ca2+ signalling.

  2. T cell receptor (TCR)-transgenic CD8 lymphocytes rendered insensitive to transforming growth factor beta (TGFβ) signaling mediate superior tumor regression in an animal model of adoptive cell therapy

    PubMed Central

    2012-01-01

    Tumor antigen-reactive T cells must enter into an immunosuppressive tumor microenvironment, continue to produce cytokine and deliver apoptotic death signals to affect tumor regression. Many tumors produce transforming growth factor beta (TGFβ), which inhibits T cell activation, proliferation and cytotoxicity. In a murine model of adoptive cell therapy, we demonstrate that transgenic Pmel-1 CD8 T cells, rendered insensitive to TGFβ by transduction with a TGFβ dominant negative receptor II (DN), were more effective in mediating regression of established B16 melanoma. Smaller numbers of DN Pmel-1 T cells effectively mediated tumor regression and retained the ability to produce interferon-γ in the tumor microenvironment. These results support efforts to incorporate this DN receptor in clinical trials of adoptive cell therapy for cancer. PMID:22713761

  3. Programmed Cell Death of Dendritic Cells in Immune Regulation

    PubMed Central

    Chen, Min; Wang, Jin

    2010-01-01

    Summary Programmed cell death is essential for the maintenance of lymphocyte homeostasis and immune tolerance. Dendritic cells (DCs), the most efficient antigen presenting cells, represent a small cell population in the immune system. However, DCs play major roles in the regulation of both innate and adaptive immune responses. Programmed cell death in DCs is essential for regulating DC homeostasis and consequently, the scope of immune responses. Interestingly, different DC subsets show varied turnover rates in vivo. The conventional DCs are relatively short-lived in most lymphoid organs, while plasmacytoid DCs are long-lived cells. Mitochondrion-dependent programmed cell death plays an important role in regulating spontaneous DC turnover. Antigen-specific T cells are also capable of killing DCs, thereby providing a mechanism for negative feedback regulation of immune responses. It has been shown that a surplus of DCs due to defects in programmed cell death leads to overactivation of lymphocytes and the onset of autoimmunity. Studying programmed cell death in DCs will shed light on the roles for DC turnover in the regulation of the duration and magnitude of immune responses in vivo, and in the maintenance of immune tolerance. PMID:20636805

  4. Autoreactive Human TCR Initiate Insulitis and Impaired Glucose Tolerance in HLA DR4 Transgenic Mice

    PubMed Central

    Gebe, John A.; Unrath, Kellee A.; Yue, Betty B.; Miyake, Tom; Falk, Ben A.; Nepom, Gerald T.

    2008-01-01

    A human TcR derived from an autoreactive T cell specific for GAD65, from a subject at high risk for autoimmune diabetes, was introduced into HLA-DR4 transgenic mice. The source of TCR was a CD4+ TH1+T cell clone which responded to an immunodominant epitope of the human islet protein GAD65, an epitope shared with both GAD65 and GAD67 in the mouse. The resulting HLA-DR4/GAD-TcR transgenic mice on a Rag2o/o/I-Abo/o/B6 background exhibited a CD4+ infiltrate into pancreatic islets that correlated with a loss of insulin in infiltrated islets. These mice also exhibited a subclinical impaired tolerance to exogenously fed glucose as assayed by an intraperitoneal glucose tolerance test. T cells containing the GAD65/67 (555–567) responsive TcR undergo strong negative selection as evidenced by a 10-fold lower thymocyte cellularity compared to non-TcR transgenic mice, and clonotype peripheral T cells represented approximately 1 percent of CD4+ T cells in Rag2 sufficient mice. Upon in vitro stimulation, GAD65/67 555–567 responsive T cells secrete IFN-γ, minimal IL2 and TNFα and no IL4, IL5, IL10, or IL17, consistent with a TH1 profile. These data demonstrate that CD4+ T cells specific for a naturally processed epitope within GAD can specifically home to pancreatic islets and lead to impaired islet beta cell function in diabetes-associated HLA-DR4 transgenic mice on the relatively non-autoimmune C57BL/6 background. The relatively slow progression and patchy insulitis are reminiscent of the chronic pre-clinical phase similar to a majority of human at-risk subjects, and models these indolent features of human T1D. PMID:17949947

  5. Epitope flexibility and dynamic footprint revealed by molecular dynamics of a pMHC-TCR complex.

    PubMed

    Reboul, Cyril F; Meyer, Grischa R; Porebski, Benjamin T; Borg, Natalie A; Buckle, Ashley M

    2012-01-01

    The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent 'snapshots' and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6-9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into 'scanning' mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2β loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography.

  6. Epitope Flexibility and Dynamic Footprint Revealed by Molecular Dynamics of a pMHC-TCR Complex

    PubMed Central

    Porebski, Benjamin T.; Borg, Natalie A.; Buckle, Ashley M.

    2012-01-01

    The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent ‘snapshots’ and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6–9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into ‘scanning’ mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2β loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography. PMID:22412359

  7. Thyroid Hormones as Renal Cell Cancer Regulators

    PubMed Central

    Matak, Damian; Bartnik, Ewa; Szczylik, Cezary; Czarnecka, Anna M.

    2016-01-01

    It is known that thyroid hormone is an important regulator of cancer development and metastasis. What is more, changes across the genome, as well as alternative splicing, may affect the activity of the thyroid hormone receptors. Mechanism of action of the thyroid hormone is different in every cancer; therefore in this review thyroid hormone and its receptor are presented as a regulator of renal cell carcinoma. PMID:27034829

  8. Cell Cycle Regulators and Cell Death in Immunity

    PubMed Central

    Zebell, Sophia G.; Dong, Xinnian

    2015-01-01

    Summary Various cell death mechanisms are integral to host defense in both plants and mammals. Plant defense against biotrophic pathogens is associated with programmed cell death (PCD) of the infected cell. This effector-triggered PCD is partly analogous to pyroptosis, an inflammatory host cell death process that plays a crucial role in defense against microbial infections in mammals. Plant effector-triggered PCD also shares with mammalian apoptosis the involvement of cell cycle regulators as signaling components. Here we explore the similarities between these different cell death programs as they relate to host defense and their relationship to the cell-cycle. PMID:26468745

  9. Tip cells: master regulators of tubulogenesis?

    PubMed

    Weavers, Helen; Skaer, Helen

    2014-07-01

    The normal development of an organ depends on the coordinated regulation of multiple cell activities. Focusing on tubulogenesis, we review the role of specialised cells or groups of cells that are selected from within tissue primordia and differentiate at the outgrowing tips or leading edge of developing tubules. Tip or leading cells develop distinctive patterns of gene expression that enable them to act both as sensors and transmitters of intercellular signalling. This enables them to explore the environment, respond to both tissue intrinsic signals and extrinsic cues from surrounding tissues and to regulate the behaviour of their neighbours, including the setting of cell fate, patterning cell division, inducing polarity and promoting cell movement and cell rearrangements by neighbour exchange. Tip cells are also able to transmit mechanical tension to promote tissue remodelling and, by interacting with the extracellular matrix, they can dictate migratory pathways and organ shape. Where separate tubular structures fuse to form networks, as in the airways of insects or the vascular system of vertebrates, specialised fusion tip cells act to interconnect disparate elements of the developing network. Finally, we consider their importance in the maturation of mature physiological function and in the development of disease.

  10. Caspase-8 regulation by direct interaction with TRAF6 in T cell receptor-induced NF-kappaB activation.

    PubMed

    Bidère, Nicolas; Snow, Andrew L; Sakai, Keiko; Zheng, Lixin; Lenardo, Michael J

    2006-08-22

    Triggering of lymphocyte antigen receptors is the critical first step in the adaptive immune response against pathogens. T cell receptor (TCR) ligation assembles a large membrane signalosome, culminating in NF-kappaB activation [1,2]. Recently, caspase-8 was found to play a surprisingly prominent role in lymphocyte activation in addition to its well-known role in apoptosis [3]. Caspase-8 is activated after TCR stimulation and nucleates a complex with B cell lymphoma 10 (BCL10), paracaspase MALT1, and the inhibitors of kappaB kinase (IKK) complex [4]. We now report that the ubiquitin ligase TRAF6 binds to active caspase-8 upon TCR stimulation and facilitates its movement into lipid rafts. We identified in silico two putative TRAF6 binding motifs in the caspase-8 sequence and found that mutation of critical residues within these sites abolished TRAF6 binding and diminished TCR-induced NF-kappaB activation. Moreover, RNAi-mediated silencing of TRAF6 abrogated caspase-8 recruitment to the lipid rafts. Protein kinase Ctheta (PKCtheta), CARMA1, and BCL10 are also required for TCR-induced caspase-8 relocation, but only PKCtheta and BCL10 control caspase-8 activation. Our results suggest that PKCtheta independently controls CARMA1 phosphorylation and BCL10-dependent caspase-8 activation and unveil an essential role for TRAF6 as a critical adaptor linking these two convergent signaling events.

  11. Epigenetic Mechanisms Regulating Mesenchymal Stem Cell Differentiation

    PubMed Central

    Pérez-Campo, Flor M.; Riancho, José A.

    2015-01-01

    Human Mesenchymal Stem Cells (hMSCs) have emerged in the last few years as one of the most promising therapeutic cell sources and, in particular, as an important tool for regenerative medicine of skeletal tissues. Although they present a more restricted potency than Embryonic Stem (ES) cells, the use of hMCS in regenerative medicine avoids many of the drawbacks characteristic of ES cells or induced pluripotent stem cells. The challenge in using these cells lies into developing precise protocols for directing cellular differentiation to generate a specific cell lineage. In order to achieve this goal, it is of the upmost importance to be able to control de process of fate decision and lineage commitment. This process requires the coordinate regulation of different molecular layers at transcriptional, posttranscriptional and translational levels. At the transcriptional level, switching on and off different sets of genes is achieved not only through transcriptional regulators, but also through their interplay with epigenetic modifiers. It is now well known that epigenetic changes take place in an orderly way through development and are critical in the determination of lineage-specific differentiation. More importantly, alteration of these epigenetic changes would, in many cases, lead to disease generation and even tumour formation. Therefore, it is crucial to elucidate how epigenetic factors, through their interplay with transcriptional regulators, control lineage commitment in hMSCs. PMID:27019612

  12. Operationally tolerant and minimally immunosuppressed kidney recipients display strongly altered blood T-cell clonal regulation.

    PubMed

    Brouard, Sophie; Dupont, Alexandre; Giral, Magali; Louis, Stéphanie; Lair, David; Braudeau, Cécile; Degauque, Nicolas; Moizant, Frédérique; Pallier, Annaick; Ruiz, Catherine; Guillet, Marina; Laplaud, David; Soulillou, Jean-Paul

    2005-02-01

    Most kidney transplant recipients who discontinue immunosuppression reject their graft. Nevertheless, a small number do not, suggesting that allogeneic tolerance state (referred to operational tolerance) is achievable in humans. So far, however, the rarity of such patients has limited their study. Because operational tolerance could be linked to anergy, ignorance or to an active regulatory mechanism, we analyzed the blood T-cell repertoire usage of these patients. We report on comparison of T-cell selection in drug-free operationally tolerant kidney recipients (or with minimal immunosuppression), recipients with stable graft function, chronic rejection and healthy individuals. The blood T cells of operationally tolerant patients display two major characteristics: an unexpected strongly altered T-cell receptor (TCR) Vbeta usage and high TCR transcript accumulation in selected T cells. The cytokine transcriptional patterns of sorted T cells with altered TCR usage show no accumulation of cytokine transcripts (IL10, IL2, IL13, IFN-gamma), suggesting a state of hyporesponsiveness in these patients. Identification of such a potential surrogate pattern of operational tolerance in transplant recipients under life-long immunosuppression may provide a new basis and rationale for exploration of tolerance state. However, these data obtained in a limited number of patients require further confirmation on larger series.

  13. DNA asymmetry and cell fate regulation in stem cells.

    PubMed

    Yennek, Siham; Tajbakhsh, Shahragim

    2013-01-01

    The semi-conservative nature of DNA replication has suggested that identical DNA molecules within chromatids are inherited by daughter cells after cell division. Numerous reports of non-random DNA segregation in prokaryotes and eukaryotes suggest that this is not always the case, and that epigenetic marks on chromatids, if not the individual DNA strands themselves, could have distinct signatures. Their selective distribution to daughter cells provides a novel mechanism for gene and cell fate regulation by segregating chromatids asymmetrically. Here we highlight some examples and potential mechanisms that can regulate this process. We propose that cellular asymmetry is inherently present during each cell division, and that it provides an opportunity during each cell cycle for moderating cell fates.

  14. Epigenetic Regulation of the Mammalian Cell

    PubMed Central

    Baverstock, Keith; Rönkkö, Mauno

    2008-01-01

    Background Understanding how mammalian cells are regulated epigenetically to express phenotype is a priority. The cellular phenotypic transition, induced by ionising radiation, from a normal cell to the genomic instability phenotype, where the ability to replicate the genotype accurately is compromised, illustrates important features of epigenetic regulation. Based on this phenomenon and earlier work we propose a model to describe the mammalian cell as a self assembled open system operating in an environment that includes its genotype, neighbouring cells and beyond. Phenotype is represented by high dimensional attractors, evolutionarily conditioned for stability and robustness and contingent on rules of engagement between gene products encoded in the genetic network. Methodology/Findings We describe how this system functions and note the indeterminacy and fluidity of its internal workings which place it in the logical reasoning framework of predicative logic. We find that the hypothesis is supported by evidence from cell and molecular biology. Conclusions Epigenetic regulation and memory are fundamentally physical, as opposed to chemical, processes and the transition to genomic instability is an important feature of mammalian cells with probable fundamental relevance to speciation and carcinogenesis. A source of evolutionarily selectable variation, in terms of the rules of engagement between gene products, is seen as more likely to have greater prominence than genetic variation in an evolutionary context. As this epigenetic variation is based on attractor states phenotypic changes are not gradual; a phenotypic transition can involve the changed contribution of several gene products in a single step. PMID:18523589

  15. Regulation of cell division in higher plants

    SciTech Connect

    Jacobs, T.W.

    1992-01-01

    Cell division is arguably the most fundamental of all developmental processes. In higher plants, mitotic activity is largely confined to foci of patterned cell divisions called meristems. From these perpetually embryonic tissues arise the plant's essential organs of light capture, support, protection and reproduction. Once an adequate understanding of plant cell mitotic regulation is attained, unprecedented opportunities will ensue for analyzing and genetically controlling diverse aspects of development, including plant architecture, leaf shape, plant height, and root depth. The mitotic cycle in a variety of model eukaryotic systems in under the control of a regulatory network of striking evolutionary conservation. Homologues of the yeast cdc2 gene, its catalytic product, p34, and the cyclin regulatory subunits of the MPF complex have emerged as ubiquitous mitotic regulators. We have cloned cdc2-like and cyclin genes from pea. As in other eukaryotic model systems, p34 of Pisum sativum is a subunit of a high molecular weight complex which binds the fission yeast p13 protein and displays histone H1 kinase activity in vitro. Our primary objective in this study is to gain baseline information about the regulation of this higher plant cell division control complex in non-dividing, differentiated cells as well as in synchronous and asynchronous mitotic cells. We are investigating cdc2 and cyclin expression at the levels of protein abundance, protein phosphorylation and quaternary associations.

  16. Stem cell technologies: regulation, patents and problems.

    PubMed

    Then, Shih-Ning

    2004-11-01

    Human embryonic stem cell research promises to deliver in the future a whole range of therapeutic treatments, but currently governments in different jurisdictions must try to regulate this burgeoning area. Part of the problem has been, and continues to be, polarised community opinion on the use of human embryonic stem cells for research. This article compares the approaches of the Australian, United Kingdom and United States governments in regulating human embryonic stem cell research. To date, these governments have approached the issue through implementing legislation or policy to control research. Similarly, the three jurisdictions have viewed the patentability of human embryonic stem cell technologies in their own ways with different policies being adopted by the three patent offices. This article examines these different approaches and discusses the inevitable concerns that have been raised due to the lack of a universal approach in relation to the regulation of research; the patenting of stem cell technologies; and the effects patents granted are having on further human embryonic stem cell research.

  17. Cell Polarity As A Regulator of Cancer Cell Behavior Plasticity

    PubMed Central

    Muthuswamy, Senthil K; Xue, Bin

    2013-01-01

    Cell polarization is an evolutionarily conserved process that facilitates asymmetric distribution of organelles and proteins, is an evolutionarily conserved property that is modified dynamically during physiological processes such as cell division, migration, and morphogenesis. The plasticity with which cells change their behavior and phenotype in response to cell intrinsic and extrinsic cues is an essential feature of normal physiology. In disease states such as cancer, cells lose their ability to behave normally in response to physiological cues. A molecular understanding of mechanisms that alter the behavior of cancer cells is limited. Cell polarity proteins are an recognized class of molecules that can receive and interpret both intrinsic and extrinsic signals to modulate cell behavior. In this review, we discuss how cell polarity proteins regulate a diverse array of biological processes and how they can contribute to alterations in the behavior of cancer cells. PMID:22881459

  18. Redox regulation in cancer stem cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reactive oxygen species (ROS) and ROS-dependent (redox regulation) signaling pathways and transcriptional activities are thought to be critical in stem cell self-renewal and differentiation during growth and organogenesis. Aberrant ROS burst and dysregulation of those ROS-dependent cellular processe...

  19. Cell cycle regulation of hematopoietic stem or progenitor cells.

    PubMed

    Hao, Sha; Chen, Chen; Cheng, Tao

    2016-05-01

    The highly regulated process of blood production is achieved through the hierarchical organization of hematopoietic stem cell (HSC) subsets and their progenies, which differ in self-renewal and differentiation potential. Genetic studies in mice have demonstrated that cell cycle is tightly controlled by the complex interplay between extrinsic cues and intrinsic regulatory pathways involved in HSC self-renewal and differentiation. Deregulation of these cellular programs may transform HSCs or hematopoietic progenitor cells (HPCs) into disease-initiating stem cells, and can result in hematopoietic malignancies such as leukemia. While previous studies have shown roles for some cell cycle regulators and related signaling pathways in HSCs and HPCs, a more complete picture regarding the molecular mechanisms underlying cell cycle regulation in HSCs or HPCs is lacking. Based on accumulated studies in this field, the present review introduces the basic components of the cell cycle machinery and discusses their major cellular networks that regulate the dormancy and cell cycle progression of HSCs. Knowledge on this topic would help researchers and clinicians to better understand the pathogenesis of relevant blood disorders and to develop new strategies for therapeutic manipulation of HSCs.

  20. Topological regulation of lipid balance in cells.

    PubMed

    Drin, Guillaume

    2014-01-01

    Lipids are unevenly distributed within and between cell membranes, thus defining organelle identity. Such distribution relies on local metabolic branches and mechanisms that move lipids. These processes are regulated by feedback mechanisms that decipher topographical information in organelle membranes and then regulate lipid levels or flows. In the endoplasmic reticulum, the major lipid source, transcriptional regulators and enzymes sense changes in membrane features to modulate lipid production. At the Golgi apparatus, lipid-synthesizing, lipid-flippase, and lipid-transport proteins (LTPs) collaborate to control lipid balance and distribution within the membrane to guarantee remodeling processes crucial for vesicular trafficking. Open questions exist regarding LTPs, which are thought to be lipid sensors that regulate lipid synthesis or carriers that transfer lipids between organelles across long distances or in contact sites. A novel model is that LTPs, by exchanging two different lipids, exploit one lipid gradient between two distinct membranes to build a second lipid gradient.

  1. Regulation of ceramide synthase 6 in a spontaneous experimental autoimmune encephalomyelitis model is sex dependent.

    PubMed

    Eberle, Max; Ebel, Philipp; Wegner, Marthe-Susanna; Männich, Julia; Tafferner, Nadja; Ferreiros, Nerea; Birod, Kerstin; Schreiber, Yannick; Krishnamoorthy, Gurumoorthy; Willecke, Klaus; Geisslinger, Gerd; Grösch, Sabine; Schiffmann, Susanne

    2014-11-15

    Ceramides (Cer) are mediators of inflammatory processes. In a chronic experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), we observed a significant elevation of C16-Cer and its synthesizing enzyme, ceramide synthase(CerS)6, in the lumbar spinal cord. In the present study, we have confirmed that C16-Cer and CerS6 are also upregulated in the lumbar spinal cord in a spontaneous relapse-remitting EAE model, using SJL mice overexpressing a transgenic T cell receptor (TCR1640). CerS6 was found to be expressed in macrophages, T cells and B cells in EAE lesions. In macrophages, we demonstrated that interferon gamma (IFN-γ)-induced CerS6 upregulation was amplified by 17ß-estradiol, an action that was further accompanied by increased upregulation of tumor necrosis factor alpha (TNF-α). Accordingly, CerS6 and TNF-α expression was upregulated predominantly in the spinal cord in female TCR1640 mice, which usually develop the relapse-remitting form of EAE, while male TCR1640 mice showed an attenuated regulation of CerS6 and TNF-α and exhibit mostly chronic disease progression. Furthermore, expression of TNFR2, one of two receptors of TNF-α, which is linked to neuroprotection and remyelination, was also upregulated to a greater extent during EAE in female TCR1640 mice in comparison to male TCR1640 mice. Taken together, our results confirm the upregulation of CerS6 and C16-Cer in an adjuvant-independent, physiological EAE model and further suggest an anti-inflammatory role of CerS6 in the regulation of the disease course in female TCR1640 mice via TNF-α/TNFR2.

  2. Triiodothyronine regulates cell growth and survival in renal cell cancer.

    PubMed

    Czarnecka, Anna M; Matak, Damian; Szymanski, Lukasz; Czarnecka, Karolina H; Lewicki, Slawomir; Zdanowski, Robert; Brzezianska-Lasota, Ewa; Szczylik, Cezary

    2016-10-01

    Triiodothyronine plays an important role in the regulation of kidney cell growth, differentiation and metabolism. Patients with renal cell cancer who develop hypothyreosis during tyrosine kinase inhibitor (TKI) treatment have statistically longer survival. In this study, we developed cell based model of triiodothyronine (T3) analysis in RCC and we show the different effects of T3 on renal cell cancer (RCC) cell growth response and expression of the thyroid hormone receptor in human renal cell cancer cell lines from primary and metastatic tumors along with human kidney cancer stem cells. Wild-type thyroid hormone receptor is ubiquitously expressed in human renal cancer cell lines, but normalized against healthy renal proximal tube cell expression its level is upregulated in Caki-2, RCC6, SKRC-42, SKRC-45 cell lines. On the contrary the mRNA level in the 769-P, ACHN, HKCSC, and HEK293 cells is significantly decreased. The TRβ protein was abundant in the cytoplasm of the 786-O, Caki-2, RCC6, and SKRC-45 cells and in the nucleus of SKRC-42, ACHN, 769-P and cancer stem cells. T3 has promoting effect on the cell proliferation of HKCSC, Caki-2, ASE, ACHN, SK-RC-42, SMKT-R2, Caki-1, 786-0, and SK-RC-45 cells. Tyrosine kinase inhibitor, sunitinib, directly inhibits proliferation of RCC cells, while thyroid hormone receptor antagonist 1-850 (CAS 251310‑57-3) has less significant inhibitory impact. T3 stimulation does not abrogate inhibitory effect of sunitinib. Renal cancer tumor cells hypostimulated with T3 may be more responsive to tyrosine kinase inhibition. Moreover, some tumors may be considered as T3-independent and present aggressive phenotype with thyroid hormone receptor activated independently from the ligand. On the contrary proliferation induced by deregulated VHL and or c-Met pathways may transgress normal T3 mediated regulation of the cell cycle.

  3. Characteristics of TCR/CD3 complex CD3{varepsilon} chains of regulatory CD4+ T (Treg) lymphocytes: role in Treg differentiation in vitro and impact on Treg in vivo.

    PubMed

    Rojo, Jose M; Ojeda, Gloria; Acosta, Yenny Y; Montes-Casado, Maria; Criado, Gabriel; Portolés, Pilar

    2014-03-01

    Tregs are anergic CD4(+)CD25(+)Foxp3(+) T lymphocytes exerting active suppression to control immune and autoimmune responses. However, the factors in TCR recognition underlying Treg differentiation are unclear. Based on our previous data, we hypothesized that Treg TCR/CD3 antigen receptor complexes might differ from those of CD4(+)CD25(-) Tconv. Expression levels of TCR/CD3, CD3ε,ζ chains, or other molecules involved in antigen signaling and the characteristics of CD3ε chains were analyzed in thymus or spleen Treg cells from normal mice. Tregs had quantitative and qualitatively distinct TCR/CD3 complexes and CD3ε chains. They expressed significantly lower levels of the TCR/CD3 antigen receptor, CD3ε chains, TCR-ζ chain, or the CD4 coreceptor than Tconv. Levels of kinases, adaptor molecules involved in TCR signaling, and early downstream activation pathways were also lower in Tregs than in Tconv. Furthermore, TCR/CD3 complexes in Tregs were enriched in CD3ε chains conserving their N-terminal, negatively charged amino acid residues; this trait is linked to a higher activation threshold. Transfection of mutant CD3ε chains lacking these residues inhibited the differentiation of mature CD4(+)Foxp3(-) T lymphocytes into CD4(+)Foxp3(+) Tregs, and differences in CD3ε chain recognition by antibodies could be used to enrich for Tregs in vivo. Our results show quantitative and qualitative differences in the TCR/CD3 complex, supporting the hyporesponsive phenotype of Tregs concerning TCR/CD3 signals. These differences might reconcile avidity and flexible threshold models of Treg differentiation and be used to implement therapeutic approaches involving Treg manipulation.

  4. "Natural Regulators": NK Cells as Modulators of T Cell Immunity.

    PubMed

    Schuster, Iona S; Coudert, Jerome D; Andoniou, Christopher E; Degli-Esposti, Mariapia A

    2016-01-01

    Natural killer (NK) cells are known as frontline responders capable of rapidly mediating a response upon encountering transformed or infected cells. Recent findings indicate that NK cells, in addition to acting as innate effectors, can also regulate adaptive immune responses. Here, we review recent studies on the immunoregulatory function of NK cells with a specific focus on their ability to affect the generation of early, as well as long-term antiviral T cell responses, and their role in modulating immune pathology and disease. In addition, we summarize the current knowledge of the factors governing regulatory NK cell responses and discuss origin, tissue specificity, and open questions about the classification of regulatory NK cells as classical NK cells versus group 1 innate lymphoid cells.

  5. Mitochondrial Regulation of Cell Cycle and Proliferation

    PubMed Central

    Antico Arciuch, Valeria Gabriela; Elguero, María Eugenia; Poderoso, Juan José

    2012-01-01

    Abstract Eukaryotic mitochondria resulted from symbiotic incorporation of α-proteobacteria into ancient archaea species. During evolution, mitochondria lost most of the prokaryotic bacterial genes and only conserved a small fraction including those encoding 13 proteins of the respiratory chain. In this process, many functions were transferred to the host cells, but mitochondria gained a central role in the regulation of cell proliferation and apoptosis, and in the modulation of metabolism; accordingly, defective organelles contribute to cell transformation and cancer, diabetes, and neurodegenerative diseases. Most cell and transcriptional effects of mitochondria depend on the modulation of respiratory rate and on the production of hydrogen peroxide released into the cytosol. The mitochondrial oxidative rate has to remain depressed for cell proliferation; even in the presence of O2, energy is preferentially obtained from increased glycolysis (Warburg effect). In response to stress signals, traffic of pro- and antiapoptotic mitochondrial proteins in the intermembrane space (B-cell lymphoma-extra large, Bcl-2-associated death promoter, Bcl-2 associated X-protein and cytochrome c) is modulated by the redox condition determined by mitochondrial O2 utilization and mitochondrial nitric oxide metabolism. In this article, we highlight the traffic of the different canonical signaling pathways to mitochondria and the contributions of organelles to redox regulation of kinases. Finally, we analyze the dynamics of the mitochondrial population in cell cycle and apoptosis. Antioxid. Redox Signal. 16, 1150–1180. PMID:21967640

  6. Activated skin γδ T-cells regulate T-cell infiltration of the wound site after burn.

    PubMed

    Rani, Meenakshi; Zhang, Qiong; Scherer, Michael R; Cap, Andrew P; Schwacha, Martin G

    2015-02-01

    Burn induces an immunopathological response involving multiple immune cell types that includes γδ T-cells. Nonetheless, the role of γδ T-cells at the wound site after burn is not clearly defined. Wild type and γδ T-cell receptor deficient (δ TCR(-/-)) mice were subjected to a major burn or sham procedure. At 1-7 d thereafter, skin samples were collected and T-cell populations analyzed. The majority of T-cells in the skin of sham mice were γδ T-cells. After burn, however, an increase in the total T-cells was observed at the wound site and these cells were predominantly αβ T-cells. Their influx was γδ T-cell dependent, as it was markedly reduced in injured δ TCR(-/-) mice. Burn wound γδ T-cells were activated with increased expression of TLRs and CD69. In contrast, the infiltrating αβ T-cells TLR and CD69 expressions were attenuated after burn. Thus, burn is associated with of γδ T-cell activation at the injury site, which initiates a massive infiltration of the wound with αβ T-cells that likely facilitate the transition from the inflammatory to the proliferative phase of healing.

  7. Epigenetic Regulation of Hematopoietic Stem Cells

    PubMed Central

    Sharma, Shilpa; Gurudutta, Gangenahalli

    2016-01-01

    Hematopoietic stem cells are endowed with a distinct potential to bolster self-renewal and to generate progeny that differentiate into mature cells of myeloid and lymphoid lineages. Both hematopoietic stem cells and mature cells have the same genome, but their gene expression is controlled by an additional layer of epigenetics such as DNA methylation and post-translational histone modifications, enabling each cell-type to acquire various forms and functions. Until recently, several studies have largely focussed on the transcription factors andniche factors for the understanding of the molecular mechanisms by which hematopoietic cells replicate and differentiate. Several lines of emerging evidence suggest that epigenetic modifications eventually result in a defined chromatin structure and an “individual” gene expression pattern, which play an essential role in the regulation of hematopoietic stem cell self-renewal and differentiation. Distinct epigenetic marks decide which sets of genes may be expressed and which genes are kept silent. Epigenetic mechanisms are interdependent and ensure lifelong production of blood and bone marrow, thereby contributing to stem cell homeostasis. The epigenetic analysis of hematopoiesis raises the exciting possibility that chromatin structure is dynamic enough for regulated expression of genes. Though controlled chromatin accessibility plays an essential role in maintaining blood homeostasis; mutations in chromatin impacts on the regulation of genes critical to the development of leukemia. In this review, we explored the contribution of epigenetic machinery which has implications for the ramification of molecular details of hematopoietic self-renewal for normal development and underlying events that potentially co-operate to induce leukemia. PMID:27426084

  8. Cell cycle regulation of glucocorticoid receptor function.

    PubMed Central

    Hsu, S C; Qi, M; DeFranco, D B

    1992-01-01

    Glucocorticoid receptor (GR) nuclear translocation, transactivation and phosphorylation were examined during the cell cycle in mouse L cell fibroblasts. Glucocorticoid-dependent transactivation of the mouse mammary tumor virus promoter was observed in G0 and S phase synchronized L cells, but not in G2 synchronized cells. G2 effects were selective on the glucocorticoid hormone signal transduction pathway, since glucocorticoid but not heavy metal induction of the endogenous Metallothionein-1 gene was also impaired in G2 synchronized cells. GRs that translocate to the nucleus of G2 synchronized cells in response to dexamethasone treatment were not efficiently retained there and redistributed to the cytoplasmic compartment. In contrast, GRs bound by the glucocorticoid antagonist RU486 were efficiently retained within nuclei of G2 synchronized cells. Inefficient nuclear retention was observed for both dexamethasone- and RU486-bound GRs in L cells that actively progress through G2 following release from an S phase arrest. Finally, site-specific alterations in GR phosphorylation were observed in G2 synchronized cells suggesting that cell cycle regulation of specific protein kinases and phosphatases could influence nuclear retention, recycling and transactivation activity of the GR. Images PMID:1505524

  9. Extrinsic regulation of satellite cell specification.

    PubMed

    Bentzinger, C Florian; von Maltzahn, Julia; Rudnicki, Michael A

    2010-08-26

    Cellular commitment during vertebrate embryogenesis is controlled by an interplay of intrinsic regulators and morphogenetic signals. These mechanisms recruit a subset of cells in the developing organism to become the ancestors of skeletal muscle. Signals that control progression through the myogenic lineage converge on a battery of hierarchically organized transcription factors which modulate the cells to either remain in a primitive state or allow their commitment and differentiation into skeletal muscle fibers. A small population of cells will retain a largely unspecified state throughout development. Such stem cells, in conjunction with more committed myogenic progenitors, form a heterogeneous population that colonizes adult skeletal muscle as satellite cells. The satellite cell pool is responsible for the remarkable regenerative capacity of skeletal muscle. Similar to their counterparts during embryonic development, satellite cells are capable of self-renewal and can give rise to myogenic progeny. Impaired satellite cell homeostasis has been associated with numerous muscular disorders. Due to intense research efforts in the past two decades, the complex biology of muscle stem cells has now revealed some of its secrets and new avenues for the development of therapeutic molecules have emerged. In the present review we focus on the extrinsic mechanisms that control self-renewal, specification and differentiation of satellite cells and their significance for the development of biologic drugs.

  10. The endocytosis and signaling of the γδ T cell coreceptor WC1 are regulated by a dileucine motif.

    PubMed

    Hsu, Haoting; Baldwin, Cynthia L; Telfer, Janice C

    2015-03-01

    WC1 proteins, which are specifically expressed by bovine γδ T cells from a gene array containing 13 members, are part of the scavenger receptor cysteine-rich family. WC1 cytoplasmic domains contains multiple tyrosines, one of which is required to be phosphorylated for TCR coreceptor activity, and a dileucine endocytosis motif. Like the TCR coreceptor CD4, WC1 is endocytosed in response to PMA. Because WC1 endocytosis may play a role in the activation of γδ T cells, we examined WC1 endocytosis in the adherent cell 293T and Jurkat T cell lines using a fusion protein of extracellular CD4 and the transmembrane and cytoplasmic domain of WC1. Individual mutation of the two leucine residues of the endocytic dileucine motif in the WC1 cytoplasmic domain significantly reduced PMA-induced endocytosis in both cell types and enhanced IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1 in Jurkat cells, suggesting that the sustained membrane coligation of CD3/TCR with WC1 caused by a decrease in endocytosis increases T cell activation. Mutation of two serines upstream of the endocytic dileucine motif affected endocytosis only in adherent 293T cells. Although the two upstream serines were not required for WC1 endocytosis in Jurkat cells, the pan-protein kinase C inhibitor Gö6983 blocked endocytosis of CD4/WC1, and mutation of the upstream serines in WC1 inhibited IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1. These studies provide insights into the signaling of WC1 gene arrays that are present in most mammals and play critical roles in γδ T cell responses to bacterial pathogens.

  11. T cell receptor signals to NF-κB are transmitted by a cytosolic p62-Bcl10-Malt1-IKK signalosome.

    PubMed

    Paul, Suman; Traver, Maria K; Kashyap, Anuj K; Washington, Michael A; Latoche, Joseph R; Schaefer, Brian C

    2014-05-13

    Antigen-mediated stimulation of the T cell receptor (TCR) triggers activation of nuclear factor κB (NF-κB), a key transcriptional regulator of T cell proliferation and effector cell differentiation. TCR signaling to NF-κB requires both the Carma1-Bcl10-Malt1 (CBM) complex and the inhibitor of κB (IκB) kinase (IKK) complex; however, the molecular mechanisms connecting the CBM complex to activation of IKK are incompletely defined. We found that the active IKK complex is a component of a TCR-dependent cytosolic Bcl10-Malt1 signalosome containing the adaptor protein p62, which forms in effector T cells. Phosphorylated IκBα and NF-κB were transiently recruited to this signalosome before NF-κB translocated to the nucleus. Inhibiting the activity of the kinase TAK1 or IKK blocked the phosphorylation of IKK, but not the formation of p62-Bcl10-Malt1 clusters, suggesting that activation of IKK occurs after signalosome assembly. Furthermore, analysis of T cells from p62-deficient mice demonstrated that the p62-dependent clustering of signaling components stimulated activation of NF-κB in effector T cells. Thus, TCR-stimulated activation of NF-κB requires the assembly of cytosolic p62-Bcl10-Malt1-IKK signalosomes, which may ensure highly regulated activation of NF-κB in response to TCR engagement.

  12. Inhibition of T cell receptor signaling by cholesterol sulfate, a naturally occurring derivative of membrane cholesterol

    PubMed Central

    Wang, Feng; Beck-García, Katharina; Zorzin, Carina; Schamel, Wolfgang W. A.; Davis, Mark M.

    2016-01-01

    Most adaptive immune responses require the activation of specific T cells through the T cell antigen receptor–CD3 complex (TCR). Here we show that cholesterol sulfate (CS), a naturally occurring analog of cholesterol, inhibits CD3 ITAM phosphorylation, a crucial first step in T cell activation. Biochemical studies show that CS disrupted TCR multimers, apparently by displacing cholesterol, known to bind TCRβ. Moreover, CS-deficient mice displayed a heightened sensitivity to a self-antigen, whereas increasing CS content by intrathymic injection inhibited thymic selection, indicating that this molecule is an intrinsic regulator of thymocyte development. These results reveal a regulatory role for CS in TCR signaling and thymic selection, highlighting the importance of the membrane microenvironment in modulating cell surface receptor activation. PMID:27213689

  13. Total Coliform Rule (TCR) Federal Register Notice

    EPA Pesticide Factsheets

    This document provides the FR notice to 40 CFR Parts 141 and 142 Drinking Water: National Primary Drinking Water Regulations; Total Coliforms (Including Fecal Coliforms and E. Coli); Final Rule (26 pp, 5 M).

  14. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-01-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

  15. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    PubMed

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-04-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T