T Cell Receptor Engineering and Analysis Using the Yeast Display Platform

PubMed Central

Smith, Sheena N.; Harris, Daniel T.; Kranz, David M.

2017-01-01

The αβ heterodimeric T cell receptor (TCR) recognizes peptide antigens that are transported to the cell surface as a complex with a protein encoded by the major histocompatibility complex (MHC). T cells thus evolved a strategy to sense these intracellular antigens, and to respond either by eliminating the antigen-presenting cell (e.g. a virus-infected cell) or by secreting factors that recruit the immune system to the site of the antigen. The central role of the TCR in the binding of antigens as peptide-MHC (pepMHC) ligands has now been studied thoroughly. Interestingly, despite their exquisite sensitivity (e.g. T cell activation by as few as 1 to 3 pepMHC complexes on a single target cell), TCRs are known to have relatively low affinities for pepMHC, with KD values in the micromolar range. There has been interest in engineering the affinity of TCRs in order to use this class of molecules in ways similar to now done with antibodies. By doing so, it would be possible to harness the potential of TCRs as therapeutics against a much wider array of antigens that include essentially all intracellular targets. To engineer TCRs, and to analyze their binding features more rapidly, we have used a yeast display system as a platform. Expression and engineering of a single-chain form of the TCR, analogous to scFv fragments from antibodies, allow the TCR to be affinity matured with a variety of possible pepMHC ligands. In addition, the yeast display platform allows one to rapidly generate TCR variants with diverse binding affinities and to analyze specificity and affinity without the need for purification of soluble forms of the TCRs. The present chapter describes the methods for engineering and analyzing single-chain TCRs using yeast display. PMID:26060072

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 Central

Witte, Amelie; Meineke, Bernhard; Sticht, Jana; Philipsen, Lars; Kuropka, Benno; Müller, Andreas J.; Freund, Christian

2017-01-01

ABSTRACT 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. PMID:28052935

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

PubMed Central

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

2016-01-01

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

Distinct 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.

WAVE2 Regulates High-Affinity Integrin Binding by Recruiting Vinculin and Talin to the Immunological Synapse▿

PubMed Central

Nolz, Jeffrey C.; Medeiros, Ricardo B.; Mitchell, Jason S.; Zhu, Peimin; Freedman, Bruce D.; Shimizu, Yoji; Billadeau, Daniel D.

2007-01-01

T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin. PMID:17591693

WAVE2 regulates high-affinity integrin binding by recruiting vinculin and talin to the immunological synapse.

PubMed

Nolz, Jeffrey C; Medeiros, Ricardo B; Mitchell, Jason S; Zhu, Peimin; Freedman, Bruce D; Shimizu, Yoji; Billadeau, Daniel D

2007-09-01

T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin.

T Cell Receptor-Major Histocompatibility Complex Interaction Strength Defines Trafficking and CD103+ Memory Status of CD8 T Cells in the Brain.

PubMed

Sanecka, Anna; Yoshida, Nagisa; Kolawole, Elizabeth Motunrayo; Patel, Harshil; Evavold, Brian D; Frickel, Eva-Maria

2018-01-01

T cell receptor-major histocompatibility complex (TCR-MHC) affinities span a wide range in a polyclonal T cell response, yet it is undefined how affinity shapes long-term properties of CD8 T cells during chronic infection with persistent antigen. Here, we investigate how the affinity of the TCR-MHC interaction shapes the phenotype of memory CD8 T cells in the chronically Toxoplasma gondii- infected brain. We employed CD8 T cells from three lines of transnuclear (TN) mice that harbor in their endogenous loci different T cell receptors specific for the same Toxoplasma antigenic epitope ROP7. The three TN CD8 T cell clones span a wide range of affinities to MHCI-ROP7. These three CD8 T cell clones have a distinct and fixed hierarchy in terms of effector function in response to the antigen measured as proliferation capacity, trafficking, T cell maintenance, and memory formation. In particular, the T cell clone of lowest affinity does not home to the brain. The two higher affinity T cell clones show differences in establishing resident-like memory populations (CD103 + ) in the brain with the higher affinity clone persisting longer in the host during chronic infection. Transcriptional profiling of naïve and activated ROP7-specific CD8 T cells revealed that Klf2 encoding a transcription factor that is known to be a negative marker for T cell trafficking is upregulated in the activated lowest affinity ROP7 clone. Our data thus suggest that TCR-MHC affinity dictates memory CD8 T cell fate at the site of infection.

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

PubMed

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

2015-01-15

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

CD3-T cell receptor modulation is selectively induced in CD8 but not CD4 lymphocytes cultured in agar.

PubMed Central

Oudrhiri, N; Farcet, J P; Gourdin, M F; M'Bemba, E; Gaulard, P; Katz, A; Divine, M; Galazka, A; Reyes, F

1990-01-01

The CD3-T cell receptor (TcR) complex is central to the immune response. Upon binding by specific ligands, internalized CD3-TcR molecules increase, and either T cell response or unresponsiveness may ensue depending on the triggering conditions. Using semi-solid agar culture, we have shown previously that quiescent CD4 but not CD8 lymphocytes generate clonal colonies under phytohaemagglutinin stimulation. Here we have demonstrated that the agar induces selective CD3-TcR modulation in the CD8 and not in the CD4 subset. CD8 lymphocytes preactivated in liquid culture and recultured in agar with exogenous recombinant interleukin-2 generate colonies with a modulated CD3-TcR surface expression. The peptides composing the CD3-TcR complex are synthesized in CD8 colonies as well as in CD4; however, the CD3 gamma chain is phosphorylated at a higher level in CD8 colonies. A component of the agar polymer, absent in agarose, appears to be the ligand that induces differential CD3-TcR modulation in the CD8 subset. In contrast to agar culture, CD8 colonies can be derived from quiescent CD8 lymphocytes in agarose. These CD8 colonies express unmodulated CD-TcR. CD3-TcR modulation with anti-CD3 monoclonal antibody prior to culturing in agarose inhibits the colony formation. We conclude that given triggering conditions can result in both CD3-TcR modulation and inhibition of the proliferative response selectively in the CD8 lymphocyte subset and not in the CD4. Images Fig. 3 Fig. 4 Fig. 5 PMID:2146997

Atomic structure of an alphabeta T cell receptor (TCR) heterodimer in complex with an anti-TCR fab fragment derived from a mitogenic antibody.

PubMed Central

Wang, J; Lim, K; Smolyar, A; Teng, M; Liu, J; Tse, A G; Liu, J; Hussey, R E; Chishti, Y; Thomson, C T; Sweet, R M; Nathenson, S G; Chang, H C; Sacchettini, J C; Reinherz, E L

1998-01-01

Each T cell receptor (TCR) recognizes a peptide antigen bound to a major histocompatibility complex (MHC) molecule via a clonotypic alphabeta heterodimeric structure (Ti) non-covalently associated with the monomorphic CD3 signaling components. A crystal structure of an alphabeta TCR-anti-TCR Fab complex shows an Fab fragment derived from the H57 monoclonal antibody (mAb), interacting with the elongated FG loop of the Cbeta domain, situated beneath the Vbeta domain. This loop, along with the partially exposed ABED beta sheet of Cbeta, and glycans attached to both Cbeta and Calpha domains, forms a cavity of sufficient size to accommodate a single non-glycosylated Ig domain such as the CD3epsilon ectodomain. That this asymmetrically localized site is embedded within the rigid constant domain module has implications for the mechanism of signal transduction in both TCR and pre-TCR complexes. Furthermore, quaternary structures of TCRs vary significantly even when they bind the same MHC molecule, as manifested by a unique twisting of the V module relative to the C module. PMID:9427737

A mechanism for expansion of regulatory T-cell repertoire and its role in self-tolerance.

PubMed

Feng, Yongqiang; van der Veeken, Joris; Shugay, Mikhail; Putintseva, Ekaterina V; Osmanbeyoglu, Hatice U; Dikiy, Stanislav; Hoyos, Beatrice E; Moltedo, Bruno; Hemmers, Saskia; Treuting, Piper; Leslie, Christina S; Chudakov, Dmitriy M; Rudensky, Alexander Y

2015-12-03

T-cell receptor (TCR) signalling has a key role in determining T-cell fate. Precursor cells expressing TCRs within a certain low-affinity range for complexes of self-peptide and major histocompatibility complex (MHC) undergo positive selection and differentiate into naive T cells expressing a highly diverse self-MHC-restricted TCR repertoire. In contrast, precursors displaying TCRs with a high affinity for 'self' are either eliminated through TCR-agonist-induced apoptosis (negative selection) or restrained by regulatory T (Treg) cells, whose differentiation and function are controlled by the X-chromosome-encoded transcription factor Foxp3 (reviewed in ref. 2). Foxp3 is expressed in a fraction of self-reactive T cells that escape negative selection in response to agonist-driven TCR signals combined with interleukin 2 (IL-2) receptor signalling. In addition to Treg cells, TCR-agonist-driven selection results in the generation of several other specialized T-cell lineages such as natural killer T cells and innate mucosal-associated invariant T cells. Although the latter exhibit a restricted TCR repertoire, Treg cells display a highly diverse collection of TCRs. Here we explore in mice whether a specialized mechanism enables agonist-driven selection of Treg cells with a diverse TCR repertoire, and the importance this holds for self-tolerance. We show that the intronic Foxp3 enhancer conserved noncoding sequence 3 (CNS3) acts as an epigenetic switch that confers a poised state to the Foxp3 promoter in precursor cells to make Treg cell lineage commitment responsive to a broad range of TCR stimuli, particularly to suboptimal ones. CNS3-dependent expansion of the TCR repertoire enables Treg cells to control self-reactive T cells effectively, especially when thymic negative selection is genetically impaired. Our findings highlight the complementary roles of these two main mechanisms of self-tolerance.

Differential TCR signals for T helper cell programming.

PubMed

Morel, Penelope A

2018-05-02

Upon encounter with their cognate antigen naïve CD4 T cells become activated and are induced to differentiate into several possible T helper (Th) cell subsets. This differentiation depends on a number of factors including antigen presenting cells, cytokines and costimulatory molecules. The strength of the T cell receptor (TCR) signal, related to the affinity of TCR for antigen and antigen dose, has emerged as a dominant factor in determining Th cell fate. Recent studies have revealed that TCR signals of high or low strength do not simply induce quantitatively different signals in the T cells, but rather qualitatively distinct pathways can be induced based on TCR signal strength. This review examines the recent literature in this area and highlights important new developments in our understanding of Th cell differentiation and TCR signal strength. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Mutation in Fas Ligand Impairs Maturation of Thymocytes Bearing Moderate Affinity T Cell Receptors

PubMed Central

Boursalian, Tamar E.; Fink, Pamela J.

2003-01-01

Fas ligand, best known as a death-inducer, is also a costimulatory molecule required for maximal proliferation of mature antigen-specific CD4+ and CD8+ T cells. We now extend the role of Fas ligand by showing that it can also influence thymocyte development. T cell maturation in some, but not all, strains of TCR transgenic mice is severely impaired in thymocytes expressing mutant Fas ligand incapable of interacting with Fas. Mutant Fas ligand inhibits neither negative selection nor death by neglect. Instead, it appears to modulate positive selection of thymocytes expressing both class I– and class II–restricted T cell receptors of moderate affinity for their positively selecting ligands. Fas ligand is therefore an inducer of death, a costimulator of peripheral T cell activation, and an accessory molecule in positive selection. PMID:12860933

Dimeric MHC-peptides inserted into an immunoglobulin scaffold as new immunotherapeutic agents

PubMed Central

Goldberg, Burt; Bona, Constantin

2011-01-01

Abstract The interactions of the T cell receptor (TCR) with cognate MHC-peptide and co-stimulatory molecules expressed at surface of antigen presenting cells (APC) leads to activation or tolerance of T cells. The development of molecular biological tools allowed for the preparation of soluble MHC-peptide molecules as surrogate for the APC. A decade ago a monomeric class II MHC molecule in which the peptide was covalently linked to β-chain of class II molecule was generated. This type of molecule had a low-binding affinity and did not cause the multimerization of TCR. The requirement of multimerization of TCR led to development of a new class of reagents, chimeric peptides covalently linked to MHC that was dimerized via Fc fragment of an immunoglobulin and linked to 3′ end of the β-chain of MHC class II molecule. These soluble dimerized MHC-peptide chimeric molecules display high affinity for the TCR and caused multimerization of TCR without processing by an APC. Because dimeric molecules are devoid of co-stimulatory molecules interacting with CD28, a second signal, they induce anergy rather the activation of T cells. In this review, we compare the human and murine dimerized MHC class II-peptides and their effect on CD4+ T cells, particularly the generation of T regulatory cells, which make these chimeric molecules an appealing approach for the treatment of autoimmune diseases. PMID:21435177

Immunotherapy expands and maintains the function of high affinity tumor infiltrating CD8 T cells in situ

PubMed Central

Moran, Amy E.; Polesso, Fanny; Weinberg, Andrew D.

2016-01-01

Cancer cells harbor high affinity tumor-associated antigens capable of eliciting potent anti-tumor T cell responses yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and PD-1 have been used. We report here that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor-antigen specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Co-expression of Nur77GFP and OX40 identifies a polyclonal population of high affinity tumor-associated antigen-specific CD8+ T cells, which produce more IFNγ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or PD-L1. Moreover, expansion of these high affinity CD8 T cells prolongs survival of tumor bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor resident CD8 T cells thereby increasing the frequency of cytolytic, high affinity, tumor-associated antigen-specific cells. PMID:27503208

Narrowing the Gap: Preserving Repertoire Diversity Despite Clonal Selection during the CD4 T Cell Response.

PubMed

Merkenschlager, Julia; Kassiotis, George

2015-01-01

T cell immunity relies on the generation and maintenance of a diverse repertoire of T cell antigen receptors (TCRs). The strength of signaling emanating from the TCR dictates the fate of T cells during development, as well as during the immune response. Whereas development of new T cells in the thymus increases the available TCR repertoire, clonal selection during the immune response narrows TCR diversity through the outgrowth of clonotypes with the fittest TCR. To ensure maintenance of TCR diversity in the antigen-selected repertoire, specific mechanisms can be envisaged that facilitate the participation of T cell clonotypes with less than best fit TCRs. Here, we summarize the evidence for the existence of such mechanisms that can prevent the loss of diversity. A number of T cell-autonomous or extrinsic factors can reverse clonotypic hierarchies set by TCR affinity for given antigen. Although not yet complete, understanding of these factors and their mechanism of action will be critical in interventional attempts to mold the antigen-selected TCR repertoire.

Dynamical footprint of cross-reactivity in a human autoimmune T-cell receptor

NASA Astrophysics Data System (ADS)

Kumar, Amit; Delogu, Francesco

2017-02-01

The present work focuses on the dynamical aspects of cross-reactivity between myelin based protein (MBP) self-peptide and two microbial peptides (UL15, PMM) for Hy.1B11 T-cell receptor (TCR). This same TCR was isolated from a patient suffering from multiple sclerosis (MS). The study aims at highlighting the chemical interactions underlying recognition mechanisms between TCR and the peptides presented by Major Histocompatibility Complex (MHC) proteins, which form a crucial component in adaptive immune response against foreign antigens. Since the ability of a TCR to recognize different peptide antigens presented by MHC depends on its cross-reactivity, we used molecular dynamics methods to obtain atomistic detail on TCR-peptide-MHC complexes. Our results show how the dynamical basis of Hy.1B11 TCR’s cross-reactivity is rooted in a similar bridging interaction pattern across the TCR-peptide-MHC interface. Our simulations confirm the importance of TCR CDR3α E98 residue interaction with MHC and a predominant role of P6 peptide residue in MHC binding affinity. Altogether, our study provides energetic and dynamical insights into factors governing peptide recognition by the cross-reactive Hy.1B11 TCR, found in MS patient.

pMHC affinity controls duration of CD8+ T cell–DC interactions and imprints timing of effector differentiation versus expansion

PubMed Central

Sharpe, James; Zehn, Dietmar; Kreutzfeldt, Mario

2016-01-01

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity–primed T cells acquired cytotoxic activity earlier than high affinity–primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity–stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. PMID:27799622

Reduced TCR signaling potential impairs negative selection but does not result in autoimmune disease

PubMed Central

Hwang, SuJin; Song, Ki-Duk; Lesourne, Renaud; Lee, Jan; Pinkhasov, Julia; Li, LiQi; El-Khoury, Dalal

2012-01-01

Negative selection and regulatory T (T reg) cell development are two thymus-dependent processes necessary for the enforcement of self-tolerance, and both require high-affinity interactions between the T cell receptor (TCR) and self-ligands. However, it remains unclear if they are similarly impacted by alterations in TCR signaling potential. We generated a knock-in allele (6F) of the TCR ζ chain gene encoding a mutant protein lacking signaling capability whose expression is controlled by endogenous ζ regulatory sequences. Although negative selection was defective in 6F/6F mice, leading to the survival of autoreactive T cells, 6F/6F mice did not develop autoimmune disease. We found that 6F/6F mice generated increased numbers of thymus-derived T reg cells. We show that attenuation of TCR signaling potential selectively impacts downstream signaling responses and that this differential effect favors Foxp3 expression and T reg cell lineage commitment. These results identify a potential compensatory pathway for the enforcement of immune tolerance in response to defective negative selection caused by reduced TCR signaling capability. PMID:22945921

NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability

PubMed Central

Sommermeyer, Daniel; Conrad, Heinke; Krönig, Holger; Gelfort, Haike; Bernhard, Helga; Uckert, Wolfgang

2013-01-01

The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy. PMID:22907642

Modulation of TCRβ surface expression during TCR revision.

PubMed

Simmons, Kalynn B; Wubeshet, Maramawit; Ames, Kristina T; McMahan, Catherine J; Hale, J Scott; Fink, Pamela J

2012-01-01

TCR revision is a tolerance mechanism by which self-reactive TCRs expressed by mature CD4(+) peripheral T cells are replaced by receptors encoded by genes generated by post-thymic DNA rearrangement. The downmodulation of surface TCR expression initiates TCR revision, and serves as a likely trigger for the induction of the recombinase machinery. We show here in a Vβ5 transgenic mouse model system that downregulation of the self-reactive transgene-encoded TCR is not maintained by transgene loss or diminished transcription or translation. The downregulation of surface TCR expression likely occurs in two stages, only one of which requires tolerogen expression. Copyright © 2011 Elsevier Inc. All rights reserved.

First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases.

PubMed

Borroto, Aldo; Reyes-Garau, Diana; Jiménez, M Angeles; Carrasco, Esther; Moreno, Beatriz; Martínez-Pasamar, Sara; Cortés, José R; Perona, Almudena; Abia, David; Blanco, Soledad; Fuentes, Manuel; Arellano, Irene; Lobo, Juan; Heidarieh, Haleh; Rueda, Javier; Esteve, Pilar; Cibrián, Danay; Martinez-Riaño, Ana; Mendoza, Pilar; Prieto, Cristina; Calleja, Enrique; Oeste, Clara L; Orfao, Alberto; Fresno, Manuel; Sánchez-Madrid, Francisco; Alcamí, Antonio; Bovolenta, Paola; Martín, Pilar; Villoslada, Pablo; Morreale, Antonio; Messeguer, Angel; Alarcon, Balbino

2016-12-21

Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC 50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases. Copyright © 2016, American Association for the Advancement of Science.

Adoptive immunotherapy using PRAME-specific T cells in medulloblastoma.

PubMed

Orlando, Domenico; Miele, Evelina; De Angelis, Biagio; Guercio, Marika; Boffa, Iolanda; Sinibaldi, Matilde; Po, Agnese; Caruana, Ignazio; Abballe, Luana; Carai, Andrea; Caruso, Simona; Camera, Antonio; Moseley, Annemarie; Hagedoorn, Renate S; Heemskerk, Mirjam H M; Giangaspero, Felice; Mastronuzzi, Angela; Ferretti, Elisabetta; Locatelli, Franco; Quintarelli, Concetta

2018-04-03

Medulloblastoma is the most frequent malignant childhood brain tumor with a high morbidity. Identification of new therapeutic targets would be instrumental in improving patient outcomes. We evaluated the expression of the tumor-associated antigen PRAME in biopsies from 60 medulloblastoma patients. PRAME expression was detectable in 82% of tissues independent of molecular and histopathologic subgroups. High PRAME expression also correlated with worse overall survival. We next investigated the relevance of PRAME as a target for immunotherapy. Medulloblastoma cells were targeted using genetically modified T cells with a PRAME-specific TCR (SLL TCR T cells). SLL TCR T cells efficiently killed medulloblastoma HLA-A*02+ DAOY cells as well as primary HLA-A*02+ medulloblastoma cells. Moreover, SLL TCR T cells controlled tumor growth in an orthotopic mouse model of medulloblastoma. To prevent unexpected T cell-related toxicity,an inducible caspase 9 (iC9) gene was introduced in frame with the SLL TCR; this safety switch triggered prompt elimination of genetically-modified T cells. Altogether, these data indicate that T cells genetically modified with a high-affinity, PRAME-specific TCR and iC9 may represent a promising innovative approach for treating HLA-A*02+ medulloblastoma patients. Copyright ©2018, American Association for Cancer Research.

Subcellular distribution of Lck during CD4 T-cell maturation in the thymic medulla regulates the T-cell activation threshold.

PubMed

Stephen, Tom Li; Wilson, Bridget S; Laufer, Terri M

2012-05-08

Mature peripheral T cells respond to foreign but not to self-antigens. During development in the thymus, deletion of high-affinity self-reactive immature thymocytes contributes to tolerance of mature T cells. However, double-positive thymocytes are positively selected to survive if they respond to self-peptide-MHC complexes; thus, there must be mechanisms to prevent overt reactivity to those same complexes in the periphery. "Developmental tuning" is the active process through which T-cell receptor (TCR)-associated signaling pathways of single-positive (SP) thymocytes are attenuated to respond appropriately to self-peptide-MHC complexes in the periphery. We previously showed that MHC class II expression in the thymic medulla was necessary to tune CD4(+) SP (CD4 SP) thymocytes. CD4 SP thymocytes from mice lacking medullary MHC class II expression had inappropriately enhanced proximal TCR signaling to low-affinity self-ligands that was associated with altered cellular distribution of the tyrosine kinase Lck. Now, we report that activation of both tuned and untuned CD4 SP thymocytes is Lck-dependent. Untuned CD4 SP cells contain a pool of Lck with increased basal phosphorylation that is not associated with the CD4 coreceptor. Phosphorylation of this pool of Lck decreases with tuning. Immunogold transmission electron microscopy of membrane sheets permitted direct visualization of Lck. In the absence of tuning, a significant proportion of Lck and the TCR subunit CD3ζ are expressed on the same protein island; this close association of Lck and the TCR probably explains the enhanced activation of untuned CD4 SP cells. Thus, changes in membrane topography during thymic maturation determine the set point for TCR responsiveness.

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

PubMed

Neumann, Frank; Sturm, Christine; Hülsmeyer, Martin; Dauth, Nina; Guillaume, Philippe; Luescher, Immanuel F; Pfreundschuh, Michael; Held, Gerhard

2009-08-15

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane.

PubMed

James, Scott E; Greenberg, Philip D; Jensen, Michael C; Lin, Yukang; Wang, Jinjuan; Till, Brian G; Raubitschek, Andrew A; Forman, Stephen J; Press, Oliver W

2008-05-15

We have targeted CD22 as a novel tumor-associated Ag for recognition by human CTL genetically modified to express chimeric TCR (cTCR) recognizing this surface molecule. CD22-specific cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR(+) CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR(+) CTL exhibited lower levels of maximum lysis and lower Ag sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of Ag engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope, but constructed as a truncated CD22 molecule to approximate the length of a TCR:peptide-MHC complex. The reduced sensitivity of CD22-specific cTCR(+) CTL for Ag-induced triggering of effector functions has potential therapeutic applications, because such cells selectively lysed B cell lymphoma lines expressing high levels of CD22, but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength, and consequently Ag sensitivity, can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate Ag density.

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.

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

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. ©2015 American Association for Cancer Research.

Analysis of the Expression of Peptide–Major Histocompatibility Complexes Using High Affinity Soluble Divalent T Cell Receptors

PubMed Central

O'Herrin, Sean M.; Lebowitz, Michael S.; Bieler, Joan G.; al-Ramadi, Basel K.; Utz, Ursula; Bothwell, Alfred L.M.; Schneck, Jonathan P.

1997-01-01

Understanding the regulation of cell surface expression of specific peptide–major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide–MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR–Ig) that have high affinity for their cognate peptide–MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR–Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus–1 presented by human histocompatibility leukocyte antigens–A2. Further, using 2C TCR– Ig, a more detailed analysis of the interaction with cognate peptide–MHC complexes revealed several interesting findings. Soluble divalent 2C TCR–Ig detected significant changes in the level of specific antigenic–peptide MHC cell surface expression in cells treated with γ-interferon (γ-IFN). Interestingly, the effects of γ-IFN on expression of specific peptide–MHC complexes recognized by 2C TCR–Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of γ-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide–MHC complexes. Analysis of the binding of 2C TCR–Ig for specific peptide–MHC ligands unexpectedly revealed that the affinity of the 2C TCR–Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8–H-2 Kbm3, is very low, weaker than 71 μM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca–H-2 Ld, is ∼1000-fold higher. Thus, negatively selecting peptide–MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis, indicate the importance of using high affinity biologically relevant cognates, such as soluble divalent TCR, in furthering our understanding of immune responses. PMID:9334373

Nuclear pore complex-mediated modulation of TCR signaling is required for naïve CD4+ T cell homeostasis.

PubMed

Borlido, Joana; Sakuma, Stephen; Raices, Marcela; Carrette, Florent; Tinoco, Roberto; Bradley, Linda M; D'Angelo, Maximiliano A

2018-06-01

Nuclear pore complexes (NPCs) are channels connecting the nucleus with the cytoplasm. We report that loss of the tissue-specific NPC component Nup210 causes a severe deficit of naïve CD4 + T cells. Nup210-deficient CD4 + T lymphocytes develop normally but fail to survive in the periphery. The decreased survival results from both an impaired ability to transmit tonic T cell receptor (TCR) signals and increased levels of Fas, which sensitize Nup210 -/- naïve CD4 + T cells to Fas-mediated cell death. Mechanistically, Nup210 regulates these processes by modulating the expression of Cav2 (encoding Caveolin-2) and Jun at the nuclear periphery. Whereas the TCR-dependent and CD4 + T cell-specific upregulation of Cav2 is critical for proximal TCR signaling, cJun expression is required for STAT3-dependent repression of Fas. Our results uncover an unexpected role for Nup210 as a cell-intrinsic regulator of TCR signaling and T cell homeostasis and expose NPCs as key players in the adaptive immune system.

Structural basis of human β-cell killing by CD8+ T cells in Type 1 diabetes

PubMed Central

Bulek, Anna M.; Cole, David K.; Skowera, Ania; Dolton, Garry; Gras, Stephanie; Madura, Florian; Fuller, Anna; Miles, John J.; Gostick, Emma; Price, David A.; Drijfhout, Jan W.; Knight, Robin R.; Huang, Guo C.; Lissin, Nikolai; Molloy, Peter E.; Wooldridge, Linda; Jakobsen, Bent K.; Rossjohn, Jamie; Peakman, Mark; Rizkallah, Pierre J.; Sewell, Andrew K.

2011-01-01

The structural characteristics of autoreactive-T cell receptor (TCR) engagement of major histocompatability (MHC) class II-restricted self-antigens is established, but how autoimmune-TCRs interact with self-MHC class I has been unclear. We examined how CD8+ T cells kill human islet β-cells, in Type-1 diabetes, via autoreactive-TCR (1E6) recognition of an HLA-A*0201-restricted glucose-sensitive preproinsulin peptide. Rigid ‘lock-and-key’ binding underpinned the 1E6-HLA-A*0201-peptide interaction, whereby 1E6 docked similarly to most MHCI-restricted TCRs. However, this interaction was extraordinarily weak, due to limited contacts with MHCI. TCR binding was highly peptide-centric, dominated by two CDR3-loop-encoded residues, acting as an ‘aromatic-cap’, over the peptide MHCI (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8+ T cell-mediated autoreactivity. PMID:22245737

Monoclonal TCR-redirected tumor cell killing.

PubMed

Liddy, Nathaniel; Bossi, Giovanna; Adams, Katherine J; Lissina, Anna; Mahon, Tara M; Hassan, Namir J; Gavarret, Jessie; Bianchi, Frayne C; Pumphrey, Nicholas J; Ladell, Kristin; Gostick, Emma; Sewell, Andrew K; Lissin, Nikolai M; Harwood, Naomi E; Molloy, Peter E; Li, Yi; Cameron, Brian J; Sami, Malkit; Baston, Emma E; Todorov, Penio T; Paston, Samantha J; Dennis, Rebecca E; Harper, Jane V; Dunn, Steve M; Ashfield, Rebecca; Johnson, Andy; McGrath, Yvonne; Plesa, Gabriela; June, Carl H; Kalos, Michael; Price, David A; Vuidepot, Annelise; Williams, Daniel D; Sutton, Deborah H; Jakobsen, Bent K

2012-06-01

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

PubMed Central

Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

2014-01-01

Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

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

PubMed Central

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

2010-01-01

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

Molecular Mechanisms of Bcl10-Mediated NF-kB Signal Transduction

DTIC Science & Technology

2006-03-08

recruiting and activating the kinase, Akt , which is a critical mediator of pro-survival signals (3) (Figure 3). Figure 3. TCR-induced signaling...kinase and Akt rather than through upstream intermediates initiated by TCR ligation (34, 70). This suggests that TCR stimulation and CD28 co...P. Vito. 2004. Physical and functional interaction of CARMA1 and CARMA3 with Ikappa kinase gamma- NFkappaB essential modulator. J Biol Chem 279

Multisite Phosphorylation Modulates the T Cell Receptor ζ-Chain Potency but not the Switchlike Response.

PubMed

Mukhopadhyay, Himadri; de Wet, Ben; Clemens, Lara; Maini, Philip K; Allard, Jun; van der Merwe, P Anton; Dushek, Omer

2016-04-26

Multisite phosphorylation is ubiquitous in cellular signaling and is thought to provide signaling proteins with additional regulatory mechanisms. Indeed, mathematical models have revealed a large number of mechanisms by which multisite phosphorylation can produce switchlike responses. The T cell antigen receptor (TCR) is a multisubunit receptor on the surface of T cells that is a prototypical multisite substrate as it contains 20 sites that are distributed on 10 conserved immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR ζ-chain is a homodimer subunit that contains six ITAMs (12 sites) and exhibits a number of properties that are predicted to be sufficient for a switchlike response. We have used cellular reconstitution to systematically study multisite phosphorylation of the TCR ζ-chain. We find that multisite phosphorylation proceeds by a nonsequential random mechanism, and find no evidence that multiple ITAMs modulate a switchlike response but do find that they alter receptor potency and maximum phosphorylation. Modulation of receptor potency can be explained by a reduction in molecular entropy of the disordered ζ-chain upon phosphorylation. We further find that the tyrosine kinase ZAP-70 increases receptor potency but does not modulate the switchlike response. In contrast to other multisite proteins, where phosphorylations act in strong concert to modulate protein function, we suggest that the multiple ITAMs on the TCR function mainly to amplify subsequent signaling. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Mitochondria-derived hydrogen peroxide selectively enhances T cell receptor-initiated signal transduction.

PubMed

Gill, Tejpal; Levine, Alan D

2013-09-06

T cell receptor (TCR)-initiated signal transduction is reported to increase production of intracellular reactive oxygen species, such as superoxide (O2˙(-)) and hydrogen peroxide (H2O2), as second messengers. Although H2O2 can modulate signal transduction by inactivating protein phosphatases, the mechanism and the subcellular localization of intracellular H2O2 as a second messenger of the TCR are not known. The antioxidant enzyme superoxide dismutase (SOD) catalyzes the dismutation of highly reactive O2˙(-) into H2O2 and thus acts as an intracellular generator of H2O2. As charged O2˙(-) is unable to diffuse through intracellular membranes, cells express distinct SOD isoforms in the cytosol (Cu,Zn-SOD) and mitochondria (Mn-SOD), where they locally scavenge O2˙(-) leading to production of H2O2. A 2-fold organelle-specific overexpression of either SOD in Jurkat T cell lines increases intracellular production of H2O2 but does not alter the levels of intracellular H2O2 scavenging enzymes such as catalase, membrane-bound peroxiredoxin1 (Prx1), and cytosolic Prx2. We report that overexpression of Mn-SOD enhances tyrosine phosphorylation of TCR-associated membrane proximal signal transduction molecules Lck, LAT, ZAP70, PLCγ1, and SLP76 within 1 min of TCR cross-linking. This increase in mitochondrial H2O2 specifically modulates MAPK signaling through the JNK/cJun pathway, whereas overexpressing Cu,Zn-SOD had no effect on any of these TCR-mediated signaling molecules. As mitochondria translocate to the immunological synapse during TCR activation, we hypothesize this translocation provides the effective concentration of H2O2 required to selectively modulate downstream signal transduction pathways.

Antigen sensitivity of CD22-specific chimeric T cell receptors is modulated by target epitope distance from the cell membrane

PubMed Central

James, Scott E.; Greenberg, Philip D.; Jensen, Michael C.; Lin, Yukang; Wang, Jinjuan; Till, Brian G.; Raubitschek, Andrew A.; Forman, Stephen J.; Press, Oliver W.

2008-01-01

We have targeted CD22 as a novel tumor-associated antigen for recognition by human CTL genetically modified to express chimeric T cell receptors (cTCR) recognizing this surface molecule. CD22-specifc cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR+ CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR+ CTL exhibited lower levels of maximum lysis and lower antigen sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of antigen engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope but constructed as a truncated CD22 molecule to approximate the length of a TCR:pMHC complex. The reduced sensitivity of CD22-specific cTCR+ CTL for antigen-induced triggering of effector functions has potential therapeutic applications, as such cells selectively lysed B cell lymphoma lines expressing high levels of CD22 but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength – and consequently antigen sensitivity – can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate antigen density. PMID:18453625

PU.1 regulates TCR expression by modulating GATA-3 activity

PubMed Central

Chang, Hua-Chen; Han, Ling; Jabeen, Rukhsana; Carotta, Sebastian; Nutt, Stephen L.; Kaplan, Mark H.

2009-01-01

The Ets transcription factor PU.1 is a master regulator for the development of multiple lineages during hematopoiesis. The expression pattern of PU.1 is dynamically regulated during early T lineage development in the thymus. We previously revealed that PU.1 delineates heterogeneity of effector Th2 populations. In this study, we further define the function of PU.1 on the Th2 phenotype using mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1lck-/-). While deletion of PU.1 by the lck-Cre transgene does not affect T cell development, Sfpi1lck-/- T cells have a lower activation threshold than wild type T cells. When TCR engagement is limiting, Sfpi1lck-/- T cells cultured in Th2 polarizing conditions secrete higher levels of Th2 cytokines and have greater cytokine homogeneity than wild type cells. We show that PU.1 modulates the levels of TCR expression in CD4+ T cells by regulating the DNA-binding activity of GATA-3 and limiting GATA-3 regulation of TCR gene expression. GATA-3 dependent regulation of TCR expression is also observed in Th1 and Th2 cells. In CD4+ T cells, PU.1 expression segregates into subpopulations of cells that have lower levels of surface TCR, suggesting that PU.1 contributes to the heterogeneity of TCR expression. Thus, we have identified a mechanism whereby increased GATA-3 function in the absence of the antagonizing activity of PU.1 leads to increased TCR expression, a reduced activation threshold and increased homogeneity in Th2 populations. PMID:19801513

Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity

PubMed Central

Saitakis, Michael; Dogniaux, Stéphanie; Goudot, Christel; Bufi, Nathalie; Asnacios, Sophie; Maurin, Mathieu; Randriamampita, Clotilde; Asnacios, Atef; Hivroz, Claire

2017-01-01

T cells are mechanosensitive but the effect of stiffness on their functions is still debated. We characterize herein how human primary CD4+ T cell functions are affected by stiffness within the physiological Young’s modulus range of 0.5 kPa to 100 kPa. Stiffness modulates T lymphocyte migration and morphological changes induced by TCR/CD3 triggering. Stiffness also increases TCR-induced immune system, metabolism and cell-cycle-related genes. Yet, upon TCR/CD3 stimulation, while cytokine production increases within a wide range of stiffness, from hundreds of Pa to hundreds of kPa, T cell metabolic properties and cell cycle progression are only increased by the highest stiffness tested (100 kPa). Finally, mechanical properties of adherent antigen-presenting cells modulate cytokine production by T cells. Together, these results reveal that T cells discriminate between the wide range of stiffness values found in the body and adapt their responses accordingly. DOI: http://dx.doi.org/10.7554/eLife.23190.001 PMID:28594327

Proline substitution independently enhances H-2D(b) complex stabilization and TCR recognition of melanoma-associated peptides.

PubMed

Uchtenhagen, Hannes; Abualrous, Esam T; Stahl, Evi; Allerbring, Eva B; Sluijter, Marjolein; Zacharias, Martin; Sandalova, Tatyana; van Hall, Thorbald; Springer, Sebastian; Nygren, Per-Åke; Achour, Adnane

2013-11-01

The immunogenicity of H-2D(b) (D(b)) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T-cell cross-reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D(b) in complex with an optimized version of the melanoma-associated epitope gp10025-33 . Furthermore, the p3P modification directly increased the affinity of the D(b)/gp10025-33 -specific T-cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D(b)/gp10025-33 complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Impact of lipid rafts on the T -cell-receptor and peptide-major-histocompatibility-complex interactions under different measurement conditions

NASA Astrophysics Data System (ADS)

Li, Long; Xu, Guang-Kui; Song, Fan

2017-01-01

The interactions between T-cell receptor (TCR) and peptide-major-histocompatibility complex (pMHC), which enable T-cell development and initiate adaptive immune responses, have been intensively studied. However, a central issue of how lipid rafts affect the TCR-pMHC interactions remains unclear. Here, by using a statistical-mechanical membrane model, we show that the binding affinity of TCR and pMHC anchored on two apposing cell membranes is significantly enhanced because of the lipid raft-induced signaling protein aggregation. This finding may provide an alternative insight into the mechanism of T-cell activation triggered by very low densities of pMHC. In the case of cell-substrate adhesion, our results indicate that the loss of lateral mobility of the proteins on the solid substrate leads to the inhibitory effect of lipid rafts on TCR-pMHC interactions. Our findings help to understand why different experimental methods for measuring the impact of lipid rafts on the receptor-ligand interactions have led to contradictory conclusions.

Chimeric PD-1:28 Receptor Upgrades Low-Avidity T cells and Restores Effector Function of Tumor-Infiltrating Lymphocytes for Adoptive Cell Therapy.

PubMed

Schlenker, Ramona; Olguín-Contreras, Luis Felipe; Leisegang, Matthias; Schnappinger, Julia; Disovic, Anja; Rühland, Svenja; Nelson, Peter J; Leonhardt, Heinrich; Harz, Hartmann; Wilde, Susanne; Schendel, Dolores J; Uckert, Wolfgang; Willimsky, Gerald; Noessner, Elfriede

2017-07-01

Inherent intermediate- to low-affinity T-cell receptors (TCR) that develop during the natural course of immune responses may not allow sufficient activation for tumor elimination, making the majority of T cells suboptimal for adoptive T-cell therapy (ATT). TCR affinity enhancement has been implemented to provide stronger T-cell activity but carries the risk of creating undesired cross-reactivity leading to potential serious adverse effects in clinical application. We demonstrate here that engineering of low-avidity T cells recognizing a naturally processed and presented tumor-associated antigen with a chimeric PD-1:28 receptor increases effector function to levels seen with high-avidity T cells of identical specificity. Upgrading the function of low-avidity T cells without changing the TCR affinity will allow a large arsenal of low-avidity T cells previously thought to be therapeutically inefficient to be considered for ATT. PD-1:28 engineering reinstated Th1 function in tumor-infiltrating lymphocytes that had been functionally disabled in the human renal cell carcinoma environment without unleashing undesired Th2 cytokines or IL10. Involved mechanisms may be correlated to restoration of ERK and AKT signaling pathways. In mouse tumor models of ATT, PD-1:28 engineering enabled low-avidity T cells to proliferate stronger and prevented PD-L1 upregulation and Th2 polarization in the tumor milieu. Engineered T cells combined with checkpoint blockade secreted significantly more IFNγ compared with T cells without PD-1:28, suggesting a beneficial combination with checkpoint blockade therapy or other therapeutic strategies. Altogether, the supportive effects of PD-1:28 engineering on T-cell function make it an attractive tool for ATT. Cancer Res; 77(13); 3577-90. ©2017 AACR . ©2017 American Association for Cancer Research.

Phosphatase CD45 Both Positively and Negatively Regulates T Cell Receptor Phosphorylation in Reconstituted Membrane Protein Clusters*♦

PubMed Central

Furlan, Gabriela; Minowa, Takashi; Hanagata, Nobutaka; Kataoka-Hamai, Chiho; Kaizuka, Yoshihisa

2014-01-01

T cell receptor (TCR) phosphorylation requires the kinase Lck and phosphatase CD45. CD45 activates Lck by dephosphorylating an inhibitory tyrosine of Lck to relieve autoinhibition. However, CD45 also dephosphorylates the TCR, and the spatial exclusion of CD45 from TCR clustering in the plasma membrane appears to attenuate this negative effect of CD45. To further investigate the role of CD45 in signal initiation, we reconstituted membrane TCR clusters in vitro on supported lipid bilayers. Fluorescence microscopy of single clusters showed that incorporation of CD45 enhanced phosphorylation of TCR clusters, but only when Lck co-clustered with TCR. We found that clustered Lck autophosphorylated the inhibitory tyrosine and thus could be activated by CD45, whereas diffusive Lck molecules did not. In the TCR-Lck clusters and at low CD45 density, we speculate that the effect of Lck activation may overcome dephosphorylation of TCR, resulting in a net positive regulation. The CD45 density in physiological TCR clusters is also low because of the exclusion of CD45. Thus, we propose that the spatial organization of TCR/Lck/CD45 in T cell membranes is important not only for modulating the negative role of CD45 but also for creating conditions in which CD45 has a positive role in signal initiation. PMID:25128530

The natural antibody repertoire of sharks and humans recognizes the potential universe of antigens.

PubMed

Adelman, Miranda K; Schluter, Samuel F; Marchalonis, John J

2004-02-01

In ancestral sharks, a rapid emergence in the evolution of the immune system occurred, giving jawed-vertebrates the necessary components for the combinatorial immune response (CIR). To compare the natural antibody (NAb) repertoires of the most divergent vertebrates with the capacity to produce antibodies, we isolated NAbs to the same set of antigens by affinity chromatography from two species of Carcharhine sharks and from human polyclonal IgG and IgM antibody preparations. The activities of the affinity-purified anti-T-cell receptor (anti-TCR) NAbs were compared with those of monoclonal anti-TCR NAbs that were generated from a systemic lupus erythematosus patient. We report that sharks and humans, representing the evolutionary extremes of vertebrate species sharing the CIR, have NAbs to human TCRs, Igs, the human senescent cell antigen, and to numerous retroviral antigens, indicating that essential features of the combinatorial repertoire and the capacity to recognize the potential universe of antigens is shared among all jawed-vertebrates.

CD8+ recent thymic emigrants exhibit increased responses to low affinity ligands and improved access to peripheral sites of inflammation

PubMed Central

Berkley, Amy M.; Fink, Pamela J.

2014-01-01

To explore the TCR sensitivity of recent thymic emigrants (RTEs), we triggered T cells with altered peptide ligands (APLs). Upon peptide stimulation in vitro, RTEs exhibited increased TCR signal transduction, and following infection in vivo with APL-expressing bacteria, CD8 RTEs expanded to a greater extent in response to low affinity antigens than their mature T cell counterparts. RTEs skewed to short-lived effector cells in response to all APLs but were also characterized by diminished cytokine production. RTEs responding to infection expressed increased levels of VLA-4, with consequent improved entry into inflamed tissue and pathogen clearance. These positive outcomes were offset by the capacity of RTEs to elicit autoimmunity. Overall, salient features of CD8 RTE biology should inform strategies to improve neonatal vaccination and therapies for cancer and HIV, as RTEs make up a large proportion of the T cells in lymphodepleted environments. PMID:25172492

Cutting edge: CD8+ recent thymic emigrants exhibit increased responses to low-affinity ligands and improved access to peripheral sites of inflammation.

PubMed

Berkley, Amy M; Fink, Pamela J

2014-10-01

To explore the TCR sensitivity of recent thymic emigrants (RTEs), we triggered T cells with altered peptide ligands (APLs). Upon peptide stimulation in vitro, RTEs exhibited increased TCR signal transduction, and following infection in vivo with APL-expressing bacteria, CD8 RTEs expanded to a greater extent in response to low-affinity Ags than did their mature T cell counterparts. RTEs skewed to short-lived effector cells in response to all APLs but also were characterized by diminished cytokine production. RTEs responding to infection expressed increased levels of VLA-4, with consequent improved entry into inflamed tissue and pathogen clearance. These positive outcomes were offset by the capacity of RTEs to elicit autoimmunity. Overall, salient features of CD8 RTE biology should inform strategies to improve neonatal vaccination and therapies for cancer and HIV, because RTEs make up a large proportion of the T cells in lymphodepleted environments. Copyright © 2014 by The American Association of Immunologists, Inc.

T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters

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

Manz, Boryana N.; Jackson, Bryan L.; Petit, Rebecca S.

2011-05-31

T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. In this study, we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC contentmore » within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. In conclusion, this threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.« less

Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

PubMed

Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

2017-01-01

CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

Retinoic Acid Modulates Interferon-γ Production by Hepatic Natural Killer T Cells via Phosphatase 2A and the Extracellular Signal-Regulated Kinase Pathway

PubMed Central

Chang, Heng-Kwei

2015-01-01

Retinoic acid (RA), an active metabolite converted from vitamin A, plays an active role in immune function, such as defending against infections and immune regulation. Although RA affects various types of immune cells, including antigen-presenting cells, B lymphocytes, and T lymphocytes, whether it affects natural killer T (NKT) cells remain unknown. In this study, we found that RA decreased interferon (IFN)-γ production by activated NKT cells through T-cell receptor (TCR) and CD28. We also found that RA reduced extracellular signal-regulated kinase (ERK) phosphorylation, but increased phosphatase 2A (PP2A) activity in TCR/CD28-stimulated NKT cells. The increased PP2A activity, at least partly, contributed to the reduction of ERK phosphorylation. Since inhibition of ERK activation decreases IFN-γ production by TCR/CD28-stimulated NKT cells, RA may downregulate IFN-γ production by TCR/CD28-stimulated NKT cells through the PP2A-ERK pathway. Our results demonstrated a novel function of RA in modulating the IFN-γ expression by activated NKT cells. PMID:25343668

Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions.

PubMed

Swee, Lee Kim; Tan, Zhen Wei; Sanecka, Anna; Yoshida, Nagisa; Patel, Harshil; Grotenbreg, Gijsbert; Frickel, Eva-Maria; Ploegh, Hidde L

2016-11-01

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-2 b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 L d -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. © 2016 The Authors.

SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1

PubMed Central

Côte, Marjorie; Fos, Camille; Canonigo-Balancio, Ann J.; Ley, Klaus; Bécart, Stéphane; Altman, Amnon

2015-01-01

ABSTRACT SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca2+ signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4+ T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4+ T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6−/−) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling. PMID:26483383

SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1.

PubMed

Côte, Marjorie; Fos, Camille; Canonigo-Balancio, Ann J; Ley, Klaus; Bécart, Stéphane; Altman, Amnon

2015-12-01

SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca(2+) signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4(+) T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4(+) T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6(-/-)) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling. © 2015. Published by The Company of Biologists Ltd.

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

PubMed Central

Friend, Samantha F.; Peterson, Lisa K.; Kedl, Ross M.; Dragone, Leonard L.

2014-01-01

How T cell receptor (TCR) avidity influences CD8+ T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP−/− mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP−/− Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8+ T cell development and repertoire selection. In comparing SLAP−/− OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP−/− Vβ5 mice. We have found that SLAP−/− OT-1 mice have fewer CD8+ thymocytes but have increased CD5 expression. SLAP−/− OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8+ splenocytes upon tetramer staining. Our data demonstrate that SLAP−/− Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8+ T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8+ T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8+ T cell development influences repertoire selection. PMID:22956467

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells.

PubMed

Friend, Samantha F; Peterson, Lisa K; Kedl, Ross M; Dragone, Leonard L

2013-03-01

How T cell receptor (TCR) avidity influences CD8(+) T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP(-/-) mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP(-/-) Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8(+) T cell development and repertoire selection. In comparing SLAP(-/-) OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP(-/-) Vβ5 mice. We have found that SLAP(-/-) OT-1 mice have fewer CD8(+) thymocytes but have increased CD5 expression. SLAP(-/-) OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8(+) splenocytes upon tetramer staining. Our data demonstrate that SLAP(-/-) Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8(+) T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8(+) T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8(+) T cell development influences repertoire selection.

Src homology 2-domain containing leukocyte-specific phosphoprotein of 76 kDa is mandatory for TCR-mediated inside-out signaling, but dispensable for CXCR4-mediated LFA-1 activation, adhesion, and migration of T cells.

PubMed

Horn, Jessica; Wang, Xiaoqian; Reichardt, Peter; Stradal, Theresia E; Warnecke, Nicole; Simeoni, Luca; Gunzer, Matthias; Yablonski, Deborah; Schraven, Burkhart; Kliche, Stefanie

2009-11-01

Engagement of the TCR or of chemokine receptors such as CXCR4 induces adhesion and migration of T cells via so-called inside-out signaling pathways. The molecular processes underlying inside-out signaling events are as yet not completely understood. In this study, we show that TCR- and CXCR4-mediated activation of integrins critically depends on the membrane recruitment of the adhesion- and degranulation-promoting adapter protein (ADAP)/Src kinase-associated phosphoprotein of 55 kDa (SKAP55)/Rap1-interacting adapter protein (RIAM)/Rap1 module. We further demonstrate that the Src homology 2 domain containing leukocyte-specific phosphoprotein of 76 kDa (SLP76) is crucial for TCR-mediated inside-out signaling and T cell/APC interaction. Besides facilitating membrane recruitment of ADAP, SKAP55, and RIAM, SLP76 regulates TCR-mediated inside-out signaling by controlling the activation of Rap1 as well as Rac-mediated actin polymerization. Surprisingly, however, SLP76 is not mandatory for CXCR4-mediated inside-out signaling. Indeed, both CXCR4-induced T cell adhesion and migration are not affected by loss of SLP76. Moreover, after CXCR4 stimulation, the ADAP/SKAP55/RIAM/Rap1 module is recruited to the plasma membrane independently of SLP76. Collectively, our data indicate a differential requirement for SLP76 in TCR- vs CXCR4-mediated inside-out signaling pathways regulating T cell adhesion and migration.

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development.

PubMed

Wang, Haopeng; Holst, Jeff; Woo, Seng-Ryong; Guy, Cliff; Bettini, Matt; Wang, Yao; Shafer, Aaron; Naramura, Mayumi; Mingueneau, Michaël; Dragone, Leonard L; Hayes, Sandra M; Malissen, Bernard; Band, Hamid; Vignali, Dario A A

2010-04-07

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3varepsilon proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3zeta-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development

PubMed Central

Wang, Haopeng; Holst, Jeff; Woo, Seng-Ryong; Guy, Cliff; Bettini, Matt; Wang, Yao; Shafer, Aaron; Naramura, Mayumi; Mingueneau, Michaël; Dragone, Leonard L; Hayes, Sandra M; Malissen, Bernard; Band, Hamid; Vignali, Dario A A

2010-01-01

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ɛ proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development. PMID:20150895

Human intraepithelial lymphocytes.

PubMed

Mayassi, Toufic; Jabri, Bana

2018-04-20

The location of intraepithelial lymphocytes (IEL) between epithelial cells, their effector memory, cytolytic and inflammatory phenotype positions them to kill infected epithelial cells and protect the intestine against pathogens. Human TCRαβ + CD8αβ + IEL have the dual capacity to recognize modified self via natural killer (NK) receptors (autoreactivity) as well as foreign antigen via the T cell receptor (TCR), which is accomplished in mouse by two cell subsets, the naturally occurring TCRαβ + CD8αα + and adaptively induced TCRαβ + CD8αβ + IEL subsets, respectively. The private/oligoclonal nature of the TCR repertoire of both human and mouse IEL suggests local environmental factors dictate the specificity of IEL responses. The line between sensing of foreign antigens and autoreactivity is blurred for IEL in celiac disease, where recognition of stress ligands by induced activating NK receptors in conjunction with inflammatory signals such as IL-15 can result in low-affinity TCR/non-cognate antigen and NK receptor/stress ligand interactions triggering destruction of intestinal epithelial cells.

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition

PubMed Central

Nicholls, Sarah; Piper, Karen P.; Mohammed, Fiyaz; Dafforn, Timothy R.; Tenzer, Stefan; Salim, Mahboob; Mahendra, Premini; Craddock, Charles; van Endert, Peter; Schild, Hansjörg; Cobbold, Mark; Engelhard, Victor H.; Moss, Paul A. H.; Willcox, Benjamin E.

2009-01-01

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1H versus HA-1R) in the HMHA1 gene. The HA-1H peptide is restricted by HLA-A2 and is immunogenic in HA-1R/R into HA-1H transplants, while HA-1R has been suggested to be a “null allele” in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1R variant. To understand these findings, we determined the structure of an HLA-A2-HA-1H complex to 1.3Å resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1H-specific T-cells bound HA-1H peptide with moderate affinity but failed to bind HA-1R, indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition. PMID:19234124

The activation threshold of CD4+ T cells is defined by TCR/peptide-MHC class II interactions in the thymic medulla.

PubMed

Stephen, Tom Li; Tikhonova, Anastasia; Riberdy, Janice M; Laufer, Terri M

2009-11-01

Immature thymocytes that are positively selected based upon their response to self-peptide-MHC complexes develop into mature T cells that are not overtly reactive to those same complexes. Developmental tuning is the active process through which TCR-associated signaling pathways of single-positive thymocytes are attenuated to respond appropriately to the peptide-MHC molecules that will be encountered in the periphery. In this study, we explore the mechanisms that regulate the tuning of CD4(+) single-positive T cells to MHC class II encountered in the thymic medulla. Experiments with murine BM chimeras demonstrate that tuning can be mediated by MHC class II expressed by either thymic medullary epithelial cells or thymic dendritic cells. Tuning does not require the engagement of CD4 by MHC class II on stromal cells. Rather, it is mediated by interactions between MHC class II and the TCR. To understand the molecular changes that distinguish immature hyperactive T cells from tuned mature CD4(+) T cells, we compared their responses to TCR stimulation. The altered response of mature CD4 single-positive thymocytes is characterized by the inhibition of ERK activation by low-affinity self-ligands and increased expression of the inhibitory tyrosine phosphatase SHP-1. Thus, persistent TCR engagement by peptide-MHC class II on thymic medullary stroma inhibits reactivity to self-Ags and prevents autoreactivity in the mature repertoire.

Eradication of Large Solid Tumors by Gene Therapy with a T-Cell Receptor Targeting a Single Cancer-Specific Point Mutation.

PubMed

Leisegang, Matthias; Engels, Boris; Schreiber, Karin; Yew, Poh Yin; Kiyotani, Kazuma; Idel, Christian; Arina, Ainhoa; Duraiswamy, Jaikumar; Weichselbaum, Ralph R; Uckert, Wolfgang; Nakamura, Yusuke; Schreiber, Hans

2016-06-01

Cancers usually contain multiple unique tumor-specific antigens produced by single amino acid substitutions (AAS) and encoded by somatic nonsynonymous single nucleotide substitutions. We determined whether adoptively transferred T cells can reject large, well-established solid tumors when engineered to express a single type of T-cell receptor (TCR) that is specific for a single AAS. By exome and RNA sequencing of an UV-induced tumor, we identified an AAS in p68 (mp68), a co-activator of p53. This AAS seemed to be an ideal tumor-specific neoepitope because it is encoded by a trunk mutation in the primary autochthonous cancer and binds with highest affinity to the MHC. A high-avidity mp68-specific TCR was used to genetically engineer T cells as well as to generate TCR-transgenic mice for adoptive therapy. When the neoepitope was expressed at high levels and by all cancer cells, their direct recognition sufficed to destroy intratumor vessels and eradicate large, long-established solid tumors. When the neoepitope was targeted as autochthonous antigen, T cells caused cancer regression followed by escape of antigen-negative variants. Escape could be thwarted by expressing the antigen at increased levels in all cancer cells or by combining T-cell therapy with local irradiation. Therapeutic efficacies of TCR-transduced and TCR-transgenic T cells were similar. Gene therapy with a single TCR targeting a single AAS can eradicate large established cancer, but a uniform expression and/or sufficient levels of the targeted neoepitope or additional therapy are required to overcome tumor escape. Clin Cancer Res; 22(11); 2734-43. ©2015 AACRSee related commentary by Liu, p. 2602. ©2015 American Association for Cancer Research.

Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity

PubMed Central

Cole, David K.; Bulek, Anna M.; Dolton, Garry; Schauenberg, Andrea J.; Szomolay, Barbara; Trimby, Andrew; Jothikumar, Prithiviraj; Fuller, Anna; Skowera, Ania; Rossjohn, Jamie; Zhu, Cheng; Miles, John J.; Wooldridge, Linda; Rizkallah, Pierre J.; Sewell, Andrew K.

2016-01-01

The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease. PMID:27183389

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

PubMed Central

Nunes-Alves, Cláudio; Booty, Matthew G.; Carpenter, Stephen M.; Rothchild, Alissa C.; Martin, Constance J.; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N.; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M.

2015-01-01

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity. PMID:25945999

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection.

PubMed

Nunes-Alves, Cláudio; Booty, Matthew G; Carpenter, Stephen M; Rothchild, Alissa C; Martin, Constance J; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M

2015-05-01

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

TCR signaling by conventional CD4+ T cells is required for optimal maintenance of peripheral regulatory T cell numbers.

PubMed

Leichner, Theresa M; Satake, Atsushi; Kambayashi, Taku

2016-06-01

To maintain immune tolerance, regulatory T cell (Treg) numbers must be closely indexed to the number of conventional T cells (Tconvs) so that an adequate Treg:Tconv ratio can be maintained. Two factors important in this process are the cytokine interleukin-2 (IL-2) and T cell receptor (TCR) stimulation by major histocompatibility complex class II (MHC-II). Here, we report that in addition to TCR stimulation of Tregs themselves, the maintenance of Tregs also requires TCR signaling by Tconvs. We found that Tconvs produce IL-2 in response to self-peptide-MHC-II complexes and that Tconvs possessing more highly self-reactive TCRs express more IL-2 at baseline. Furthermore, selective disruption of TCR signaling in Tconvs led to a trend toward decreased expression of IL-2 and attenuated their ability to maintain Treg numbers. These data suggest that in order to maintain an adequate Treg:Tconv ratio, Tregs are continuously indexed to self-peptide-MHC-II-induced TCR signaling of Tconvs. These results have implications in attempts to modulate immune tolerance, as Treg numbers adjust to the self-reactivity, and ultimately IL-2 production by the T cells around them.

Specific T-cell activation in an unspecific T-cell repertoire.

PubMed

Van Den Berg, Hugo A; Molina-París, Carmen; Sewell, Andrew K

2011-01-01

T-cells are a vital type of white blood cell that circulate around our bodies, scanning for cellular abnormalities and infections. They recognise disease-associated antigens via a surface receptor called the T-cell antigen receptor (TCR). If there were a specific TCR for every single antigen, no mammal could possibly contain all the T-cells it needs. This is clearly absurd and suggests that T-cell recognition must, to the contrary, be highly degenerate. Yet highly promiscuous TCRs would appear to be equally impossible: they are bound to recognise self as well as non-self antigens. We review how contributions from mathematical analysis have helped to resolve the paradox of the promiscuous TCR. Combined experimental and theoretical work shows that TCR degeneracy is essentially dynamical in nature, and that the T-cell can differentially adjust its functional sensitivity to the salient epitope, "tuning up" sensitivity to the antigen associated with disease and "tuning down" sensitivity to antigens associated with healthy conditions. This paradigm of continual modulation affords the TCR repertoire, despite its limited numerical diversity, the flexibility to respond to almost any antigenic challenge while avoiding autoimmunity.

The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

NASA Astrophysics Data System (ADS)

Xia, Zhen; Chen, Huabiao; Kang, Seung-Gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

2014-02-01

Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function.

The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

PubMed Central

Xia, Zhen; Chen, Huabiao; Kang, Seung-gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

2014-01-01

Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function. PMID:24522437

Proteasome inhibition blocks NF-κB and ERK1/2 pathways, restores antigen expression and sensitizes resistant human melanoma to TCR-engineered CTLs

PubMed Central

Jazirehi, Ali R.; Economou, James S.

2012-01-01

Adoptive cell transfer (ACT) of ex vivo engineered autologous lymphocytes encoding high-affinity MART-1/HLA-A*0201-specific T-cell receptor (TCR) α/β chains (F5 CTL), densely infiltrate into sites of metastatic disease, mediating dramatic but partial clinical responses in melanoma patients. We hypothesized that MART-1 down-modulation in addition to aberrant apoptotic/survival signaling could confer resistance to death signals delivered by transgenic CTLs. To explore this hypothesis, we established an in vitro model of resistant (R) lines from MART-1+/HLA-A*0201+ F5 CTL-sensitive parental (P) lines under serial F5 CTL-selective pressure. We have recently reported that several melanoma R lines, while retaining MART-1 expression, exhibited constitutive NF-κB activation and over-expression of NF-κB-dependent resistance factors. Another established melanoma cell line M244, otherwise sensitive to F5 CTL, yielded R lines after serial F5 CTL selective pressure which had both reduced MART-1 expression levels, thus, could not be recognized, and were resistant to CTL-delivered apoptotic death signals. The proteasome inhibitor bortezomib blocked NF-κB activity, decreased phopspho-ERK1/2, increased phospho-JNK levels, reduced expression of resistance-factors, restored MART-1 expression to sufficient levels, which in combination allowed M244R lines be sensitized to F5 CTL-killing. These findings suggest that proteasome inhibition in immune resistant tumors can restore proapoptotic signaling and improve tumor antigen expression. PMID:22532603

Sodium chloride inhibits IFN-γ, but not IL-4, production by invariant NKT cells.

PubMed

Jeong, Dongjin; Kim, Hye Young; Chung, Doo Hyun

2018-01-01

Invariant NKT (iNKT) cells are a distinct subset of T cells that exert Janus-like functions in vivo by producing IFN-γ and IL-4. Sodium chloride modulates the functions of various immune cells, including conventional CD4 + T cells and macrophages. However, it is not known whether sodium chloride affects iNKT cell function, so we addressed this issue. Sodium chloride inhibited IFN-γ, but not IL-4, production by iNKT cells upon TCR or TCR-independent (IL-12 and IL-18) stimulation in a dose-dependent manner. Consistently, sodium chloride reduced the expression level of tbx21, but not gata-3, in iNKT cells stimulated with TCR engagement or IL-12 + IL-18. Sodium chloride increased phosphorylated p38 expression in iNKT cells and inhibitors of p38, NFAT5, SGK1, and TCF-1 restored IFN-γ production by iNKT cells stimulated with sodium chloride and TCR engagement. Furthermore, adoptive transfer of iNKT cells pretreated with sodium chloride restored antibody-induced joint inflammation to a lesser extent than for untreated iNKT cells in Jα18 knockout mice. These findings suggest that sodium chloride inhibits IFN-γ production by iNKT cells in TCR-dependent and TCR-independent manners, which is dependent on p38, NFAT5, SGK1, and TCF-1. These findings highlight the functional role of sodium chloride in iNKT cell-mediated inflammatory diseases. ©2017 Society for Leukocyte Biology.

HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation.

PubMed

Abraham, Libin; Bankhead, Peter; Pan, Xiaoyu; Engel, Ulrike; Fackler, Oliver T

2012-08-15

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.

How nonuniform contact profiles of T cell receptors modulate thymic selection outcomes

NASA Astrophysics Data System (ADS)

Chen, Hanrong; Chakraborty, Arup K.; Kardar, Mehran

2018-03-01

T cell receptors (TCRs) bind foreign or self-peptides attached to major histocompatibility complex (MHC) molecules, and the strength of this interaction determines T cell activation. Optimizing the ability of T cells to recognize a diversity of foreign peptides yet be tolerant of self-peptides is crucial for the adaptive immune system to properly function. This is achieved by selection of T cells in the thymus, where immature T cells expressing unique, stochastically generated TCRs interact with a large number of self-peptide-MHC; if a TCR does not bind strongly enough to any self-peptide-MHC, or too strongly with at least one self-peptide-MHC, the T cell dies. Past theoretical work cast thymic selection as an extreme value problem and characterized the statistical enrichment or depletion of amino acids in the postselection TCR repertoire, showing how T cells are selected to be able to specifically recognize peptides derived from diverse pathogens yet have limited self-reactivity. Here, we investigate how the diversity of the postselection TCR repertoire is modified when TCRs make nonuniform contacts with peptide-MHC. Specifically, we were motivated by recent experiments showing that amino acids at certain positions of a TCR sequence have large effects on thymic selection outcomes, and crystal structure data that reveal a nonuniform contact profile between a TCR and its peptide-MHC ligand. Using a representative TCR contact profile as an illustration, we show via simulations that the statistical enrichment or depletion of amino acids now varies by position according to the contact profile, and, importantly, it depends on the implementation of nonuniform contacts during thymic selection. We explain these nontrivial results analytically. Our study has implications for understanding the selection forces that shape the functionality of the postselection TCR repertoire.

TCRs Used in Cancer Gene Therapy Cross-React with MART-1/Melan-A Tumor Antigens via Distinct Mechanisms

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

Borbulevych, Oleg Y.; Santhanagopolan, Sujatha M.; Hossain, Moushumi

2013-09-18

T cells engineered to express TCRs specific for tumor Ags can drive cancer regression. The first TCRs used in cancer gene therapy, DMF4 and DMF5, recognize two structurally distinct peptide epitopes of the melanoma-associated MART-1/Melan-A protein, both presented by the class I MHC protein HLA-A*0201. To help understand the mechanisms of TCR cross-reactivity and provide a foundation for the further development of immunotherapy, we determined the crystallographic structures of DMF4 and DMF5 in complex with both of the MART-1/Melan-A epitopes. The two TCRs use different mechanisms to accommodate the two ligands. Although DMF4 binds the two with a different orientation,more » altering its position over the peptide/MHC, DMF5 binds them both identically. The simpler mode of cross-reactivity by DMF5 is associated with higher affinity toward both ligands, consistent with the superior functional avidity of DMF5. More generally, the observation of two diverging mechanisms of cross-reactivity with the same Ags and the finding that TCR-binding orientation can be determined by peptide alone extend our understanding of the mechanisms underlying TCR cross-reactivity.« less

T cell receptors used in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens via distinct mechanisms1

PubMed Central

Borbulevych, Oleg Y.; Santhanagopolan, Sujatha M.; Hossain, Moushumi; Baker, Brian M.

2011-01-01

T cells engineered to express T cell receptors (TCRs) specific for tumor antigens can drive cancer regression. The first TCRs used in cancer gene therapy, DMF4 and DMF5, recognize two structurally distinct peptide epitopes of the melanoma-associated MART-1/Melan-A protein, both presented by the class I MHC protein HLA-A*0201. To help understand the mechanisms of TCR cross-reactivity and provide a foundation for the further development of immunotherapy, we determined the crystallographic structures of DMF4 and DMF5 in complex with both of the MART-1/Melan-A epitopes. The two TCRs use different mechanisms to accommodate the two ligands. Whereas DMF4 binds the two with a different orientation, altering its position over the peptide/MHC, DMF5 binds them both identically. The simpler mode of cross-reactivity by DMF5 is associated with higher affinity towards both ligands, consistent with the superior functional avidity of DMF5. More generally, the observation of two diverging mechanisms of cross-reactivity with the same antigens and the finding that TCR binding orientation can be determined by peptide alone extend our understanding of the mechanisms underlying TCR cross-reactivity. PMID:21795600

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. Copyright © 2016 by The American Association of Immunologists, Inc.

Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

PubMed

Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Vázquez-Chávez, Elena; Lasserre, Rémi; Agüera-González, Sonia; Cuche, Céline; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

2017-04-01

The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production. Copyright © 2017 by The American Association of Immunologists, Inc.

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

PubMed Central

Dunn, Steven M.; Rizkallah, Pierre J.; Baston, Emma; Mahon, Tara; Cameron, Brian; Moysey, Ruth; Gao, Feng; Sami, Malkit; Boulter, Jonathan; Li, Yi; Jakobsen, Bent K.

2006-01-01

The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential. PMID:16600963

Functional and molecular alterations in T Cells induced by CCL5.

PubMed

Cridge, T J; Horowitz, K M; Marinucci, M N; Rose, K M; Wells, M; Werner, M T; Kurt, Robert A

2006-01-01

To delineate whether, and the extent to which, CCL5 could impact T cell function we examined cytokine production and proliferative ability following CCL5 treatment in vitro. We report a decreased ability of splenic T cells to produce IFN-? and TNF-a as well as proliferate in response to crosslinking with antibody to CD3 after 72, but not 24 hours of CCL5 exposure. To identify a mechanism by which CCL5 modulated T cell function, we examined T cell receptor translocation and lipid raft clustering. After exposure to CCL5, T cells were less efficient at translocating the TCR and clustering lipid rafts. Since TCR translocation and lipid raft clustering are required for creation of an immunological synapse, these data suggest that extended exposure to CCL5 may impact T cell effector function by modulating the ability to create a functional immunological synapse.

Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules.

PubMed

Jin, Jianjian; Gkitsas, Nikolaos; Fellowes, Vicki S; Ren, Jiaqiang; Feldman, Steven A; Hinrichs, Christian S; Stroncek, David F; Highfill, Steven L

2018-01-24

Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64-92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.

In-depth Characterization of a TCR-specific Tracer for Sensitive Detection of Tumor-directed Transgenic T Cells by Immuno-PET.

PubMed

Yusufi, Nahid; Mall, Sabine; Bianchi, Henrique de Oliveira; Steiger, Katja; Reder, Sybille; Klar, Richard; Audehm, Stefan; Mustafa, Mona; Nekolla, Stephan; Peschel, Christian; Schwaiger, Markus; Krackhardt, Angela M; D'Alessandria, Calogero

2017-01-01

A number of different technologies have been developed to monitor in vivo the distribution of gene-modified T cells used in immunotherapy. Nevertheless, in-depth characterization of novel approaches with respect to sensitivity and clinical applicability are so far missing. We have previously described a novel method to track engineered human T cells in tumors using 89 Zr-Df-aTCRmu-F(ab') 2 targeting the murinized part of the TCR beta domain (TCRmu) of a transgenic TCR. Here, we performed an in-depth in vitro characterization of the tracer in terms of antigen affinity, immunoreactivity, influence on T-cell functionality and stability in vitro and in vivo . Of particular interest, we have developed diverse experimental settings to quantify TCR-transgenic T cells in vivo . Local application of 89 Zr-Df-aTCRmu-F(ab') 2 -labeled T cells in a spot-assay revealed signal detection down to approximately 1.8x10 4 cells. In a more clinically relevant model, NSG mice were intravenously injected with different numbers of transgenic T cells, followed by injection of the 89 Zr-Df-aTCRmu-F(ab') 2 tracer, PET/CT imaging and subsequent ex vivo T-cell quantification in the tumor. Using this setting, we defined a comparable detection limit of 1.0x10 4 T cells. PET signals correlated well to total numbers of transgenic T cells detected ex vivo independently of the engraftment rates observed in different individual experiments. Thus, these findings confirm the high sensitivity of our novel PET/CT T-cell tracking method and provide critical information about the quantity of transgenic T cells in the tumor environment suggesting our technology being highly suitable for further clinical translation.

Receptor revision in CD4 T cells is influenced by follicular helper T cell formation and germinal-center interactions.

PubMed

Higdon, Lauren E; Deets, Katherine A; Friesen, Travis J; Sze, Kai-Yin; Fink, Pamela J

2014-04-15

Peripheral CD4 T cells in Vβ5 transgenic (Tg) C57BL/6J mice undergo tolerance to an endogenous superantigen encoded by mouse mammary tumor virus 8 (Mtv-8) by either deletion or T-cell receptor (TCR) revision. Revision is a process by which surface expression of the Vβ5(+) TCR is down-regulated in response to Mtv-8 and recombination activating genes are expressed to drive rearrangement of the endogenous TCRβ locus, effecting cell rescue through the expression of a newly generated, non-self-reactive TCR. In an effort to identify the microenvironment in which revision takes place, we show here that the proportion of T follicular helper cells (Tfh) and production of high-affinity antibody during a primary response are increased in Vβ5 Tg mice in an Mtv-8-dependent manner. Revising T cells have a Tfh-like surface phenotype and transcription factor profile, with elevated expression of B-cell leukemia/lymphoma 6 (Bcl-6), CXC chemokine receptor 5, programmed death-1, and other Tfh-associated markers. Efficient revision requires Bcl-6 and is inhibited by B lymphocyte-induced maturation protein-1. Revision completes less efficiently in the absence of signaling lymphocytic activation molecule-associated protein although initiation proceeds normally. These data indicate that Tfh formation is required for the initiation of revision and germinal-center interactions for its completion. The germinal center is known to provide a confined space in which B-cell antigen receptors undergo selection. Our data extend the impact of this selective microenvironment into the arena of T cells, suggesting that this fluid structure also provides a regulatory environment in which TCR revision can safely take place.

Molecular analysis of antigen-independent adhesion forces between T and B lymphocytes.

PubMed Central

Amblard, F; Auffray, C; Sekaly, R; Fischer, A

1994-01-01

The low-affinity interactions underlying antigen recognition by T-cell receptors (TCRs) are thought to involve antigen-independent adhesion mechanisms. Using a hydrodynamic approach, we found that antigen-independent adhesion occurred between human B cells and resting T cells in a transient and temperature-dependent fashion. The mean cell-cell adhesion force was 0.32 x 10(-9) N and was generated by similar contributions (0.16 x 10(-9) N) of the LFA-1- and CD2-dependent adhesion pathways. After T-cell stimulation with a phorbol ester, the force contributed by LFA-1 was drastically increased, while that of CD2 was unaffected. We propose that weak receptor-mediated adhesion initiates antigen-independent intercellular contacts required for antigen recognition by the TCR and is upregulated following TCR engagement. The method used permits adhesion forces between living cells to be resolved at the molecular level and should prove valuable for the rapid assessment of interaction forces between various types of cells and cell-sized particles. Images PMID:7909604

Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling.

PubMed

Brdicka, Tomás; Imrich, Martin; Angelisová, Pavla; Brdicková, Nadezda; Horváth, Ondrej; Spicka, Jirí; Hilgert, Ivan; Lusková, Petra; Dráber, Petr; Novák, Petr; Engels, Niklas; Wienands, Jürgen; Simeoni, Luca; Osterreicher, Jan; Aguado, Enrique; Malissen, Marie; Schraven, Burkhart; Horejsí, Václav

2002-12-16

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.

Minocycline blocks asthma-associated inflammation in part by interfering with the T cell receptor-nuclear factor κB-GATA-3-IL-4 axis without a prominent effect on poly(ADP-ribose) polymerase.

PubMed

Naura, Amarjit S; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C; Jordan, Joaquin; Catling, Andrew D; Rezk, Bashir M; Abd Elmageed, Zakaria Y; Pyakurel, Kusma; Tarhuni, Abdelmetalab F; Abughazleh, Mohammad Q; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C; Boulares, A Hamid

2013-01-18

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

Minocycline Blocks Asthma-associated Inflammation in Part by Interfering with the T Cell Receptor-Nuclear Factor κB-GATA-3-IL-4 Axis without a Prominent Effect on Poly(ADP-ribose) Polymerase*

PubMed Central

Naura, Amarjit S.; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C.; Jordan, Joaquin; Catling, Andrew D.; Rezk, Bashir M.; Elmageed, Zakaria Y. Abd; Pyakurel, Kusma; Tarhuni, Abdelmetalab F.; Abughazleh, Mohammad Q.; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C.; Boulares, A. Hamid

2013-01-01

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N′-nitro-N-nitroso-guanidine-treated mice or H2O2-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production. PMID:23184953

A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14-3-3 proteins on SLP-76.

PubMed

Di Bartolo, Vincenzo; Montagne, Benjamin; Salek, Mogjiborahman; Jungwirth, Britta; Carrette, Florent; Fourtane, Julien; Sol-Foulon, Nathalie; Michel, Frédérique; Schwartz, Olivier; Lehmann, Wolf D; Acuto, Oreste

2007-03-19

The SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is a pivotal element of the signaling machinery controlling T cell receptor (TCR)-mediated activation. Here, we identify 14-3-3epsilon and zeta proteins as SLP-76 binding partners. This interaction was induced by TCR ligation and required phosphorylation of SLP-76 at serine 376. Ribonucleic acid interference and in vitro phosphorylation experiments showed that serine 376 is the target of the hematopoietic progenitor kinase 1 (HPK-1). Interestingly, either S376A mutation or HPK-1 knockdown resulted in increased TCR-induced tyrosine phosphorylation of SLP-76 and phospholipase C-gamma1. Moreover, an SLP-76-S376A mutant induced higher interleukin 2 gene transcription than wild-type SLP-76. These data reveal a novel negative feedback loop involving HPK-1-dependent serine phosphorylation of SLP-76 and 14-3-3 protein recruitment, which tunes T cell activation.

The immunological synapse as a pharmacological target.

PubMed

Francesca, Finetti; Baldari, Cosima T

2018-06-10

The development of T cell mediated immunity relies on the assembly of a highly specialized interface between T cell and antigen presenting cell (APC), known as the immunological synapse (IS). IS assembly is triggered when the T cell receptor (TCR) binds to specific peptide antigen presented in association to the major histocompatibility complex (MHC) by the APC, and is followed by the spatiotemporal dynamic redistribution of TCR, integrins, co-stimulatory receptors and signaling molecules, allowing for the fine-tuning and integration of the signals that lead to T cell activation. The knowledge acquired to date about the mechanisms of IS assembly underscores this structure as a robust pharmacological target. The activity of molecules involved in IS assembly and function can be targeted by specific compounds to modulate the immune response in a number of disorders, including cancers and autoimmune diseases, or in transplanted patients. Here, we will review the state-of-the art of the current therapies which exploit the IS to modulate the immune response. Copyright © 2018. Published by Elsevier Ltd.

Enhanced Detection of Antigen-Specific CD4+ T Cells Using Altered Peptide Flanking Residue Peptide–MHC Class II Multimers

PubMed Central

Holland, Christopher J.; Dolton, Garry; Scurr, Martin; Ladell, Kristin; Schauenburg, Andrea J.; Miners, Kelly; Madura, Florian; Sewell, Andrew K.; Price, David A.

2015-01-01

Fluorochrome-conjugated peptide–MHC (pMHC) class I multimers are staple components of the immunologist’s toolbox, enabling reliable quantification and analysis of Ag-specific CD8+ T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4+ T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II–bound peptides, can enhance TCR–pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4+ T cells, highlighting an unappreciated feature of TCR–pMHC-II interactions. PMID:26553072

Activation-induced proteolysis of cytoplasmic domain of zeta in T cell receptors and Fc receptors.

PubMed

Taupin, J L; Anderson, P

1994-12-01

The CD3-T cell receptor (TCR) complex on T cells and the Fc gamma receptor type III (Fc gamma RIII)-zeta-gamma complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits zeta and gamma. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized zeta-p14 heterodimer. zeta-p14 is a component of both CD3-TCR and Fc gamma RIII-zeta-gamma. Peptide mapping analysis shows that p14 is structurally related to zeta, suggesting that it is either: (i) derived from zeta proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding zeta express zeta-p14 supports the former possibility. The expression of CD3-TCR complexes including zeta-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of zeta may contribute to receptor modulation or desensitization.

HCV T Cell Receptor Chain Modifications to Enhance Expression, Pairing, and Antigen Recognition in T Cells for Adoptive Transfer.

PubMed

Foley, Kendra C; Spear, Timothy T; Murray, David C; Nagato, Kaoru; Garrett-Mayer, Elizabeth; Nishimura, Michael I

2017-06-16

T cell receptor (TCR)-gene-modified T cells for adoptive cell transfer can mediate objective clinical responses in melanoma and other malignancies. When introducing a second TCR, mispairing between the endogenous and introduced α and β TCR chains limits expression of the introduced TCR, which can result in impaired efficacy or off-target reactivity and autoimmunity. One approach to promote proper TCR chain pairing involves modifications of the introduced TCR genes: introducing a disulfide bridge, substituting murine for human constant regions, codon optimization, TCR chain leucine zipper fusions, and a single-chain TCR. We have introduced these modifications into our hepatitis C virus (HCV) reactive TCR and utilize a marker gene, CD34t, which allows us to directly compare transduction efficiency with TCR expression and T cell function. Our results reveal that of the TCRs tested, T cells expressing the murine Cβ2 TCR or leucine zipper TCR have the highest levels of expression and the highest percentage of lytic and interferon-γ (IFN-γ)-producing T cells. Our studies give us a better understanding of how TCR modifications impact TCR expression and T cell function that may allow for optimization of TCR-modified T cells for adoptive cell transfer to treat patients with malignancies.

The inability to disrupt the immunological synapse between infected human T cells and APCs distinguishes HIV-1 from most other primate lentiviruses

PubMed Central

Arhel, Nathalie; Lehmann, Martin; Clauß, Karen; Nienhaus, G. Ulrich; Piguet, Vincent; Kirchhoff, Frank

2009-01-01

Viruses that infect T cells, including those of the lentivirus genus, such as HIV-1, modulate the responsiveness of infected T cells to stimulation by interacting APCs in a manner that renders the T cells more permissive for viral replication. HIV-1 and other primate lentiviruses use their Nef proteins to manipulate the T cell/APC contact zone, the immunological synapse (IS). It is known that primate lentiviral Nef proteins differ substantially in their ability to modulate cell surface expression of the TCR-CD3 and CD28 receptors critical for the formation and function of the IS. However, the impact of these differences in Nef function on the interaction and communication between virally infected T cells and primary APCs has not been investigated. Here we have used primary human cells to show that Nef proteins encoded by HIV-2 and most SIVs, which downmodulate cell surface expression of TCR-CD3, disrupt formation of the IS between infected T cells and Ag-presenting macrophages or DCs. In contrast, nef alleles from HIV-1 and its simian precursor SIVcpz failed to suppress synapse formation and events downstream of TCR signaling. Our data suggest that most primate lentiviruses disrupt communication between virally infected CD4+ Th cells and APCs, whereas HIV-1 and its SIV precursor have largely lost this capability. The resulting differences in the levels of T cell activation and apoptosis may play a role in the pathogenesis of AIDS. PMID:19759518

Molecular basis of mycobacterial lipid antigen presentation by CD1c and its recognition by αβ T cells

PubMed Central

Roy, Sobhan; Ly, Dalam; Li, Nan-Sheng; Altman, John D.; Piccirilli, Joseph A.; Moody, D. Branch; Adams, Erin J.

2014-01-01

CD1c is a member of the group 1 CD1 family of proteins that are specialized for lipid antigen presentation. Despite high cell surface expression of CD1c on key antigen-presenting cells and the discovery of its mycobacterial lipid antigen presentation capability, the molecular basis of CD1c recognition by T cells is unknown. Here we present a comprehensive functional and molecular analysis of αβ T-cell receptor (TCR) recognition of CD1c presenting mycobacterial phosphomycoketide antigens. Our structure of CD1c with the mycobacterial phosphomycoketide (PM) shows similarities to that of CD1c-mannosyl-β1-phosphomycoketide in that the A' pocket accommodates the mycoketide alkyl chain; however, the phosphate head-group of PM is shifted ∼6 Å in relation to that of mannosyl-β1-PM. We also demonstrate a bona fide interaction between six human TCRs and CD1c-mycoketide complexes, measuring high to moderate affinities. The crystal structure of the DN6 TCR and mutagenic studies reveal a requirement of five complementarity determining region (CDR) loops for CD1c recognition. Furthermore, mutagenesis of CD1c reveals residues in both the α1 and α2 helices involved in TCR recognition, yet not entirely overlapping among the examined TCRs. Unlike patterns for MHC I, no archetypical binding footprint is predicted to be shared by CD1c-reactive TCRs, even when recognizing the same or similar antigens. PMID:25298532

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

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

Bhattacharya, Monolekha; Das, Amit Kumar, E-mail: amitk@hijli.iitkgp.ernet.in

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 notmore » 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.« less

Modulation of T-cell receptor functional sensitivity via the opposing actions of protein tyrosine kinases and phosphatases: a mathematical model.

PubMed

Szomolay, Barbara; van den Berg, Hugo A

2014-12-01

Combining receptor kinetics and stochastic modelling of receptor activation, we show that a T-cell can specifically augment its functional sensitivity to one particular peptide ligand while simultaneously decreasing its sensitivity to other ligands, by coordinating the expression levels of the co-receptor CD8 and the relative activities of kinases and phosphatases in the vicinity of the T-cell receptor (TCR). We propose that this focusable degeneracy of epitope recognition allows a TCR to have a wide range of potential ligands but be specifically sensitive to only one or a few of these at any one time, which resolves the paradox of how a relatively small number of clones (∼10(6)) can maintain the potential to respond to a vast space of ligands (∼20(9)) whilst avoiding auto-immunity. We validate the model against experimental data and predict shifts in functional sensitivity following a shift in the kinase/phosphatase balance (which could in principle be induced by experimental means). Moreover, we propose that in vivo, the T-cell gauges ligand quality by monitoring changes in TCR triggering rate concomitant with shifts in this balance, for instance as the immunological synapse matures.

T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy.

PubMed

Roszik, János; Sebestyén, Zsolt; Govers, Coen; Guri, Yakir; Szöor, Arpád; Pályi-Krekk, Zsuzsanna; Vereb, György; Nagy, Peter; Szöllosi, János; Debets, Reno

2011-05-01

T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and β chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Affine cellularity of finite type KLR algebras, and homomorphisms between Specht modules for KLR algebras in affine type A

NASA Astrophysics Data System (ADS)

Loubert, Joseph William

This thesis consists of two parts. In the first we prove that the Khovanov-Lauda-Rouquier algebras Ralpha of finite type are (graded) affine cellular in the sense of Koenig and Xi. In fact, we establish a stronger property, namely that the affine cell ideals in Ralpha are generated by idempotents. This in particular implies the (known) result that the global dimension of Ralpha is finite. In the second part we use the presentation of the Specht modules given by Kleshchev-Mathas-Ram to derive results about Specht modules. In particular, we determine all homomorphisms from an arbitrary Specht module to a fixed Specht module corresponding to any hook partition. Along the way, we give a complete description of the action of the standard KLR generators on the hook Specht module. This work generalizes a result of James. This dissertation includes previously published coauthored material.

Nasal-type NK/T-cell lymphomas are more frequently T rather than NK lineage based on T-cell receptor gene, RNA, and protein studies: lineage does not predict clinical behavior.

PubMed

Hong, Mineui; Lee, Taehee; Young Kang, So; Kim, Suk-Jin; Kim, Wonseog; Ko, Young-Hyeh

2016-05-01

Extranodal natural killer (NK)/T-cell lymphoma (ENKTL), nasal type, comprises NK or cytotoxic T cells. We evaluated the clinical impact of cell type and the usefulness of T-cell receptor (TCR) gene transcripts in distinguishing cell lineage. One hundred and eight cases of ENKTL were analyzed for TCR gene rearrangements using the BIOMED-2 protocol and for TCR gene expression using immunohistochemistry for TCR-βF1 and TCR-cγM1, and RNA in situ hybridization for TCR gene transcripts. Prognostic factors were analyzed. Among the 108 cases, 44 were monoclonal for a TCR rearrangement (40%) while 64 (60%) were undefinable. The monoclonal cases expressed TCR-βF1 in 14 out of 40 cases (35%) and TCR-cγM1 in 1 out of 44 cases (2%). The 64 undetermined cases expressed TCR-βF1 in 15 cases (23%) and TCR-cγM1 in 1 (2%). Thirteen of 40 TCR-β constant gene transcript-positive cases (33%) expressed TCR-βF1 and one of nine TCR-γ constant gene transcript-positive cases (11%) expressed TCR-cγM1. TCR gene transcripts were not useful in the distinction of cell lineages. TCR gene transcripts were positive in ENKTLs as well as in normal B cells and aggressive NK-cell leukemia. Based on gene rearrangements and immunohistochemistry for TCR, there were 60 T-cell type cases (56%), 32 NK-cell type cases (30%), and 16 cases with an undetermined cell type (14%). TCR protein was expressed in 30/60 T-ENKTLs (50%) in a variable fraction of tumor cells. There were no significant differences in clinical findings or overall patient survival between T- or NK-cell types of ENKTL, although those with a T-cell type tended to show a better prognosis for those with localized nasal lymphomas. Univariate and multivariate analysis showed that a non-nasal ENKTL, age >60 years, high level of lactate dehydrogenase, bone marrow involvement, and the absence of radiotherapy were independent prognostic factors.

A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

PubMed Central

Thie, Holger

2017-01-01

ABSTRACT Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs. PMID:28055297

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.

Biologically relevant conformational features of linear and cyclic proteolipid protein (PLP) peptide analogues obtained by high-resolution nuclear magnetic resonance and molecular dynamics

NASA Astrophysics Data System (ADS)

Kordopati, Golfo G.; Tzoupis, Haralambos; Troganis, Anastassios N.; Tsivgoulis, Gerasimos M.; Golic Grdadolnik, Simona; Simal, Carmen; Tselios, Theodore V.

2017-09-01

Proteolipid protein (PLP) is one of the main proteins of myelin sheath that are destroyed during the progress of multiple sclerosis (MS). The immunodominant PLP139-151 epitope is known to induce experimental autoimmune encephalomyelitis (EAE, animal model of MS), wherein residues 144 and 147 are recognized by T cell receptor (TCR) during the formation of trimolecular complex with peptide-antigen and major histocompability complex. The conformational behavior of linear and cyclic peptide analogues of PLP, namely PLP139-151 and cyclic (139-151) (L144, R147) PLP139-151, have been studied in solution by means of nuclear magnetic resonance (NMR) methods in combination with unrestrained molecular dynamics simulations. The results indicate that the side chains of mutated amino acids in the cyclic analogue have different spatial orientation compared with the corresponding side chains of the linear analogue, which can lead to reduced affinity to TCR. NMR experiments combined with theoretical calculations pave the way for the design and synthesis of potent restricted peptides of immunodominant PLP139-151 epitope as well as non peptide mimetics that rises as an ultimate goal.

Modulation of DNA binding by gene-specific transcription factors.

PubMed

Schleif, Robert F

2013-10-01

The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

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

Immunochemical Proof that a Novel Rearranging Gene Encodes the T Cell Receptor δ Subunit

NASA Astrophysics Data System (ADS)

Band, Hamid; Hochstenbach, Frans; McLean, Joanne; Hata, Shingo; Krangel, Michael S.; Brenner, Michael B.

1987-10-01

The T cell receptor (TCR) δ protein is expressed as part of a heterodimer with TCR γ , in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR δ was produced that binds specifically to the surface of several TCR γ δ cell lines and immunoprecipitates the TCR γ δ as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR δ subunit alone after chain separation. A candidate human TCR δ complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR γ δ cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR δ . This complementary DNA clone thus corresponds to the gene that encodes the TCR δ subunit.

Cutting Edge: TCR Revision Affects Predominantly Foxp3− Cells and Skews Them toward the Th17 Lineage1

PubMed Central

Zehn, Dietmar; Bevan, Michael J.; Fink, Pamela J.

2009-01-01

CD4+ T cells respond to peripheral endogenous superantigen stimulation by undergoing deletion or TCR revision. The latter involves RAG re-expression, TCR gene rearrangement, and expression of a novel TCR. TCR-revised T cells are functional and express a diverse TCR repertoire. Because TCR revision harbors the potential to create self-reactivity, it is important to explore whether T cells known to be self-reactive (regulatory T cells) or those involved in autoimmunity (Th17 cells) arise from TCR revision. Interestingly, we observed that Foxp3+ cells are excluded from revising their TCR and that only a small fraction of postrevision cells expresses Foxp3. In contrast, Th17 cells are 20 times more frequent among revised than among C57BL/6 CD4+ T cells, indicating that postrevision cells are biased toward the Th17 lineage. The link between Th17 differentiation and TCR revision might be highly relevant to the role of Th17 cells in promoting autoimmunity. PMID:17947636

Cell surface control of the multiubiquitination and deubiquitination of high-affinity immunoglobulin E receptors.

PubMed Central

Paolini, R; Kinet, J P

1993-01-01

Multiubiquitination of proteins is a critical step leading to selective degradation for many polypeptides. Therefore, activation-induced multiubiquitination of cell surface receptors, such as the platelet-derived growth factor (PDGF) receptor and the T cell antigen (TCR) receptor, may correspond to a degradation pathway for ligand-receptor complexes. Here we show that the antigen-induced engagement of high-affinity immunoglobulin E receptors (Fc epsilon RI) results in the immediate multiubiquitination of Fc epsilon RI beta and gamma chains. This ubiquitination is independent of receptor phosphorylation and is restricted to activated receptors. Surprisingly, receptor multiubiquitination is immediately reversible when receptors are disengaged. Therefore, multiubiquitination and deubiquitination of Fc epsilon RI receptors is controlled at the cell surface by receptor engagement and disengagement. The rapidity, specificity and, most importantly, the reversibility of the activation-induced receptor multiubiquitination suggest that this process may turn on/off a cell surface receptor signaling function thus far unsuspected. Images PMID:8382611

Dominant role of antigen dose in CD4+Foxp3+ regulatory T cell induction and expansion1

PubMed Central

Turner, Michael S.; Kane, Lawrence P.; Morel, Penelope A.

2009-01-01

The definitions of tolerogenic vs. immunogenic dendritic cells (DC) remain controversial. Immature DC have been shown to induce T regulatory cells (Treg) specific for foreign and allo-antigens. However, we have previously reported that mature DC (G4DC) prevented the onset of autoimmune diabetes whereas immature DC (GMDC) were therapeutically ineffective. In this study, islet-specific CD4+ T cells from BDC2.5 TCR Tg mice were stimulated, in the absence of exogenous cytokine, with GMDC or G4DC pulsed with high- or low-affinity antigenic peptides and examined for Treg induction. Both GMDC and G4DC presenting low peptide doses induced weak TCR signaling via the Akt/mTOR pathway, resulting in significant expansion of Foxp3+ Treg. Furthermore, unpulsed G4DC, but not GMDC, also induced Treg. High peptide doses induced strong Akt/mTOR signaling and favored the expansion of Foxp3neg Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 and OT-II TCR-Tg T cells and was recapitulated with anti-CD3/CD28 stimulation in the absence of DC. IL-6 production in these cultures correlated positively with antigen dose and inversely with Treg expansion. Studies with T cells or DC from IL-6−/− mice revealed that IL-6 production by T cells was more important in the inhibition of Treg induction at low antigen doses. These studies indicate that strength of Akt/mTOR signaling, a critical T cell intrinsic determinant for Treg vs Th induction, can be controlled by adjusting the dose of antigenic peptide. Furthermore, this operates in a dominant fashion over DC phenotype and cytokine production. PMID:19801514

iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo

PubMed Central

Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M.; Brennan, Patrick J.; Banerjee, Pinaki P.; Wiener, Susan J.; Orange, Jordan S.; Brenner, Michael B.; Grupp, Stephan A.; Nichols, Kim E.

2013-01-01

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we find that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially-induced by iNKT cell agonists of varying TCR affinities, such as OCH, α-galactosyl ceramide and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of T cell receptor (TCR) signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell-deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T-lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T-lymphoma. PMID:24563871

iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo .

PubMed

Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M; Brennan, Patrick J; Banerjee, Pinaki P; Wiener, Susan J; Orange, Jordan S; Brenner, Michael B; Grupp, Stephan A; Nichols, Kim E

2014-01-01

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we found that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially induced by iNKT cell agonists of varying T-cell receptor (TCR) affinities, such as OCH, α-galactosyl ceramide, and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of TCR signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell–deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T lymphoma. ©2013 AACR.

Asymptotic Representations of Quantum Affine Superalgebras

NASA Astrophysics Data System (ADS)

Zhang, Huafeng

2017-08-01

We study representations of the quantum affine superalgebra associated with a general linear Lie superalgebra. In the spirit of Hernandez-Jimbo, we construct inductive systems of Kirillov-Reshetikhin modules based on a cyclicity result that we established previously on tensor products of these modules, and realize their inductive limits as modules over its Borel subalgebra, the so-called q-Yangian. A new generic asymptotic limit of the same inductive systems is proposed, resulting in modules over the full quantum affine superalgebra. We derive generalized Baxter's relations in the sense of Frenkel-Hernandez for representations of the full quantum group.

MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells.

PubMed

Mukherjee, P; Tinder, T L; Basu, G D; Gendler, S J

2005-01-01

MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex.

NKT Cell Subsets Can Exert Opposing Effects in Autoimmunity, Tumor Surveillance and Inflammation

PubMed Central

Viale, Rachael; Ware, Randle; Maricic, Igor; Chaturvedi, Varun; Kumar, Vipin

2014-01-01

The innate-like natural killer T (NKT) cells are essential regulators of immunity. These cells comprise at least two distinct subsets and recognize different lipid antigens presented by the MHC class I like molecules CD1d. The CD1d-dependent recognition pathway of NKT cells is highly conserved from mouse to humans. While most type I NKT cells can recognize αGalCer and express a semi-invariant T cell receptor (TCR), a major population of type II NKT cells reactive to sulfatide utilizes an oligoclonal TCR. Furthermore TCR recognition features of NKT subsets are also distinctive with almost parallel as opposed to perpendicular footprints on the CD1d molecules for the type I and type II NKT cells respectively. Here we present a view based upon the recent studies in different clinical and experimental settings that while type I NKT cells are more often pathogenic, they may also be regulatory. On the other hand, sulfatide-reactive type II NKT cells mostly play an inhibitory role in the control of autoimmune and inflammatory diseases. Since the activity and cytokine secretion profiles of NKT cell subsets can be modulated differently by lipid ligands or their analogs, novel immunotherapeutic strategies are being developed for their differential activation for potential intervention in inflammatory diseases. PMID:25288922

CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells.

PubMed

Legut, Mateusz; Dolton, Garry; Mian, Afsar Ali; Ottmann, Oliver G; Sewell, Andrew K

2018-01-18

Adoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-β is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αβ and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR-modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer-reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αβ TCR resulted in more efficient redirection of CD4 + and CD8 + T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies. © 2018 by The American Society of Hematology.

Crystal structure of a complete ternary complex of T-cell receptor, peptide-MHC, and CD4

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

Yin, Yiyuan; Wang, Xin Xiang; Mariuzza, Roy A

2012-07-11

Adaptive immunity depends on specific recognition by a T-cell receptor (TCR) of an antigenic peptide bound to a major histocompatibility complex (pMHC) molecule on an antigen-presenting cell (APC). In addition, T-cell activation generally requires binding of this same pMHC to a CD4 or CD8 coreceptor. Here, we report the structure of a complete TCR-pMHC-CD4 ternary complex involving a human autoimmune TCR, a myelin-derived self-peptide bound to HLA-DR4, and CD4. The complex resembles a pointed arch in which TCR and CD4 are each tilted ~65° relative to the T-cell membrane. By precluding direct contacts between TCR and CD4, the structure explainsmore » how TCR and CD4 on the T cell can simultaneously, yet independently, engage the same pMHC on the APC. The structure, in conjunction with previous mutagenesis data, places TCR-associated CD3εγ and CD3εδ subunits, which transmit activation signals to the T cell, inside the TCR-pMHC-CD4 arch, facing CD4. By establishing anchor points for TCR and CD4 on the T-cell membrane, the complex provides a basis for understanding how the CD4 coreceptor focuses TCR on MHC to guide TCR docking on pMHC during thymic T-cell selection.« less

Cutting edge: rescue of pre-TCR but not mature TCR signaling in mice expressing membrane-targeted SLP-76.

PubMed

Bezman, Natalie A; Baker, Rebecca G; Lenox, Laurie E; Jordan, Martha S; Koretzky, Gary A

2009-05-01

SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) organizes signaling from immunoreceptors, including the platelet collagen receptor, the pre-TCR, and the TCR, and is required for T cell development. In this study we examine a mouse in which wild-type SLP-76 is replaced with a mutant constitutively targeted to the cell membrane. Membrane-targeted SLP-76 (MTS) supports ITAM signaling in platelets and from the pre-TCR. Signaling from the mature TCR, however, is defective in MTS thymocytes, resulting in failed T cell differentiation. Defective thymic selection by MTS is not rescued by a SLP-76 mutant whose localization is restricted to the cytosol. Thus, fixed localization of SLP-76 reveals differential requirements for the subcellular localization of signaling complexes downstream of the pre-TCR vs mature TCR.

γδT cells but not αβT cells contribute to sepsis-induced white matter injury and motor abnormalities in mice.

PubMed

Zhang, Xiaoli; Rocha-Ferreira, Eridan; Li, Tao; Vontell, Regina; Jabin, Darakhshan; Hua, Sha; Zhou, Kai; Nazmi, Arshed; Albertsson, Anna-Maj; Sobotka, Kristina; Ek, Joakim; Thornton, Claire; Hagberg, Henrik; Mallard, Carina; Leavenworth, Jianmei W; Zhu, Changlian; Wang, Xiaoyang

2017-12-20

Infection and sepsis are associated with brain white matter injury in preterm infants and the subsequent development of cerebral palsy. In the present study, we used a neonatal mouse sepsis-induced white matter injury model to determine the contribution of different T cell subsets (αβT cells and γδT cells) to white matter injury and consequent behavioral changes. C57BL/6J wild-type (WT), T cell receptor (TCR) δ-deficient (Tcrd -/- , lacking γδT cells), and TCRα-deficient (Tcra -/- , lacking αβT cells) mice were administered with lipopolysaccharide (LPS) at postnatal day (PND) 2. Brain myelination was examined at PNDs 12, 26, and 60. Motor function and anxiety-like behavior were evaluated at PND 26 or 30 using DigiGait analysis and an elevated plus maze. White matter development was normal in Tcrd -/- and Tcrα -/- compared to WT mice. LPS exposure induced reductions in white matter tissue volume in WT and Tcrα -/- mice, but not in the Tcrd -/- mice, compared with the saline-treated groups. Neither LPS administration nor the T cell deficiency affected anxiety behavior in these mice as determined with the elevated plus maze. DigiGait analysis revealed motor function deficiency after LPS-induced sepsis in both WT and Tcrα -/- mice, but no such effect was observed in Tcrd -/- mice. Our results suggest that γδT cells but not αβT cells contribute to sepsis-induced white matter injury and subsequent motor function abnormalities in early life. Modulating the activity of γδT cells in the early stages of preterm white matter injury might represent a novel therapeutic strategy for the treatment of perinatal brain injury.

STCRDab: the structural T-cell receptor database

PubMed Central

de Oliveira, Saulo H P; Krawczyk, Konrad

2018-01-01

Abstract The Structural T–cell Receptor Database (STCRDab; http://opig.stats.ox.ac.uk/webapps/stcrdab) is an online resource that automatically collects and curates TCR structural data from the Protein Data Bank. For each entry, the database provides annotations, such as the α/β or γ/δ chain pairings, major histocompatibility complex details, and where available, antigen binding affinities. In addition, the orientation between the variable domains and the canonical forms of the complementarity-determining region loops are also provided. Users can select, view, and download individual or bulk sets of structures based on these criteria. Where available, STCRDab also finds antibody structures that are similar to TCRs, helping users explore the relationship between TCRs and antibodies. PMID:29087479

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

PubMed Central

Hemmers, Saskia; Schizas, Michail; Faire, Mehlika B.; Konopacki, Catherine; Schmidt-Supprian, Marc; Germain, Ronald N.

2017-01-01

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. PMID:28130403

Ci8 short, a novel LPS-induced peptide from the ascidian Ciona intestinalis, modulates responses of the human immune system.

PubMed

Bonura, Angela; Vizzini, Aiti; Vlah, Sara; Gervasi, Francesco; Longo, Alessandra; Melis, Mario R; Schildberg, Frank A; Colombo, Paolo

2018-02-01

The selective modulation of immunity is an emerging concept driven by the vast advances in our understanding of this crucial host defense system. Invertebrates have raised researchers' interest as potential sources of new bioactive molecules owing to their antibacterial, anticancer and immunomodulatory activities. A LipoPolySaccharide (LPS) challenge in the ascidian Ciona intestinalis generates the transcript, Ci8 short, with cis-regulatory elements in the 3' UTR region that are essential for shaping innate immune responses. The derived amino acidic sequence in silico analysis showed specific binding to human Major Histocompatibility Complex (MHC) Class I and Class II alleles. The role of Ci8 short peptide was investigated in a more evolved immune system using human Peripheral Blood Mononuclear Cells (PBMCs) as in vitro model. The biological activities of this molecule include the activation of 70kDa TCR ζ chain Associated Protein kinase (ZAP-70) and T Cell Receptor (TCR) Vβ oligo clonal selection on CD4 + T lymphocytes as well as increased proliferation and IFN-γ secretion. Furthermore Ci8 short affects CD4 + /CD25 high induced regulatory T cells (iTreg) subset selection which co-expressed the functional markers TGF-β1/Latency Associated Protein (LAP) and CD39/CD73. This paper describes a new molecule that modulates important responses of the human adaptive immune system. Copyright © 2017 Elsevier GmbH. All rights reserved.

Pre-T Cell Receptors (Pre-TCRs) Leverage Vβ Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands.

PubMed

Das, Dibyendu Kumar; Mallis, Robert J; Duke-Cohan, Jonathan S; Hussey, Rebecca E; Tetteh, Paul W; Hilton, Mark; Wagner, Gerhard; Lang, Matthew J; Reinherz, Ellis L

2016-12-02

The pre-T cell receptor (pre-TCR) is a pTα-β heterodimer functioning in early αβ T cell development. Although once thought to be ligand-autonomous, recent studies show that pre-TCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the αβTCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the Cβ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCRαβ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic Vβ patch apposition. This patch functions in the pre-TCR as a surrogate Vα domain, fostering ligand promiscuity to favor development of β chains with self-reactivity but is occluded by α subunit replacement of pTα upon αβTCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor αβTCR repertoire tuning via the pre-TCR. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Pre-T Cell Receptors (Pre-TCRs) Leverage Vβ Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands*♦

PubMed Central

Das, Dibyendu Kumar; Mallis, Robert J.; Duke-Cohan, Jonathan S.; Hussey, Rebecca E.; Tetteh, Paul W.; Hilton, Mark; Wagner, Gerhard; Lang, Matthew J.; Reinherz, Ellis L.

2016-01-01

The pre-T cell receptor (pre-TCR) is a pTα-β heterodimer functioning in early αβ T cell development. Although once thought to be ligand-autonomous, recent studies show that pre-TCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the αβTCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the Cβ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCRαβ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic Vβ patch apposition. This patch functions in the pre-TCR as a surrogate Vα domain, fostering ligand promiscuity to favor development of β chains with self-reactivity but is occluded by α subunit replacement of pTα upon αβTCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor αβTCR repertoire tuning via the pre-TCR. PMID:27707880

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

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. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Affinity maturation of a portable Fab–RNA module for chaperone-assisted RNA crystallography

PubMed Central

Koirala, Deepak; Shelke, Sandip A; Dupont, Marcel; Ruiz, Stormy; DasGupta, Saurja; Bailey, Lucas J; Benner, Steven A; Piccirilli, Joseph A

2018-01-01

Abstract Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography. PMID:29309709

Ikaros promotes rearrangement of TCR alpha genes in an Ikaros null thymoma cell line

PubMed Central

Collins, Bernard; Clambey, Eric T.; Scott-Browne, James; White, Janice; Marrack, Philippa; Hagman, James; Kappler, John W.

2014-01-01

Summary Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4+CD8+ thymocyte using an Ikaros null CD4−CD8− mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR β gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry non-functional rearrangements on both alleles of their TCR α loci. Retroviral re-introduction of Ikaros dramatically increased the rate of transcription in the α locus and TCR Vα/Jα recombination resulting in the appearance of many new αβTCR+ cells. The process is RAG dependent, requires SWI/SNF chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR α enhancer. Furthermore, knockdown of Mi2/NuRD complexes increased the frequency of TCR α rearrangement. Our data suggest that Ikaros controls Vα/Jα recombination in T cells by controlling access of the transcription and recombination machinery to the TCR α loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to αβTCR+ CD4+CD8+ thymocyte transition. PMID:23172374

Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex.

PubMed

Myers, Margaret D; Sosinowski, Tomasz; Dragone, Leonard L; White, Carmen; Band, Hamid; Gu, Hua; Weiss, Arthur

2006-01-01

The adaptor molecule SLAP and E3 ubiquitin ligase c-Cbl each regulate expression of T cell receptor (TCR)-CD3 on thymocytes. Here we provide genetic and biochemical evidence that both molecules function in the same pathway. TCR-CD3 expression was similar in the absence of SLAP and/or c-Cbl. SLAP and c-Cbl were found to interact, and their expression together downregulated CD3epsilon. This required multiple domains in SLAP and the ring finger of c-Cbl. Furthermore, expression of SLAP and c-Cbl together induced TCRzeta ubiquitination and degradation, preventing the accumulation of fully assembled recycling TCR complexes. These studies indicate that SLAP links the E3 ligase activity of c-Cbl to the TCR, allowing for stage-specific regulation of TCR expression.

Energetic and flexibility properties captured by long molecular dynamics simulations of a membrane-embedded pMHCII-TCR complex.

PubMed

Bello, Martiniano; Correa-Basurto, José

2016-04-01

Although crystallographic data have provided important molecular insight into the interactions in the pMHC-TCR complex, the inherent features of this structural approach cause it to only provide a static picture of the interactions. While unbiased molecular dynamics simulations (UMDSs) have provided important information about the dynamic structural behavior of the pMHC-TCR complex, most of them have modeled the pMHC-TCR complex as soluble, when in physiological conditions, this complex is membrane bound; therefore, following this latter UMDS protocol might hamper important dynamic results. In this contribution, we performed three independent 300 ns-long UMDSs of the pMHCII-TCR complex anchored in two opposing membranes to explore the structural and energetic properties of the recognition of pMHCII by the TCR. The conformational ensemble generated through UMDSs was subjected to clustering and Cartesian principal component analyses (cPCA) to explore the dynamical behavior of the pMHCII-TCR association. Furthermore, based on the conformational population sampled through UMDSs, the effective binding free energy, per-residue free energy decomposition, and alanine scanning mutations were explored for the native pMHCII-TCR complex, as well as for 12 mutations (p1-p12MHCII-TCR) introduced in the native peptide. Clustering analyses and cPCA provide insight into the rocking motion of the TCR onto pMHCII, together with the presence of new electrostatic interactions not observed through crystallographic methods. Energetic results provide evidence of the main contributors to the pMHC-TCR complex formation as well as the key residues involved in this molecular recognition process.

Mfd translocase is necessary and sufficient for transcription-coupled repair in Escherichia coli.

PubMed

Adebali, Ogun; Sancar, Aziz; Selby, Christopher P

2017-11-10

Nucleotide excision repair in Escherichia coli is stimulated by transcription, specifically in the transcribed strand. Previously, it was shown that this transcription-coupled repair (TCR) is mediated by the Mfd translocase. Recently, it was proposed that in fact the majority of TCR in E. coli is catalyzed by a second pathway ("backtracking-mediated TCR") that is dependent on the UvrD helicase and the guanosine pentaphosphate (ppGpp) alarmone/stringent response regulator. Recently, we reported that as measured by the excision repair-sequencing (XR-seq), UvrD plays no role in TCR genome-wide. Here, we tested the role of ppGpp and UvrD in TCR genome-wide and in the lacZ operon using the XR-seq method, which directly measures repair. We found that the mfd mutation abolishes TCR genome-wide and in the lacZ operon. In contrast, the relA - spoT - mutant deficient in ppGpp synthesis carries out normal TCR. We conclude that UvrD and ppGpp play no role in TCR in E. coli . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

CD8+ TCR repertoire formation is guided primarily by the peptide component of the antigenic complex.

PubMed

Koning, Dan; Costa, Ana I; Hoof, Ilka; Miles, John J; Nanlohy, Nening M; Ladell, Kristin; Matthews, Katherine K; Venturi, Vanessa; Schellens, Ingrid M M; Borghans, Jose A M; Kesmir, Can; Price, David A; van Baarle, Debbie

2013-02-01

CD8(+) T cells recognize infected or dysregulated cells via the clonotypically expressed αβ TCR, which engages Ag in the form of peptide bound to MHC class I (MHC I) on the target cell surface. Previous studies have indicated that a diverse Ag-specific TCR repertoire can be beneficial to the host, yet the determinants of clonotypic diversity are poorly defined. To better understand the factors that govern TCR repertoire formation, we conducted a comprehensive clonotypic analysis of CD8(+) T cell populations directed against epitopes derived from EBV and CMV. Neither pathogen source nor the restricting MHC I molecule were linked with TCR diversity; indeed, both HLA-A and HLA-B molecules were observed to interact with an overlapping repertoire of expressed TRBV genes. Peptide specificity, however, markedly impacted TCR diversity. In addition, distinct peptides sharing HLA restriction and viral origin mobilized TCR repertoires with distinct patterns of TRBV gene usage. Notably, no relationship was observed between immunodominance and TCR diversity. These findings provide new insights into the forces that shape the Ag-specific TCR repertoire in vivo and highlight a determinative role for the peptide component of the peptide-MHC I complex on the molecular frontline of CD8(+) T cell-mediated immune surveillance.

Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision

PubMed Central

Hale, J. Scott; Nelson, Lisa T.; Simmons, Kalynn B.; Fink, Pamela J.

2010-01-01

Peripheral CD4+Vβ5+ T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of post-revision CD4+Vβ5− T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the post-revision population. Here, we investigate the role of Bim-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4+ T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR expressing cells are TCR revision intermediates, and that the population of RAG-expressing, revising CD4+ T cells is increased in Bim deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the post-revision repertoire. PMID:21148799

Analysis of the CDR3 length repertoire and the diversity of TCR alpha chain in human peripheral blood T lymphocytes.

PubMed

Yao, Xin Sheng; Diao, Ying; Sun, Wan Bang; Luo, Jun Min; Qin, Ming; Tang, Xian Ying

2007-06-01

Analysis of complementarity determining region 3 (CDR3) length of T lymphocyte receptors (TCRs) by immunoscope spectratyping technique has been used successfully to investigate the diversity of TCR in autoimmune diseases and infection diseases. In this study, we investigated the patterns of CDR3 length distribution for all 32 TCR AV gene families in human peripheral blood lymphocytes of four normal volunteers by the immunoscope spectratyping technique. It was found that PCR products exhibited an obscure band on 1.5% agarose gel electrophoresis. Each TCR AV family exhibited more than 8 bands on 6% sequencing gel electrophoresis. The CDR3 spectratyping of all TCR AV families showed a standard Gaussian distribution with different CDR3 length, and the expression frequency of CDR3 was similar among the gene families. Most of CDR3 in TCR AV family recombine in frame. However, some of the CDR3 showed out-of frame gene rearrangement. Additionally, we found that in some of TCR AV families there were 18 amino acid discrepancies between the longest CDR3 and shortest CDR3. These results may be helpful to further study the recombination mechanism of human TCR genes, the TCR CDR3 gene repertoire, and the repertoire drift in health people and disease state.

Noncore RAG1 regions promote Vβ rearrangements and αβ T cell development by overcoming inherent inefficiency of Vβ recombination signal sequences.

PubMed

Horowitz, Julie E; Bassing, Craig H

2014-02-15

The RAG proteins are comprised of core endonuclease domains and noncore regions that modulate endonuclease activity. Mutation or deletion of noncore RAG regions in humans causes immunodeficiency and altered TCR repertoire, and mice expressing core but not full-length Rag1 (Rag1(C/C)) or Rag2 (Rag2(C/C)) exhibit lymphopenia, reflecting impaired V(D)J recombination and lymphocyte development. Rag1(C/C) mice display reduced D-to-J and V-to-DJ rearrangements of TCRβ and IgH loci, whereas Rag2(C/C) mice show decreased V-to-DJ rearrangements and altered Vβ/VH repertoire. Because Vβs/VHs only recombine to DJ complexes, the Rag1(C/C) phenotype could reflect roles for noncore RAG1 regions in promoting recombination during only the D-to-J step or during both steps. In this study, we demonstrate that a preassembled TCRβ gene, but not a preassembled DβJβ complex or the prosurvival BCL2 protein, completely rescues αβ T cell development in Rag1(C/C) mice. We find that Rag1(C/C) mice exhibit altered Vβ utilization in Vβ-to-DJβ rearrangements, increased usage of 3'Jα gene segments in Vα-to-Jα rearrangements, and abnormal changes in Vβ repertoire during αβ TCR selection. Inefficient Vβ/VH recombination signal sequences (RSSs) have been hypothesized to cause impaired V-to-DJ recombination on the background of a defective recombinase as in core-Rag mice. We show that replacement of the Vβ14 RSS with a more efficient RSS increases Vβ14 recombination and rescues αβ T cell development in Rag1(C/C) mice. Our data indicate that noncore RAG1 regions establish a diverse TCR repertoire by overcoming Vβ RSS inefficiency to promote Vβ recombination and αβ T cell development, and by modulating TCRβ and TCRα gene segment utilization.

Ikaros promotes rearrangement of TCR α genes in an Ikaros null thymoma cell line.

PubMed

Collins, Bernard; Clambey, Eric T; Scott-Browne, James; White, Janice; Marrack, Philippa; Hagman, James; Kappler, John W

2013-02-01

Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4(+) CD8(+) thymocyte using an Ikaros null CD4(-) CD8(-) mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR β gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry nonfunctional rearrangements on both alleles of their TCR α loci. Retroviral reintroduction of Ikaros dramatically increased the rate of transcription in the α locus and TCR Vα/Jα recombination resulting in the appearance of many new αβTCR(+) cells. The process is RAG dependent, requires switch/sucrose nonfermentable chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR α enhancer. Furthermore, knockdown of Mi2/nucleosome remodeling and deacetylase complexes increased the frequency of TCR α rearrangement. Our data suggest that Ikaros controls Vα/Jα recombination in T cells by controlling access of the transcription and recombination machinery to the TCR α loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to αβTCR(+) CD4(+) CD8(+) thymocyte transition. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Anesthetic influence on occurrence and treatment of the trigemino-cardiac reflex: a systematic literature review.

PubMed

Meuwly, Cyrill; Chowdhury, Tumul; Sandu, Nora; Reck, Martin; Erne, Paul; Schaller, Bernhard

2015-05-01

Trigeminocardiac reflex (TCR) is defined as sudden onset of parasympathetic dysrhythmia including hypotension, apnea, and gastric hypermotility during stimulation of any branches of the trigeminal nerve. Previous publications imply a relation between TCR and depth of anesthesia. To gain more detailed insights into this hypothesis, we performed a systematic literature review.Literature about occurrence of TCR was systematically identified through searching in Cochrane Central Register of Controlled Trials (CENTRAL), PubMed (MEDLINE), EMBASE (Ovid SP), and the Institute for Scientific Information (ISI Web of Sciences) databases until June 2013, as well as reference lists of articles for risk calculation. In this study, TCR was defined as drop in mean arterial blood pressure and heart rate, both >20% to baseline. We calculated intraoperative cerebral state index (CSI) of each TCR-case using a newly developed method. These data were further divided into 3 subgroups: CSI <40 (deep anesthesia), CSI 40-60 (regular anesthesia), and CSI >60 (slight anesthesia).Including 45 studies with 910 patients, 140 (15%) presented with TCR, and 770 (85%) without TCR during operation. TCR occurrence showed a 1.2-fold higher pooled risk slighter anesthesia (CSI <40: 13%, at CSI 40-60: 21%, and at CSI >60: 27%) compared with deeper anesthesia. In addition, we could discover a 1.3-fold higher pooled risk of higher MABP drop with a strong negative correlation (r = -0.935; r = 0.89) and a 4.5-fold higher pooled risk of asystole during TCR under slight anesthesia compared with deeper anesthesia.Our work is the first systematic review about TCR and demonstrates clear evidence for TCR occurrence and a more severe course of the TCR in slight anesthesia underlying the importance of skills in anesthesia management during skull base surgery. Furthermore, we have introduced a new standard method to calculate the depth of anesthesia.

Selection of High-Affinity Peptidic Serine Protease Inhibitors with Increased Binding Entropy from a Back-Flip Library of Peptide-Protease Fusions.

PubMed

Sørensen, Hans Peter; Xu, Peng; Jiang, Longguang; Kromann-Hansen, Tobias; Jensen, Knud J; Huang, Mingdong; Andreasen, Peter A

2015-09-25

We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the concept by fusing a 10-residue-long, disulfide-bond-constrained inhibitory peptide, randomized in selected positions, to the catalytic domain of the serine protease murine urokinase-type plasminogen activator. High-affinity inhibitory peptide variants were identified as those that conferred to the fusion protease the lowest activity for substrate hydrolysis. The usefulness of the strategy was demonstrated by the selection of peptidic inhibitors of murine urokinase-type plasminogen activator with a low nanomolar affinity. The high affinity could not have been predicted by rational considerations, as the high affinity was associated with a loss of polar interactions and an increased binding entropy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modulating the selectivity of affinity absorbents to multi-phosphopeptides by a competitive substitution strategy.

PubMed

Liu, Zheyi; Wang, Fangjun; Chen, Jin; Zhou, Ye; Zou, Hanfa

2016-08-26

Although many affinity adsorbents have been developed for phosphopeptides enrichment, high-specifically capturing the multi-phosphopeptides is still a big challenge. Here, we investigated the mechanism of phosphate ion coordination and substitution on affinity adsorbents surfaces and modulated the selectivity of affinity adsorbents to multi-phosphopeptides based on the different capability of mono- and multi-phosphopeptides in competitively substituting the pre-coordinated phosphate ions at strong acidic condition. We demonstrated both the species of pre-coordinated phosphate ions and the substituting conditions played crucial roles in modulating the enrichment selectivity to multi-phosphopeptides, and the pre-coordinated affinity materials with relative more surfaces positive charges exhibited better enrichment efficiency due to the cooperative effect of electrostatic interaction and competitive substitution. Finally, an enrichment selectivity of 85% to multi-phosphopeptides was feasibly achieved with 66% improvement in identification numbers for complex protein sample extracted from HepG2 cells. Data are available via ProteomeXchange with identifier PXD004252. Copyright © 2016 Elsevier B.V. All rights reserved.

Blood T-cell receptor diversity decreases during the course of HIV infection, but the potential for a diverse repertoire persists

PubMed Central

Young, Jennifer J.; Schmidt, Diane; Zhang, Qianjun; Hoh, Rebecca; Busch, Michael; Martin, Jeffrey; Deeks, Steven; McCune, Joseph M.

2012-01-01

HIV infection results in a decrease in circulating CD4+ T-cell and naive T-cell numbers. If such losses were associated with an erosion of T-cell receptor (TCR) repertoire diversity in the peripheral T-cell pool, this might exacerbate the state of persistent immunodeficiency. Existing methods for the analysis of the TCR repertoire have demonstrated skewed distributions of TCR genes in HIV-infected subjects but cannot directly measure TCR diversity. Here we used AmpliCot, a quantitative assay based on DNA hybridization kinetics, to measure TCR diversity in a cross-sectional comparison of 19 HIV-infected persons to 18 HIV-uninfected controls. HIV-infected persons had a 10-fold decrease in total TCR repertoire diversity in 1.5 mL of blood compared with uninfected controls, with decreased diversity correlating most closely with a lower CD4+ T-cell percentage. Nonetheless, the TCR repertoire diversity of sort-purified T-cell subpopulations in HIV-infected and HIV-uninfected subjects was comparable. These observations suggest that the TCR repertoire diversity changes in whole blood during HIV disease progression are primarily the result of changes in the number and proportion of T-cell subpopulations and that most HIV-infected persons may retain a sufficiently diverse TCR repertoire to permit immune reconstitution with antiretroviral therapy alone, without thymopoiesis. PMID:22371879

Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision.

PubMed

Hale, J Scott; Nelson, Lisa T; Simmons, Kalynn B; Fink, Pamela J

2011-01-15

Peripheral CD4(+)Vβ5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of postrevision CD4(+)Vβ5(-) T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study, we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing, revising CD4(+) T cells is increased in Bim-deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the postrevision repertoire.

Calcium-mediated shaping of naive CD4 T-cell phenotype and function

PubMed Central

Guichard, Vincent; Bonilla, Nelly; Durand, Aurélie; Audemard-Verger, Alexandra; Guilbert, Thomas; Martin, Bruno

2017-01-01

Continuous contact with self-major histocompatibility complex ligands is essential for the survival of naive CD4 T cells. We have previously shown that the resulting tonic TCR signaling also influences their fate upon activation by increasing their ability to differentiate into induced/peripheral regulatory T cells. To decipher the molecular mechanisms governing this process, we here focus on the TCR signaling cascade and demonstrate that a rise in intracellular calcium levels is sufficient to modulate the phenotype of mouse naive CD4 T cells and to increase their sensitivity to regulatory T-cell polarization signals, both processes relying on calcineurin activation. Accordingly, in vivo calcineurin inhibition leads the most self-reactive naive CD4 T cells to adopt the phenotype of their less self-reactive cell-counterparts. Collectively, our findings demonstrate that calcium-mediated activation of the calcineurin pathway acts as a rheostat to shape both the phenotype and effector potential of naive CD4 T cells in the steady-state. PMID:29239722

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. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

High-throughput identification of antigen-specific TCRs by TCR gene capture.

PubMed

Linnemann, Carsten; Heemskerk, Bianca; Kvistborg, Pia; Kluin, Roelof J C; Bolotin, Dmitriy A; Chen, Xiaojing; Bresser, Kaspar; Nieuwland, Marja; Schotte, Remko; Michels, Samira; Gomez-Eerland, Raquel; Jahn, Lorenz; Hombrink, Pleun; Legrand, Nicolas; Shu, Chengyi Jenny; Mamedov, Ilgar Z; Velds, Arno; Blank, Christian U; Haanen, John B A G; Turchaninova, Maria A; Kerkhoven, Ron M; Spits, Hergen; Hadrup, Sine Reker; Heemskerk, Mirjam H M; Blankenstein, Thomas; Chudakov, Dmitriy M; Bendle, Gavin M; Schumacher, Ton N M

2013-11-01

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

Rag Deletion in Peripheral T Cells Blocks TCR Revision

PubMed Central

Hale, J. Scott; Ames, Kristina T.; Boursalian, Tamar E.; Fink, Pamela J.

2010-01-01

Mature CD4+Vβ5+ T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or T cell receptor (TCR) revision. In Vβ5 transgenic mice, this latter tolerance pathway results in the appearance of CD4+Vβ5−TCRβ+ T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of Vβ-DJβ recombination intermediates in peripheral CD4+ T cells. Because post-thymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We now show that Rag deletion in post-positive selection T cells in Vβ5 transgenic mice blocks TCR revision in vivo, and that mature peripheral T cells sorted to remove cells bearing endogenous TCRβ chains can express newly generated TCRβ molecules in adoptive hosts. These findings unambiguously demonstrate post-thymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4+ T cells. PMID:20435935

Cutting Edge: Rag deletion in peripheral T cells blocks TCR revision.

PubMed

Hale, J Scott; Ames, Kristina T; Boursalian, Tamar E; Fink, Pamela J

2010-06-01

Mature CD4(+)Vbeta5(+) T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or TCR revision. In Vbeta5 transgenic mice, this latter tolerance pathway results in the appearance of CD4(+)Vbeta5(-)TCRbeta(+) T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of V(beta)-DJ(beta) recombination intermediates in peripheral CD4(+) T cells. Because postthymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We show in this study that Rag deletion in post-positive selection T cells in Vbeta5 transgenic mice blocks TCR revision in vivo and that mature peripheral T cells sorted to remove cells bearing endogenous TCRbeta-chains can express newly generated TCRbeta molecules in adoptive hosts. These findings unambiguously demonstrate postthymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4(+) T cells.

Structural basis for clonal diversity of the human T-cell response to a dominant influenza virus epitope

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

Yang, Xinbo; Chen, Guobing; Weng, Nan-ping

Influenza A virus (IAV) causes an acute infection in humans that is normally eliminated by CD8+ cytotoxic T lymphocytes. Individuals expressing the MHC class I molecule HLA-A2 produce cytotoxic T lymphocytes bearing T-cell receptors (TCRs) that recognize the immunodominant IAV epitope GILGFVFTL (GIL). Most GIL-specific TCRs utilize α/β chain pairs encoded by the TRAV27/TRBV19 gene combination to recognize this relatively featureless peptide epitope (canonical TCRs). However, ~40% of GIL-specific TCRs express a wide variety of other TRAV/TRBV combinations (non-canonical TCRs). To investigate the structural underpinnings of this remarkable diversity, we determined the crystal structure of a non-canonical GIL-specific TCR (F50)more » expressing the TRAV13-1/TRBV27 gene combination bound to GIL–HLA-A2 to 1.7 Å resolution. Comparison of the F50–GIL–HLA-A2 complex with the previously published complex formed by a canonical TCR (JM22) revealed that F50 and JM22 engage GIL–HLA-A2 in markedly different orientations. These orientations are distinguished by crossing angles of TCR to peptide–MHC of 29° for F50 versus 69° for JM22 and by a focus by F50 on the C terminus rather than the center of the MHC α1 helix for JM22. In addition, F50, unlike JM22, uses a tryptophan instead of an arginine to fill a critical notch between GIL and the HLA-A2 α2 helix. The F50–GIL–HLA-A2 complex shows that there are multiple structurally distinct solutions to recognizing an identical peptide–MHC ligand with sufficient affinity to elicit a broad anti-IAV response that protects against viral escape and T-cell clonal loss.« less

End-binding protein 1 controls signal propagation from the T cell receptor

PubMed Central

Martín-Cófreces, Noa B; Baixauli, Francesc; López, María J; Gil, Diana; Monjas, Alicia; Alarcón, Balbino; Sánchez-Madrid, Francisco

2012-01-01

The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unresolved. Here, we show that T cell activation requires the growth of MTs mediated by the plus-end specific protein end-binding 1 (EB1). A direct interaction of the T cell receptor (TCR) complex with EB1 provides the molecular basis for EB1 activity promoting TCR encounter with signalling vesicles at the IS. EB1 knockdown alters TCR dynamics at the IS and prevents propagation of the TCR activation signal to LAT, thus inhibiting activation of PLCγ1 and its localization to the IS. These results identify a role for EB1 interaction with the TCR in controlling TCR sorting and its connection with the LAT/PLCγ1 signalosome. PMID:22922463

Tumour necrosis factors modulate the affinity state of the leukotriene B4 receptor on human neutrophils.

PubMed Central

Brom, J; Knöller, J; Köller, M; König, W

1988-01-01

Pre-incubation of human polymorphonuclear granulocytes with recombinant human tumour necrosis factors (TNF) revealed a time- and dose-dependent reduction of the expression of leukotriene B4-receptor sites. Analysis of the binding data by Scatchard plots showed a shift from a heterologous receptor population (indicating high- and low-affinity subsets) to a homologous population. From the results it is considered that TNF can influence host defence through the modulation of leukotriene B4 receptor affinity. PMID:2851543

Inhibition of Gαs/cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4(+) T Helper Cells.

PubMed

Hynes, Thomas R; Yost, Evan A; Yost, Stacy M; Hartle, Cassandra M; Ott, Braden J; Berlot, Catherine H

2015-07-06

The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gαs and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. The effect of blocking Gs-coupled receptor (GsPCR)-mediated Gs activation on TCR-stimulated IL-2 mRNA levels in CD4(+) T cells was compared with that of knocking down Gαs expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gαs expression on TCR-stimulated cAMP accumulation was compared with that of blocking GsPCR signaling. ZM-241385, an antagonist to the Gs-coupled A2A adenosine receptor (A2AR), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4(+) T helper cells and in Jurkat T cells. A dominant negative Gαs construct, GαsDN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to GsPCR antagonists, GαsDN3 blocked GsPCR-dependent activation of both Gαs and Gβγ. In contrast, Gαs siRNA and 2',5'-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gαs siRNA, but not GαsDN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. GsPCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gαs and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gαs does not appear to involve GsPCRs. These results suggest that the context of Gαs/cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels.

SDF-1 signaling via the CXCR4-TCR heterodimer requires PLC-β3 and PLC-γ1 for distinct cellular responses 1

PubMed Central

Kremer, Kimberly N.; Clift, Ian C.; Miamen, Alexander G.; Bamidele, Adebowale O.; Qian, Nan-Xin; Humphreys, Troy D.; Hedin, Karen E.

2011-01-01

The CXCR4 chemokine receptor is a G protein-coupled receptor (GPCR) that signals in T lymphocytes by forming a heterodimer with the T cell antigen receptor (TCR). CXCR4 and TCR functions are consequently highly cross-regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAP kinase and downstream AP-1-dependent cytokine transcription in response to SDF-1, the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP-70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate GPCR-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. Here, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-β3 to activate the Ras-ERK pathway and increase intracellular Ca2+ concentrations, while PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-β3, is required for SDF-1-mediated migration, via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-β3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors in order to distinctly regulate migration versus other signaling functions. PMID:21705626

Selection of Fecal Enterococci Exhibiting tcrB-Mediated Copper Resistance in Pigs Fed Diets Supplemented with Copper † ▿

PubMed Central

Amachawadi, R. G.; Shelton, N. W.; Shi, X.; Vinasco, J.; Dritz, S. S.; Tokach, M. D.; Nelssen, J. L.; Scott, H. M.; Nagaraja, T. G.

2011-01-01

Copper, as copper sulfate, is increasingly used as an alternative to in-feed antibiotics for growth promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance (tcrB) gene, has been reported in Enterococcus faecium and E. faecalis. A longitudinal field study was undertaken to determine the relationship between copper supplementation and the prevalence of tcrB-positive enterococci in piglets. The study was done with weaned piglets, housed in 10 pens with 6 piglets per pen, fed diets supplemented with a normal (16.5 ppm; control) or an elevated (125 ppm) level of copper. Fecal samples were randomly collected from three piglets per pen on days 0, 14, 28, and 42 and plated on M-Enterococcus agar, and three enterococcal isolates were obtained from each sample. The overall prevalence of tcrB-positive enterococci was 21.1% (38/180) in piglets fed elevated copper and 2.8% (5/180) in the control. Among the 43 tcrB-positive isolates, 35 were E. faecium and 8 were E. faecalis. The mean MICs of copper for tcrB-negative and tcrB-positive enterococci were 6.2 and 22.2 mM, respectively. The restriction digestion of the genomic DNA of E. faecium or E. faecalis with S1 nuclease yielded a band of ∼194-kbp size to which both tcrB and the erm(B) gene probes hybridized. A conjugation assay demonstrated cotransfer of tcrB and erm(B) genes between E. faecium and E. faecalis strains. The higher prevalence of tcrB-positive enterococci in piglets fed elevated copper compared to that in piglets fed normal copper suggests that supplementation of copper in swine diets selected for resistance. PMID:21705534

Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis

PubMed Central

1996-01-01

The importance of cytotoxic T lymphocytes (CTLs) in the immunosurveillance of Epstein-Barr virus (EBV)-infected B cells is firmly established, and the viral antigens of CTL recognition in latent infection are well defined. The epitopes targeted by CTLs during primary infection have not been identified, however, and there is only limited information about T cell receptor (TCR) selection. In the present report, we have monitored the development of memory TCR-beta clonotypes selected in response to natural EBV infection in a longitudinal study of an HLA-B8+ individual with acute infectious mononucleosis (IM). By stimulating peripheral blood lymphocytes with HLA-B8+ EBV-transformed B lymphoblastoid cells, the primary virus- specific CTL response was shown to include specificities for two HLA-B8- restricted antigenic determinants, FLRGRAYGL and QAKWRLQTL, which are encoded within the latent EBV nuclear antigen EBNA-3. TCR-beta sequence analysis of CTL clones specific for each epitope showed polyclonal TCR- beta repertoire selection, with structural restrictions on recognition that indicated antigen-driven selection. Furthermore, longitudinal repertoire analysis revealed long-term preservation of a multiclonal effector response throughout convalescence, with the reemergence of distinct memory T cell clonotypes sharing similar structural restrictions. Tracking the progression of specific TCR-beta clonotypes and antigen-specific TCR-V beta family gene expression in the peripheral repertoire ex vivo using semiquantitative PCR strongly suggested that selective TCR-beta expansions were present at the clonotype level, but not at the TCR-V beta family level. Overall, in this first analysis of antigen-specific TCR development in IM, a picture of polyclonal TCR stimulation is apparent. This diversity may be especially important in the establishment of an effective CTL control during acute EBV infection and in recovery from disease. PMID:8920869

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

PubMed Central

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

2013-01-01

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. PMID:23758320

Dissection of the IgNAR V domain: molecular scanning and orthologue database mining define novel IgNAR hallmarks and affinity maturation mechanisms.

PubMed

Fennell, B J; Darmanin-Sheehan, A; Hufton, S E; Calabro, V; Wu, L; Müller, M R; Cao, W; Gill, D; Cunningham, O; Finlay, W J J

2010-07-09

The shark antigen-binding V(NAR) domain has the potential to provide an attractive alternative to traditional biotherapeutics based on its small size, advantageous physiochemical properties, and unusual ability to target clefts in enzymes or cell surface molecules. The V(NAR) shares many of the properties of the well-characterised single-domain camelid V(H)H but is much less understood at the molecular level. We chose the hen-egg-lysozyme-specific archetypal Type I V(NAR) 5A7 and used ribosome display in combination with error-prone mutagenesis to interrogate the entire sequence space. We found a high level of mutational plasticity across the V(NAR) domain, particularly within the framework 2 and hypervariable region 2 regions. A number of residues important for affinity were identified, and a triple mutant combining A1D, S61R, and G62R resulted in a K(D) of 460 pM for hen egg lysozyme, a 20-fold improvement over wild-type 5A7, and the highest K(D) yet reported for V(NAR)-antigen interactions. These findings were rationalised using structural modelling and indicate the importance of residues outside the classical complementarity determining regions in making novel antigen contacts that modulate affinity. We also located two solvent-exposed residues (G15 and G42), distant from the V(NAR) paratope, which retain function upon mutation to cysteine and have the potential to be exploited as sites for targeted covalent modification. Our findings with 5A7 were extended to all known NAR structures using an in-depth bioinformatic analysis of sequence data available in the literature and a newly generated V(NAR) database. This study allowed us to identify, for the first time, both V(NAR)-specific and V(NAR)/Ig V(L)/TCR V(alpha) overlapping hallmark residues, which are critical for the structural and functional integrity of the single domain. Intriguingly, each of our designated V(NAR)-specific hallmarks align precisely with previously defined mutational 'cold spots' in natural nurse shark cDNA sequences. These findings will aid future V(NAR) engineering and optimisation studies towards the development of V(NAR) single-domain proteins as viable biotherapeutics. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modulation of Memory T Cells to Control Acquired Bone Marrow Failure

DTIC Science & Technology

2016-01-01

Representative images show the tissues from one of 6 recipients in each group at day 7 after transplantation. Images were obtained with an OlympusBX41...alloreactive effector T cells capable of mediating host tissue injury and could be beneficial targets for improving the efficacy of allogeneic HSCT...leukemia (GVL) effect, but showed impaired expansion in local tissues .69-72 This nTEM pool might have less diverse T cell receptor (TCR) repertoire

Protein kinase D2 is a digital amplifier of T cell receptor–stimulated diacylglycerol signaling in naïve CD8+ T cells

PubMed Central

Navarro, María N.; Feijoo-Carnero, Carmen; Arandilla, Alba Gonzalez; Trost, Matthias; Cantrell, Doreen A.

2016-01-01

Protein kinase D2 (PKD2) is a serine and threonine kinase that is activated in T cells by diacylglycerol and protein kinase C in response to stimulation of the T cell receptor (TCR) by antigen. We quantified the activation of PKD2 at the single-cell level and found that this kinase acts as a sensitive digital amplifier of TCR engagement, enabling CD8+ T cells to match the production of inflammatory cytokines to the quality and quantity of TCR ligands. There was a digital response pattern of PKD2 activation in response to TCR engagement, such that increasing the concentration and potency of TCR ligands increased the number of cells that exhibited activated PKD2. However, for each cell that responded to TCR stimulation, the entire cellular pool of PKD2 (~400,000 molecules) was activated. Moreover, PKD2 acted as an amplification checkpoint for antigen-stimulated digital cytokine responses and translated the differential strength of TCR signaling to determine the number of naïve CD8+ T cells that became effector cells. Together, these results provide insights into PKD family kinases and how they act digitally to amplify signaling networks controlled by the TCR. PMID:25336615

TCR tuning of T cell subsets.

PubMed

Cho, Jae-Ho; Sprent, Jonathan

2018-05-01

After selection in the thymus, the post-thymic T cell compartments comprise heterogenous subsets of naive and memory T cells that make continuous T cell receptor (TCR) contact with self-ligands bound to major histocompatibility complex (MHC) molecules. T cell recognition of self-MHC ligands elicits covert TCR signaling and is particularly important for controlling survival of naive T cells. Such tonic TCR signaling is tightly controlled and maintains the cells in a quiescent state to avoid autoimmunity. Here, we review how naive and memory T cells are differentially tuned and wired for TCR sensitivity to self and foreign ligands. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

T-cell receptor gene therapy: critical parameters for clinical success.

PubMed

Linnemann, Carsten; Schumacher, Ton N M; Bendle, Gavin M

2011-09-01

T-cell receptor (TCR) gene therapy aims to induce immune reactivity against tumors by introducing genes encoding a tumor-reactive TCR into patient T cells. This approach has been extensively tested in preclinical mouse models, and initial clinical trials have demonstrated the feasibility and potential of TCR gene therapy as a cancer treatment. However, data obtained from preclinical and clinical studies suggest that both the therapeutic efficacy and the safety of TCR gene therapy can be and needs to be further enhanced. This review highlights those strategies that can be followed to develop TCR gene therapy into a clinically relevant treatment option for cancer patients.

The signaling symphony: T cell receptor tunes cytokine-mediated T cell differentiation

PubMed Central

Huang, Weishan; August, Avery

2015-01-01

T cell development, differentiation, and maintenance are orchestrated by 2 key signaling axes: the antigen-specific TCR and cytokine-mediated signals. The TCR signals the recognition of self- and foreign antigens to control T cell homeostasis for immune tolerance and immunity, which is regulated by a variety of cytokines to determine T cell subset homeostasis and differentiation. TCR signaling can synergize with or antagonize cytokine-mediated signaling to fine tune T cell fate; however, the latter is less investigated. Murine models with attenuated TCR signaling strength have revealed that TCR signaling can function as regulatory feedback machinery for T cell homeostasis and differentiation in differential cytokine milieus, such as IL-2-mediated Treg development; IL-7-mediated, naïve CD8+ T cell homeostasis; and IL-4-induced innate memory CD8+ T cell development. In this review, we discuss the symphonic cross-talk between TCR and cytokine-mediated responses that differentially control T cell behavior, with a focus on the negative tuning by TCR activation on the cytokine effects. PMID:25525115

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. Copyright © 2014 by The American Association of Immunologists, Inc.

Cardioprotective effect of tincture of Crataegus on isoproterenol-induced myocardial infarction in rats.

PubMed

Jayalakshmi, R; Niranjali Devaraj, S

2004-07-01

Tincture of Crataegus (TCR), an alcoholic extract of the berries of hawthorn (Crataegus oxycantha), is used in herbal and homeopathic medicine. The present study was done to investigate the protective effect of TCR on experimentally induced myocardial infarction in rats. Pretreatment of TCR, at a dose of 0.5 mL/100 g bodyweight per day, orally for 30 days, prevented the increase in lipid peroxidation and activity of marker enzymes observed in isoproterenol-induced rats (85 mg kg(-1) s. c. for 2 days at an interval of 24 h). TCR prevented the isoproterenol-induced decrease in antioxidant enzymes in the heart and increased the rate of ADP-stimulated oxygen uptake and respiratory coupling ratio. TCR protected against pathological changes induced by isoproterenol in rat heart. The results show that pretreatment with TCR may be useful in preventing the damage induced by isoproterenol in rat heart.

Thermodynamics of T cell receptor – peptide/MHC interactions: progress and opportunities

PubMed Central

Armstrong, Kathryn M.; Insaidoo, Francis K.; Baker, Brian M.

2013-01-01

αβ T cell receptors (TCR) recognize peptide antigens presented by class I or class II major histocompatibility complex molecules (pMHC). Here we review the use of thermodynamic measurements in the study of TCR-pMHC interactions, with attention to the diversity in binding thermodynamics and how this is related to the variation in TCR-pMHC interfaces. We show that there is no enthalpic or entropic signature for TCR binding; rather, enthalpy and entropy changes vary in a compensatory manner that reflects a narrow free energy window for the interactions that have been characterized. Binding enthalpy and entropy changes do not correlate with structural features such as buried surface area or the number of hydrogen bonds within TCR-pMHC interfaces, possibly reflecting the myriad of contributors to binding thermodynamics, but likely also reflecting a reliance on van’t Hoff over calorimetric measurements and the unaccounted influence of equilibria linked to binding. TCR-pMHC binding heat capacity changes likewise vary considerably. In some cases the heat capacity changes are consistent with conformational differences between bound and free receptors, but there is little data indicating these conformational differences represent the need to organize commonly disordered CDR loops. In this regard, we discuss how thermodynamics may provide additional insight into conformational changes occurring upon TCR binding. Finally, we highlight opportunities for the further use of thermodynamic measurements in the study of TCR-pMHC interactions, not only for understanding TCR binding in general, but for understanding specifics of individual interactions and the engineering of T cell receptors with desired molecular recognition properties. PMID:18496839

Reduction of T cell receptor diversity in NOD mice prevents development of type 1 diabetes but not Sjögren's syndrome.

PubMed

Kern, Joanna; Drutel, Robert; Leanhart, Silvia; Bogacz, Marek; Pacholczyk, Rafal

2014-01-01

Non-obese diabetic (NOD) mice are well-established models of independently developing spontaneous autoimmune diseases, Sjögren's syndrome (SS) and type 1 diabetes (T1D). The key determining factor for T1D is the strong association with particular MHCII molecule and recognition by diabetogenic T cell receptor (TCR) of an insulin peptide presented in the context of I-Ag7 molecule. For SS the association with MHCII polymorphism is weaker and TCR diversity involved in the onset of the autoimmune phase of SS remains poorly understood. To compare the impact of TCR diversity reduction on the development of both diseases we generated two lines of TCR transgenic NOD mice. One line expresses transgenic TCRβ chain originated from a pathogenically irrelevant TCR, and the second line additionally expresses transgenic TCRαmini locus. Analysis of TCR sequences on NOD background reveals lower TCR diversity on Treg cells not only in the thymus, but also in the periphery. This reduction in diversity does not affect conventional CD4+ T cells, as compared to the TCRmini repertoire on B6 background. Interestingly, neither transgenic TCRβ nor TCRmini mice develop diabetes, which we show is due to lack of insulin B:9-23 specific T cells in the periphery. Conversely SS develops in both lines, with full glandular infiltration, production of autoantibodies and hyposalivation. It shows that SS development is not as sensitive to limited availability of TCR specificities as T1D, which suggests wider range of possible TCR/peptide/MHC interactions driving autoimmunity in SS.

Fc gamma RII/III and CD2 expression mark distinct subpopulations of immature CD4-CD8- murine thymocytes: in vivo developmental kinetics and T cell receptor beta chain rearrangement status.

PubMed

Rodewald, H R; Awad, K; Moingeon, P; D'Adamio, L; Rabinowitz, D; Shinkai, Y; Alt, F W; Reinherz, E L

1993-04-01

We have recently identified a dominant wave of CD4-CD8- (double-negative [DN]) thymocytes in early murine fetal development that express low affinity Fc gamma receptors (Fc gamma RII/III) and contain precursors for Ti alpha/beta lineage T cells. Here we show that Fc gamma RII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that Fc gamma RII/III expression is downregulated within the DN subpopulation and before the CD3-CD8low SP stage in T cell receptor (TCR)-alpha/beta lineage-committed thymocytes. DN Fc gamma RII/III+ thymocytes also contain a small fraction of TCR-gamma/delta lineage cells in addition to TCR-alpha/beta progenitors. Fetal day 15.5 DN TCR-alpha/beta lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of Fc gamma RII/III vs. CD2 (Fc gamma RII/III+CD2-, Fc gamma RII/III+CD2+, Fc gamma RII/III-CD2+). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57B1/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (Fc gamma RII/III+CD2(-)-->Fc gamma RII/III+CD2(+)-->Fc gamma RII/III-CD2+) that precedes the entry of DN thymocytes into the CD4+CD8+ double-positive (DP) TCRlow/- stage. Unseparated day 15.5 fetal thymocytes develop into DP thymocytes within 2.5 d and remain at the DP stage for > 48 h before being selected into either CD4+ or CD8+ SP thymocytes. In contrast, Fc gamma RII/III+CD2- DN thymocytes follow this same developmental pathway but are delayed by approximately 24 h before entering the DP compartment, while Fc gamma RII/III-CD2+ display accelerated development by approximately 24 h compared with total day 15.5 thymocytes. Fc gamma RII/III-CD2+ are also more developmentally advanced than Fc gamma RII/III+CD2- fetal thymocytes with respect to their TCR beta chain V(D)J rearrangement. At day 15.5 in gestation, beta chain V(D)J rearrangement is mostly, if not entirely, restricted to the Fc gamma RII/III-CD2+ subset of DN fetal thymocytes. Consistent with this analysis in fetal thymocytes, > 90% of adult thymocytes derived from mice carrying a disrupting mutation at the recombination-activating gene 2 locus (RAG-2-/-) on both alleles are developmentally arrested at the DN CD2- stage. In addition, there is a fivefold increase in the relative percentage of thymocytes expressing Fc gamma RII/III in TCR and immunoglobulin gene rearrangement-incompetent homozygous RAG-2-/- mice (15% Fc gamma RII/III+) versus rearrangement-competent heterozygous RAG-2+/- mice (< 3% Fc gamma RII/III+). Thus, Fc gamma RII/III expression defines an early DN stage preceding V beta(D beta)I beta rearrangement, which in turn is followed by surface expression of CD2. Loss of Fc gamma RII/III and acquisition of CD2 expression characterize a late DN stage immediately before the conversion into DP thymocytes.

Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription.

PubMed

Nadkarni, Aditi; Burns, John A; Gandolfi, Alberto; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E; Scicchitano, David A

2016-01-08

DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription*

PubMed Central

Nadkarni, Aditi; Burns, John A.; Gandolfi, Alberto; Chowdhury, Moinuddin A.; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E.; Scicchitano, David A.

2016-01-01

DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. PMID:26559971

Characterisation of exposure to total and hexavalent chromium of welders using biological monitoring.

PubMed

Scheepers, P T J; Heussen, G A H; Peer, P G M; Verbist, K; Anzion, R; Willems, J

2008-05-30

Inhalation exposure to total and hexavalent chromium (TCr and HCr) was assessed by personal air sampling and biological monitoring in 53 welders and 20 references. Median inhalation exposure levels of TCr were 1.3, 6.0, and 5.4 microg/m(3) for welders of mild steel (MS, <5% alloys), high alloy steel (HAS, >5% alloys), and stainless steel (SS, >26% alloys), respectively. The median exposures to HCr compounds were 0.23, 0.20, and 0.08 microg/m(3), respectively. Median concentrations of TCr in urine, blood plasma and erythrocytes were elevated in all welders, compared with the corresponding median concentrations in the reference group (p<0.005). The TCr levels observed in plasma were two-fold higher in welders of SS and HAS than in welders of MS (p<0.01). Exposure to HCr as indicated by median total content of Cr in erythrocytes was 10 microg/L in welders of SS, MS and HAS. Uptake of TCr during the shift was confirmed for welders of SS by a median increase of urinary TCr from pre- to post-shift of 0.30 microg/g creatinine. For welders of MS and HAS as a group TCr was not increased.

The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

PubMed Central

Awerkiew, Sabine; Schmidt, Annette; Hombach, Andreas A.; Pfister, Herbert; Abken, Hinrich

2012-01-01

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1+ CD57+ CD7− phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter. PMID:22292024

A Jurkat 76 based triple parameter reporter system to evaluate TCR functions and adoptive T cell strategies.

PubMed

Rosskopf, Sandra; Leitner, Judith; Paster, Wolfgang; Morton, Laura T; Hagedoorn, Renate S; Steinberger, Peter; Heemskerk, Mirjam H M

2018-04-03

Adoptive T cell therapy using TCR transgenic autologous T cells has shown great potential for the treatment of tumor patients. Thorough characterization of genetically reprogrammed T cells is necessary to optimize treatment success. Here, we describe the generation of triple parameter reporter T cells based on the Jurkat 76 T cell line for the evaluation of TCR and chimeric antigen receptor functions as well as adoptive T cell strategies. This Jurkat subline is devoid of endogenous TCR alpha and TCR beta chains, thereby circumventing the problem of TCR miss-pairing and unexpected specificities. The resultant reporter cells allow simultaneous determination of the activity of the transcription factors NF-κB, NFAT and AP-1 that play key roles in T cell activation. Human TCRs directed against tumor and virus antigens were introduced and reporter responses were determined using tumor cell lines endogenously expressing the antigens of interest or via addition of antigenic peptides. Finally, we demonstrate that coexpression of adhesion molecules like CD2 and CD226 as well as CD28 chimeric receptors represents an effective strategy to augment the response of TCR-transgenic reporters to cells presenting cognate antigens.

Evaluating tear clearance rate with optical coherence tomography.

PubMed

Garaszczuk, Izabela K; Mousavi, Maryam; Cervino Exposito, Alejandro; Bartuzel, Maciej M; Montes-Micó, Robert; Iskander, D Robert

2018-02-01

To assess the early-phase of tear clearance rate (TCR) with anterior segment optical coherence tomography (OCT) and to determine the association between TCR and other clinical measures of the tear film in a group of young subjects with different levels of tear film quality. TCR was classified as the percentage decrease of subject's inferior tear meniscus height 30s after instillation of 5μl 0.9% saline solution. Fifty subjects (32F and 18M) aged (mean±standard deviation) 25.5±4.3 years volunteered for the study. It consisted of a review of medical history, Ocular Surface Disease Index (OSDI) questionnaire, tear film osmolarity measurements, slit lamp examination and TCR estimation based on dynamic measurements of the lower tear meniscus with OCT. Estimates of TCR were contrasted against subject age and tear film measures commonly used for dry eye diagnosis, which includes OSDI score, fluorescein tear film break-up time (FBUT), tear meniscus height (TMH), blinking frequency, tear film osmolarity and corneal staining. The group mean TCR was 29±13% and 36±19% respectively after 30 and 60s margin after saline solution instillation. Statistically significant correlations were found between TCR and FBUT (r 2 =0.319, p<0.001), blinking frequency (r 2 =0.138, p<0.01), tear film osmolarity (r 2 =0.133, p<0.01) and subject's age (r 2 =0.095, p<0.05). Anterior segment optical coherence tomography allows following changes of tear meniscus morphology post saline solution instillation and evaluating the TCR. OCT based TCR might be used as additional measure of the lacrimal functional unit. Copyright © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

Influences of Histidine-1 and Azaphenylalanine-4 on the Affinity, Anti-inflammatory, and Antiangiogenic Activities of Azapeptide Cluster of Differentiation 36 Receptor Modulators.

PubMed

Chignen Possi, Kelvine; Mulumba, Mukandila; Omri, Samy; Garcia-Ramos, Yesica; Tahiri, Houda; Chemtob, Sylvain; Ong, Huy; Lubell, William D

2017-11-22

Azapeptide analogues of growth hormone releasing peptide-6 (GHRP-6) exhibit promising affinity, selectivity, and modulator activity on the cluster of differentiation 36 receptor (CD36). For example, [A 1 , azaF 4 ]- and [azaY 4 ]-GHRP-6 (1a and 2b) were previously shown to bind selectively to CD36 and exhibited respectively significant antiangiogenic and slight angiogenic activities in a microvascular sprouting assay using choroid explants. The influences of the 1- and 4-position residues on the affinity, anti-inflammatory, and antiangiogenic activity of these azapeptides have now been studied in detail by the synthesis and analysis of a set of 25 analogues featuring Ala 1 or His 1 and a variety of aromatic side chains at the aza-amino acid residue in the 4-position. Although their binding affinities differed only by a factor of 17, the analogues exhibited significant differences in ability to modulate production of nitric oxide (NO) in macrophages and choroidal neovascularization.

Genetic engineering with T cell receptors.

PubMed

Zhang, Ling; Morgan, Richard A

2012-06-01

In the past two decades, human gene transfer research has been translated from a laboratory technology to clinical evaluation. The success of adoptive transfer of tumor-reactive lymphocytes to treat the patients with metastatic melanoma has led to new strategies to redirect normal T cells to recognize tumor antigens by genetic engineering with tumor antigen-specific T cell receptor (TCR) genes. This new strategy can generate large numbers of defined antigen-specific cells for therapeutic application. Much progress has been made to TCR gene transfer systems by optimizing gene expression and gene transfer protocols. Vector and protein modifications have enabled excellent expression of introduced TCR chains in human lymphocytes with reduced mis-pairing between the introduced and endogenous TCR chains. Initial clinical studies have demonstrated that TCR gene-engineered T cells could mediate tumor regression in vivo. In this review, we discuss the progress and prospects of TCR gene-engineered T cells as a therapeutic strategy for treating patients with melanoma and other cancers. Published by Elsevier B.V.

T Cell Receptor Engagement Triggers Its CD3ε and CD3ζ Subunits to Adopt a Compact, Locked Conformation

PubMed Central

Risueño, Ruth M.; Schamel, Wolfgang W. A.; Alarcón, Balbino

2008-01-01

How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood. One current model proposes that strong pMHC ligands, but not weak ones, induce a conformational change in the TCR. Evidence supporting this comes from a pull-down assay that detects ligand-induced binding of the TCR to the N-terminal SH3 domain of the adapter protein Nck, and also from studies with a neoepitope-specific antibody. Both methods rely on the exposure of a polyproline sequence in the CD3ε subunit of the TCR, and neither indicates whether the conformational change is transmitted to other CD3 subunits. Using a protease-sensitivity assay, we now show that the cytoplasmic tails of CD3ε and CD3ζ subunits become fully protected from degradation upon TCR triggering. These results suggest that the TCR conformational change is transmitted to the tails of CD3ε and CD3ζ, and perhaps all CD3 subunits. Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR. These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails. PMID:18320063

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.

A TNF-Regulated Recombinatorial Macrophage Immune Receptor Implicated in Granuloma Formation in Tuberculosis

PubMed Central

Streich, Roswita; Breysach, Caroline; Raddatz, Dirk; Oniga, Septimia; Peccerella, Teresa; Findeisen, Peter; Kzhyshkowska, Julia; Gratchev, Alexei; Schweyer, Stefan; Saunders, Bernadette; Wessels, Johannes T.; Möbius, Wiebke; Keane, Joseph; Becker, Heinz; Ganser, Arnold; Neumaier, Michael; Kaminski, Wolfgang E.

2011-01-01

Macrophages play a central role in host defense against mycobacterial infection and anti- TNF therapy is associated with granuloma disorganization and reactivation of tuberculosis in humans. Here, we provide evidence for the presence of a T cell receptor (TCR) αβ based recombinatorial immune receptor in subpopulations of human and mouse monocytes and macrophages. In vitro, we find that the macrophage-TCRαβ induces the release of CCL2 and modulates phagocytosis. TNF blockade suppresses macrophage-TCRαβ expression. Infection of macrophages from healthy individuals with mycobacteria triggers formation of clusters that express restricted TCR Vβ repertoires. In vivo, TCRαβ bearing macrophages abundantly accumulate at the inner host-pathogen contact zone of caseous granulomas from patients with lung tuberculosis. In chimeric mouse models, deletion of the variable macrophage-TCRαβ or TNF is associated with structurally compromised granulomas of pulmonary tuberculosis even in the presence of intact T cells. These results uncover a TNF-regulated recombinatorial immune receptor in monocytes/macrophages and demonstrate its implication in granuloma formation in tuberculosis. PMID:22114556

Galectin-3 Shapes Antitumor Immune Responses by Suppressing CD8+ T Cells via LAG-3 and Inhibiting Expansion of Plasmacytoid Dendritic Cells.

PubMed

Kouo, Theodore; Huang, Lanqing; Pucsek, Alexandra B; Cao, Minwei; Solt, Sara; Armstrong, Todd; Jaffee, Elizabeth

2015-04-01

Galectin-3 is a 31-kDa lectin that modulates T-cell responses through several mechanisms, including apoptosis, T-cell receptor (TCR) cross-linking, and TCR downregulation. We found that patients with pancreatic ductal adenocarcinoma (PDA) who responded to a granulocyte-macrophage colony-stimulating factor-secreting allogeneic PDA vaccine developed neutralizing antibodies to galectin-3 after immunization. We show that galectin-3 binds activated antigen-committed CD8(+) T cells only in the tumor microenvironment. Galectin-3-deficient mice exhibit improved CD8(+) T-cell effector function and increased expression of several inflammatory genes. Galectin-3 binds to LAG-3, and LAG-3 expression is necessary for galectin-3-mediated suppression of CD8(+) T cells in vitro. Lastly, galectin-3-deficient mice have elevated levels of circulating plasmacytoid dendritic cells, which are superior to conventional dendritic cells in activating CD8(+) T cells. Thus, inhibiting galectin-3 in conjunction with CD8(+) T-cell-directed immunotherapies should enhance the tumor-specific immune response. ©2015 American Association for Cancer Research.

Identification of a regulatory T cell specific cell surface molecule that mediates suppressive signals and induces Foxp3 expression.

PubMed

Wang, Rui; Wan, Qi; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2008-07-16

Regulatory T (T(reg)) cells control immune activation and maintain tolerance. How T(regs) mediate their suppressive function is unclear. Here we identified a cell surface molecule, called GARP, (or LRRC32), which within T cells is specifically expressed in T(regs) activated through the T cell receptor (TCR). Ectopic expression of GARP in human naïve T (T(N)) cells inhibited their proliferation and cytokine secretion upon TCR activation. Remarkably, GARP over-expression in T(N) cells induced expression of T(reg) master transcription factor Foxp3 and endowed them with a partial suppressive function. The extracellular but not the cytoplasmic region of GARP, was necessary for these functions. Silencing Foxp3 in human T(reg) cells reduced expression of GARP and attenuated their suppressive function. However, GARP function was not affected when Foxp3 was downregulated in GARP-overexpressing cells, while silencing GARP in Foxp3-overexpressing cells reduced their suppressive activity. These findings reveal a novel cell surface molecule-mediated regulatory mechanism, with implications for modulating aberrant immune responses.

Incorporating Transformative Consumer Research into the Consumer Behavior Course Experience

ERIC Educational Resources Information Center

Petkus, Ed, Jr.

2010-01-01

In contrast to understanding consumer behavior for the benefit of business organizations, transformative consumer research (TCR) seeks to understand consumer behavior for the benefit of consumers themselves. Following Mari's (2008) call for the incorporation of TCR in doctoral programs in marketing, this article outlines the relevance of TCR to…

Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation

PubMed Central

Alcázar, Isabela; Marqués, Miriam; Kumar, Amit; Hirsch, Emilio; Wymann, Matthias; Carrera, Ana C.; Barber, Domingo F.

2007-01-01

Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation. PMID:17998387

Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling.

PubMed

Liston, Adrian; Hardy, Kristine; Pittelkow, Yvonne; Wilson, Susan R; Makaroff, Lydia E; Fahrer, Aude M; Goodnow, Christopher C

2007-01-01

T cells in the thymus undergo opposing positive and negative selection processes so that the only T cells entering circulation are those bearing a T cell receptor (TCR) with a low affinity for self. The mechanism differentiating negative from positive selection is poorly understood, despite the fact that inherited defects in negative selection underlie organ-specific autoimmune disease in AIRE-deficient people and the non-obese diabetic (NOD) mouse strain Here we use homogeneous populations of T cells undergoing either positive or negative selection in vivo together with genome-wide transcription profiling on microarrays to identify the gene expression differences underlying negative selection to an Aire-dependent organ-specific antigen, including the upregulation of a genomic cluster in the cytogenetic band 2F. Analysis of defective negative selection in the autoimmune-prone NOD strain demonstrates a global impairment in the induction of the negative selection response gene set, but little difference in positive selection response genes. Combining expression differences with genetic linkage data, we identify differentially expressed candidate genes, including Bim, Bnip3, Smox, Pdrg1, Id1, Pdcd1, Ly6c, Pdia3, Trim30 and Trim12. The data provide a molecular map of the negative selection response in vivo and, by analysis of deviations from this pathway in the autoimmune susceptible NOD strain, suggest that susceptibility arises from small expression differences in genes acting at multiple points in the pathway between the TCR and cell death.

Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling

PubMed Central

Liston, Adrian; Hardy, Kristine; Pittelkow, Yvonne; Wilson, Susan R; Makaroff, Lydia E; Fahrer, Aude M; Goodnow, Christopher C

2007-01-01

Background T cells in the thymus undergo opposing positive and negative selection processes so that the only T cells entering circulation are those bearing a T cell receptor (TCR) with a low affinity for self. The mechanism differentiating negative from positive selection is poorly understood, despite the fact that inherited defects in negative selection underlie organ-specific autoimmune disease in AIRE-deficient people and the non-obese diabetic (NOD) mouse strain Results Here we use homogeneous populations of T cells undergoing either positive or negative selection in vivo together with genome-wide transcription profiling on microarrays to identify the gene expression differences underlying negative selection to an Aire-dependent organ-specific antigen, including the upregulation of a genomic cluster in the cytogenetic band 2F. Analysis of defective negative selection in the autoimmune-prone NOD strain demonstrates a global impairment in the induction of the negative selection response gene set, but little difference in positive selection response genes. Combining expression differences with genetic linkage data, we identify differentially expressed candidate genes, including Bim, Bnip3, Smox, Pdrg1, Id1, Pdcd1, Ly6c, Pdia3, Trim30 and Trim12. Conclusion The data provide a molecular map of the negative selection response in vivo and, by analysis of deviations from this pathway in the autoimmune susceptible NOD strain, suggest that susceptibility arises from small expression differences in genes acting at multiple points in the pathway between the TCR and cell death. PMID:17239257

Induction of peripheral T cell tolerance and allo/xenoimmunity.

PubMed

Charpentier, B; Alard, P; Hiesse, C; Lantz, O

1994-03-01

It is theoretically impossible or at least difficult to tolerize animals in xenogeneic situation, particularly in non-concordant species. Both humoral defense and several cellular components (T and non T) are offensive weapons which can be directed at myriad of self-antigens in a normal situation, at allo-antigens in abnormal situations, at xeno-antigens in exceptional situations. In either cases, the fine epitopic definition of allo/xeno antigens seen by the TCR is far from being totally known. From recent studies showing the precise requirement of peptides assembly composition in the MHC class I and II groove it should be theoretically possible to tolerize any group of peptides if they are presented, some other, because not presented, may remain ignored, thus apparently tolerized. It is also know, that transplantation tolerance is not a law of "either nothing or all" but a multiple process depending of both the affinity of the TCR and the nature/compositions of the target. In view of the complex array of factors influencing the pathway of T cell activation, three forms of T cell non responsiveness may be suggested in the context of xenogeneic recognition: physical deletion of potentially reactive T cells occurring predominantly in the thymus, non reactivity of T cells resulting from their failure to be influenced by antigens, and anergy possibly due to inappropriate signals from non professional antigen presenting cells. Future investigations must elucidate the requirements for inducing these events in a purpose of xenogeneic organ transplantation.

Accelerated proton echo planar spectroscopic imaging (PEPSI) using GRAPPA with a 32-channel phased-array coil.

PubMed

Tsai, Shang-Yueh; Otazo, Ricardo; Posse, Stefan; Lin, Yi-Ru; Chung, Hsiao-Wen; Wald, Lawrence L; Wiggins, Graham C; Lin, Fa-Hsuan

2008-05-01

Parallel imaging has been demonstrated to reduce the encoding time of MR spectroscopic imaging (MRSI). Here we investigate up to 5-fold acceleration of 2D proton echo planar spectroscopic imaging (PEPSI) at 3T using generalized autocalibrating partial parallel acquisition (GRAPPA) with a 32-channel coil array, 1.5 cm(3) voxel size, TR/TE of 15/2000 ms, and 2.1 Hz spectral resolution. Compared to an 8-channel array, the smaller RF coil elements in this 32-channel array provided a 3.1-fold and 2.8-fold increase in signal-to-noise ratio (SNR) in the peripheral region and the central region, respectively, and more spatial modulated information. Comparison of sensitivity-encoding (SENSE) and GRAPPA reconstruction using an 8-channel array showed that both methods yielded similar quantitative metabolite measures (P > 0.1). Concentration values of N-acetyl-aspartate (NAA), total creatine (tCr), choline (Cho), myo-inositol (mI), and the sum of glutamate and glutamine (Glx) for both methods were consistent with previous studies. Using the 32-channel array coil the mean Cramer-Rao lower bounds (CRLB) were less than 8% for NAA, tCr, and Cho and less than 15% for mI and Glx at 2-fold acceleration. At 4-fold acceleration the mean CRLB for NAA, tCr, and Cho was less than 11%. In conclusion, the use of a 32-channel coil array and GRAPPA reconstruction can significantly reduce the measurement time for mapping brain metabolites. (c) 2008 Wiley-Liss, Inc.

Allelic polymorphism in the T cell receptor and its impact on immune responses.

PubMed

Gras, Stephanie; Chen, Zhenjun; Miles, John J; Liu, Yu Chih; Bell, Melissa J; Sullivan, Lucy C; Kjer-Nielsen, Lars; Brennan, Rebekah M; Burrows, Jacqueline M; Neller, Michelle A; Khanna, Rajiv; Purcell, Anthony W; Brooks, Andrew G; McCluskey, James; Rossjohn, Jamie; Burrows, Scott R

2010-07-05

In comparison to human leukocyte antigen (HLA) polymorphism, the impact of allelic sequence variation within T cell receptor (TCR) loci is much less understood. Particular TCR loci have been associated with autoimmunity, but the molecular basis for this phenomenon is undefined. We examined the T cell response to an HLA-B*3501-restricted epitope (HPVGEADYFEY) from Epstein-Barr virus (EBV), which is frequently dominated by a TRBV9*01(+) public TCR (TK3). However, the common allelic variant TRBV9*02, which differs by a single amino acid near the CDR2beta loop (Gln55-->His55), was never used in this response. The structure of the TK3 TCR, its allelic variant, and a nonnaturally occurring mutant (Gln55-->Ala55) in complex with HLA-B*3501(HPVGEADYFEY) revealed that the Gln55-->His55 polymorphism affected the charge complementarity at the TCR-peptide-MHC interface, resulting in reduced functional recognition of the cognate and naturally occurring variants of this EBV peptide. Thus, polymorphism in the TCR loci may contribute toward variability in immune responses and the outcome of infection.

A conserved αβ transmembrane interface forms the core of a compact T-cell receptor-CD3 structure within the membrane.

PubMed

Krshnan, Logesvaran; Park, Soohyung; Im, Wonpil; Call, Melissa J; Call, Matthew E

2016-10-25

The T-cell antigen receptor (TCR) is an assembly of eight type I single-pass membrane proteins that occupies a central position in adaptive immunity. Many TCR-triggering models invoke an alteration in receptor complex structure as the initiating event, but both the precise subunit organization and the pathway by which ligand-induced alterations are transferred to the cytoplasmic signaling domains are unknown. Here, we show that the receptor complex transmembrane (TM) domains form an intimately associated eight-helix bundle organized by a specific interhelical TCR TM interface. The salient features of this core structure are absolutely conserved between αβ and γδ TCR sequences and throughout vertebrate evolution, and mutations at key interface residues caused defects in the formation of stable TCRαβ:CD3δε:CD3γε:ζζ complexes. These findings demonstrate that the eight TCR-CD3 subunits form a compact and precisely organized structure within the membrane and provide a structural basis for further investigation of conformationally regulated models of transbilayer TCR signaling.

Actin polymerization‐dependent activation of Cas‐L promotes immunological synapse stability

PubMed Central

Santos, Luís C; Blair, David A; Kumari, Sudha; Cammer, Michael; Iskratsch, Thomas; Herbin, Olivier; Alexandropoulos, Konstantina

2016-01-01

The immunological synapse formed between a T‐cell and an antigen‐presenting cell is important for cell–cell communication during T‐cell‐mediated immune responses. Immunological synapse formation begins with stimulation of the T‐cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization‐dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte‐specific Crk‐associated substrate (Cas‐L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas‐L is phosphorylated at TCR microclusters in an actin polymerization‐dependent fashion. Furthermore, Cas‐L participates in a positive feedback loop leading to amplification of Ca2+ signaling, inside–out integrin activation, and actomyosin contraction. We propose a new role for Cas‐L in T‐cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin‐dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T‐cell‐mediated immune responses. PMID:27359298

T cell receptor repertoires of mice and humans are clustered in similarity networks around conserved public CDR3 sequences

PubMed Central

Madi, Asaf; Poran, Asaf; Shifrut, Eric; Reich-Zeliger, Shlomit; Greenstein, Erez; Zaretsky, Irena; Arnon, Tomer; Laethem, Francois Van; Singer, Alfred; Lu, Jinghua; Sun, Peter D; Cohen, Irun R; Friedman, Nir

2017-01-01

Diversity of T cell receptor (TCR) repertoires, generated by somatic DNA rearrangements, is central to immune system function. However, the level of sequence similarity of TCR repertoires within and between species has not been characterized. Using network analysis of high-throughput TCR sequencing data, we found that abundant CDR3-TCRβ sequences were clustered within networks generated by sequence similarity. We discovered a substantial number of public CDR3-TCRβ segments that were identical in mice and humans. These conserved public sequences were central within TCR sequence-similarity networks. Annotated TCR sequences, previously associated with self-specificities such as autoimmunity and cancer, were linked to network clusters. Mechanistically, CDR3 networks were promoted by MHC-mediated selection, and were reduced following immunization, immune checkpoint blockade or aging. Our findings provide a new view of T cell repertoire organization and physiology, and suggest that the immune system distributes its TCR sequences unevenly, attending to specific foci of reactivity. DOI: http://dx.doi.org/10.7554/eLife.22057.001 PMID:28731407

The chemokine CXCL12 generates costimulatory signals in T cells to enhance phosphorylation and clustering of the adaptor protein SLP-76.

PubMed

Smith, Xin; Schneider, Helga; Köhler, Karsten; Liu, Hebin; Lu, Yuning; Rudd, Christopher E

2013-07-30

The CXC chemokine CXCL12 mediates the chemoattraction of T cells and enhances the stimulation of T cells through the T cell receptor (TCR). The adaptor SLP-76 [Src homology 2 (SH2) domain-containing leukocyte protein of 76 kD] has two key tyrosine residues, Tyr(113) and Tyr(128), that mediate signaling downstream of the TCR. We investigated the effect of CXCL12 on SLP-76 phosphorylation and the TCR-dependent formation of SLP-76 microclusters. Although CXCL12 alone failed to induce SLP-76 cluster formation, it enhanced the number, stability, and phosphorylation of SLP-76 microclusters formed in response to stimulation of the TCR by an activating antibody against CD3, a component of the TCR complex. Addition of CXCL12 to anti-CD3-stimulated cells resulted in F-actin polymerization that stabilized SLP-76 microclusters in the cells' periphery at the interface with antibody-coated coverslips and increased the interaction between SLP-76 clusters and those containing ZAP-70, the TCR-associated kinase that phosphorylates SLP-76, as well as increased TCR-dependent gene expression. Costimulation with CXCL12 and anti-CD3 increased the extent of phosphorylation of SLP-76 at Tyr(113) and Tyr(128), but not that of other TCR-proximal components, and mutation of either one of these residues impaired the CXCL12-dependent effect on SLP-76 microcluster formation, F-actin polymerization, and TCR-dependent gene expression. The effects of CXCL12 on SLP-76 microcluster formation were dependent on the coupling of its receptor CXCR4 to G(i)-family G proteins (heterotrimeric guanine nucleotide-binding proteins). Thus, we identified a costimulatory mechanism by which CXCL12 and antigen converge at SLP-76 microcluster formation to enhance T cell responses.

Mycobacterium bovis BCG Scar Status and HLA Class II Alleles Influence Purified Protein Derivative-Specific T-Cell Receptor Vβ Expression in Pulmonary Tuberculosis Patients from Southern India

PubMed Central

Shanmugalakshmi, S.; Dheenadhayalan, V.; Muthuveeralakshmi, P.; Arivarignan, G.; Pitchappan, R.M.

2003-01-01

Purified protein derivative (PPD) RT23-recalled T-cell receptor (TCR) Vβ expression was studied in the peripheral blood of 42 pulmonary tuberculosis patients and 44 healthy controls from southern India, a region where tuberculosis is endemic. Forty-eight-hour whole-blood cultures in the presence or absence of PPD-RT23 were set up, and at the end of the culture period total RNA was extracted and cDNA was synthesized. Expression of various TCR Vβ families was assessed by using family-specific primers. PPD-specific expression (usage) of TCR Vβ families 4, 6, 8 to 12, and 14 was found in more controls than patients. Among the responders (individuals who showed PPD-specific expression), endemic controls had significantly higher responses than the patients had for TCR Vβ families 2, 3, 7, 13, and 17. The majority of the patients did not show usage of most of the TCR Vβ families, and this was attributed to T-cell downregulation. A four-way nested classification analysis revealed that TCR Vβ family 1, 5, 9, 12, and 13 usage in the context of HLA class II high-risk alleles (DRB1*1501, DRB1*08, and DQB1*0601) and Mycobacterium bovis BCG scar status were the determining factors in susceptibility and resistance to tuberculosis. The healthier status of controls was attributed to the wider usage of many TCR Vβ families readily recalled by PPD, while the disease status of the patients was attributed to TCR Vβ downregulation and the resultant T-cell (memory cell?) unresponsiveness. Host genetics (HLA status) and BCG vaccination (scar status) seem to play important roles in skewing the immune response in adult susceptibility to pulmonary tuberculosis through TCR Vβ usage. PMID:12874334

Differences in TCR-Vβ Repertoire and Effector Phenotype between Tumor Infiltrating Lymphocytes and Peripheral Blood Lymphocytes Increase with Age

PubMed Central

Wang, Teng; Shen, Han; Wu, Fenglin; Zhang, Wenfeng; Tao, Changli; Yuan, Yin; Bo, Huaben; Wang, Hui; Huang, Shulin

2014-01-01

Tumor infiltrating lymphocytes (TIL) reflect the host's anti-tumor immune response, and can be a valuable predictor of prognosis. However, many properties of TIL are not fully understood. In the present study, TCR-Vβ repertoires of cancer patients were primarily analyzed by flow cytometry. Abnormally expressed TCR-Vβ subfamilies were generally found in both TIL and peripheral blood lymphocytes (PBL) of each patient. Of note, increased patient age was associated with increasingly biased TCR-Vβ repertoire in TIL but not in PBL, and the dispersion degree of the differences of TCR-Vβ subfamilies between TIL and PBL correlated positively with age (P = 0.007). Utilizing immunoscope analysis, we identified the age-related reduction in TCR-Vβ diversity, but polyclonal pattern was predominant in significantly expanded TCR-Vβ subfamilies. In addition, we found that older patients possessed a decreased ratio of CD8+CD62L+ non-effector cells in TIL compared to PBL, implying age-related increase of CD8+CD62L− effector cells in TIL. The colocalization analysis of CD8 and CD3, however, suggested the suppressed activity of these effector cells in tumor microenvironment. These findings further elucidate the properties of TIL, showing an increasing difference between TIL and PBL with age, which may provide insight for the development of effective immunotherapies for cancer patients of different ages. PMID:25019226

Evidences in Neurological Surgery and a Cutting Edge Classification of the Trigeminocardiac Reflex: A Systematic Review.

PubMed

Leon-Ariza, Daniel S; Leon-Ariza, Juan S; Nangiana, Jasvinder; Grau, Gabriel Vargas; Leon-Sarmiento, Fidias E; Quiñones-Hinojosa, Alfredo

2018-06-05

The trigeminocardiac reflex (TCR) is characterized by bradycardia, decrease of main arterial blood pressure (MABP), and sometimes, asystole during surgery. We critically reviewed TCR studies and devised a novel classification scheme for assessing the reflex. and Methods: A comprehensive systematic literature review was performed using PubMed, MEDLINE, Web of Science, EMBASE, and Scielo databases. Eligible studies were extracted based on stringent inclusion and exclusion criteria. Statistical analyses were used to assess cardiovascular variables. TCR was classified according to morphophysiological aspects involved with reflex elicitation. 575 patients were included in this study. TCR was found in 8.9% of patients. The reflex was more often triggered by interventions made within the anterior cranial fossa. The maxillary branch (type II in the new classification) was the most prevalent nerve branch found to trigger the TCR. Heart rate (HR) and mean arterial blood pressure (MABP) were similarly altered (p = 0.06; F = 0.3912809), covaried with age (p = 0.012; F = 9.302), and inversely correlated to each other (r = -0.27). TCR is a critical cardiovascular phenomenon that must be quickly identified, efficiently classified, and should trigger vigilance. Prompt therapeutic measures during neurosurgical procedures should be carefully addressed to avoid unwanted complications. Accurate categorization using the new classification scheme will help to improve understanding and guide the management of TCR in the perioperative period. Copyright © 2018 Elsevier Inc. All rights reserved.

Modulators of haemocyanin oxygen affinity in the hypoxia- and sulphide-tolerant Baltic isopod Saduria entomon (L.).

PubMed

Hagerman, L; Vismann, B

2001-11-01

Dialysed haemocyanin from the isopod Saduria entomon had a considerably increased oxygen affinity (lower P50) and Bohr factor (-1.71) compared to native haemocyanin (Bohr factor -1.36) indicating that dialysis removes a small molecule size modulating factor decreasing the affinity of native haemolymph. Dialysed haemocyanin had a slightly lower co-operativity (2.42 +/- 0.3) than native haemocyanin (2.9 +/- 0.2). L-Lactate (10 mmol l(-1)) improved oxygen affinity by 1-1.5 torr while urate had no effect. Mg2+ affected affinity in a pH-dependent manner (Bohr-factor increased to -1.67) while Ca2+ had no effect on the Bohr factor but increased affinity with ca 1 torr. Thiosulphate changed the Bohr factor to -1.75 to -1.82, similar to dialysed blood. Co-operativity was in neither case affected. The haemocyanin characteristics of S. entomon are similar to those of crustaceans from hydrothermal vents. These characteristics are probably general for crustaceans that are more or less permanently exposed to sulphide.

Synthesis of (nor)tropeine (di)esters and allosteric modulation of glycine receptor binding.

PubMed

Maksay, Gábor; Nemes, Péter; Vincze, Zoltán; Bíró, Timea

2008-02-15

(Hetero)aromatic mono- and diesters of tropine and nortropine were prepared. Modulation of [3H]strychnine binding to glycine receptors of rat spinal cord was examined with a ternary allosteric model. The esters displaced [3H]strychnine binding with nano- or micromolar potencies and strong negative cooperativity. Coplanarity and distance of the ester moieties of diesters affected the binding affinity being nanomolar for isophthaloyl-bistropane and nortropeines. Nortropisetron had the highest affinity (K(A) approximately 10 nM). Two esters displayed negative cooperativity with glycine in displacement, while three esters of low-affinity and nortropisetron exerted positive cooperativity with glycine.

MHC class II/ESO tetramer-based generation of in vitro primed anti-tumor T-helper lines for adoptive cell therapy of cancer.

PubMed

Poli, Caroline; Raffin, Caroline; Dojcinovic, Danijel; Luescher, Immanuel; Ayyoub, Maha; Valmori, Danila

2013-02-01

Generation of tumor-antigen specific CD4(+) T-helper (T(H)) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4(+) T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific T(H) lines. We isolated phenotypically defined CD4(+) T-cell subpopulations from circulating lymphocytes of DR52b(+) healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO(119-143), autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO(119-143) tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer(+) cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer(+) cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific T(H) lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer(+) T(H) lines from conventional CD4(+)CD25(-) naïve and central memory populations, but not from effector memory populations or CD4(+)CD25(+) Treg. In vitro primed T(H) lines recognized ESO with affinities comparable to ESO-tetramer(+) cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO-monospecific polyclonal T(H) lines from non-immune individuals. This is an approach that is of potential interest for adoptive cell therapy of patients bearing ESO-expressing cancers.

Occurrence of the Transferable Copper Resistance Gene tcrB among Fecal Enterococci of U.S. Feedlot Cattle Fed Copper-Supplemented Diets

PubMed Central

Amachawadi, R. G.; Alvarado, C. A.; Mainini, T. R.; Vinasco, J.; Drouillard, J. S.; Nagaraja, T. G.

2013-01-01

Copper, an essential micronutrient, is supplemented in the diet at elevated levels to reduce morbidity and mortality and to promote growth in feedlot cattle. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a transferable copper resistance gene (tcrB) borne on a plasmid. The present study was undertaken to investigate whether the feeding of copper at levels sufficient to promote growth increases the prevalence of the tcrB gene among the fecal enterococci of feedlot cattle. The study was performed with 261 crossbred yearling heifers housed in 24 pens, with pens assigned randomly to a 2×2 factorial arrangement of treatments consisting of dietary copper and a commercial linseed meal-based energy protein supplement. A total of 22 isolates, each identified as Enterococcus faecium, were positive for tcrB with an overall prevalence of 3.8% (22/576). The prevalence was higher among the cattle fed diets supplemented with copper (6.9%) compared to normal copper levels (0.7%). The tcrB-positive isolates always contained both erm(B) and tet(M) genes. Median copper MICs for tcrB-positive and tcrB-negative enterococci were 22 and 4 mM, respectively. The transferability of the tcrB gene was demonstrated via a filter-mating assay. Multilocus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci remain potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut. PMID:23666328

Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCRzeta for degradation.

PubMed

Myers, Margaret D; Dragone, Leonard L; Weiss, Arthur

2005-07-18

Src-like adaptor protein (SLAP) down-regulates expression of the T cell receptor (TCR)-CD3 complex during a specific stage of thymocyte development when the TCR repertoire is selected. Consequently, SLAP-/- thymocytes display alterations in thymocyte development. Here, we have studied the mechanism of SLAP function. We demonstrate that SLAP-deficient thymocytes have increased TCRzeta chain expression as a result of a defect in TCRzeta degradation. Failure to degrade TCRzeta leads to an increased pool of fully assembled TCR-CD3 complexes that are capable of recycling back to the cell surface. We also provide evidence that SLAP functions in a pathway that requires the phosphorylated TCRzeta chain and the Src family kinase Lck, but not ZAP-70 (zeta-associated protein of 70 kD). These studies reveal a unique mechanism by which SLAP contributes to the regulation of TCR expression during a distinct stage of thymocyte development.

T follicular helper and T follicular regulatory cells have different TCR specificity

PubMed Central

Maceiras, Ana Raquel; Almeida, Silvia Cristina Paiva; Mariotti-Ferrandiz, Encarnita; Chaara, Wahiba; Jebbawi, Fadi; Six, Adrien; Hori, Shohei; Klatzmann, David; Faro, Jose; Graca, Luis

2017-01-01

Immunization leads to the formation of germinal centres (GCs) that contain both T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. Whether T-cell receptor (TCR) specificity defines the differential functions of Tfh and Tfr cells is unclear. Here we show that antigen-specific T cells after immunization are preferentially recruited to the GC to become Tfh cells, but not Tfr cells. Tfh cells, but not Tfr cells, also proliferate efficiently on restimulation with the same immunizing antigen in vitro. Ex vivo TCR repertoire analysis shows that immunization induces oligoclonal expansion of Tfh cells. By contrast, the Tfr pool has a TCR repertoire that more closely resembles that of regulatory T (Treg) cells. Our data thus indicate that the GC Tfh and Tfr pools are generated from distinct TCR repertoires, with Tfh cells expressing antigen-responsive TCRs to promote antibody responses, and Tfr cells expressing potentially autoreactive TCRs to suppress autoimmunity. PMID:28429709

T-cell receptor revision: friend or foe?

PubMed

Hale, J Scott; Fink, Pamela J

2010-04-01

T-cell receptor (TCR) revision is a process of tolerance induction by which peripheral T cells lose surface expression of an autoreactive TCR, reinduce expression of the recombinase machinery, rearrange genes encoding extrathymically generated TCRs for antigen, and express these new receptors on the cell surface. We discuss the evidence for this controversial tolerance mechanism below. Despite the apparent heresy of post-thymic gene rearrangement, we argue here that TCR revision follows the rules obeyed by maturing thymocytes undergoing gene recombination. Expression of the recombinase is carefully controlled both spatially and temporally, and may be initiated by loss of signals through surface TCRs. The resulting TCR repertoire is characterized by its diversity, self major histocompatibility complex restriction, self tolerance, and ability to mount productive immune responses specific for foreign antigens. Hence, TCR revision is a carefully regulated process of tolerance induction that can contribute to the protection of the individual against invading pathogens while preserving the integrity of self tissue.

Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

PubMed

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling.

Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications

PubMed Central

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

Changing physiological status predicts severe injury and need for specialized trauma center resources.

PubMed

Talbert, Steven

2009-01-01

This study evaluated the association between changing physiological status (delta data) with severe injury (SI) or need for trauma center resources (TCR). Prehospital and emergency department arrival weighted RTS (RTSw) were computed for patients with complete records entered into the registry from 2002 to 2004 (n = 23,753). Physiological change was classified as unchanged, deteriorated, or improved (PreRTSw vs EDRTSw). Performance of delta data was evaluated using standard epidemiological approaches and multiple logistic regression. Deterioration status predicted SI (operating room [OR] = 1.38) and TCR (OR = 2.09). Improved status predicted TCR (OR = 1.27). Delta data independently predicted both SI and TCR.

The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms

PubMed Central

Ophir, Michael J.; Liu, Beiyun C.

2013-01-01

The T cell receptor (TCR) triggers the assembly of “SLP-76 microclusters,” which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase–associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A “tandem dimer” containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP–interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and “inside-out” signaling to β1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and β1 integrins. PMID:24368808

The three-dimensional structure of a T-cell antigen receptor V alpha V beta heterodimer reveals a novel arrangement of the V beta domain.

PubMed Central

Housset, D; Mazza, G; Grégoire, C; Piras, C; Malissen, B; Fontecilla-Camps, J C

1997-01-01

The crystal structure of a mouse T-cell antigen receptor (TCR) Fv fragment complexed to the Fab fragment of a specific anti-clonotypic antibody has been determined to 2.6 A resolution. The polypeptide backbone of the TCR V alpha domain is very similar to those of other crystallographically determined V alphas, whereas the V beta structure is so far unique among TCR V beta domains in that it displays a switch of the c" strand from the inner to the outer beta-sheet. The beta chain variable region of this TCR antigen-binding site is characterized by a rather elongated third complementarity-determining region (CDR3beta) that packs tightly against the CDR3 loop of the alpha chain, without leaving any intervening hydrophobic pocket. Thus, the conformation of the CDR loops with the highest potential diversity distinguishes the structure of this TCR antigen-binding site from those for which crystallographic data are available. On the basis of all these results, we infer that a significant conformational change of the CDR3beta loop found in our TCR is required for binding to its cognate peptide-MHC ligand. PMID:9250664

Intimate association of Thy-1 and the T-cell antigen receptor with the CD45 tyrosine phosphatase

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

Volarevic, S.; Burns, C.M.; Sussman, J.J.

1990-09-01

Immunoprecipitation of Thy-1 from Triton X-100 detergent lysates of surface-iodinated and chemically cross-linked T cells precipitated at least first major and discrete bands. Four of these bands were identified as Thy-1, CD45 (a trasmembrane tyrosine phosphatase), a major histocompatibility complex-encoded class I molecule, and {beta}{sub 2}-microglobulin. Similar analyses revealed that CD45 was coprecipitated from lysates of cross-linker-treated cells by antibodies to the T-cell antigen receptor (TCR). The same pattern of coprecipitated bands was observed when digitonin was used to lyse untreated cells. Immunoprecipitation of Thy-1 or the TCR from lysates of cross-linked T cells precipitated CD45 tyrosine phosphatase activity. Calculationsmore » based upon the amounts of coprecipitated enzymatic activity or TCR {zeta} chain indicate that a substantial fraction of Thy-1 and TCR complexes can be cross-linked to CD45. These data support a model in which the dependence of Thy-1 signaling on TCR coexpression is due to their common interaction with a tyrosine phosphatase and provide a possible structural basis for the influence of CD45 on TCR-mediated signaling.« less

Propensity of a single-walled carbon nanotube-peptide to mimic a KK10 peptide in an HLA-TCR complex

NASA Astrophysics Data System (ADS)

Feng, Mei; Bell, David R.; Zhou, Ruhong

2017-12-01

The application of nanotechnology to improve disease diagnosis, treatment, monitoring, and prevention is the goal of nanomedicine. We report here a theoretical study of a functionalized single-walled carbon nanotube (CNT) mimic binding to a human leukocyte antigen-T cell receptor (HLA-TCR) immune complex as a first attempt of a potential nanomedicine for human immunodeficiency virus (HIV) vaccine development. The carbon nanotube was coated with three arginine residues to imitate the HIV type 1 immunodominant viral peptide KK10 (gag 263-272: KRWIILGLNK), named CNT-peptide hereafter. Through molecular dynamics simulations, we explore the CNT-peptide and KK10 binding to an important HLA-TCR complex. Our results suggest that the CNT-peptide and KK10 bind comparably to the HLA-TCR complex, but the CNT-peptide forms stronger interactions with the TCR. Desorption simulations highlight the innate flexibility of KK10 over the CNT-peptide, resulting in a slightly higher desorption energy required for KK10 over the CNT-peptide. Our findings indicate that the designed CNT-peptide mimic has favorable propensity to activate TCR pathways and should be further explored to understand therapeutic potential.

Evolutionarily conserved TCR binding sites, identification of T cells in primary lymphoid tissues, and surprising trans-rearrangements in nurse shark.

PubMed

Criscitiello, Michael F; Ohta, Yuko; Saltis, Mark; McKinney, E Churchill; Flajnik, Martin F

2010-06-15

Cartilaginous fish are the oldest animals that generate RAG-based Ag receptor diversity. We have analyzed the genes and expressed transcripts of the four TCR chains for the first time in a cartilaginous fish, the nurse shark (Ginglymostoma cirratum). Northern blotting found TCR mRNA expression predominantly in lymphoid and mucosal tissues. Southern blotting suggested translocon-type loci encoding all four chains. Based on diversity of V and J segments, the expressed combinatorial diversity for gamma is similar to that of human, alpha and beta may be slightly lower, and delta diversity is the highest of any organism studied to date. Nurse shark TCRdelta have long CDR3 loops compared with the other three chains, creating binding site topologies comparable to those of mammalian TCR in basic paratope structure; additionally, nurse shark TCRdelta CDR3 are more similar to IgH CDR3 in length and heterogeneity than to other TCR chains. Most interestingly, several cDNAs were isolated that contained IgM or IgW V segments rearranged to other gene segments of TCRdelta and alpha. Finally, in situ hybridization experiments demonstrate a conservation of both alpha/beta and gamma/delta T cell localization in the thymus across 450 million years of vertebrate evolution, with gamma/delta TCR expression especially high in the subcapsular region. Collectively, these data make the first cellular identification of TCR-expressing lymphocytes in a cartilaginous fish.

Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation.

PubMed

Link, C S; Eugster, A; Heidenreich, F; Rücker-Braun, E; Schmiedgen, M; Oelschlägel, U; Kühn, D; Dietz, S; Fuchs, Y; Dahl, A; Domingues, A M J; Klesse, C; Schmitz, M; Ehninger, G; Bornhäuser, M; Schetelig, J; Bonifacio, E

2016-06-01

Allogeneic stem cell transplantation is potentially curative, but associated with post-transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR-α repertoire and its clinical relevance in patients following stem cell transplantation. Using next-generation sequencing we examined the TCR-α repertoire of CD8(+) T cells and CMV-specific CD8(+) T cells in four patients. Additionally, we performed single-cell TCR-αβ sequencing of CMV-specific CD8(+) T cells. The TCR-α composition of human leucocyte antigen (HLA)-A*0201 CMVpp65- and CMVIE -specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8(+) T cells and half of the total CD8(+) T cell repertoire was dominated by few CMV-reactive clonotypes. Some TCR-α clonotypes were shared between patients. Gene expression of the circulating CMV-specific CD8(+) T cells was consistent with chronically activated effector memory T cells. The CD8(+) T cell response to CMV reactivation resulted in an expansion of a few TCR-α clonotypes to dominate the CD8(+) repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti-viral T cell response in this setting. © 2016 British Society for Immunology.

Yellow Fever Virus, but Not Zika Virus or Dengue Virus, Inhibits T-Cell Receptor-Mediated T-Cell Function by an RNA-Based Mechanism.

PubMed

McLinden, James H; Bhattarai, Nirjal; Stapleton, Jack T; Chang, Qing; Kaufman, Thomas M; Cassel, Suzanne L; Sutterwala, Fayyaz S; Haim, Hillel; Houtman, Jon C; Xiang, Jinhua

2017-11-27

The Flavivirus genus within the Flaviviridae family is comprised of many important human pathogens including yellow fever virus (YFV), dengue virus (DENV), and Zika virus (ZKV), all of which are global public health concerns. Although the related flaviviruses hepatitis C virus and human pegivirus (formerly named GBV-C) interfere with T-cell receptor (TCR) signaling by novel RNA and protein-based mechanisms, the effect of other flaviviruses on TCR signaling is unknown. Here, we studied the effect of YFV, DENV, and ZKV on TCR signaling. Both YFV and ZKV replicated in human T cells in vitro; however, only YFV inhibited TCR signaling. This effect was mediated at least in part by the YFV envelope (env) protein coding RNA. Deletion mutagenesis studies demonstrated that expression of a short, YFV env RNA motif (vsRNA) was required and sufficient to inhibit TCR signaling. Expression of this vsRNA and YFV infection of T cells reduced the expression of a Src-kinase regulatory phosphatase (PTPRE), while ZKV infection did not. YFV infection in mice resulted in impaired TCR signaling and PTPRE expression, with associated reduction in murine response to experimental ovalbumin vaccination. Together, these data suggest that viruses within the flavivirus genus inhibit TCR signaling in a species-dependent manner. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Cloning of a promoter-like soybean DNA sequence responding to IAA induction in Escherichia coli K12.

PubMed

Kline, E L; Chiang, S J; Lattora, D; Chaung, W

1992-02-01

We have constructed a soybean genomic DNA library in Escherichia coli K12 strain KC13 using plasmid pPV33, which consists of a promoter-less tetracycline resistance (Tcr) gene. A recombinant clone, KC13(pAU-SB1)+, was obtained by selecting for resistance to tetracycline in the presence of indole-3-acetic acid (IAA). Restriction enzyme cleavage and Southern hybridization analysis revealed that the pAU-SB1 plasmid has a 250 bp soybean DNA insert fused with the Tcr gene. In the presence of a selected group of auxins, induction of the Tcr phenotype and mRNA synthesis of the Tcr gene are observed only in KC13(pAU-SB1)+ cultures. On the other hand, induction of the Tcr phenotype and mRNA synthesis of the Tcr gene are absent in cells harboring the cloning vector pPV33 or a recombinant plasmid containing the 250 bp insert in the reverse orientation, pAU-SB1ro. This demonstrated a need for the insertion of the 250 bp soybean DNA and the specificity of its orientation in response to IAA induction. The start point of mRNA transcription in response to IAA, IBA, IPA, 2,4,5-T, and a-NAP is at base pair -96 or -95 upstream of the translational start site of the Tcr gene and base pair -98 with 2,4-D.

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

Takahashi, Takeshi, E-mail: takeshi-takahashi@ciea.or.jp; Katano, Ikumi; Ito, Ryoji

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.more » 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.« less

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

An alternative mode of CD43 signal transduction activates pro-survival pathways of T lymphocytes.

PubMed

Bravo-Adame, Maria Elena; Vera-Estrella, Rosario; Barkla, Bronwyn J; Martínez-Campos, Cecilia; Flores-Alcantar, Angel; Ocelotl-Oviedo, Jose Pablo; Pedraza-Alva, Gustavo; Rosenstein, Yvonne

2017-01-01

CD43 is one of the most abundant co-stimulatory molecules on a T-cell surface; it transduces activation signals through its cytoplasmic domain, contributing to modulation of the outcome of T-cell responses. The aim of this study was to uncover new signalling pathways regulated by this sialomucin. Analysis of changes in protein abundance allowed us to identify pyruvate kinase isozyme M2 (PKM2), an enzyme of the glycolytic pathway, as an element potentially participating in the signalling cascade resulting from the engagement of CD43 and the T-cell receptor (TCR). We found that the glycolytic activity of this enzyme was not significantly increased in response to TCR+CD43 co-stimulation, but that PKM2 was tyrosine phosphorylated, suggesting that it was performing moonlight functions. We report that phosphorylation of both Y 105 of PKM2 and of Y 705 of signal transducer and activator of transcription 3 was induced in response to TCR+CD43 co-stimulation, resulting in activation of the mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) pathway. ERK5 and the cAMP response element binding protein (CREB) were activated, and c-Myc and nuclear factor-κB (p65) nuclear localization, as well as Bad phosphorylation, were augmented. Consistent with this, expression of human CD43 in a murine T-cell hybridoma favoured cell survival. Altogether, our data highlight novel signalling pathways for the CD43 molecule in T lymphocytes, and underscore a role for CD43 in promoting cell survival through non-glycolytic functions of metabolic enzymes. © 2016 John Wiley & Sons Ltd.

Pirfenidone Inhibits T Cell Activation, Proliferation, Cytokine and Chemokine Production, and Host Alloresponses

PubMed Central

Visner, Gary A.; Liu, Fengzhi; Bizargity, Peyman; Liu, Hanzhong; Liu, Kaifeng; Yang, Jun; Wang, Liqing; Hancock, Wayne W.

2009-01-01

Background We previously showed that pirfenidone, an anti-fibrotic agent, reduces lung allograft injury/rejection. In this study, we tested the hypothesis that pirfenidone has immune modulating activities and evaluated its effects on the function of T cell subsets, which play important roles in allograft rejection. Method We first evaluated whether pirfenidone alters T cell proliferation and cytokine release in response to T cell receptor (TCR) activation, and whether pirfenidone alters regulatory T cells (CD4+CD25+) suppressive effects using an in vitro assay. Additionally, pirfenidone effects on alloantigen-induced T cell proliferation in vivo were assessed by adoptive transfer of CFSE-labeled T cells across a parent->F1 MHC mismatch, as well as using a murine heterotopic cardiac allograft model (BALB/c->C57BL/6). Results Pirfenidone was found to inhibit the responder frequency of TCR-stimulated CD4+ cell total proliferation in vitro and in vivo, whereas both CD4 and CD8 proliferation index were reduced by pirfenidone. Additionally, pirfenidone inhibited TCR-induced production of multiple pro-inflammatory cytokines and chemokines. Interestingly, there was no change on TGF-β production by purified T cells, and pirfenidone had no effect on the suppressive properties of naturally occurring regulatory T cells. Pirfenidone alone showed a small but significant (p < 0.05) effect on the in vivo allogeneic response while the combination of pirfenidone and low dose rapamycin had more remarkable effect in reducing the alloantigen response with prolonged graft survival. Conclusion Pirfenidone may be an important new agent in transplantation, with particular relevance to combating chronic rejection by inhibiting both fibroproliferative and alloimmune responses. PMID:19667934

Proteomic and Mutant Analysis of the CO 2 Concentrating Mechanism of Hydrothermal Vent Chemolithoautotroph Thiomicrospira crunogena

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

Mangiapia, Mary; Brown, Terry-René W.; Chaput, Dale

Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO 2+ HCO 3 -+ CO 3 2-) with CO 2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotrophThiomicrospira crunogenahas a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes ofT. crunogenacultivated under low- and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, weremore » at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded byTcr_0854; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853, required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853 -encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla.DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among theBacteriaandArchaea. In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotrophT. crunogenawere identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla inBacteriaand also in one phylum ofArchaea, theEuryarchaeota. Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genusThiomicrospira.« less

Proteomic and Mutant Analysis of the CO 2 Concentrating Mechanism of Hydrothermal Vent Chemolithoautotroph Thiomicrospira crunogena

DOE PAGES

Mangiapia, Mary; Brown, Terry-René W.; Chaput, Dale; ...

2017-01-23

Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO 2+ HCO 3 -+ CO 3 2-) with CO 2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotrophThiomicrospira crunogenahas a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes ofT. crunogenacultivated under low- and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, weremore » at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded byTcr_0854; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853, required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853 -encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla.DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among theBacteriaandArchaea. In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotrophT. crunogenawere identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla inBacteriaand also in one phylum ofArchaea, theEuryarchaeota. Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genusThiomicrospira.« less

Trial shows partial caries removal is an effective technique in primary molars.

PubMed

Santamaria, Ruth; Innes, Nicola

2014-09-01

Randomised controlled trial in a university setting. Children aged three to eight years, with at least one molar with an acute, deep carious lesion into the dentine were recruited. Treatment took place under rubber dam with decayed dentine being removed completely from the lateral walls of cavities in both groups using round burs operated at low speed. TCR or PCR was then performed in the pulpal wall of each tooth. After caries removal teeth were restored with calcium hydroxide cement and composite resin. Teeth with pulpal exposure were pulpotomised using ferric sulphate. The presence of a fistula, swelling, spontaneous pain and mobility not compatible with root resorption were considered to be clinical signs of failure. Radiolucency at the furcation or in the periapical region and internal or external pathological resorption were considered to be radiographic signs of failure. One hundred and twenty-four teeth in 51 patients were randomised. In the TCR group there were 57 teeth and 38 patients, with 41 patients and 67 teeth in the PCR group. Three patients (four teeth; one PCR and three TCR) dropped out leaving 120 teeth (PCR: n = 66; TCR: n = 54) for analysis. In the TCR group 27.5% (15) teeth in 13 children had pulp exposure compared with one tooth in one child in the PCR group (2%). The mean operative time was significantly higher for TCR (28.1 min; 95% CI: 23.6-32.6 min) than for PCR (17.9 min; 95% CI: 16.3-19.5 min). There was no statistical difference in success rates at 24 months between the groups. The success rate in the TCR group was 96%; (95% CI: 85-99%) compared with 92%; (95% CI: 81-96%) in the PCR group. The clinical and radiographic success rates of PCR and TCR in primary teeth with deep carious lesions were high and did not differ significantly, indicating that PCR is a reliable minimally invasive approach in primary teeth and that the retention of carious dentine does not interfere with pulp vitality. Moreover, PCR provided other clinically relevant advantages over TCR, especially lower incidence of pulp exposure and lower operative time.

Proteomic and Mutant Analysis of the CO2 Concentrating Mechanism of Hydrothermal Vent Chemolithoautotroph Thiomicrospira crunogena

PubMed Central

Mangiapia, Mary; Brown, Terry-René W.; Chaput, Dale; Haller, Edward; Harmer, Tara L.; Hashemy, Zahra; Keeley, Ryan; Leonard, Juliana; Mancera, Paola; Nicholson, David; Stevens, Stanley; Wanjugi, Pauline; Zabinski, Tania; Pan, Chongle

2017-01-01

ABSTRACT Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO2 + HCO3− + CO32−) with CO2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotroph Thiomicrospira crunogena has a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes of T. crunogena cultivated under low- and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, were at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded by Tcr_0854; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853, required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853-encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla. IMPORTANCE DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among the Bacteria and Archaea. In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotroph T. crunogena were identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla in Bacteria and also in one phylum of Archaea, the Euryarchaeota. Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genus Thiomicrospira. PMID:28115547

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*

PubMed Central

Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.

2015-01-01

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638

Should modulation of p50 be a therapeutic target in the critically ill?

PubMed

Srinivasan, Amudan J; Morkane, Clare; Martin, Daniel S; Welsby, Ian J

2017-05-01

A defining feature of human hemoglobin is its oxygen binding affinity, quantified by the partial pressure of oxygen at which hemoglobin is 50% saturated (p50), and the variability of this parameter over a range of physiological and environmental states. Modulation of this property of hemoglobin can directly affect the degree of peripheral oxygen offloading and tissue oxygenation. Areas covered: This review summarizes the role of hemoglobin oxygen affinity in normal and abnormal physiology and discusses the current state of the literature regarding artificial modulation of p50. Hypoxic tumors, sickle cell disease, heart failure, and transfusion medicine are discussed in the context of recent advances in hemoglobin oxygen affinity manipulation. Expert commentary: Of particular clinical interest is the possibility of maintaining adequate end-organ oxygen availability in patients with anemia or compromised cardiac function via an increase in systemic p50. This increase in systemic p50 can be achieved with small molecule drugs or a packed red blood cell unit processing variant called rejuvenation, and human trials are needed to better understand the potential clinical benefits to modulating p50.

Functional hierarchy of the N-terminal tyrosines of SLP-76.

PubMed

Jordan, Martha S; Sadler, Jeffrey; Austin, Jessica E; Finkelstein, Lisa D; Singer, Andrew L; Schwartzberg, Pamela L; Koretzky, Gary A

2006-02-15

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a central role in T cell activation and T cell development. SLP-76 has three functional modules: an acidic domain with three key tyrosines, a central proline-rich domain, and a C-terminal Src homology 2 domain. Of these, mutation of the three N-terminal tyrosines (Y112, Y128, and Y145) results in the most profound effects on T cell development and function. Y112 and Y128 associate with Vav and Nck, two proteins shown to be important for TCR-induced phosphorylation of proximal signaling substrates, Ca(2+) flux, and actin reorganization. Y145 has been shown to be important for optimal association of SLP-76 with inducible tyrosine kinase, a key regulator of T cell function. To investigate further the role of the phosphorylatable tyrosines of SLP-76 in TCR signaling, cell lines and primary T cells expressing SLP-76 with mutations in individual or paired tyrosine residues were analyzed. These studies show that Tyr(145) of SLP-76 is the most critical tyrosine for both T cell function in vitro and T cell development in vivo.

Transcription Factor IRF4 Promotes CD8+ T Cell Exhaustion and Limits the Development of Memory-like T Cells during Chronic Infection.

PubMed

Man, Kevin; Gabriel, Sarah S; Liao, Yang; Gloury, Renee; Preston, Simon; Henstridge, Darren C; Pellegrini, Marc; Zehn, Dietmar; Berberich-Siebelt, Friederike; Febbraio, Mark A; Shi, Wei; Kallies, Axel

2017-12-19

During chronic stimulation, CD8 + T cells acquire an exhausted phenotype characterized by expression of inhibitory receptors, down-modulation of effector function, and metabolic impairments. T cell exhaustion protects from excessive immunopathology but limits clearance of virus-infected or tumor cells. We transcriptionally profiled antigen-specific T cells from mice infected with lymphocytic choriomeningitis virus strains that cause acute or chronic disease. T cell exhaustion during chronic infection was driven by high amounts of T cell receptor (TCR)-induced transcription factors IRF4, BATF, and NFATc1. These regulators promoted expression of inhibitory receptors, including PD-1, and mediated impaired cellular metabolism. Furthermore, they repressed the expression of TCF1, a transcription factor required for memory T cell differentiation. Reducing IRF4 expression restored the functional and metabolic properties of antigen-specific T cells and promoted memory-like T cell development. These findings indicate that IRF4 functions as a central node in a TCR-responsive transcriptional circuit that establishes and sustains T cell exhaustion during chronic infection. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells.

PubMed

Jensen, Michael C; Riddell, Stanley R

2014-01-01

A major advance in adoptive T-cell therapy (ACT) is the ability to efficiently endow patient's T cells with reactivity for tumor antigens through the stable or regulated introduction of genes that encode high affinity tumor-targeting T-cell receptors (TCRs) or synthetic chimeric antigen receptors (CARs). Case reports and small series of patients treated with TCR- or CAR-modified T cells have shown durable responses in a subset of patients, particularly with B-cell malignancies treated with T cells modified to express a CAR that targets the CD19 molecule. However, many patients do not respond to therapy and serious on and off-target toxicities have been observed with TCR- and CAR-modified T cells. Thus, challenges remain to make ACT with gene-modified T cells a reproducibly effective and safe therapy and to expand the breadth of patients that can be treated to include those with common epithelial malignancies. This review discusses research topics in our laboratories that focus on the design and implementation of ACT with CAR-modified T cells. These include cell intrinsic properties of distinct T-cell subsets that may facilitate preparing therapeutic T-cell products of defined composition for reproducible efficacy and safety, the design of tumor targeting receptors that optimize signaling of T-cell effector functions and facilitate tracking of migration of CAR-modified T cells in vivo, and novel CAR designs that have alternative ligand binding domains or confer regulated function and/or survival of transduced T cells. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Expitope: a web server for epitope expression.

PubMed

Haase, Kerstin; Raffegerst, Silke; Schendel, Dolores J; Frishman, Dmitrij

2015-06-01

Adoptive T cell therapies based on introduction of new T cell receptors (TCRs) into patient recipient T cells is a promising new treatment for various kinds of cancers. A major challenge, however, is the choice of target antigens. If an engineered TCR can cross-react with self-antigens in healthy tissue, the side-effects can be devastating. We present the first web server for assessing epitope sharing when designing new potential lead targets. We enable the users to find all known proteins containing their peptide of interest. The web server returns not only exact matches, but also approximate ones, allowing a number of mismatches of the users choice. For the identified candidate proteins the expression values in various healthy tissues, representing all vital human organs, are extracted from RNA Sequencing (RNA-Seq) data as well as from some cancer tissues as control. All results are returned to the user sorted by a score, which is calculated using well-established methods and tools for immunological predictions. It depends on the probability that the epitope is created by proteasomal cleavage and its affinities to the transporter associated with antigen processing and the major histocompatibility complex class I alleles. With this framework, we hope to provide a helpful tool to exclude potential cross-reactivity in the early stage of TCR selection for use in design of adoptive T cell immunotherapy. The Expitope web server can be accessed via http://webclu.bio.wzw.tum.de/expitope. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

PubMed

Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

2017-05-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

T-cell receptor revision: friend or foe?

PubMed Central

Hale, J Scott; Fink, Pamela J

2010-01-01

T-cell receptor (TCR) revision is a process of tolerance induction by which peripheral T cells lose surface expression of an autoreactive TCR, reinduce expression of the recombinase machinery, rearrange genes encoding extrathymically generated TCRs for antigen, and express these new receptors on the cell surface. We discuss the evidence for this controversial tolerance mechanism below. Despite the apparent heresy of post-thymic gene rearrangement, we argue here that TCR revision follows the rules obeyed by maturing thymocytes undergoing gene recombination. Expression of the recombinase is carefully controlled both spatially and temporally, and may be initiated by loss of signals through surface TCRs. The resulting TCR repertoire is characterized by its diversity, self major histocompatibility complex restriction, self tolerance, and ability to mount productive immune responses specific for foreign antigens. Hence, TCR revision is a carefully regulated process of tolerance induction that can contribute to the protection of the individual against invading pathogens while preserving the integrity of self tissue. PMID:20201984

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

[Experimental analysis of some determinants of inductive reasoning].

PubMed

Ono, K

1989-02-01

Three experiments were conducted from a behavioral perspective to investigate the determinants of inductive reasoning and to compare some methodological differences. The dependent variable used in these experiments was the threshold of confident response (TCR), which was defined as "the minimal sample size required to establish generalization from instances." Experiment 1 examined the effects of population size on inductive reasoning, and the results from 35 college students showed that the TCR varied in proportion to the logarithm of population size. In Experiment 2, 30 subjects showed distinct sensitivity to both prior probability and base-rate. The results from 70 subjects who participated in Experiment 3 showed that the TCR was affected by its consequences (risk condition), and especially, that humans were sensitive to a loss situation. These results demonstrate the sensitivity of humans to statistical variables in inductive reasoning. Furthermore, methodological comparison indicated that the experimentally observed values of TCR were close to, but not as precise as the optimal values predicted by Bayes' model. On the other hand, the subjective TCR estimated by subjects was highly discrepant from the observed TCR. These findings suggest that various aspects of inductive reasoning can be fruitfully investigated not only from subjective estimations such as probability likelihood but also from an objective behavioral perspective.

KIAA1530 Protein Is Recruited by Cockayne Syndrome Complementation Group Protein A (CSA) to Participate in Transcription-coupled Repair (TCR)

PubMed Central

Fei, Jia; Chen, Junjie

2012-01-01

Transcription-coupled repair (TCR) is the major pathway involved in the removal of UV-induced photolesions from the transcribed strand of active genes. Two Cockayne syndrome (CS) complementation group proteins, CSA and CSB, are important for TCR repair. The molecular mechanisms by which CS proteins regulate TCR remain elusive. Here, we report the characterization of KIAA1530, an evolutionarily conserved protein that participates in this pathway through its interaction with CSA and the TFIIH complex. We found that UV irradiation led to the recruitment of KIAA1530 onto chromatin in a CSA-dependent manner. Cells lacking KIAA1530 were highly sensitive to UV irradiation and displayed deficiency in TCR. In addition, KIAA1530 depletion abrogated stability of the CSB protein following UV irradiation. More excitingly, we found that a unique CSA mutant (W361C), which was previously identified in a patient with UVsS syndrome, showed defective KIAA1530 binding and resulted in a failure of recruiting KIAA1530 and stabilizing CSB after UV treatment. Together, our data not only reveal that KIAA1530 is an important player in TCR but also lead to a better understanding of the molecular mechanism underlying UVsS syndrome. PMID:22902626

αβ T cell receptors as predictors of health and disease

PubMed Central

Attaf, Meriem; Huseby, Eric; Sewell, Andrew K

2015-01-01

The diversity of antigen receptors and the specificity it underlies are the hallmarks of the cellular arm of the adaptive immune system. T and B lymphocytes are indeed truly unique in their ability to generate receptors capable of recognizing virtually any pathogen. It has been known for several decades that T lymphocytes recognize short peptides derived from degraded proteins presented by major histocompatibility complex (MHC) molecules at the cell surface. Interaction between peptide-MHC (pMHC) and the T cell receptor (TCR) is central to both thymic selection and peripheral antigen recognition. It is widely assumed that TCR diversity is required, or at least highly desirable, to provide sufficient immune coverage. However, a number of immune responses are associated with the selection of predictable, narrow, or skewed repertoires and public TCR chains. Here, we summarize the current knowledge on the formation of the TCR repertoire and its maintenance in health and disease. We also outline the various molecular mechanisms that govern the composition of the pre-selection, naive and antigen-specific TCR repertoires. Finally, we suggest that with the development of high-throughput sequencing, common TCR ‘signatures' raised against specific antigens could provide important diagnostic biomarkers and surrogate predictors of disease onset, progression and outcome. PMID:25619506

The molecular determinants of CD8 co-receptor function.

PubMed

Cole, David K; Laugel, Bruno; Clement, Mathew; Price, David A; Wooldridge, Linda; Sewell, Andrew K

2012-10-01

CD8(+) T cells respond to signals mediated through a specific interaction between the T-cell receptor (TCR) and a composite antigen in the form of an epitopic peptide bound between the polymorphic α1 and α2 helices of an MHC class I (MHCI) molecule. The CD8 glycoprotein 'co-receives' antigen by binding to an invariant region of the MHCI molecule and can enhance ligand recognition by up to 1 million-fold. In recent years, a number of structural and biophysical investigations have shed light on the role of the CD8 co-receptor during T-cell antigen recognition. Here, we provide a collated resource for these data, and discuss how the structural and biophysical parameters governing CD8 co-receptor function further our understanding of T-cell cross-reactivity and the productive engagement of low-affinity antigenic ligands. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

Evolution of the antigen-specific CD8+ TCR repertoire across the life span: evidence for clonal homogenization of the old TCR repertoire.

PubMed

Rudd, Brian D; Venturi, Vanessa; Davenport, Miles P; Nikolich-Zugich, Janko

2011-02-15

Defects in T cell responses against pathogens and reduced diversity of TCRs have been described at both extremes of the life span. Yet, we still lack information on how Ag-specific T cell populations are maintained and/or altered from birth to old age. In this study, for the first time to our knowledge, we provide insight into Ag-specific TCR repertoire changes over the life span at the single-cell level. We have examined the TCR diversity of the primary CD8(+) T cell response to the immunodominant HSV-1 epitope HSV glycoprotein B 495-502 (HSV gB(498-505); SSIEFARL) (gB-8p) in neonatal, adult, and old C57BL/6 mice. The global distinctive features of the gB-8p-specific TCR repertoire were preserved in mice of different ages. However, both old and especially neonatal mice exhibited significant decreases in TCR diversity compared with that of adult mice. Still, although the neonatal Ag-specific repertoire comprised expectedly shorter germline-biased CDR3β lengths, the repertoire was surprisingly complex, and only a minority of responding cells lacked random nucleotide additions. Changes with aging included increased use of the already dominant TCRVβ10 family, a trend for lower content of the TCR containing the germline WG motif in the CDR3, and a remarkable sharing of one dominant clonotype between individual old mice, implying operation of selective mechanisms. Implications for the rational design of vaccines for neonates and the elderly are discussed.

CD28 co-stimulation restores T cell responsiveness in NOD mice by overcoming deficiencies in Rac-1/p38 mitogen-activated protein kinase signaling and IL-2 and IL-4 gene transcription.

PubMed

Zhang, J; Salojin, K V; Delovitch, T L

2001-03-01

Previously, we reported that T cell hyporesponsiveness induced by TCR ligation is causal to autoimmune diabetes in NOD mice. Neonatal CD28 co-stimulation reverses T cell hyporesponsiveness and protects NOD mice from diabetes by an IL-4-mediated mechanism, indicating that a deficiency in TCR signaling may be overcome by CD28/B7-2 co-stimulation in NOD T cells. To investigate which co-stimulation-induced signaling events mediate this protection, we analyzed the activity of Ras, Rac-1, mitogen-activated protein kinases (MAPK) and several transcription factors in TCR-activated NOD T cells in the presence or absence of CD28 co-stimulation. We show that CD28 co-stimulation restores normal TCR-induced activation of Rac-1 and p38 MAPK in NOD T cells. Deficiencies in TCR-induced nuclear expression of activating protein (AP)-1 binding proteins as well as activation of AP-1 and NF-AT in the IL-2 and IL-4 P1 promoters are also corrected by CD28 co-stimulation. Thus, CD28 co-stimulation reverses NOD T cell hyporesponsiveness by restoring TCR signaling leading to the activation of AP-1 and NF-AT during IL-2 and IL-4 gene transcription. Our findings provide additional evidence that CD28 co-stimulation amplifies signals delivered by the TCR and further explain the mechanism by which CD28 co-stimulation may protect against autoimmune diabetes.

Intrathymic selection of NK1.1+α/β T cell antigen receptor (TCR)+ cells in transgenic mice bearing TCR specific for chicken ovalbumin and restricted to I-Ad

PubMed Central

Iwabuchi, Chikako; Iwabuchi, Kazuya; Nakagawa, Ken-ichi; Takayanagi, Toshiaki; Nishihori, Hiroki; Tone, Saori; Ogasawara, Kazumasa; Good, Robert A.; Onoé, Kazunori

1998-01-01

Generation and negative selection of NK1.1+α/β T cell receptor (TCR)+ thymocytes were analyzed using TCR-transgenic (B10.D2 × DO10)F1 and (C57BL/6 × DO10)F1 mice and Rag-1−/−/DO10 mice, which had been established by breeding and backcrossing between Rag-1−/− and DO10 mice. Almost all T cells from these mice were shown to bear Vα13/Vβ8.2 that is specific for chicken ovalbumin (cOVA) and restricted to I-Ad. A normal proportion of the NK1.1+ Vα13/Vβ8.2+ thymocytes was generated in these mice. However, the actual cell number of both NK1.1+ and NK1.1− thymocytes in I-Ad/d mice (positive selecting background) was larger than that in I-Ab/d mice (negative selecting background). Markedly low but significant proportions of NK1.1+ Vα13/Vβ8.2+ cells were detected in the spleens from I-Ad/d and I-Ab/d mice. It was shown that the splenic NK1.1+ T cells of the I-Ab/d mice were anergized against stimulation through TCR. When (B10.D2 × DO10)F1 and (C57BL/6 × DO10)F1 mice were given cOVA, extensive or intermediate elimination of NK1.1+α/βTCR+ thymocytes was induced in I-Ad/d or I-Ab/d mice, respectively. However, the clonal elimination was not as complete as that seen in the major NK1.1− thymocyte population. The present findings indicate that normal generation of NK1.1+α/βTCR+ thymocytes occurs in the absence of Vα14-Jα281 and that substantial negative selection operates on the NK1.1+α/βTCR+ cells. PMID:9653164

Alteration of the gene expression profile of T-cell receptor αβ-modified T-cells with diffuse large B-cell lymphoma specificity.

PubMed

Zha, Xianfeng; Yin, Qingsong; Tan, Huo; Wang, Chunyan; Chen, Shaohua; Yang, Lijian; Li, Bo; Wu, Xiuli; Li, Yangqiu

2013-05-01

Antigen-specific, T-cell receptor (TCR)-modified cytotoxic T lymphocytes (CTLs) that target tumors are an attractive strategy for specific adoptive immunotherapy. Little is known about whether there are any alterations in the gene expression profile after TCR gene transduction in T cells. We constructed TCR gene-redirected CTLs with specificity for diffuse large B-cell lymphoma (DLBCL)-associated antigens to elucidate the gene expression profiles of TCR gene-redirected T-cells, and we further analyzed the gene expression profile pattern of these redirected T-cells by Affymetrix microarrays. The resulting data were analyzed using Bioconductor software, a two-fold cut-off expression change was applied together with anti-correlation of the profile ratios to render the microarray analysis set. The fold change of all genes was calculated by comparing the three TCR gene-modified T-cells and a negative control counterpart. The gene pathways were analyzed using Bioconductor and Kyoto Encyclopedia of Genes and Genomes. Identical genes whose fold change was greater than or equal to 2.0 in all three TCR gene-redirected T-cell groups in comparison with the negative control were identified as the differentially expressed genes. The differentially expressed genes were comprised of 33 up-regulated genes and 1 down-regulated gene including JUNB, FOS, TNF, INF-γ, DUSP2, IL-1B, CXCL1, CXCL2, CXCL9, CCL2, CCL4, and CCL8. These genes are mainly involved in the TCR signaling, mitogen-activated protein kinase signaling, and cytokine-cytokine receptor interaction pathways. In conclusion, we characterized the gene expression profile of DLBCL-specific TCR gene-redirected T-cells. The changes corresponded to an up-regulation in the differentiation and proliferation of the T-cells. These data may help to explain some of the characteristics of the redirected T-cells.

The affine cohomology spaces and its applications

NASA Astrophysics Data System (ADS)

Fraj, Nizar Ben; Laraiedh, Ismail

2016-12-01

We compute the nth cohomology space of the affine Lie superalgebra 𝔞𝔣𝔣(1) on the (1,1)-dimensional real superspace with coefficient in a large class of 𝔞𝔣𝔣(1)-modules M. We apply our results to the module of weight densities and the module of linear differential operators acting on a superspace of weighted densities. This work is the generalization of a result by Basdouri et al. [The linear 𝔞𝔣𝔣(n|1)-invariant differential operators on weighted densities on the superspace ℝ1|n and 𝔞𝔣𝔣(n|1)-relative cohomology, Int. J. Geom. Meth. Mod. Phys. 10 (2013), Article ID: 1320004, 9 pp.

Broad spectrum antibiotic enrofloxacin modulates contact sensitivity through gut microbiota in a murine model.

PubMed

Strzępa, Anna; Majewska-Szczepanik, Monika; Lobo, Francis M; Wen, Li; Szczepanik, Marian

2017-07-01

Medical advances in the field of infection therapy have led to an increasing use of antibiotics, which, apart from eliminating pathogens, also partially eliminate naturally existing commensal bacteria. It has become increasingly clear that less exposure to microbiota early in life may contribute to the observed rise in "immune-mediated" diseases, including autoimmunity and allergy. We sought to test whether the change of gut microbiota with the broad spectrum antibiotic enrofloxacin will modulate contact sensitivity (CS) in mice. Natural gut microbiota were modified by oral treatment with enrofloxacin prior to sensitization with trinitrophenyl chloride followed by CS testing. Finally, adoptive cell transfers were performed to characterize the regulatory cells that are induced by microbiota modification. Oral treatment with enrofloxacin suppresses CS and production of anti-trinitrophenyl chloride IgG1 antibodies. Adoptive transfer experiments show that antibiotic administration favors induction of regulatory cells that suppress CS. Flow cytometry and adoptive transfer of purified cells show that antibiotic-induced suppression of CS is mediated by TCR αβ + CD4 + CD25 + FoxP3 + Treg, CD19 + B220 + CD5 + IL-10 + , IL-10 + Tr1, and IL-10 + TCR γδ + cells. Treatment with the antibiotic induces dysbiosis characterized by increased proportion of Clostridium coccoides (cluster XIVa), C coccoides-Eubacterium rectale (cluster XIVab), Bacteroidetes, and Bifidobacterium spp, but decreased segmented filamentous bacteria. Transfer of antibiotic-modified gut microbiota inhibits CS, but this response can be restored through oral transfer of control gut bacteria to antibiotic-treated animals. Oral treatment with a broad spectrum antibiotic modifies gut microbiota composition and promotes anti-inflammatory response, suggesting that manipulation of gut microbiota can be a powerful tool to modulate the course of CS. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

PubMed Central

Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

2012-01-01

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

PubMed Central

Li, Qiongshu; Liu, Muyun; Wu, Man; Zhou, Xin; Wang, Shaobin; Hu, Yuan; Wang, Youfu; He, Yixin; Zeng, Xiaoping; Chen, Junhui; Liu, Qubo; Xiao, Dong; Hu, Xiang; Liu, Weibin

2018-01-01

Placenta-specific 1 (PLAC1), a novel cancer-testis antigen (CTA), is expressed in a number of different human malignancies. It is frequently produced in breast cancer, serving a function in tumorigenesis. Adoptive immunotherapy using T cell receptor (TCR)-engineered T cells against CTA mediates objective tumor regression; however, to the best of our knowledge, targeting PLAC1 using engineered T cells has not yet been attempted. In the present study, the cDNAs encoding TCRα- and β-chains specific for human leukocyte antigen (HLA)-A*0201-restricted PLAC1 were cloned from a cytotoxic T-lymphocyte, generated by in vitro by the stimulation of CD8+ T cells using autologous HLA-A2+ dendritic cells loaded with a PLAC1-specific peptide (p28-36, VLCSIDWFM). The TCRα/β-chains were linked by a 2A peptide linker (TCRα-Thosea asigna virus-TCRβ), and the constructs were cloned into the lentiviral vector, followed by transduction into human cytotoxic (CD8+) T cells. The efficiency of transduction was up to 25.16%, as detected by PLAC1 multimers. TCR-transduced CD8+ T cells, co-cultured with human non-metastatic breast cancer MCF-7 cells (PLAC1+, HLA-A2+) and triple-negative breast cancer MDAMB-231 cells (PLAC1+, HLA-A2+), produced interferon γ and tumor necrosis factor α, suggesting TCR activation. Furthermore, the PLAC1 TCR-transduced CD8+ T cells efficiently and specifically identified and annihilated the HLA-A2+/PLAC1+ breast cancer cell lines in a lactate dehydrogenase activity assay. Western blot analysis demonstrated that TCR transduction stimulated the production of mitogen-activated protein kinase signaling molecules, extracellular signal-regulated kinases 1/2 and nuclear factor-κB, through phosphoinositide 3-kinase γ-mediated phosphorylation of protein kinase B in CD8+ T cells. Xenograft mouse assays revealed that PLAC1 TCR-transduced CD8+T cells significantly delayed the tumor progression in mice-bearing breast cancer compared with normal saline or negative control-transduced groups. In conclusion, a novel HLA-A2-restricted and PLAC1-specific TCR was identified. The present study demonstrated PLAC1 to be a potential target for breast cancer treatment; and the usage of PLAC1-specific TCR-engineered T cells may be a novel strategy for PLAC1-positive breast cancer treatment. PMID:29556312

LAPTM5 promotes lysosomal degradation of intracellular CD3ζ but not of cell surface CD3ζ.

PubMed

Kawai, Yohei; Ouchida, Rika; Yamasaki, Sho; Dragone, Leonard; Tsubata, Takeshi; Wang, Ji-Yang

2014-07-01

The lysosomal protein LAPTM5 has been shown to negatively regulate cell surface T cell receptor (TCR) expression and T-cell activation by promoting CD3ζ degradation in lysosomes, but the mechanism remains largely unknown. Here we show that LAPTM5 promotes lysosomal translocation of intracellular CD3ζ but not of the cell surface CD3ζ associated with the mature TCR complex. Kinetic analysis of the subcellular localization of the newly synthesized CD3ζ suggests that LAPTM5 targets CD3ζ in the Golgi apparatus and promotes its lysosomal translocation. Consistently, a Golgi-localizing mutant CD3ζ can be transported to and degraded in the lysosome by LAPTM5. A CD3ζ YF mutant in which all six tyrosine residues in the immunoreceptor tyrosine-based activation motif are mutated to phenylalanines is degraded as efficiently as is wild type CD3ζ, further suggesting that TCR signaling-triggered tyrosine phosphorylation of CD3ζ is dispensable for LAPTM5-mediated degradation. Previously, Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl have been shown to mediate the ubiquitination of CD3ζ in the internalized TCR complex and its subsequent lysosomal degradation. We show that LAPTM5 and SLAP/c-Cbl function in distinct genetic pathways to negatively regulate TCR expression. Collectively, these results suggest that CD3ζ can be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3ζ dependent on TCR signaling, and LAPTM5, which targets intracellular CD3ζ independent of TCR signaling.

The pool of preactivated Lck in the initiation of T-cell signaling: a critical re-evaluation of the Lck standby model.

PubMed

Ballek, Ondřej; Valečka, Jan; Manning, Jasper; Filipp, Dominik

2015-04-01

The initiation of T-cell receptor (TCR) signaling, based on the cobinding of TCR and CD4-Lck heterodimer to a peptide-major histocompatibility complex II on antigen presenting cells, represents a classical model of T-cell signaling. What is less clear however, is the mechanism which translates TCR engagement to the phosphorylation of immunoreceptor tyrosine-based activation motifs on CD3 chains and how this event is coupled to the delivery of Lck function. Recently proposed 'standby model of Lck' posits that resting T-cells contain an abundant pool of constitutively active Lck (pY394(Lck)) required for TCR triggering, and this amount, upon TCR engagement, remains constant. Here, we show that although maintenance of the limited pool of pY394(Lck) is necessary for the generation of TCR proximal signals in a time-restricted fashion, the total amount of this pool, ~2%, is much smaller than previously reported (~40%). We provide evidence that this dramatic discrepancy in the content of pY394(Lck)is likely the consequence of spontaneous phosphorylation of Lck that occurred after cell solubilization. Additional discrepancies can be accounted for by the sensitivity of different pY394(Lck)-specific antibodies and the type of detergents used. These data suggest that reagents and conditions used for the quantification of signaling parameters must be carefully validated and interpreted. Thus, the limited size of pY394(Lck) pool in primary T-cells invites a discussion regarding the adjustment of the quantitative parameters of the standby model of Lck and reevaluation of the mechanism by which this pool contributes to the generation of proximal TCR signaling.

T-Cell Receptors Binding Orientation over Peptide/MHC Class I Is Driven by Long-Range Interactions

PubMed Central

Ferber, Mathias; Zoete, Vincent; Michielin, Olivier

2012-01-01

Crystallographic data about T-Cell Receptor – peptide – major histocompatibility complex class I (TCRpMHC) interaction have revealed extremely diverse TCR binding modes triggering antigen recognition. Understanding the molecular basis that governs TCR orientation over pMHC is still a considerable challenge. We present a simplified rigid approach applied on all non-redundant TCRpMHC crystal structures available. The CHARMM force field in combination with the FACTS implicit solvation model is used to study the role of long-distance interactions between the TCR and pMHC. We demonstrate that the sum of the coulomb interactions and the electrostatic solvation energies is sufficient to identify two orientations corresponding to energetic minima at 0° and 180° from the native orientation. Interestingly, these results are shown to be robust upon small structural variations of the TCR such as changes induced by Molecular Dynamics simulations, suggesting that shape complementarity is not required to obtain a reliable signal. Accurate energy minima are also identified by confronting unbound TCR crystal structures to pMHC. Furthermore, we decompose the electrostatic energy into residue contributions to estimate their role in the overall orientation. Results show that most of the driving force leading to the formation of the complex is defined by CDR1,2/MHC interactions. This long-distance contribution appears to be independent from the binding process itself, since it is reliably identified without considering neither short-range energy terms nor CDR induced fit upon binding. Ultimately, we present an attempt to predict the TCR/pMHC binding mode for a TCR structure obtained by homology modeling. The simplicity of the approach and the absence of any fitted parameters make it also easily applicable to other types of macromolecular protein complexes. PMID:23251658

T-cell receptors binding orientation over peptide/MHC class I is driven by long-range interactions.

PubMed

Ferber, Mathias; Zoete, Vincent; Michielin, Olivier

2012-01-01

Crystallographic data about T-Cell Receptor - peptide - major histocompatibility complex class I (TCRpMHC) interaction have revealed extremely diverse TCR binding modes triggering antigen recognition. Understanding the molecular basis that governs TCR orientation over pMHC is still a considerable challenge. We present a simplified rigid approach applied on all non-redundant TCRpMHC crystal structures available. The CHARMM force field in combination with the FACTS implicit solvation model is used to study the role of long-distance interactions between the TCR and pMHC. We demonstrate that the sum of the coulomb interactions and the electrostatic solvation energies is sufficient to identify two orientations corresponding to energetic minima at 0° and 180° from the native orientation. Interestingly, these results are shown to be robust upon small structural variations of the TCR such as changes induced by Molecular Dynamics simulations, suggesting that shape complementarity is not required to obtain a reliable signal. Accurate energy minima are also identified by confronting unbound TCR crystal structures to pMHC. Furthermore, we decompose the electrostatic energy into residue contributions to estimate their role in the overall orientation. Results show that most of the driving force leading to the formation of the complex is defined by CDR1,2/MHC interactions. This long-distance contribution appears to be independent from the binding process itself, since it is reliably identified without considering neither short-range energy terms nor CDR induced fit upon binding. Ultimately, we present an attempt to predict the TCR/pMHC binding mode for a TCR structure obtained by homology modeling. The simplicity of the approach and the absence of any fitted parameters make it also easily applicable to other types of macromolecular protein complexes.

T-cell receptor variable genes and genetic susceptibility to celiac disease: an association and linkage study.

PubMed

Roschmann, E; Wienker, T F; Gerok, W; Volk, B A

1993-12-01

Genetic susceptibility of celiac disease is primarily associated with a particular combination of and HLA-DQA1/DQB1 gene; however, this does not fully account for the genetic predisposition. Therefore, the aim of this study was to examine whether T-cell receptor (TCR) genes may be susceptibility genes in celiac disease. HLA class II typing was performed by polymerase chain reaction amplification in combination with sequence-specific oligonucleotide hybridization. TCR alpha (TCRA), TCR gamma (TCRG), and TCR beta (TCRB) loci were investigated by restriction fragment length polymorphism analysis. Allelic frequencies of TCRA, TCRG, and TCRB variable genes were compared between patients with celiac disease (n = 53) and control patients (n = 67), and relative risk (RR) estimates were calculated. The RR was 1.67 for allele C1 at TCRA1, 3.35 for allele D2 at TCRA2, 1.66 for allele B2 at TCRG, and 1.35 for allele B at TCRB, showing no significant association. Additionally, linkage analysis was performed in 23 families. The logarithm of odd scores for celiac disease vs. the TCR variable genes at TCRA, TCRG, and TCRB showed no significant linkage. These data suggest that the analyzed TCR variable gene segments V alpha 1.2, V gamma 11, and V beta 8 do not play a major role in susceptibility to celiac disease.

Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

PubMed

Langerak, A W; Molina, T J; Lavender, F L; Pearson, D; Flohr, T; Sambade, C; Schuuring, E; Al Saati, T; van Dongen, J J M; van Krieken, J H J M

2007-02-01

Lymphoproliferations are generally diagnosed via histomorphology and immunohistochemistry. Although mostly conclusive, occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult. In such cases molecular clonality studies of immunoglobulin (Ig)/T-cell receptor (TCR) rearrangements can be useful. Here we address the issue of clonality assessment in 106 histologically defined reactive lesions, using the standardized BIOMED-2 Ig/TCR multiplex polymerase chain reaction (PCR) heteroduplex and GeneScan assays. Samples were reviewed nationally, except 10% random cases and cases with clonal results selected for additional international panel review. In total 75% (79/106) only showed polyclonal Ig/TCR targets (type I), whereas another 15% (16/106) represent probably polyclonal cases, with weak Ig/TCR (oligo)clonality in an otherwise polyclonal background (type II). Interestingly, in 10% (11/106) clear monoclonal Ig/TCR products were observed (types III/IV), which prompted further pathological review. Clonal cases included two missed lymphomas in national review and nine cases that could be explained as diagnostically difficult cases or probable lymphomas upon additional review. Our data show that the BIOMED-2 Ig/TCR multiplex PCR assays are very helpful in confirming the polyclonal character in the vast majority of reactive lesions. However, clonality detection in a minority should lead to detailed pathological review, including close interaction between pathologist and molecular biologist.

Deconstructing Ras Signaling in the Thymus

PubMed Central

Kortum, Robert L.; Sommers, Connie L.; Pinski, John M.; Alexander, Clayton P.; Merrill, Robert K.; Li, Wenmei; Love, Paul E.

2012-01-01

Thymocytes must transit at least two distinct developmental checkpoints, governed by signals that emanate from either the pre-T cell receptor (pre-TCR) or the TCR to the small G protein Ras before emerging as functional T lymphocytes. Recent studies have shown a role for the Ras guanine exchange factor (RasGEF) Sos1 at the pre-TCR checkpoint. At the second checkpoint, the quality of signaling through the TCR is interrogated to ensure the production of an appropriate T cell repertoire. Although RasGRP1 is the only confirmed RasGEF required at the TCR checkpoint, current models suggest that the intensity and character of Ras activation, facilitated by both Sos and RasGRP1, will govern the boundary between survival (positive selection) and death (negative selection) at this stage. Using mouse models, we have assessed the independent and combined roles for the RasGEFs Sos1, Sos2, and RasGRP1 during thymocyte development. Although Sos1 was the dominant RasGEF at the pre-TCR checkpoint, combined Sos1/RasGRP1 deletion was required to effectively block development at this stage. Conversely, while RasGRP1 deletion efficiently blocked positive selection, combined RasGRP1/Sos1 deletion was required to block negative selection. This functional redundancy in RasGEFs during negative selection may act as a failsafe mechanism ensuring appropriate central tolerance. PMID:22586275

Targeted reconstruction of T cell receptor sequence from single cell RNA-seq links CDR3 length to T cell differentiation state

PubMed Central

Yates, Kathleen B.; Bi, Kevin; Darko, Samuel; Godec, Jernej; Gerdemann, Ulrike; Swadling, Leo; Douek, Daniel C.; Klenerman, Paul; Barnes, Eleanor J.; Sharpe, Arlene H.

2017-01-01

Abstract The T cell compartment must contain diversity in both T cell receptor (TCR) repertoire and cell state to provide effective immunity against pathogens. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current methods for TCR inference from scRNA-seq are limited in their sensitivity and require long sequencing reads, thus increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present TRAPeS, a publicly available tool that can efficiently extract TCR sequence information from short-read scRNA-seq libraries. We apply it to investigate heterogeneity in the CD8+ T cell response in humans and mice, and show that it is accurate and more sensitive than existing approaches. Coupling TRAPeS with transcriptome analysis of CD8+ T cells specific for a single epitope from Yellow Fever Virus (YFV), we show that the recently described ‘naive-like’ memory population have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype cells. This suggests that TCR usage is associated with the differentiation state of the CD8+ T cell response to YFV. PMID:28934479

Tumor vessel-injuring ability improves antitumor effect of cytotoxic T lymphocytes in adoptive immunotherapy.

PubMed

Kanagawa, N; Yanagawa, T; Nakagawa, T; Okada, N; Nakagawa, S

2013-01-01

Angiogenesis is required for normal physiologic processes, but it is also involved in tumor growth, progression and metastasis. Here, we report the development of an immune-based antiangiogenic strategy based on the generation of T lymphocytes that possess killing specificity for cells expressing vascular endothelial growth factor receptor 2 (VEGFR2). To target VEGFR2-expressing cells, we engineered cytotoxic T lymphocyte (CTL) expressing chimeric T-cell receptors (cTCR-CTL) comprised of a single-chain variable fragment (scFv) against VEGFR2 linked to an intracellular signaling sequence derived from the CD3ζ chain of the TCR and CD28 by retroviral gene transduction methods. The cTCR-CTL exhibited efficient killing specificity against VEGFR2 and a tumor-targeting function in vitro and in vivo. Reflecting such abilities, we confirmed that the cTCR-CTL strongly inhibited the growth of a variety of syngeneic tumors after adoptive transfer into tumor-bearing mice without consequent damage to normal tissue. In addition, CTL expressing both cTCR and tumor-specific TCR induced complete tumor regression due to enhanced tumor infiltration by the CTL and long-term antigen-specific function. These findings provide evidence that the tumor vessel-injuring ability improved the antitumor effect of CTLs in adoptive immunotherapy for a broad range of cancers by inducing immune-mediated destruction of the tumor neovasculature.

A conserved αβ transmembrane interface forms the core of a compact T-cell receptor–CD3 structure within the membrane

PubMed Central

Krshnan, Logesvaran; Park, Soohyung; Im, Wonpil; Call, Melissa J.; Call, Matthew E.

2016-01-01

The T-cell antigen receptor (TCR) is an assembly of eight type I single-pass membrane proteins that occupies a central position in adaptive immunity. Many TCR-triggering models invoke an alteration in receptor complex structure as the initiating event, but both the precise subunit organization and the pathway by which ligand-induced alterations are transferred to the cytoplasmic signaling domains are unknown. Here, we show that the receptor complex transmembrane (TM) domains form an intimately associated eight-helix bundle organized by a specific interhelical TCR TM interface. The salient features of this core structure are absolutely conserved between αβ and γδ TCR sequences and throughout vertebrate evolution, and mutations at key interface residues caused defects in the formation of stable TCRαβ:CD3δε:CD3γε:ζζ complexes. These findings demonstrate that the eight TCR–CD3 subunits form a compact and precisely organized structure within the membrane and provide a structural basis for further investigation of conformationally regulated models of transbilayer TCR signaling. PMID:27791034

The adapter protein SLP-76 mediates "outside-in" integrin signaling and function in T cells.

PubMed

Baker, R G; Hsu, C J; Lee, D; Jordan, M S; Maltzman, J S; Hammer, D A; Baumgart, T; Koretzky, G A

2009-10-01

The adapter protein SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an essential mediator of signaling from the T-cell antigen receptor (TCR). We report here that SLP-76 also mediates signaling downstream of integrins in T cells and that SLP-76-deficient T cells fail to support adhesion to integrin ligands. In response to both TCR and integrin stimulation, SLP-76 relocalizes to surface microclusters that colocalize with phosphorylated signaling proteins. Disruption of SLP-76 recruitment to the protein named LAT (linker for activation of T cells) inhibits SLP-76 clustering downstream of the TCR but not downstream of integrins. Conversely, an SLP-76 mutant unable to bind ADAP (adhesion and degranulation-promoting adapter protein) forms clusters following TCR but not integrin engagement and fails to support T-cell adhesion to integrin ligands. These findings demonstrate that SLP-76 relocalizes to integrin-initiated signaling complexes by a mechanism different from that employed during TCR signaling and that SLP-76 relocalization corresponds to SLP-76-dependent integrin function in T cells.

Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells

NASA Astrophysics Data System (ADS)

Bertoletti, Antonio; Sette, Alessandro; Chisari, Francis V.; Penna, Amalia; Levrero, Massimo; Carli, Marco De; Fiaccadori, Franco; Ferrari, Carlo

1994-06-01

IT has been suggested that mutations within immunodominant cytotoxic T-lymphocyte (CTL) epitopes may be exploited by viruses to evade protective immune responses critical for clearance1-4. Viral escape could originate from passive mechanisms, such as mutations within crucial CTL epitopes, either affecting major histocompatibility complex binding or T-cell antigen receptor (TCR) recognition. Additionally, it has recently been shown that substitutions of TCR contact sites can yield analogue peptides that can still interact with the T-cell receptor but be unable to deliver a full stimulatory signal, thus inducing anergy5 or acting as an antagonist for the TCR6-8. We report here that hepatitis B virus isolates derived from two chronically infected patients display variant epitopes that act as natural TCR antagonists with the capacity to inhibit the CTL response to the wild-type epitope. During natural infection, TCR antagonist mutations of CTL epitopes could contribute to the development of viral persistence, especially if the antiviral CTL response is monospecific or the epitope is strongly immunodominant.

A Unique T-Cell Receptor Amino Acid Sequence Selected by Human T-Cell Lymphotropic Virus Type 1 Tax301-309-Specific Cytotoxic T Cells in HLA-A24:02-Positive Asymptomatic Carriers and Adult T-Cell Leukemia/Lymphoma Patients.

PubMed

Ishihara, Yuko; Tanaka, Yukie; Kobayashi, Seiichiro; Kawamura, Koji; Nakasone, Hideki; Gomyo, Ayumi; Hayakawa, Jin; Tamaki, Masaharu; Akahoshi, Yu; Harada, Naonori; Kusuda, Machiko; Kameda, Kazuaki; Ugai, Tomotaka; Wada, Hidenori; Sakamoto, Kana; Sato, Miki; Terasako-Saito, Kiriko; Kikuchi, Misato; Kimura, Shun-Ichi; Tanihara, Aki; Kako, Shinichi; Uchimaru, Kaoru; Kanda, Yoshinobu

2017-10-01

We previously reported that the T-cell receptor (TCR) repertoire of human T-cell lymphotropic virus type 1 (HTLV-1) Tax 301-309 -specific CD8 + cytotoxic T cells (Tax 301-309 -CTLs) was highly restricted and a particular amino acid sequence motif, the PDR motif, was conserved among HLA-A*24:02-positive (HLA-A*24:02 + ) adult T-cell leukemia/lymphoma (ATL) patients who had undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR + CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-1. On the other hand, the TCR repertoires in Tax 301-309 -CTLs of asymptomatic HTLV-1 carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity-determining region 3 (CDR3) of the TCR-β chain of Tax 301-309 -CTLs at the single-cell level and compared not only the TCR repertoires but also the frequencies and phenotypes of Tax 301-309 -CTLs between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax 301-309 -CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax 301-309 -CTLs, 1,458 Tax 301-309 -CTLs and 140 clones were identified in this cohort. Tax 301-309 -CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR + CTL frequencies and HTLV-1 proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, PDR, as a public TCR-CDR3 motif against Tax in HLA-A*24:02 + HTLV-1-infected individuals. Further investigations are warranted to elucidate the role of the PDR + CTL response in the progression from carrier state to ATL. IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-1 infection. The HTLV-1 regulatory protein Tax aggressively promotes the proliferation of HTLV-1-infected lymphocytes and is also a major target antigen for CD8 + CTLs. In our previous evaluation of Tax 301-309 -CTLs, we found that a unique amino acid sequence motif, PDR, in CDR3 of the TCR-β chain of Tax 301-309 -CTLs was conserved among ATL patients after allo-HSCT. Furthermore, the PDR + Tax 301-309 -CTL clones selectively expanded and showed strong cytotoxic activities against HTLV-1. On the other hand, it remains unclear how Tax 301-309 -CTL repertoire exists in ACs. In this study, we comprehensively compared Tax-specific TCR repertoires at the single-cell level between ACs and ATL patients. Tax 301-309 -CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was conserved in all ACs and ATL patients, regardless of clinical subtype in HTLV-1 infection. Copyright © 2017 American Society for Microbiology.

A Unique T-Cell Receptor Amino Acid Sequence Selected by Human T-Cell Lymphotropic Virus Type 1 Tax301-309-Specific Cytotoxic T Cells in HLA-A24:02-Positive Asymptomatic Carriers and Adult T-Cell Leukemia/Lymphoma Patients

PubMed Central

Ishihara, Yuko; Tanaka, Yukie; Kobayashi, Seiichiro; Kawamura, Koji; Nakasone, Hideki; Gomyo, Ayumi; Hayakawa, Jin; Tamaki, Masaharu; Akahoshi, Yu; Harada, Naonori; Kusuda, Machiko; Kameda, Kazuaki; Ugai, Tomotaka; Wada, Hidenori; Sakamoto, Kana; Sato, Miki; Terasako-Saito, Kiriko; Kikuchi, Misato; Kimura, Shun-ichi; Tanihara, Aki; Kako, Shinichi; Uchimaru, Kaoru

2017-01-01

ABSTRACT We previously reported that the T-cell receptor (TCR) repertoire of human T-cell lymphotropic virus type 1 (HTLV-1) Tax301-309-specific CD8+ cytotoxic T cells (Tax301-309-CTLs) was highly restricted and a particular amino acid sequence motif, the PDR motif, was conserved among HLA-A*24:02-positive (HLA-A*24:02+) adult T-cell leukemia/lymphoma (ATL) patients who had undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR+ CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-1. On the other hand, the TCR repertoires in Tax301-309-CTLs of asymptomatic HTLV-1 carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity-determining region 3 (CDR3) of the TCR-β chain of Tax301-309-CTLs at the single-cell level and compared not only the TCR repertoires but also the frequencies and phenotypes of Tax301-309-CTLs between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax301-309-CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax301-309-CTLs, 1,458 Tax301-309-CTLs and 140 clones were identified in this cohort. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR+ CTL frequencies and HTLV-1 proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, PDR, as a public TCR-CDR3 motif against Tax in HLA-A*24:02+ HTLV-1-infected individuals. Further investigations are warranted to elucidate the role of the PDR+ CTL response in the progression from carrier state to ATL. IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-1 infection. The HTLV-1 regulatory protein Tax aggressively promotes the proliferation of HTLV-1-infected lymphocytes and is also a major target antigen for CD8+ CTLs. In our previous evaluation of Tax301-309-CTLs, we found that a unique amino acid sequence motif, PDR, in CDR3 of the TCR-β chain of Tax301-309-CTLs was conserved among ATL patients after allo-HSCT. Furthermore, the PDR+ Tax301-309-CTL clones selectively expanded and showed strong cytotoxic activities against HTLV-1. On the other hand, it remains unclear how Tax301-309-CTL repertoire exists in ACs. In this study, we comprehensively compared Tax-specific TCR repertoires at the single-cell level between ACs and ATL patients. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was conserved in all ACs and ATL patients, regardless of clinical subtype in HTLV-1 infection. PMID:28724766

Reducing FLI1 Levels in the MRL/lpr Lupus Mouse Model Impacts T Cell Function by Modulating Glycosphingolipid Metabolism

PubMed Central

Richard, Erin Morris; Thiyagarajan, Thirumagal; Bunni, Marlene A.; Basher, Fahmin; Roddy, Patrick O.; Siskind, Leah J.; Nietert, Paul J.; Nowling, Tamara K.

2013-01-01

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1 +/+ or Fli1 +/- T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1 +/- lupus T cells compared to animals receiving Fli1 +/+ lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1 +/- T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1 +/+ T cells. Moreover, the Fli1 +/- T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1 +/+ T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus. PMID:24040398

Reducing FLI1 levels in the MRL/lpr lupus mouse model impacts T cell function by modulating glycosphingolipid metabolism.

PubMed

Richard, Erin Morris; Thiyagarajan, Thirumagal; Bunni, Marlene A; Basher, Fahmin; Roddy, Patrick O; Siskind, Leah J; Nietert, Paul J; Nowling, Tamara K

2013-01-01

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1(+/+) or Fli1(+/-) T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1(+/-) lupus T cells compared to animals receiving Fli1(+/+) lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1(+/-) T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1(+/+) T cells. Moreover, the Fli1(+/-) T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1(+/+) T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus.

Eupatilin inhibits T-cell activation by modulation of intracellular calcium flux and NF-kappaB and NF-AT activity.

PubMed

Kim, Young-Dae; Choi, Suck-Chei; Oh, Tae-Young; Chun, Jang-Soo; Jun, Chang-Duk

2009-09-01

Eupatilin, one of the pharmacologically active ingredients of Artemisia princeps, exhibits a potent anti-ulcer activity, but its effects on T-cell immunity have not been investigated. Here, we show that eupatilin has a profound inhibitory effect on IL-2 production in Jurkat T cells as well as in human peripheral blood leukocytes. Eupatilin neither influenced clustering of CD3 and LFA-1 to the immunological synapse nor inhibited conjugate formation between T cells and B cells in the presence or absence of superantigen (SEE). Eupatilin also failed to inhibit T-cell receptor (TCR) internalization, thereby, suggesting that eupatilin does not interfere with TCR-mediated signals on the membrane proximal region. In unstimulated T cells, eupatilin significantly induced apoptotic cell death, as evidenced by an increased population of annexin V(+)/PI(+) cells and cleavage of caspase-3 and PARP. To our surprise, however, once cells were activated, eupatilin had little effect on apoptosis, and instead slightly protected cells from activation-induced cell death, suggesting that apoptosis also is not a mechanism for eupatilin-induced T-cell suppression. On the contrary, eupatilin dramatically inhibited I-kappaBalpha degradation and NF-AT dephosphorylation and, consequently, inhibited NF-kappaB and NF-AT promoter activities in PMA/A23187-stimulated T cells. Interestingly, intracellular calcium flux was significantly perturbed in cells pre-treated with eupatilin, suggesting that calcium-dependent cascades might be targets for eupatilin action. Collectively, our results provide evidence for dual regulatory functions of eupatilin: (1) a pro-apoptotic effect on resting T cells and (2) an immunosuppressive effect on activated T cells, presumably through modulation of Ca(2+) flux. (c) 2009 Wiley-Liss, Inc.

Modulation of Endoplasmic Reticulum Stress Controls CD4+ T-cell Activation and Antitumor Function.

PubMed

Thaxton, Jessica E; Wallace, Caroline; Riesenberg, Brian; Zhang, Yongliang; Paulos, Chrystal M; Beeson, Craig C; Liu, Bei; Li, Zihai

2017-08-01

The endoplasmic reticulum (ER) is an energy-sensing organelle with intimate ties to programming cell activation and metabolic fate. T-cell receptor (TCR) activation represents a form of acute cell stress and induces mobilization of ER Ca 2+ stores. The role of the ER in programming T-cell activation and metabolic fate remains largely undefined. Gp96 is an ER protein with functions as a molecular chaperone and Ca 2+ buffering protein. We hypothesized that the ER stress response may be important for CD4 + T-cell activation and that gp96 may be integral to this process. To test our hypothesis, we utilized genetic deletion of the gp96 gene Hsp90b1 in a CD4 + T cell-specific manner. We show that gp96-deficient CD4 + T cells cannot undergo activation-induced glycolysis due to defective Ca 2+ mobilization upon TCR engagement. We found that activating naïve CD4 + T cells while inhibiting ER Ca 2+ exchange, through pharmacological blockade of the ER Ca 2+ channel inositol trisphosphate receptor (IP 3 R), led to a reduction in cytosolic Ca 2+ content and generated a pool of CD62L high /CD44 low CD4 + T cells compared with wild-type (WT) matched controls. In vivo IP 3 R-inhibited CD4 + T cells exhibited elevated tumor control above WT T cells. Together, these data show that ER-modulated cytosolic Ca 2+ plays a role in defining CD4 + T-cell phenotype and function. Factors associated with the ER stress response are suitable targets for T cell-based immunotherapies. Cancer Immunol Res; 5(8); 666-75. ©2017 AACR . ©2017 American Association for Cancer Research.

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

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

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE PAGES

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

2016-03-09

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

Network module detection: Affinity search technique with the multi-node topological overlap measure

PubMed Central

Li, Ai; Horvath, Steve

2009-01-01

Background Many clustering procedures only allow the user to input a pairwise dissimilarity or distance measure between objects. We propose a clustering method that can input a multi-point dissimilarity measure d(i1, i2, ..., iP) where the number of points P can be larger than 2. The work is motivated by gene network analysis where clusters correspond to modules of highly interconnected nodes. Here, we define modules as clusters of network nodes with high multi-node topological overlap. The topological overlap measure is a robust measure of interconnectedness which is based on shared network neighbors. In previous work, we have shown that the multi-node topological overlap measure yields biologically meaningful results when used as input of network neighborhood analysis. Findings We adapt network neighborhood analysis for the use of module detection. We propose the Module Affinity Search Technique (MAST), which is a generalized version of the Cluster Affinity Search Technique (CAST). MAST can accommodate a multi-node dissimilarity measure. Clusters grow around user-defined or automatically chosen seeds (e.g. hub nodes). We propose both local and global cluster growth stopping rules. We use several simulations and a gene co-expression network application to argue that the MAST approach leads to biologically meaningful results. We compare MAST with hierarchical clustering and partitioning around medoid clustering. Conclusion Our flexible module detection method is implemented in the MTOM software which can be downloaded from the following webpage: PMID:19619323

Network module detection: Affinity search technique with the multi-node topological overlap measure.

PubMed

Li, Ai; Horvath, Steve

2009-07-20

Many clustering procedures only allow the user to input a pairwise dissimilarity or distance measure between objects. We propose a clustering method that can input a multi-point dissimilarity measure d(i1, i2, ..., iP) where the number of points P can be larger than 2. The work is motivated by gene network analysis where clusters correspond to modules of highly interconnected nodes. Here, we define modules as clusters of network nodes with high multi-node topological overlap. The topological overlap measure is a robust measure of interconnectedness which is based on shared network neighbors. In previous work, we have shown that the multi-node topological overlap measure yields biologically meaningful results when used as input of network neighborhood analysis. We adapt network neighborhood analysis for the use of module detection. We propose the Module Affinity Search Technique (MAST), which is a generalized version of the Cluster Affinity Search Technique (CAST). MAST can accommodate a multi-node dissimilarity measure. Clusters grow around user-defined or automatically chosen seeds (e.g. hub nodes). We propose both local and global cluster growth stopping rules. We use several simulations and a gene co-expression network application to argue that the MAST approach leads to biologically meaningful results. We compare MAST with hierarchical clustering and partitioning around medoid clustering. Our flexible module detection method is implemented in the MTOM software which can be downloaded from the following webpage: http://www.genetics.ucla.edu/labs/horvath/MTOM/

Length-Two Representations of Quantum Affine Superalgebras and Baxter Operators

NASA Astrophysics Data System (ADS)

Zhang, Huafeng

2018-03-01

Associated to quantum affine general linear Lie superalgebras are two families of short exact sequences of representations whose first and third terms are irreducible: the Baxter TQ relations involving infinite-dimensional representations; the extended T-systems of Kirillov-Reshetikhin modules. We make use of these representations over the full quantum affine superalgebra to define Baxter operators as transfer matrices for the quantum integrable model and to deduce Bethe Ansatz Equations, under genericity conditions.

Structure-activity relationships of substituted N-benzyl piperidines in the GBR series: Synthesis of 4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(2-trifluoromethylbenzyl)piperidine, an allosteric modulator of the serotonin transporter.

PubMed

Boos, Terrence L; Greiner, Elisabeth; Calhoun, W Jason; Prisinzano, Thomas E; Nightingale, Barbara; Dersch, Christina M; Rothman, Richard B; Jacobson, Arthur E; Rice, Kenner C

2006-06-01

A series of 4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-(substituted benzyl) piperidines with substituents at the ortho and meta positions in the aromatic ring of the N-benzyl side chain were synthesized and their affinities and selectivities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) were determined. One analogue, 4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(2-trifluoromethylbenzyl)piperidine (the C(2)-trifluoromethyl substituted compound), has been found to act as an allosteric modulator of hSERT binding and function. It had little affinity for any of the transporters. Several compounds showed affinity for the DAT in the low nanomolar range and displayed a broad range of SERT/DAT selectivity ratios and very little affinity for the NET. The pharmacological tools provided by the availability of compounds with varying transporter affinity and selectivity could be used to obtain additional information about the properties a compound should have to act as a useful pharmacotherapeutic agent for cocaine addiction and help unravel the pharmacological mechanisms relevant to stimulant abuse.

A HYPOTHESIS ACCOUNTING FOR THE PARADOXICAL EXPRESSION OF THE D GENE SEGMENT IN THE BCR AND THE TCR

PubMed Central

Cohn, Melvin

2009-01-01

The D gene segment expressed in both the TCR and BCR has a challenging behavior that begs interpretation. It is incorporated in three reading frames in the rearranged transcription unit but is expressed in antigen-selected cells in a preferred frame. Why was it so important to waste 2/3 of newborn cells? The hypothesis is presented that the D region is framework playing a role in both the TCR and the BCR by determining whether a signal is transmitted to the cell upon interaction with a cognate ligand. This assumption operates in determining haplotype exclusion for the BCR and in regulating the signaling orientation for the TCR. Relevant data as well as a definitive experiment challenging the validity of this hypothesis, are discussed. PMID:18546143

Proton magnetic resonance spectroscopic creatine correlates with creatine transporter protein density in rat brain.

PubMed

Sartorius, Alexander; Lugenbiel, Patrick; Mahlstedt, Magdalena M; Ende, Gabriele; Schloss, Patrick; Vollmayr, Barbara

2008-07-30

Creatine (Cr) is an amino acid, which upon phosphorylation is utilized as an energy reservoir in cells with high-energy demand. The ongoing catabolism of creatine to creatinine requires a permanent creatine replenishment into the cells. Because neurons themselves cannot synthesize creatine, they have to take it up via the creatine transporter (CrT). Thus, the concentration of intracellular Cr available for the Cr/PCr shuttle system depends on the expression level of CrT protein. The proton magnetic resonance spectroscopy (MRS) creatine peak (total creatine=tCr) constitutes of two metabolites, namely Cr and phosphocreatine (PCr). We have quantified the level of CrT protein expression with western blotting and compared it to tCr content as estimated by in vitro MRS in Sprague-Dawley rats. Under the assumption of hemispheric symmetry, we took identical samples from left and right hemisphere, which were used for in vitro MRS (tCr) and for western blotting (CrT), respectively. Altogether, it was possible to take 90 corresponding brain samples from 31 animals. A Pearson linear regression analysis for CrT and tCr revealed p<0.0001, explaining 14% of the variance. Since MR-detectable alterations of tCr in the human brain are widespread (e.g. in most major psychiatric disorders proton MRS detectable tCr alterations have been described as regionally and usually state dependent) it is stringent to elucidate their meaning. An influence of tCr on the brain's energy regulating system seems plausible.

Gamma delta T cell responses associated with the development of tuberculosis in health care workers.

PubMed

Ordway, Diane J; Pinto, Luisa; Costa, Leonor; Martins, Marta; Leandro, Clara; Viveiros, Miguel; Amaral, Leonard; Arroz, Maria J; Ventura, Fernando A; Dockrell, Hazel M

2005-03-01

This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacterium tuberculosis (Mtb) in health care workers who remained free of active tuberculosis (HCWs w/o TB), health care workers who went on to develop active TB (HCWs w/TB), non-health care workers who were TB free (Non-HCWs) and tuberculosis patients presenting with minimal (Min TB) or advanced (Adv TB) disease. Peripheral blood mononuclear cells (PBMC) were stimulated with Mtb and PPD and the expression of T cell activation markers CD25+ and HLA-DR+, intracellular IL-4 and IFN-gamma production and cytotoxic responses were evaluated. PBMC from HCWs who developed TB showed decreased percentages of cells expressing CD8+CD25+ in comparison to HCWs who remained healthy. HCWs who developed TB showed increased gammadelta TCR+ cell cytotoxicity and decreased CD3+gammadelta TCR- cell cytotoxicity in comparison to HCWs who remained healthy. PBMC from TB patients with advanced disease showed decreased percentages of CD25+CD4+ and CD25+CD8+ T cells that were associated with increased IL-4 production in CD8+ and gammadelta TCR+ phenotypes, in comparison with TB patients presenting minimal disease. TB patients with advanced disease showed increased gammadelta TCR+ cytotoxicity and reduced CD3+gammadelta TCR- cell cytotoxicity. Our results suggest that HCWs who developed TB show an early compensatory mechanism involving an increase in lytic responses of gammadelta TCR+ cells which did not prevent TB.

Current status and future challenges in T-cell receptor/peptide/MHC molecular dynamics simulations.

PubMed

Knapp, Bernhard; Demharter, Samuel; Esmaielbeiki, Reyhaneh; Deane, Charlotte M

2015-11-01

The interaction between T-cell receptors (TCRs) and major histocompatibility complex (MHC)-bound epitopes is one of the most important processes in the adaptive human immune response. Several hypotheses on TCR triggering have been proposed. Many of them involve structural and dynamical adjustments in the TCR/peptide/MHC interface. Molecular Dynamics (MD) simulations are a computational technique that is used to investigate structural dynamics at atomic resolution. Such simulations are used to improve understanding of signalling on a structural level. Here we review how MD simulations of the TCR/peptide/MHC complex have given insight into immune system reactions not achievable with current experimental methods. Firstly, we summarize methods of TCR/peptide/MHC complex modelling and TCR/peptide/MHC MD trajectory analysis methods. Then we classify recently published simulations into categories and give an overview of approaches and results. We show that current studies do not come to the same conclusions about TCR/peptide/MHC interactions. This discrepancy might be caused by too small sample sizes or intrinsic differences between each interaction process. As computational power increases future studies will be able to and should have larger sample sizes, longer runtimes and additional parts of the immunological synapse included. © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Rhinitis and sleep disorders: The trigeminocardiac reflex link?

PubMed

Bindu, Barkha; Singh, Gyaninder Pal; Chowdhury, Tumul; Schaller, Bernhard

2017-06-01

Rhinitis, allergic or non-allergic, is an inflammatory condition of the nose. It is associated with a wide range of sleep disorders that are generally attributed to nasal congestion and presence of inflammatory mediators like cytokines and interleukins. However, the pathophysiological mechanisms behind these sleep disorders remain unclear. On the other hand, the trigeminocardiac reflex (TCR) has recently been linked to various sleep disorders like obstructive sleep apnea, sleep bruxism and rapid eye movement (REM) sleep apnea. TCR can be incited by stimulation of the trigeminal nerve or the area innervated by its branches including the nasal mucosa. Trigeminal nasal afferents can be activated on exposure to noxious stimuli (mechanical or chemical) like ammonia vapors, carbon-dioxide, nicotine, hypertonic saline, air-puffs and smoke. In rhinitis, there is associated neuronal hyper-responsiveness of sensory nasal afferents due to inflammation (which can be suppressed by steroids). This may further lead to increased occurrence of TCR in rhinitis. Moreover, there is involvement of autonomic nervous system both in rhinitis and TCR. In TCR, parasympathetic over activity and sympathetic inhibition leads to sudden onset bradycardia, hypotension, apnea and gastric motility. Also, the autonomic imbalance reportedly plays a significant role in the pathophysiology of rhinitis. Thus, considering these facts we hypothesize that the TCR could be the link between rhinitis and sleep disorders and we believe that further research in this direction may yield significant development in our understanding of sleep disorders in rhinitis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Altered Actin Centripetal Retrograde Flow in Physically Restricted Immunological Synapses

PubMed Central

Yu, Cheng-han; Wu, Hung-Jen; Kaizuka, Yoshihisa; Vale, Ronald D.; Groves, Jay T.

2010-01-01

Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3ε on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network. PMID:20686692

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases.

PubMed

Chen, Hui; You, Hongqin; Wang, Lifang; Zhang, Xuan; Zhang, Jianmin; He, Wei

2016-09-16

Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorylation of a Tyrosine Residue on Zap70 by Lck and Its Subsequent Binding via an SH2 Domain May Be a Key Gatekeeper of T Cell Receptor Signaling In Vivo.

PubMed

Thill, Peter A; Weiss, Arthur; Chakraborty, Arup K

2016-09-15

The initiation of signaling in T lymphocytes in response to the binding of the T cell receptor (TCR) to cognate ligands is a key step in the emergence of adaptive immune responses. Conventional models posit that TCR signaling is initiated by the phosphorylation of receptor-associated immune receptor activation motifs (ITAMs). The cytoplasmic tyrosine kinase Zap70 binds to phosphorylated ITAMs, is subsequently activated, and then propagates downstream signaling. While evidence for such models is provided by experiments with cell lines, in vivo, Zap70 is bound to phosphorylated ITAMs in resting T cells. However, Zap70 is activated only upon TCR binding to cognate ligand. We report the results of computational studies of a new model for the initiation of TCR signaling that incorporates these in vivo observations. Importantly, the new model is shown to allow better and faster TCR discrimination between self-ligands and foreign ligands. The new model is consistent with many past experimental observations, and experiments that could further test the model are proposed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

SLAP deficiency enhances number and function of regulatory T cells preventing chronic autoimmune arthritis in SKG mice.

PubMed

Peterson, Lisa K; Shaw, Laura A; Joetham, Anthony; Sakaguchi, Shimon; Gelfand, Erwin W; Dragone, Leonard L

2011-02-15

To test if manipulating TCR complex-mediated signaling (TCR signaling) could treat autoimmune disease, we generated the double SKG Src-like adapter protein (SLAP) knockout (DSSKO) mouse model. The SKG mutation in ZAP70 and SLAP have opposing functions on the regulation of TCR signaling. The combination of these two mutations alters TCR signaling in the context of a defined genetic background, uniform environmental conditions, and a well-characterized signaling disruption. In contrast to SKG mice, DSSKO mice do not develop zymosan-induced chronic autoimmune arthritis. This arthritis prevention is not due to significant alterations in thymocyte development or repertoire selection but instead enhanced numbers of regulatory T cells (Tregs) and decreased numbers of Th17 cells skewing the ratio of Tregs to autoreactive effector T cells. Treg depletion and/or functional blockade led to the development of arthritis in DSSKO mice. In vitro suppression of effector T cell proliferation was also enhanced, demonstrating that DSSKO mice have increased numbers of Tregs with increased function. Understanding how TCR signals influence development, expansion, and function of Tregs in DSSKO mice could advance our ability to manipulate Treg biology to treat ultimately autoimmune disease.

SLAP Deficiency Enhances Number and Function of Regulatory T Cells Preventing Chronic Autoimmune Arthritis in SKG Mice

PubMed Central

Peterson, Lisa K.; Shaw, Laura A.; Joetham, Anthony; Sakaguchi, Shimon; Gelfand, Erwin W.; Dragone, Leonard L.

2011-01-01

To test if manipulating TCR complex-mediated signaling (TCR signaling) could treat autoimmune disease, we generated the double SKG Src-like adapter protein (SLAP) knockout (DSSKO) mouse model. The SKG mutation in ZAP70 and SLAP have opposing functions on the regulation of TCR signaling. The combination of these two mutations alters TCR signaling in the context of a defined genetic background, uniform environmental conditions, and a well-characterized signaling disruption. In contrast to SKG mice, DSSKO mice do not develop zymosan-induced chronic autoimmune arthritis. This arthritis prevention is not due to significant alterations in thymocyte development or repertoire selection but instead enhanced numbers of regulatory T cells (Tregs) and decreased numbers of Th17 cells skewing the ratio of Tregs to autoreactive effector T cells. Treg depletion and/or functional blockade led to the development of arthritis in DSSKO mice. In vitro suppression of effector T cell proliferation was also enhanced, demonstrating that DSSKO mice have increased numbers of Tregs with increased function. Understanding how TCR signals influence development, expansion, and function of Tregs in DSSKO mice could advance our ability to manipulate Treg biology to treat ultimately autoimmune disease. PMID:21248251

Phosphoproteomics analyses show subnetwork systems in T-cell receptor signaling.

PubMed

Hatano, Atsushi; Matsumoto, Masaki; Nakayama, Keiichi I

2016-10-01

A key issue in the study of signal transduction is how multiple signaling pathways are systematically integrated into the cell. We have now performed multiple phosphoproteomics analyses focused on the dynamics of the T-cell receptor (TCR) signaling network and its subsystem mediated by the Ca 2+ signaling pathway. Integration of these phosphoproteomics data sets and extraction of components of the TCR signaling network dependent on Ca 2+ signaling showed unexpected phosphorylation kinetics for candidate substrates of the Ca 2+ -dependent phosphatase calcineurin (CN) during TCR stimulation. Detailed characterization of the TCR-induced phosphorylation of a novel CN substrate, Itpkb, showed that phosphorylation of this protein is regulated by both CN and the mitogen-activated protein kinase Erk in a competitive manner. Phosphorylation of additional CN substrates was also found to be regulated by Erk and CN in a similar manner. The combination of multiple phosphoproteomics approaches thus showed two major subsystems mediated by Erk and CN in the TCR signaling network, with these subsystems regulating the phosphorylation of a group of proteins in a competitive manner. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Hyper-reactive cloned mice generated by direct nuclear transfer of antigen-specific CD4+ T cells.

PubMed

Kaminuma, Osamu; Katayama, Kazufumi; Inoue, Kimiko; Saeki, Mayumi; Nishimura, Tomoe; Kitamura, Noriko; Shimo, Yusuke; Tofukuji, Soichi; Ishida, Satoru; Ogonuki, Narumi; Kamimura, Satoshi; Oikawa, Mami; Katoh, Shigeki; Mori, Akio; Shichijo, Michitaka; Hiroi, Takachika; Ogura, Atsuo

2017-06-01

T-cell receptor (TCR)-transgenic mice have been employed for evaluating antigen-response mechanisms, but their non-endogenous TCR might induce immune response differently than the physiologically expressed TCR Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen-specific CD4 + T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre-rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic-type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter-regulated antigen-specific TCR are a unique animal model with allergic predisposition for investigating CD4 + T-cell-mediated pathogenesis and cellular commitment in immune diseases. © 2017 The Authors.

Impact of karyotype organization on interlocus recombination between T cell receptor genes in Equidae.

PubMed

Drbalova, Jitka; Musilova, Petra; Kubickova, Svatava; Sebestova, Hana; Vahala, Jiri; Rubes, Jiri

2014-01-01

The T cell receptor (TCR) genes (TRA, TRB, TRD and TRG) reside in 3 different chromosomal regions. During the maturation of T lymphocytes, the TCR genes are rearranged by site-specific recombination, a process that also predisposes T cells to aberrant rearrangements. Illegitimate recombination between the TCR genes occurs at a low level in healthy individuals, but this frequency may correlate with the risk of lymphoma. The aim of this work was to investigate interlocus recombination in equids. Illegitimate rearrangements were studied in peripheral blood lymphocytes by FISH with painting and BAC probes and by sequencing of PCR products, and the frequencies of recombination were assessed in horses and 4 other equids. The presence of several trans-rearrangement products between the TRA and TRG genes was verified by PCR in all investigated equids. Frequencies of trans-rearrangements in horses are higher than in humans, and colocalization of the TCR genes on the same chromosome increases the incidence of trans-rearrangements between them. The orientation of the TCR genes does not impact interlocus recombination itself but does affect the viability of cells carrying its products and consequently the number of trans-rearrangements observed in lymphocytes.

A high fat diet containing saturated but not unsaturated fatty acids enhances T cell receptor clustering on the nanoscale.

PubMed

Shaikh, Saame Raza; Boyle, Sarah; Edidin, Michael

2015-09-01

Cell culture studies show that the nanoscale lateral organization of surface receptors, their clustering or dispersion, can be altered by changing the lipid composition of the membrane bilayer. However, little is known about similar changes in vivo, which can be effected by changing dietary lipids. We describe the use of a newly developed method, k-space image correlation spectroscopy, kICS, for analysis of quantum dot fluorescence to show that a high fat diet can alter the nanometer-scale clustering of the murine T cell receptor, TCR, on the surface of naive CD4(+) T cells. We found that diets enriched primarily in saturated fatty acids increased TCR nanoscale clustering to a level usually seen only on activated cells. Diets enriched in monounsaturated or n-3 polyunsaturated fatty acids had no effect on TCR clustering. Also none of the high fat diets affected TCR clustering on the micrometer scale. Furthermore, the effect of the diets was similar in young and middle aged mice. Our data establish proof-of-principle that TCR nanoscale clustering is sensitive to the composition of dietary fat. Copyright © 2015 Elsevier Ltd. All rights reserved.

c-Cbl promotes T cell receptor-induced thymocyte apoptosis by activating the phosphatidylinositol 3-kinase/Akt pathway.

PubMed

Thien, Christine B F; Dagger, Samantha A; Steer, James H; Koentgen, Frank; Jansen, Elisa S; Scott, Clare L; Langdon, Wallace Y

2010-04-02

The ability of thymocytes to assess T cell receptor (TCR) signaling strength and initiate the appropriate downstream response is crucial for determining their fate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse, in which the E3 ubiquitin ligase activity of c-Cbl is inactivated, is highly sensitive to TCR-induced death signals that cause thymic deletion. This high intensity signal involves the enhanced tyrosine phosphorylation of the mutant c-Cbl protein promoting a marked increase in the activation of Akt. Here we show that this high intensity signal in c-Cbl RING finger mutant thymocytes also promotes the enhanced induction of two mediators of TCR-directed thymocyte apoptosis, Nur77 and the pro-apoptotic Bcl-2 family member, Bim. In contrast, a knock-in mouse harboring a mutation at Tyr-737, the site in c-Cbl that activates phosphatidylinositol 3-kinase, shows reduced TCR-mediated responses including suppression of Akt activation, a reduced induction of Nur77 and Bim, and greater resistance to thymocyte death. These findings identify tyrosine-phosphorylated c-Cbl as a critical sensor of TCR signal strength that regulates the engagement of death-promoting signals.

T lymphocyte subpopulations diverge in commercially raised chickens

PubMed Central

Bridle, Byram W.; Julian, Richard; Shewen, Patricia E.; Vaillancourt, Jean-Pierre

2006-01-01

Abstract To evaluate immunocompetence in commercially raised chickens, we immunophenotyped Dekalb Delta and H&N White Leghorn (WLH) hybrids, 20 chickens in each of 3 age groups (9 wk [juvenile], 25 wk [young adult], and 79 or 80 wk [adult]), for circulating CD3+, CD4+, CD8+, TCR1+, TCR2+, and TCR3+ lymphocytes. The proportion of CD3+ T cells, including CD4+ and CD8+ subsets, was increased in the hybrids as compared with published values for laboratory-raised outbred WLH chickens. The proportion of the TCR2+ (Vβ1) T cell subpopulation was also increased. An age-related decrease in the proportion of TCR1+ (γδ) T cells was noted in both hybrids. Further, a remarkably low CD4:CD8 ratio was evident in all age groups of both hybrids, indicating decreased immunocompetence. Overall, these experiments provide age-related proportions of various peripheral-blood T lymphocyte subpopulations in commercially raised Dekalb Delta and H&N chickens that diverge from the proportions in laboratory-raised outbred WLH chickens and suggest reduced immunocompetence. Such a decline in immunocompetence, including humoral immune capacity, could be attributed to genetic selection for production traits, environmental factors associated with commercial operations, and intense immunization. PMID:16850940

Transcription-coupled repair of UV damage in the halophilic archaea.

PubMed

Stantial, Nicole; Dumpe, Jarrod; Pietrosimone, Kathryn; Baltazar, Felicia; Crowley, David J

2016-05-01

Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) in which excision repair proteins are targeted to RNA polymerase-arresting lesions located in the transcribed strand of active genes. TCR has been documented in a variety of bacterial and eukaryotic organisms but has yet to be observed in the Archaea. We used Halobacterium sp. NRC-1 and Haloferax volcanii to determine if TCR occurs in the halophilic archaea. Following UV irradiation of exponentially growing cultures, we quantified the rate of repair of cyclobutane pyrimidine dimers in the two strands of the rpoB2B1A1A2 and the trpDFEG operons of Halobacterium sp. NRC-1 and the pts operon of H. volcanii through the use of a Southern blot assay and strand-specific probes. TCR was observed in all three operons and was dependent on the NER gene uvrA in Halobacterium sp. NRC-1, but not in H. volcanii. The halophilic archaea likely employ a novel mechanism for TCR in which an as yet unknown coupling factor recognizes the arrested archaeal RNA polymerase complex and recruits certain NER proteins to complete the process. Copyright © 2016 Elsevier B.V. All rights reserved.

Decreased SAP expression in T cells from patients with SLE contributes to early signaling abnormalities and reduced IL-2 production

PubMed Central

Karampetsou, Maria P.; Comte, Denis; Kis-Toth, Katalin; Terhorst, Cox; Kyttaris, Vasileios C.; Tsokos, George C.

2016-01-01

T cells from patients with systemic lupus erythematosus (SLE) display a number of functions including increased early signaling events following engagement of the T cell receptor (TCR). Signaling lymphocytic activation molecule family (SLAMF) cell surface receptors and the X-chromosome-defined signaling lymphocytic activation molecule-associated protein (SAP) adaptor are important in the development of several immunocyte lineages and modulating immune response. Here we present evidence that SAP protein levels are decreased in T cells and in their main subsets isolated from 32 women and 3 men with SLE independently of disease activity. In SLE T cells the SAP protein is also subject to increased degradation by a caspase-3. Forced expression of SAP in SLE T cells simultaneously heightened IL-2 production, calcium (Ca2+) responses and tyrosine phosphorylation of a number of proteins. Exposure of normal T cells to SLE serum IgG, known to contain anti-CD3/TCR antibodies, resulted in SAP downregulation. We conclude that SLE T cells display reduced levels of the adaptor protein SAP probably as a result of continuous T cell activation and degradation by caspase-3. Restoration of SAP levels in SLE T cells corrects the overexcitable lupus T cell phenotype. PMID:27183584

Superantigens Modulate Bacterial Density during Staphylococcus aureus Nasal Colonization

PubMed Central

Xu, Stacey X.; Kasper, Katherine J.; Zeppa, Joseph J.; McCormick, John K.

2015-01-01

Superantigens (SAgs) are potent microbial toxins that function to activate large numbers of T cells in a T cell receptor (TCR) Vβ-specific manner, resulting in excessive immune system activation. Staphylococcus aureus possesses a large repertoire of distinct SAgs, and in the context of host-pathogen interactions, staphylococcal SAg research has focused primarily on the role of these toxins in severe and invasive diseases. However, the contribution of SAgs to colonization by S. aureus remains unclear. We developed a two-week nasal colonization model using SAg-sensitive transgenic mice expressing HLA-DR4, and evaluated the role of SAgs using two well-studied stains of S. aureus. S. aureus Newman produces relatively low levels of staphylococcal enterotoxin A (SEA), and although we did not detect significant TCR-Vβ specific changes during wild-type S. aureus Newman colonization, S. aureus Newman Δsea established transiently higher bacterial loads in the nose. S. aureus COL produces relatively high levels of staphylococcal enterotoxin B (SEB), and colonization with wild-type S. aureus COL resulted in clear Vβ8-specific T cell skewing responses. S. aureus COL Δseb established consistently higher bacterial loads in the nose. These data suggest that staphylococcal SAgs may be involved in regulating bacterial densities during nasal colonization. PMID:26008236

SARS Coronavirus Fusion Peptide-Derived Sequence Suppresses Collagen-Induced Arthritis in DBA/1J Mice.

PubMed

Shen, Zu T; Sigalov, Alexander B

2016-06-28

During the co-evolution of viruses and their hosts, the viruses have evolved numerous strategies to counter and evade host antiviral immune responses in order to establish a successful infection, replicate and persist in the host. Recently, based on our model of immune signaling, the Signaling Chain HOmoOLigomerization (SCHOOL) model, we suggested specific molecular mechanisms used by different viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) to modulate the host immune response mediated by members of the family of multichain immune recognition receptors (MIRRs). This family includes T cell receptor (TCR) that is critically involved in immune diseases such as autoimmune arthritis. In the present study, we provide compelling experimental in vivo evidence in support of our hypothesis. Using the SCHOOL approach and the SARS-CoV fusion peptide sequence, we rationally designed a novel immunomodulatory peptide that targets TCR. We showed that this peptide ameliorates collagen-induced arthritis in DBA/1J mice and protects against bone and cartilage damage. Incorporation of the peptide into self-assembling lipopeptide nanoparticles that mimic native human high density lipoproteins significantly increases peptide dosage efficacy. Together, our data further confirm that viral immune evasion strategies that target MIRRs can be transferred to therapeutic strategies that require similar functionalities.

Decreased SAP Expression in T Cells from Patients with Systemic Lupus Erythematosus Contributes to Early Signaling Abnormalities and Reduced IL-2 Production.

PubMed

Karampetsou, Maria P; Comte, Denis; Kis-Toth, Katalin; Terhorst, Cox; Kyttaris, Vasileios C; Tsokos, George C

2016-06-15

T cells from patients with systemic lupus erythematosus (SLE) display a number of abnormalities, including increased early signaling events following engagement of the TCR. Signaling lymphocytic activation molecule family cell surface receptors and the X-chromosome-defined signaling lymphocytic activation molecule-associated protein (SAP) adaptor are important in the development of several immunocyte lineages and modulating the immune response. We present evidence that SAP protein levels are decreased in T cells and in their main subsets isolated from 32 women and three men with SLE, independent of disease activity. In SLE T cells, SAP protein is also subject to increased degradation by caspase-3. Forced expression of SAP in SLE T cells normalized IL-2 production, calcium (Ca(2+)) responses, and tyrosine phosphorylation of a number of proteins. Exposure of normal T cells to SLE serum IgG, known to contain anti-CD3/TCR Abs, resulted in SAP downregulation. We conclude that SLE T cells display reduced levels of the adaptor protein SAP, probably as a result of continuous T cell activation and degradation by caspase-3. Restoration of SAP levels in SLE T cells corrects the overexcitable lupus T cell phenotype. Copyright © 2016 by The American Association of Immunologists, Inc.

Imaging Vesicular Traffic at the Immune Synapse.

PubMed

Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Alcover, Andrés

2017-01-01

Immunological synapse formation is the result of a profound T cell polarization process that involves the coordinated action of the actin and microtubule cytoskeleton, as well as intracellular vesicle traffic. Endosomal vesicle traffic ensures the targeting of the T cell receptor (TCR) and various signaling molecules to the synapse, being necessary for the generation of signaling complexes downstream of the TCR. Here we describe the microscopy imaging methods that we currently use to unveil how TCR and signaling molecules are associated with endosomal compartments and deliver their cargo to the immunological synapse.

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

Zohar, S.; Choi, Y.; Love, D. M.

We use X-ray Excited Luminescence Microscopy to investigate the elemental and layer resolved magnetic reversal in an interlayer exchange coupled (IEC) epitaxial Fe/Cr wedge/Co heterostructure. The transition from strongly coupled parallel Co-Fe reversal for Cr thickness t(Cr) < 0.34 nm to weakly coupled layer independent reversal for t(Cr) > 1.5 nm is punctuated at 0.34 < t(Cr) < 1.5 nm by a combination of IEC guided domain wall motion and stationary zig zag domain walls. Domain walls nucleated at switching field minima are guided by IEC spatial gradients and collapse at switching field maxima.

Alterations in Intestinal Microbiota Lead to Production of Interleukin 17 by Intrahepatic γδ T-Cell Receptor-Positive Cells and Pathogenesis of Cholestatic Liver Disease.

PubMed

Tedesco, Dana; Thapa, Manoj; Chin, Chui Yoke; Ge, Yong; Gong, Minghao; Li, Jing; Gumber, Sanjeev; Speck, Patrick; Elrod, Elizabeth J; Burd, Eileen M; Kitchens, William H; Magliocca, Joseph F; Adams, Andrew B; Weiss, David S; Mohamadzadeh, Mansour; Grakoui, Arash

2018-06-01

Variants at the ABCB4 or MDR2 locus, which encodes a biliary transport protein, are associated with a spectrum of cholestatic liver diseases. Exacerbation of liver disease has been linked to increased hepatic levels of interleukin (IL) 17, yet the mechanisms of this increase are not understood. We studied mice with disruption of Mdr2 to determine how defects in liver and alteration in the microbiota contribute to production of IL17 by intrahepatic γδ T cells. We performed studies with Mdr2 -/- and littermate FVB/NJ (control) mice. IL17 was measured in serum samples by an enzyme-linked immunosorbent assay. Mice were injected with neutralizing antibodies against the γδ T-cell receptor (TCR; anti-γδ TCR) or mouse IL17A (anti-IL17A). Livers were collected and bacteria were identified in homogenates by culture procedures; TCRγδ + cells were isolated by flow cytometry. Fecal samples were collected from mice and analyzed by 16S ribosomal DNA sequencing. Cells were stimulated with antibodies or bacteria, and cytokine production was measured. We obtained tissues from 10 patients undergoing liver transplantation for primary sclerosing cholangitis or chronic hepatitis C virus infection. Tissues were analyzed for cytokine production by γδ TCR + cells. Mdr2 -/- mice had collagen deposition around hepatic bile ducts and periportal-bridging fibrosis with influx of inflammatory cells and increased serum levels of IL17 compared with control mice. Administration of anti-IL17A reduced hepatic fibrosis. Livers from Mdr2 -/- mice had increased numbers of IL17A + γδTCR + cells-particularly of IL17A + Vγ6Jγ1 γδ TCR + cells. Fecal samples from Mdr2 -/- mice were enriched in Lactobacillus, and liver tissues were enriched in Lactobacillus gasseri compared with control mice. Mdr2 -/- mice also had increased intestinal permeability. The γδ TCR + cells isolated from Mdr2 -/- livers produced IL17 in response to heat-killed L gasseri. Intraperitoneal injection of control mice with L gasseri led to increased serum levels of IL17 and liver infiltration by inflammatory cells; injection of these mice with anti-γδ TCR reduced serum level of IL17. Intravenous injections of Mdr2 -/- mice with anti-γδ TCR reduced fibrosis; liver levels of IL17, and inflammatory cells; and serum levels of IL17. γδTCR + cells isolated from livers of patients with primary sclerosing cholangitis, but not hepatitis C virus infection, produced IL17. In Mdr2 -/- mice, we found development of liver fibrosis and inflammation to require hepatic activation of γδ TCR + cells and production of IL17 mediated by exposure to L gasseri. This pathway appears to contribute to development of cholestatic liver disease in patients. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Human HLA-A*02:01/CHM1+ allo-restricted T cell receptor transgenic CD8+ T Cells specifically inhibit Ewing sarcoma growth in vitro and in vivo

PubMed Central

Kirschner, Andreas; Thiede, Melanie; Rubio, Rebeca Alba; Schirmer, David; Kirchner, Thomas; Richter, Gunther H.S.; Mall, Sabine; Klar, Richard; Riddell, Stanley; Busch, Dirk H.; Krackhardt, Angela; Grunewald, Thomas G.P.; Burdach, Stefan

2016-01-01

The endochondral bone protein Chondromodulin-I (CHM1) provides oncogene addiction in Ewing sarcoma (ES). We pre-clinically tested the targetability of CHM1 by TCR transgenic, allo-restricted, peptide specific T cells to treat ES. We previously generated allo-restricted wildtype CD8+ T cells directed against the ES specific antigen CHM1319 causing specific responses against ES. However, utilization of these cells in current therapy protocols is hampered due to high complexity in production, relatively low cell numbers, and rapid T cell exhaustion. In order to provide off-the-shelf products in the future, we successfully generated HLA-A*02:01-restricted T cell receptor (TCR) transgenic T cells directed against CHM1319 by retroviral transduction. After short-term expansion a 100% purified CHM1319-TCR-transgenic T cell population expressed a CD62L+/CD45RO and CD62L+/CD45RA+ phenotype. These cells displayed specific in vitro IFNg and granzyme B release in co-culture with HLA-A*02:01+ ES cell lines expressing CHM1. When co-injected with ES cells in Rag2−/−ɣc−/− mice, CHM1-specific TCR-transgenic T cells significantly inhibited the formation of lung and liver metastases in contrast to control mice. Lungs and livers of representative mice displayed CD8+ T cell infiltration in the presence (control group treated with unspecific T cells) and in the absence (study group) of metastatic disease, respectively. Furthermore, mice receiving unspecific T cells showed signs of graft-versus-host-disease in contrast to all mice, receiving CHM1319-TCR-transgenic T cells. CHM1319 specific TCR-transgenic T cells were successfully generated causing anti-ES responses in vitro and in vivo. In the future, CHM1319-TCR-transgenic T cells may control minimal residual disease rendering donor lymphocyte infusions more efficacious and less toxic. PMID:27281613

Lack of Heterologous Cross-reactivity toward HLA-A*02:01 Restricted Viral Epitopes Is Underpinned by Distinct αβT Cell Receptor Signatures.

PubMed

Grant, Emma J; Josephs, Tracy M; Valkenburg, Sophie A; Wooldridge, Linda; Hellard, Margaret; Rossjohn, Jamie; Bharadwaj, Mandvi; Kedzierska, Katherine; Gras, Stephanie

2016-11-18

αβT cell receptor (TCR) genetic diversity is outnumbered by the quantity of pathogenic epitopes to be recognized. To provide efficient protective anti-viral immunity, a single TCR ideally needs to cross-react with a multitude of pathogenic epitopes. However, the frequency, extent, and mechanisms of TCR cross-reactivity remain unclear, with conflicting results on anti-viral T cell cross-reactivity observed in humans. Namely, both the presence and lack of T cell cross-reactivity have been reported with HLA-A*02:01-restricted epitopes from the Epstein-Barr and influenza viruses (BMLF-1 and M1 58 , respectively) or with the hepatitis C and influenza viruses (NS3 1073 and NA 231 , respectively). Given the high sequence similarity of these paired viral epitopes (56 and 88%, respectively), the ubiquitous nature of the three viruses, and the high frequency of the HLA-A*02:01 allele, we selected these epitopes to establish the extent of T cell cross-reactivity. We combined ex vivo and in vitro functional assays, single-cell αβTCR repertoire sequencing, and structural analysis of these four epitopes in complex with HLA-A*02:01 to determine whether they could lead to heterologous T cell cross-reactivity. Our data show that sequence similarity does not translate to structural mimicry of the paired epitopes in complexes with HLA-A*02:01, resulting in induction of distinct αβTCR repertoires. The differences in epitope architecture might be an obstacle for TCR recognition, explaining the lack of T cell cross-reactivity observed. In conclusion, sequence similarity does not necessarily result in structural mimicry, and despite the need for cross-reactivity, antigen-specific TCR repertoires can remain highly specific. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

T Lymphocyte Activation Threshold and Membrane Reorganization Perturbations in Unique Culture Model

NASA Technical Reports Server (NTRS)

Adams, C. L.; Sams, C. F.

2000-01-01

Quantitative activation thresholds and cellular membrane reorganization are mechanisms by which resting T cells modulate their response to activating stimuli. Here we demonstrate perturbations of these cellular processes in a unique culture system that non-invasively inhibits T lymphocyte activation. During clinorotation, the T cell activation threshold is increased 5-fold. This increased threshold involves a mechanism independent of TCR triggering. Recruitment of lipid rafts to the activation site is impaired during clinorotation but does occur with increased stimulation. This study describes a situation in which an individual cell senses a change in its physical environment and alters its cell biological behavior.

Decommissioning strategy for liquid low-level radioactive waste surface storage water reservoir.

PubMed

Utkin, S S; Linge, I I

2016-11-22

The Techa Cascade of water reservoirs (TCR) is one of the most environmentally challenging facilities resulted from FSUE "PA "Mayak" operations. Its reservoirs hold over 360 mln m 3 of liquid radioactive waste with a total activity of some 5 × 10 15 Bq. A set of actions implemented under a special State program involving the development of a strategic plan aimed at complete elimination of TCR challenges (Strategic Master-Plan for the Techa Cascade of water reservoirs) resulted in considerable reduction of potential hazards associated with this facility. The paper summarizes the key elements of this master-plan: defining TCR final state, feasibility study of the main strategies aimed at its attainment, evaluation of relevant long-term decommissioning strategy, development of computational tools enabling the long-term forecast of TCR behavior depending on various engineering solutions and different weather conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Near-zero temperature coefficient of resistivity associated with magnetic ordering in antiperovskite Mn{sub 3+x}Ni{sub 1−x}N

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

Deng, Sihao; Sun, Ying; Wang, Lei

2016-01-25

The near-zero temperature coefficient of resistivity (NZ-TCR) behavior is reported in the antiperovskite compounds Mn{sub 3+x}Ni{sub 1−x}N (0 ≤ x ≤ 0.333). Our results indicate that the broad temperature range (above 275 K extending to above 220 K) of NZ-TCR is obtained by Mn doping at the Ni site. The short-range magnetic ordering is revealed by both neutron powder diffraction and inverse magnetic susceptibility. Further, we find a strong correlation between the anomalous resistivity change of Mn{sub 3+x}Ni{sub 1−x}N from the metal-like to the NZ-TCR behavior and the lack of the long-range magnetic ordering. The possible mechanism of NZ-TCR behavior is discussed using the spin-disorder scatteringmore » model.« less

Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering.

PubMed

Cai, Haogang; Muller, James; Depoil, David; Mayya, Viveka; Sheetz, Michael P; Dustin, Michael L; Wind, Shalom J

2018-04-30

Elucidating the rules for receptor triggering in cell-cell and cell-matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This comprises monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer. The TCR ligand could be co-planar with the supported lipid bilayer (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engineering for immunotherapy.

A T-Cell Receptor Breaks the Rules | Center for Cancer Research

Cancer.gov

Most mature T cells function immunologically when a T-cell receptor (TCR) located on the cell surface encounters and engages its ligand, a major histocompatability complex (MHC), which displays a specific part of a target protein called an antigen. This antigen-presenting complex is assembled from one of the dozen or so MHC molecules that every person inherits from their parents; and the antigen fragment, called a peptide epitope, is excised from one of thousands of possible proteins—originally part of an invading pathogen or a cancer cell—that T cells are capable of identifying and attacking. The framework of an MHC molecule holding a centrally displayed or “presented” peptide is what engages the TCR and triggers T-cell action. This role of MHC molecules presenting antigens to the TCR is a central tenet of immunology, with the fit between a TCR and the MHC framework actually “hardwired” into their three-dimensional structures.

Modular microfluidics for point-of-care protein purifications

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

Millet, L. J.; Lucheon, J. D.; Standaert, R. F.

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured tomore » suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.« less

Modular microfluidics for point-of-care protein purifications.

PubMed

Millet, L J; Lucheon, J D; Standaert, R F; Retterer, S T; Doktycz, M J

2015-04-21

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. We demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.

Modular microfluidics for point-of-care protein purifications

DOE PAGES

Millet, L. J.; Lucheon, J. D.; Standaert, R. F.; ...

2015-01-01

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured tomore » suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.« less

Thin film materials and devices for resistive temperature sensing applications

NASA Astrophysics Data System (ADS)

Basantani, Hitesh A.

Thin films of vanadium oxide (VOx) and hydrogenated amorphous silicon (a-Si:H) are the two dominant material systems used in resistive infrared radiation detectors (microbolometers) for sensing long wave infrared (LWIR) wavelengths in the 8--14 microm range. Typical thin films of VO x (x < 2) currently used in the bolometer industry have a magnitude of temperature coefficient of resistance (TCR) between 2%/K -- 3%/K. In contrast, thin films of hydrogenated germanium (SiGe:H) have |TCR| between 3%/K to 4%/K. Devices made from either of these materials have resulted in similar device performance with NETD ≈ 25 mK. The performance of the microbolometers is limited by the electronic noise, especially 1/f noise. Therefore, regardless of the choice of bolometer sensing material and read out circuitry, manufacturers are constantly striving to reduce 1/f noise while simultaneously increasing TCR to give better signal to noise ratios in their bolometers and ultimately, better image quality with more thermal information to the end user. In this work, thin films of VOx and hydrogenated germanium (Ge:H), having TCR values > 4 %/K are investigated as potential candidates for higher sensitivity next generation of microbolometers. Thin films of VO x were deposited by Biased Target Ion Beam Deposition (BTIBD) (˜85 nm thick). Electrical characterization of lateral resistor structures showed resistivity ranging from 104 O--cm to 2.1 x 104 O--cm, TCR varying from --4%/K to --5%/K, normalized Hooge parameter (alphaH/n) of 5 x 10 -21 to 5 x 10-18 cm3. Thin films of Ge:H were deposited by plasma enhanced chemical vapor deposition (PECVD) by incorporating an increasing amount of crystal fraction in the growing thin films. Thin films of Ge:H having a mixed phase, amorphous + nanocrystalline, having a |TCR| > 6 %/K were deposited with resistivity < 2,300 O--cm and a normalized Hooge's parameter 'alphaH/n' < 2 x 10-20 cm3. Higher TCR materials are desired, however, such materials have higher resistivity and therefore unacceptable large electrical resistance in a lateral resistor configuration. This work looks at an alternate bolometer device design which incorporates higher TCR materials in a vertically integrated configuration. Thin films of high TCR hydrogenated germanium (Ge:H, |TCR| > 6%/K) and vanadium oxide (VOx, TCR > 5%/K) were integrated in lateral and through film configuration. The electrical performance of the vertically integrated devices is compared with lateral resistance structures. It was confirmed experimentally that the device impedance was significantly lowered while maintaining the signal to noise ratio of the lateral resistor configuration. The vertically integrated devices allow higher device currents without any increase in self heating. These structures may help reduce integration time and may result in higher frame rate. Finally, one dimensional arrays were fabricated using both lateral and vertically integrated configurations and their performance was evaluated. It was found that the performance of the lateral devices was limited by noise floor of the measurement setup used. However, due to the lower impedance of the vertically integrated resistors, a higher signal and therefore higher signal to noise ratio could be obtained. These vertically integrated devices exhibited low RMS noise values of 12 mK.

Novel Aspects of Degradation of T Cell Receptor Subunits from the Endoplasmic Reticulum (ER) in T Cells: Importance of Oligosaccharide Processing, Ubiquitination, and Proteasome-dependent Removal from ER Membranes

PubMed Central

Yang, Mei; Omura, Satoshi; Bonifacino, Juan S.; Weissman, Allan M.

1998-01-01

Expression of the T cell antigen receptor (TCR) on the surface of thymocytes and mature T cells is dependent on the assembly of receptor subunits into TCRs in the endoplasmic reticulum (ER) and their successful traversal of the secretory pathway to the plasma membrane. TCR subunits that fail to exit the ER for the Golgi complex are degraded by nonlysosomal processes that have been referred to as “ER degradation”. The molecular basis for the loss of the TCR CD3-δ and TCR-α subunits from the ER was investigated in lymphocytes. For CD3-δ, we describe a process leading to its degradation that includes trimming of mannose residues from asparagine-linked (N-linked) oligosaccharides, generation of ubiquitinated membrane-bound intermediates, and proteasome-dependent removal from the ER membrane. When either mannosidase activity or the catalytic activity of proteasomes was inhibited, loss of CD3-δ was markedly curtailed and CD3-δ remained membrane bound in a complex with CD3-ε. TCR-α was also found to be degraded in a proteasome-dependent manner with ubiquitinated intermediates. However, no evidence of a role for mannosidases was found for TCR-α, and significant retrograde movement through the ER membrane took place even when proteasome function was inhibited. These findings provide new insights into mechanisms employed to regulate levels of TCRs, and underscore that cells use multiple mechanisms to target proteins from the ER to the cytosol for degradation. PMID:9500786

Oral-resident natural Th17 cells and γδ T cells control opportunistic Candida albicans infections.

PubMed

Conti, Heather R; Peterson, Alanna C; Brane, Lucas; Huppler, Anna R; Hernández-Santos, Nydiaris; Whibley, Natasha; Garg, Abhishek V; Simpson-Abelson, Michelle R; Gibson, Gregory A; Mamo, Anna J; Osborne, Lisa C; Bishu, Shrinivas; Ghilardi, Nico; Siebenlist, Ulrich; Watkins, Simon C; Artis, David; McGeachy, Mandy J; Gaffen, Sarah L

2014-09-22

Oropharyngeal candidiasis (OPC) is an opportunistic fungal infection caused by Candida albicans. OPC is frequent in HIV/AIDS, implicating adaptive immunity. Mice are naive to Candida, yet IL-17 is induced within 24 h of infection, and susceptibility is strongly dependent on IL-17R signaling. We sought to identify the source of IL-17 during the early innate response to candidiasis. We show that innate responses to Candida require an intact TCR, as SCID, IL-7Rα(-/-), and Rag1(-/-) mice were susceptible to OPC, and blockade of TCR signaling by cyclosporine induced susceptibility. Using fate-tracking IL-17 reporter mice, we found that IL-17 is produced within 1-2 d by tongue-resident populations of γδ T cells and CD3(+)CD4(+)CD44(hi)TCRβ(+)CCR6(+) natural Th17 (nTh17) cells, but not by TCR-deficient innate lymphoid cells (ILCs) or NK cells. These cells function redundantly, as TCR-β(-/-) and TCR-δ(-/-) mice were both resistant to OPC. Whereas γδ T cells were previously shown to produce IL-17 during dermal candidiasis and are known to mediate host defense at mucosal surfaces, nTh17 cells are poorly understood. The oral nTh17 population expanded rapidly after OPC, exhibited high TCR-β clonal diversity, and was absent in Rag1(-/-), IL-7Rα(-/-), and germ-free mice. These findings indicate that nTh17 and γδ T cells, but not ILCs, are key mucosal sentinels that control oral pathogens. © 2014 Conti et al.

Efficacy of three drugs for protecting against gentamicin-induced hair cell and hearing losses

PubMed Central

Bas, E; Van De Water, TR; Gupta, C; Dinh, J; Vu, L; Martínez-Soriano, F; Láinez, JM; Marco, J

2012-01-01

BACKGROUND AND PURPOSE Exposure to an ototoxic level of an aminoglycoside can result in hearing loss. In this we study investigated the otoprotective efficacy of dexamethasone (DXM), melatonin (MLT) and tacrolimus (TCR) in gentamicin (GM)-treated animals and cultures. EXPERIMENTAL APPROACH Wistar rats were divided into controls (treated with saline); exposed to GM only (GM); and three GM-exposed groups treated with either DXM, MLT or TCR. Auditory function and cochlear surface preparations were studied. In vitro studies of oxidative stress, pro-inflammatory cytokine mRNA levels, the MAPK pathway and caspase-3 activation were performed in organ of Corti explants from 3-day-old rats. KEY RESULTS DXM, MLT and TCR decreased levels of reactive oxygen species in GM-exposed explants. The mRNA levels of TNF-α, IL-1β and TNF-receptor type 1 were significantly reduced in GM + DXM and GM + MLT groups. Phospho-p38 MAPK levels decreased in GM + MLT and GM + TCR groups, while JNK phosphorylation was reduced in GM + DXM and GM + MLT groups. Caspase-3 activation decreased in GM + DXM, GM + MLT and GM + TCR groups. These results were consistent with in vivo results. Local treatment of GM-exposed rat cochleae with either DXM, MLT or TCR preserved auditory function and prevented auditory hair cell loss. CONCLUSIONS AND IMPLICATIONS In organ of Corti explants, GM increased oxidative stress and initiated an inflammatory response that led to the activation of MAPKs and apoptosis of hair cells. The three compounds tested demonstrated otoprotective properties that could be beneficial in the treatment of ototoxicity-induced hearing loss. PMID:22320124

T Cell Development in Mice Lacking All T Cell Receptor ζ Family Members (ζ, η, and FcεRIγ)

PubMed Central

Shores, Elizabeth W.; Ono, Masao; Kawabe, Tsutomo; Sommers, Connie L.; Tran, Tom; Lui, Kin; Udey, Mark C.; Ravetch, Jeffrey; Love, Paul E.

1998-01-01

The ζ family includes ζ, η, and FcεRIγ (Fcγ). Dimers of the ζ family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking ζ/η chains, T cell development is impaired, yet low numbers of CD4+ and CD8+ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a ζ family dimer can promote T cell maturation, or that in the absence of ζ/η, Fcγ serves as a subunit in TCR complexes. To elucidate the role of ζ family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only ζ/η or Fcγ. The data reveal that surface complexes that are expressed in the absence of ζ family dimers are capable of transducing signals required for α/β–T cell development. Strikingly, T cells generated in both ζ/η−/− and ζ/η−/−–Fcγ−/− mice exhibit a memory phenotype and elaborate interferon γ. Finally, examination of different T cell populations reveals that ζ/η and Fcγ have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of ζ family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations. PMID:9529325

Downregulation of the T-cell receptor by human immunodeficiency virus type 2 Nef does not protect against disease progression.

PubMed

Feldmann, Jérôme; Leligdowicz, Aleksandra; Jaye, Assan; Dong, Tao; Whittle, Hilton; Rowland-Jones, Sarah L

2009-12-01

Chronic immune activation is thought to play a major role in human immunodeficiency virus (HIV) pathogenesis, but the relative contributions of multiple factors to immune activation are not known. One proposed mechanism to protect against immune activation is the ability of Nef proteins from some HIV and simian immunodeficiency virus strains to downregulate the T-cell receptor (TCR)-CD3 complex of the infected cell, thereby reducing the potential for deleterious activation. HIV type 1 (HIV-1) Nef has lost this property. In contrast to HIV-1, HIV-2 infection is characterized by a marked disparity in the disease course, with most individuals maintaining a normal life span. In this study, we examined the relationship between the ability of HIV-2 Nef proteins to downregulate the TCR and immune activation, comparing progressors and nonprogressors. Representative Nef variants were isolated from 28 HIV-2-infected individuals. We assessed their abilities to downregulate the TCR from the surfaces of CD4 T cells. In the same individuals, the activation of peripheral lymphocytes was evaluated by measurement of the expression levels of HLA-DR and CD38. We observed a striking correlation of the TCR downregulation efficiency of HIV-2 Nef variants with immune activation in individuals with a low viral load. This strongly suggests that Nef expression can influence the activation state of the immune systems of infected individuals. However, the efficiency of TCR downregulation by Nef was not reduced in progressing individuals, showing that TCR downregulation does not protect against progression in HIV-2 infection.

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

PubMed Central

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.

2016-01-01

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. PMID:27689397

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.

Monitoring and source tracking of tetracycline resistance genes in lagoons and groundwater adjacent to swine production facilities over a 3-year period

USGS Publications Warehouse

Koike, S.; Krapac, I.G.; Oliver, H.D.; Yannarell, A.C.; Chee-Sanford, J. C.; Aminov, R.I.; Mackie, R.I.

2007-01-01

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tcr) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tcr genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tcr genes was quantified by real-time quantitative PCR. To confirm the Tcr gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tcr genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tcr genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool. Copyright ?? 2007, American Society for Microbiology. All Rights Reserved.

Monitoring and Source Tracking of Tetracycline Resistance Genes in Lagoons and Groundwater Adjacent to Swine Production Facilities over a 3-Year Period▿

PubMed Central

Koike, S.; Krapac, I. G.; Oliver, H. D.; Yannarell, A. C.; Chee-Sanford, J. C.; Aminov, R. I.; Mackie, R. I.

2007-01-01

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tcr) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tcr genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tcr genes was quantified by real-time quantitative PCR. To confirm the Tcr gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tcr genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tcr genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool. PMID:17545324

Efficacy of Salpingography and Transcervical Recanalization in Diagnosis, Categorization, and Treatment of Fallopian Tube Obstruction

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

Lang, Erich K.; Dunaway, Herbert E.

2000-11-15

Purpose: The efficacy of selective salpingography (SS) and transcervical recanalization (TCR) in diagnosis, categorization, and determination of optimal treatment modality for fallopian tube obstruction was investigated.Methods: SS and, in some patients, TCR was performed in 430 patients with a diagnosis of obstruction of one or both fallopian tubes, as determined by hysterosalpingograms (HSG). All patients (age 21-46 years) had an infertility problem for at least 18 months.Results: In 196 patients, 325 tubes were patent on SS. TCR recanalized 243 tubes in 176 patients. Disease of the distal tube was demonstrated in 66 patients. There were 39 live babies in amore » group of 176 patients with successful TCR. Best live birth rate was in 7 of 12 (58%) patients with underlying endometriosis, followed by postsurgical strictures in inflammatory disease, 6 of 31 (19%), and salpingitis isthmica nodosa in 25 of 168 (15%). There were no pregnancies in patients with cobblestone pattern of the distal tubes.Conclusions: SS and TCR were capable of correcting obstruction of the proximal tubes in 243 of 465 tubes in 176 of 234 patients (75%). With patency of the proximal tube restored, the distal tube could be assessed for changes indicative of damage to the ciliated epithelium which was likely to reduce the ability to become pregnant. This allowed for the triage of patients into groups benefiting from the relatively inexpensive and low complication TCR or patients in need of in vitro fertilization or similar assisted reproductive technologies.« less

Design, synthesis, and activity of 2,3-diphosphoglycerate analogs as allosteric modulators of hemoglobin O2 affinity.

PubMed

Kassa, Tigist W; Zhang, Ning; Palmer, Andre F; Matthews, Jason Shastri

2013-04-01

Four phosphonate derivates of 2,3-diphosphoglycerate (2,3-DPG), in which the phosphate group is replaced by a methylene or difluoromethylene, were successfully synthesized for use as allosteric modulators of hemoglobin (Hb) O2 affinity. The syntheses were accomplished in four steps and the reagents were converted to their potassium salts to allow for effective binding with Hb in aqueous media. O2 equilibrium measurements of the chemically modified Hbs exhibited P50 values in the range 8.9-12.8 with Hill coefficients in the range of 1.5-2.4.

The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux.

PubMed

Joseph, Noah; Reicher, Barak; Barda-Saad, Mira

2014-02-01

During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca(2+)) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca(2+) signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca(2+) from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca(2+) across the plasma membrane in a process termed store-operated Ca(2+) entry (SOCE). Because CRAC channels are themselves inhibited by Ca(2+) ions, additional factors are suggested to enable the sustained Ca(2+) influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca(2+) evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca(2+) flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca(2+) signaling by controlling the spatial and temporal distribution of Ca(2+) sources and sinks, modulating TCR-dependent Ca(2+) signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé. © 2013.

Severity of Acute Infectious Mononucleosis Correlates with Cross-Reactive Influenza CD8 T-Cell Receptor Repertoires

PubMed Central

Aslan, Nuray; Watkin, Levi B.; Gil, Anna; Mishra, Rabinarayan; Clark, Fransenio G.; Welsh, Raymond M.; Ghersi, Dario; Luzuriaga, Katherine

2017-01-01

ABSTRACT Fifty years after the discovery of Epstein-Barr virus (EBV), it remains unclear how primary infection with this virus leads to massive CD8 T-cell expansion and acute infectious mononucleosis (AIM) in young adults. AIM can vary greatly in severity, from a mild transient influenza-like illness to a prolonged severe syndrome. We questioned whether expansion of a unique HLA-A2.01-restricted, cross-reactive CD8 T-cell response between influenza virus A-M158 (IAV-M1) and EBV BMLF1280 (EBV-BM) could modulate the immune response to EBV and play a role in determining the severity of AIM in 32 college students. Only ex vivo total IAV-M1 and IAV-M1+EBV-BM cross-reactive tetramer+ frequencies directly correlated with AIM severity and were predictive of severe disease. Expansion of specific cross-reactive memory IAV-M1 T-cell receptor (TCR) Vβ repertoires correlated with levels of disease severity. There were unique profiles of qualitatively different functional responses in the cross-reactive and EBV-specific CD8 T-cell responses in each of the three groups studied, severe-AIM patients, mild-AIM patients, and seropositive persistently EBV-infected healthy donors, that may result from differences in TCR repertoire use. IAV-M1 tetramer+ cells were functionally cross-reactive in short-term cultures, were associated with the highest disease severity in AIM, and displayed enhanced production of gamma interferon, a cytokine that greatly amplifies immune responses, thus frequently contributing to induction of immunopathology. Altogether, these data link heterologous immunity via CD8 T-cell cross-reactivity to CD8 T-cell repertoire selection, function, and resultant disease severity in a common and important human infection. In particular, it highlights for the first time a direct link between the TCR repertoire with pathogenesis and the diversity of outcomes upon pathogen encounter. PMID:29208744

Cyclic citrullinated MBP87-99 peptide stimulates T cell responses: Implications in triggering disease.

PubMed

Apostolopoulos, Vasso; Deraos, George; Matsoukas, Minos-Timotheos; Day, Stephanie; Stojanovska, Lily; Tselios, Theodore; Androutsou, Maria-Eleni; Matsoukas, John

2017-01-15

Amino acid mutations to agonist peptide epitopes of myelin proteins have been used to modulate immune responses and experimental autoimmune encephalomyelitis (EAE, animal model of multiple sclerosis). Such amino acid alteration are termed, altered peptide ligands (APL). We have shown that the agonist myelin basic protein (MBP) 87-99 epitope (MBP 87-99 ) with crucial T cell receptor (TCR) substitutions at positions 91 and 96 (K 91 ,P 96 (TCR contact residues) to R 91 ,A 96 ; [R 91 ,A 96 ]MBP 87-99 ) results in altered T cell responses and inhibits EAE symptoms. In this study, the role of citrullination of arginines in [R 91 ,A 96 ]MBP 87-99 peptide analog was determined using in vivo experiments in combination with computational studies. The immunogenicity of linear [Cit 91 ,A 96 ,Cit 97 ]MBP 87-99 and its cyclic analog - cyclo(87-99)[Cit 91 ,A 96 ,Cit 97 ]MBP 87-99 when conjugated to the carrier mannan (polysaccharide) were studied in SJL/J mice. It was found that mannosylated cyclo(87-99)[Cit 91 ,A 96 ,Cit 97 ]MBP 87-99 peptide induced strong T cell proliferative responses and IFN-gamma cytokine secretion compared with the linear one. Moreover, the interaction of linear and cyclic peptide analogs with the major histocompatibility complex (MHC II, H2-IA s ) and TCR was analyzed using molecular dynamics simulations at the receptor level, in order to gain a better understanding of the molecular recognition mechanisms that underly the different immunological profiles of citrullinated peptides compared to its agonist native counterpart MBP 87-99 epitope. The results demonstrate that the citrullination of arginine in combination with the backbone conformation of mutated linear and cyclic analogs are significant elements for the immune response triggering the induction of pro-inflammatory cytokines. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Adenylate Cyclase Toxins of Bacillus anthracis and Bordetella pertussis Promote Th2 Cell Development by Shaping T Cell Antigen Receptor Signaling

PubMed Central

Rossi Paccani, Silvia; Benagiano, Marisa; Capitani, Nagaja; Zornetta, Irene; Ladant, Daniel; Montecucco, Cesare; D'Elios, Mario M.; Baldari, Cosima T.

2009-01-01

The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2–driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4+ T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2–polarizing concentrations of ET and CyaA selectively inhibit TCR–dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR–signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3ζ phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4+ T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins. PMID:19266022

High Temperature - Thin Film Strain Gages Based on Alloys of Indium Tin Oxide

NASA Technical Reports Server (NTRS)

Gregory, Otto J.; Cooke, James D.; Bienkiewicz, Joseph M.

1998-01-01

A stable, high temperature strain gage based on reactively sputtered indium tin oxide (ITO) was demonstrated at temperatures up to 1050 C. These strain sensors exhibited relatively large, negative gage factors at room temperature and their piezoresistive response was both linear and reproducible when strained up to 700 micro-in/in. When cycled between compression and tension, these sensors also showed very little hysteresis, indicating excellent mechanical stability. Thin film strain gages based on selected ITO alloys withstood more than 50,000 strain cycles of +/- 500 micro-in/in during 180 hours of testing in air at 1000 C, with minimal drift at temperature. Drift rates as low as 0.0009%/hr at 1000 C were observed for ITO films that were annealed in nitrogen at 700 C prior to strain testing. These results compare favorably with state of the art 10 micro-m thick PdCr films deposited by NASA, where drift rates of 0.047%/hr at 1050 C were observed. Nitrogen annealing not only produced the lowest drift rates to date, but also produce the largest dynamic gage factors (G = 23.5). These wide bandgap, semiconductor strain sensors also exhibited moderately low temperature coefficients of resistance (TCR) at temperatures up to 1100 C, when tested in a nitrogen ambient. A TCR of +230 ppm/C over the temperature range 200 C < T < 500 C and a TCR of -469 ppm/C over the temperature range 600 C < T < 1100 C was observed for the films tested in nitrogen. However, the resistivity behavior changed considerably when the same films were tested in oxygen ambients. A TCR of -1560 ppm/C was obtained over the temperature range of 200 C < T < 1100 C. When similar films were protected with an overcoat or when ITO films were prepared with higher oxygen contents in the plasma, two distinct TCR's were observed. At T < 800 C, a linear TCR of -210 ppm/C was observed and at T > 800 C, a linear TCR of -2170 DDm/C was observed. The combination of a moderately low TCR and a relatively large gage factor make these semiconducting oxide films promising candidates for the active strain elements in high temperature thin film strain gages, particularly in applications where static strain measurement is desired.

Successful removal of football helmet face-mask clips after 1 season of use.

PubMed

Scibek, Jason S; Gatti, Joseph M; McKenzie, Jennifer I

2012-01-01

Whereas many researchers have assessed the ability to remove loop straps in traditional face-mask attachment systems after at least 1 season of use, research in which the effectiveness of the Riddell Quick Release (QR) Face Guard Attachment System clip after 1 season has been assessed is limited. To examine the success rate of removing the QR clips after 1 season of use at the Football Championship Subdivision level. We hypothesized that 1 season of use would negatively affect the removal rate of the QR clip but repeated clip-removal trials would improve the removal rate. Retrospective, quasi-experimental design. Controlled laboratory study. Sixty-three football helmets from a National Collegiate Athletic Association Division I university located in western Pennsylvania used during the 2008 season were tested. Three certified athletic trainers (2 men, 1 woman; age = 31.3 ± 3.06 years, time certified = 9.42 ± 2.65 years) attempted to remove the QR clips from each helmet with the tool provided by the manufacturer. Helmets then were reassembled to allow each athletic trainer to attempt clip removal. The dependent variables were total left clips removed (TCR-L), total right clips removed (TCR-R), and total clips removed (TCR). Success rate of clip removal (SRCR) also was assessed. Percentages for TCR-L, TCR-R, and TCR were 100% (189 of 189), 96.30% (182 of 189), and 98.15% (371 of 378), respectively. A paired-samples t test revealed a difference between TCR-R and TCR-L (t(188) = 2.689, P = .008, μ(d) = 0.037, 95% confidence interval [CI] = 0.064, 0.010). The percentage for SRCR was 96.30% (n = 182), whereas SRCR percentages for trials 1, 2, and 3 were 95.24% (n = 60), 98.41% (n = 62), and 95.24% (n = 60), respectively, and did not represent a difference (F(2,186) = 0.588, P = .56, 95% (CI) = 0.94, 0.99). Our results indicated favorable and consistent success rates for QR clip removal after 1 season of use. Whereas the QR clip is an advancement in face-mask technology, continued examination of this system is required to ensure the highest level of function, allowing for effective management of the helmeted athlete.

Successful Removal of Football Helmet Face-Mask Clips After 1 Season of Use

PubMed Central

Scibek, Jason S.; Gatti, Joseph M.; McKenzie, Jennifer I.

2012-01-01

Context Whereas many researchers have assessed the ability to remove loop straps in traditional face-mask attachment systems after at least 1 season of use, research in which the effectiveness of the Riddell Quick Release (QR) Face Guard Attachment System clip after 1 season has been assessed is limited. Objective To examine the success rate of removing the QR clips after 1 season of use at the Football Championship Subdivision level. We hypothesized that 1 season of use would negatively affect the removal rate of the QR clip but repeated clip-removal trials would improve the removal rate. Design Retrospective, quasi-experimental design. Setting Controlled laboratory study. Patients or Other Participants Sixty-three football helmets from a National Collegiate Athletic Association Division I university located in western Pennsylvania used during the 2008 season were tested. Intervention(s) Three certified athletic trainers (2 men, 1 woman; age = 31.3 ± 3.06 years, time certified = 9.42 ± 2.65 years) attempted to remove the QR clips from each helmet with the tool provided by the manufacturer. Helmets then were reassembled to allow each athletic trainer to attempt clip removal. Main Outcome Measure(s) The dependent variables were total left clips removed (TCR-L), total right clips removed (TCR-R), and total clips removed (TCR). Success rate of clip removal (SRCR) also was assessed. Results Percentages for TCR-L, TCR-R, and TCR were 100% (189 of 189), 96.30% (182 of 189), and 98.15% (371 of 378), respectively. A paired-samples t test revealed a difference between TCR-R and TCR-L (t188 = −2.689, P = .008, μd = 0.037, 95% confidence interval [CI] = −0.064, −0.010). The percentage for SRCR was 96.30% (n = 182), whereas SRCR percentages for trials 1, 2, and 3 were 95.24% (n = 60), 98.41% (n = 62), and 95.24% (n = 60), respectively, and did not represent a difference (F2,186 = 0.588, P = .56, 95% CI = 0.94, 0.99). Conclusions Our results indicated favorable and consistent success rates for QR clip removal after 1 season of use. Whereas the QR clip is an advancement in face-mask technology, continued examination of this system is required to ensure the highest level of function, allowing for effective management of the helmeted athlete. PMID:22889659

Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics

PubMed Central

King, Amy C.; Kavosi, Mania; Wang, Mengmeng; O'Hara, Denise M.; Tchistiakova, Lioudmila; Katragadda, Madan

2018-01-01

ABSTRACT A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1–5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1–3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics. PMID:28991504

Promyelocytic leukemia zinc finger turns on the effector T cell program without requirement for agonist TCR signaling.

PubMed

Savage, Adam K; Constantinides, Michael G; Bendelac, Albert

2011-05-15

Thymocytes expressing the NKT cell semi-invariant αβ TCR are thought to undergo agonist interactions with CD1d ligands prior to expressing promyelocytic leukemia zinc finger (PLZF), a broad complex, tramtrack, bric-a-brac, poxvirus, and zinc finger transcription factor that directs acquisition of the effector program of these innate-like T cells. Whether PLZF can mediate this effector conversion independently of agonist signaling has not been investigated. We demonstrated that transgenic (Tg) expression of PLZF under the CD4 promoter induced the innate effector program in two different MHC class II-restricted TCR-Tg Rag1(-/-) models examined. In CD4 thymocytes expressing a fixed Tg TCR β-chain, the associated TCRα sequences in wild-type and PLZF-Tg mice overlapped extensively, further demonstrating that PLZF could induce the effector program in most CD4 T cells that would normally be selected as naive cells. In contrast, PLZF altered the negative selection of thymocytes expressing TCR β-chains reactive against several retroviral superantigens. Thus, PLZF is remarkable in that it is a transcription factor capable of inducing an effector program in the absence of T cell agonist interactions or cell division. Its expression may also enhance the survival of agonist-signaled thymocytes.

Differential regulation of peripheral CD4+ T cell tolerance induced by deletion and TCR revision.

PubMed

Ali, Mohamed; Weinreich, Michael; Balcaitis, Stephanie; Cooper, Cristine J; Fink, Pamela J

2003-12-01

In Vbeta5 transgenic mice, mature Vbeta5(+)CD4(+) T cells are tolerized upon recognition of a self Ag, encoded by a defective endogenous retrovirus, whose expression is confined to the lymphoid periphery. Cells are driven by the tolerogen to enter one of two tolerance pathways, deletion or TCR revision. CD4(+) T cells entering the former pathway are rendered anergic and then eliminated. In contrast, TCR revision drives gene rearrangement at the endogenous TCR beta locus and results in the appearance of Vbeta5(-), endogenous Vbeta(+), CD4(+) T cells that are both self-tolerant and functional. An analysis of the molecules that influence each of these pathways was conducted to understand better the nature of the interactions that control tolerance induction in the lymphoid periphery. These studies reveal that deletion is efficient in reconstituted radiation chimeras and is B cell, CD28, inducible costimulatory molecule, Fas, CD4, and CD8 independent. In contrast, TCR revision is radiosensitive, B cell, CD28, and inducible costimulatory molecule dependent, Fas and CD4 influenced, and CD8 independent. Our data demonstrate the differential regulation of these two divergent tolerance pathways, despite the fact that they are both driven by the same tolerogen and restricted to mature CD4(+) T cells.

Converging evolution leads to near maximal junction diversity through parallel mechanisms in B and T cell receptors

NASA Astrophysics Data System (ADS)

Benichou, Jennifer I. C.; van Heijst, Jeroen W. J.; Glanville, Jacob; Louzoun, Yoram

2017-08-01

T and B cell receptor (TCR and BCR) complementarity determining region 3 (CDR3) genetic diversity is produced through multiple diversification and selection stages. Potential holes in the CDR3 repertoire were argued to be linked to immunodeficiencies and diseases. In contrast with BCRs, TCRs have practically no Dβ germline genetic diversity, and the question emerges as to whether they can produce a diverse CDR3 repertoire. In order to address the genetic diversity of the adaptive immune system, appropriate quantitative measures for diversity and large-scale sequencing are required. Such a diversity method should incorporate the complex diversification mechanisms of the adaptive immune response and the BCR and TCR loci structure. We combined large-scale sequencing and diversity measures to show that TCRs have a near maximal CDR3 genetic diversity. Specifically, TCR have a larger junctional and V germline diversity, which starts more 5‧ in Vβ than BCRs. Selection decreases the TCR repertoire diversity, but does not affect BCR repertoire. As a result, TCR is as diverse as BCR repertoire, with a biased CDR3 length toward short TCRs and long BCRs. These differences suggest parallel converging evolutionary tracks to reach the required diversity to avoid holes in the CDR3 repertoire.

Human Immunodeficiency Virus Proteins Mimic Human T Cell Receptors Inducing Cross-Reactive Antibodies

PubMed Central

2017-01-01

Human immunodeficiency virus (HIV) hides from the immune system in part by mimicking host antigens, including human leukocyte antigens. It is demonstrated here that HIV also mimics the V-β-D-J-β of approximately seventy percent of about 600 randomly selected human T cell receptors (TCR). This degree of mimicry is greater than any other human pathogen, commensal or symbiotic organism studied. These data suggest that HIV may be evolving into a commensal organism just as simian immunodeficiency virus has done in some types of monkeys. The gp120 envelope protein, Nef protein and Pol protein are particularly similar to host TCR, camouflaging HIV from the immune system and creating serious barriers to the development of safe HIV vaccines. One consequence of HIV mimicry of host TCR is that antibodies against HIV proteins have a significant probability of recognizing the corresponding TCR as antigenic targets, explaining the widespread observation of lymphocytotoxic autoantibodies in acquired immunodeficiency syndrome (AIDS). Quantitative enzyme-linked immunoadsorption assays (ELISA) demonstrated that every HIV antibody tested recognized at least one of twelve TCR, and as many as seven, with a binding constant in the 10−8 to 10−9 m range. HIV immunity also affects microbiome tolerance in ways that correlate with susceptibility to specific opportunistic infections. PMID:28972547

Computational redesign of a protein-protein interface for high affinity and binding specificity using modular architecture and naturally occurring template fragments.

PubMed

Potapov, V; Reichmann, D; Abramovich, R; Filchtinski, D; Zohar, N; Ben Halevy, D; Edelman, M; Sobolev, V; Schreiber, G

2008-12-05

A new method is presented for the redesign of protein-protein interfaces, resulting in specificity of the designed pair while maintaining high affinity. The design is based on modular interface architecture and was carried out on the interaction between TEM1 beta-lactamase and its inhibitor protein, beta-lactamase inhibitor protein. The interface between these two proteins is composed of several mostly independent modules. We previously showed that it is possible to delete a complete module without affecting the overall structure of the interface. Here, we replace a complete module with structure fragments taken from nonrelated proteins. Nature-optimized fragments were chosen from 10(7) starting templates found in the Protein Data Bank. A procedure was then developed to identify sets of interacting template residues with a backbone arrangement mimicking the original module. This generated a final list of 361 putative replacement modules that were ranked using a novel scoring function based on grouped atom-atom contact surface areas. The top-ranked designed complex exhibited an affinity of at least the wild-type level and a mode of binding that was remarkably specific despite the absence of negative design in the procedure. In retrospect, the combined application of three factors led to the success of the design approach: utilizing the modular construction of the interface, capitalizing on native rather than artificial templates, and ranking with an accurate atom-atom contact surface scoring function.

Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays

PubMed Central

Daniels, V; Wood, M; Leclercq, K; Kaminski, R M; Gillard, M

2013-01-01

Background and Purpose Synaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889. Experimental Approach UCB1244283 was characterized in vitro using radioligand binding assays with [3H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice. Key Results Saturation binding experiments in the presence of UCB1244283 demonstrated a fivefold increase in the affinity of [3H]UCB30889 for human recombinant SV2A, combined with a twofold increase of the total number of binding sites. Similar results were obtained on rat cortex. In competition binding experiments, UCB1244283 potentiated the affinity of UCB30889 while the affinity of LEV remained unchanged. UCB1244283 significantly slowed down both the association and dissociation kinetics of [3H]UCB30889. Following i.c.v. administration in sound-sensitive mice, UCB1244283 showed a clear protective effect against both tonic and clonic convulsions. Conclusions and Implications These results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies. PMID:23530581

Creatine supplementation and oxidative stress in rat liver

PubMed Central

2013-01-01

Background The objective of this study was to determine the effects of creatine supplementation on liver biomarkers of oxidative stress in exercise-trained rats. Methods Forty 90-day-old adult male Wistar rats were assigned to four groups for the eight-week experiment. Control group (C) rats received a balanced control diet; creatine control group (CCr) rats received a balanced diet supplemented with 2% creatine; trained group (T) rats received a balanced diet and intense exercise training equivalent to the maximal lactate steady state phase; and supplemented-trained (TCr) rats were given a balanced diet supplemented with 2% creatine and subjected to intense exercise training equivalent to the maximal lactate steady state phase. At the end of the experimental period, concentrations of creatine, hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances (TBARS) were measured as well as the enzyme activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-GPx) and catalase (CAT). Liver tissue levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and the GSH/GSSG ratio were also determined. Results Hepatic creatine levels were highest in the CCr and TCr groups with increased concentration of H2O2 observed in the T and TCr animal groups. SOD activity was decreased in the TCr group. GSH-GPx activity was increased in the T and TCr groups while CAT was elevated in the CCr and TCr groups. GSH, GGS and the GSH/GSSG ratio did not differ between all animal subsets. Conclusions Our results demonstrate that creatine supplementation acts in an additive manner to physical training to raise antioxidant enzymes in rat liver. However, because markers of liver oxidative stress were unchanged, this finding may also indicate that training-induced oxidative stress cannot be ameliorated by creatine supplementation. PMID:24325803

Oral-resident natural Th17 cells and γδ T cells control opportunistic Candida albicans infections

PubMed Central

Conti, Heather R.; Peterson, Alanna C.; Brane, Lucas; Huppler, Anna R.; Hernández-Santos, Nydiaris; Whibley, Natasha; Garg, Abhishek V.; Simpson-Abelson, Michelle R.; Gibson, Gregory A.; Mamo, Anna J.; Osborne, Lisa C.; Bishu, Shrinivas; Ghilardi, Nico; Siebenlist, Ulrich; Watkins, Simon C.; Artis, David; McGeachy, Mandy J.

2014-01-01

Oropharyngeal candidiasis (OPC) is an opportunistic fungal infection caused by Candida albicans. OPC is frequent in HIV/AIDS, implicating adaptive immunity. Mice are naive to Candida, yet IL-17 is induced within 24 h of infection, and susceptibility is strongly dependent on IL-17R signaling. We sought to identify the source of IL-17 during the early innate response to candidiasis. We show that innate responses to Candida require an intact TCR, as SCID, IL-7Rα−/−, and Rag1−/− mice were susceptible to OPC, and blockade of TCR signaling by cyclosporine induced susceptibility. Using fate-tracking IL-17 reporter mice, we found that IL-17 is produced within 1–2 d by tongue-resident populations of γδ T cells and CD3+CD4+CD44hiTCRβ+CCR6+ natural Th17 (nTh17) cells, but not by TCR-deficient innate lymphoid cells (ILCs) or NK cells. These cells function redundantly, as TCR-β−/− and TCR-δ−/− mice were both resistant to OPC. Whereas γδ T cells were previously shown to produce IL-17 during dermal candidiasis and are known to mediate host defense at mucosal surfaces, nTh17 cells are poorly understood. The oral nTh17 population expanded rapidly after OPC, exhibited high TCR-β clonal diversity, and was absent in Rag1−/−, IL-7Rα−/−, and germ-free mice. These findings indicate that nTh17 and γδ T cells, but not ILCs, are key mucosal sentinels that control oral pathogens. PMID:25200028

Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice

PubMed Central

Hu, Zheng; Xia, Jinxing; Fan, Wei; Wargo, Jennifer; Yang, Yong-Guang

2016-01-01

A major factor hindering the exploration of adoptive immunotherapy in preclinical settings is the limited availability of tumor-reactive human T cells. Here we developed a humanized mouse model that permits large-scale production of human T cells expressing the engineered melanoma antigen MART-1-specific TCR. Humanized mice, made by transplantation of human fetal thymic tissue and CD34+ cells virally-transduced with HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene, showed efficient development of MART-1-TCR+ human T cells with predominantly CD8+ cells. Importantly, MART-1-TCR+CD8+ T cells developing in these mice were capable of mounting antigen-specific responses in vivo, as evidenced by their proliferation, phenotypic conversion and IFN-γ production following MART-1 peptide immunization. Moreover, these MART-1-TCR+CD8+ T cells mediated efficient killing of melanoma cells in an HLA/antigen-dependent manner. Adoptive transfer of in vitro expanded MART-1-TCR+CD8+ T cells induced potent antitumor responses that were further enhanced by IL-15 treatment in melanoma-bearing recipients. Finally, a short incubation of MART-1-specific T cells with rapamycin acted synergistically with IL-15, leading to significantly improved tumor-free survival in recipients with metastatic melanoma. These data demonstrate the practicality of using humanized mice to produce potentially unlimited source of tumor-specific human T cells for experimental and preclinical exploration of cancer immunotherapy. This study also suggests that pretreatment of tumor-reactive T cells with rapamycin in combination with IL-15 administration may be a novel strategy to improve the efficacy of adoptive T cell therapy. PMID:26824989

Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines: a case report.

PubMed

Sonntag, Katja; Hashimoto, Hisayoshi; Eyrich, Matthias; Menzel, Moritz; Schubach, Max; Döcker, Dennis; Battke, Florian; Courage, Carolina; Lambertz, Helmut; Handgretinger, Rupert; Biskup, Saskia; Schilbach, Karin

2018-02-06

Cancer vaccines can effectively establish clinically relevant tumor immunity. Novel sequencing approaches rapidly identify the mutational fingerprint of tumors, thus allowing to generate personalized tumor vaccines within a few weeks from diagnosis. Here, we report the case of a 62-year-old patient receiving a four-peptide-vaccine targeting the two sole mutations of his pancreatic tumor, identified via exome sequencing. Vaccination started during chemotherapy in second complete remission and continued monthly thereafter. We tracked IFN-γ + T cell responses against vaccine peptides in peripheral blood after 12, 17 and 34 vaccinations by analyzing T-cell receptor (TCR) repertoire diversity and epitope-binding regions of peptide-reactive T-cell lines and clones. By restricting analysis to sorted IFN-γ-producing T cells we could assure epitope-specificity, functionality, and T H 1 polarization. A peptide-specific T-cell response against three of the four vaccine peptides could be detected sequentially. Molecular TCR analysis revealed a broad vaccine-reactive TCR repertoire with clones of discernible specificity. Four identical or convergent TCR sequences could be identified at more than one time-point, indicating timely persistence of vaccine-reactive T cells. One dominant TCR expressing a dual TCRVα chain could be found in three T-cell clones. The observed T-cell responses possibly contributed to clinical outcome: The patient is alive 6 years after initial diagnosis and in complete remission for 4 years now. Therapeutic vaccination with a neoantigen-derived four-peptide vaccine resulted in a diverse and long-lasting immune response against these targets which was associated with prolonged clinical remission. These data warrant confirmation in a larger proof-of concept clinical trial.

Wrinkle-stabilized metal-graphene hybrid fibers with zero temperature coefficient of resistance.

PubMed

Fang, Bo; Xi, Jiabin; Liu, Yingjun; Guo, Fan; Xu, Zhen; Gao, Weiwei; Guo, Daoyou; Li, Peigang; Gao, Chao

2017-08-24

The interfacial adhesion between graphene and metals is poor, as metals tend to generate superlubricity on smooth graphene surface. This problem renders the free assembly of graphene and metals to be a big challenge, and therefore, some desired conducting properties (e.g., stable metal-like conductivities in air, lightweight yet flexible conductors, and ultralow temperature coefficient of resistance, TCR) likely being realized by integrating the merits of graphene and metals remains at a theoretical level. This work proposes a wrinkle-stabilized approach to address the poor adhesion between graphene surface and metals. Cyclic voltammetry (CV) tests and theoretical analysis by Scharifker-Hills models demonstrate that multiscale wrinkles effectively induce nucleation of metal particles, locking in metal nuclei and guiding the continuous growth of metal islands in an instantaneous model on rough graphene surface. The universality and practicability of the wrinkle-stabilized approach is verified by our investigation through the electrodeposition of nine kinds of metals on graphene fibers (GF). The strong interface bonding permits metal-graphene hybrid fibers to show metal-level conductivities (up to 2.2 × 10 7 S m -1 , a record high value for GF in air), reliable weatherability and favorable flexibility. Due to the negative TCR of graphene and positive TCR of metals, the TCR of Cu- and Au-coated GFs reaches zero at a wide temperature range (15 K-300 K). For this layered model, the quantitative analysis by classical theories demonstrates the suitable thickness ratio of graphene layer and metal layer to achieve zero TCR to be 0.2, agreeing well with our experimental results. This wrinkle-stabilized approach and our theoretical analysis of zero-TCR behavior of the graphene-metal system are conducive to the design of high-performance conducting materials based on graphene and metals.

Co-potentiation of antigen recognition: A mechanism to boost weak T cell responses and provide immunotherapy in vivo

PubMed Central

Hoffmann, Michele M.; Molina-Mendiola, Carlos; Nelson, Alfreda D.; Parks, Christopher A.; Reyes, Edwin E.; Hansen, Michael J.; Rajagopalan, Govindarajan; Pease, Larry R.; Schrum, Adam G.; Gil, Diana

2015-01-01

Adaptive immunity is mediated by antigen receptors that can induce weak or strong immune responses depending on the nature of the antigen that is bound. In T lymphocytes, antigen recognition triggers signal transduction by clustering T cell receptor (TCR)/CD3 multiprotein complexes. In addition, it hypothesized that biophysical changes induced in TCR/CD3 that accompany receptor engagement may contribute to signal intensity. Nonclustering monovalent TCR/CD3 engagement is functionally inert despite the fact that it may induce changes in conformational arrangement or in the flexibility of receptor subunits. We report that the intrinsically inert monovalent engagement of TCR/CD3 can specifically enhance physiologic T cell responses to weak antigens in vitro and in vivo without stimulating antigen-unengaged T cells and without interrupting T cell responses to strong antigens, an effect that we term as “co-potentiation.” We identified Mono-7D6-Fab, which biophysically altered TCR/CD3 when bound and functionally enhanced immune reactivity to several weak antigens in vitro, including a gp100-derived peptide associated with melanoma. In vivo, Mono-7D6-Fab induced T cell antigen–dependent therapeutic responses against melanoma lung metastases, an effect that synergized with other anti-melanoma immunotherapies to significantly improve outcome and survival. We conclude that Mono-7D6-Fab directly co-potentiated TCR/CD3 engagement by weak antigens and that such concept can be translated into an immunotherapeutic design. The co-potentiation principle may be applicable to other receptors that could be regulated by otherwise inert compounds whose latent potency is only invoked in concert with specific physiologic ligands. PMID:26601285

Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice

PubMed Central

Tsang, Julia Yuen-Shan; Tanriver, Yakup; Jiang, Shuiping; Xue, Shao-An; Ratnasothy, Kulachelvy; Chen, Daxin; Stauss, Hans J.; Bucy, R. Pat; Lombardi, Giovanna; Lechler, Robert

2008-01-01

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules — a process known as indirect recognition. As CD4+CD25+ Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4+CD25+ cells from C57BL/6 mice (H-2b) with allogeneic DCs from BALB/c mice (H-2d). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2Kd presented by H-2Ab MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential. PMID:18846251

Deep Sequencing of T-cell Receptor DNA as a Biomarker of Clonally Expanded TILs in Breast Cancer after Immunotherapy.

PubMed

Page, David B; Yuan, Jianda; Redmond, David; Wen, Y Hanna; Durack, Jeremy C; Emerson, Ryan; Solomon, Stephen; Dong, Zhiwan; Wong, Phillip; Comstock, Christopher; Diab, Adi; Sung, Janice; Maybody, Majid; Morris, Elizabeth; Brogi, Edi; Morrow, Monica; Sacchini, Virgilio; Elemento, Olivier; Robins, Harlan; Patil, Sujata; Allison, James P; Wolchok, Jedd D; Hudis, Clifford; Norton, Larry; McArthur, Heather L

2016-10-01

In early-stage breast cancer, the degree of tumor-infiltrating lymphocytes (TIL) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryoimmunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, T-cell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 × 10 6 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared with monotherapy, cryoablation plus ipilimumab was associated with numerically greater numbers of peripheral blood and intratumoral T-cell clones expanding robustly following therapy. In conclusion, TCR sequencing correlates with H&E lymphocyte scoring and provides additional information on clonal diversity. These findings support further study of the use of TCR sequencing as a biomarker for T-cell responses to therapy and for the study of cryoimmunotherapy in early-stage breast cancer. Cancer Immunol Res; 4(10); 835-44. ©2016 AACR. ©2016 American Association for Cancer Research.

Dissecting transcription-coupled and global genomic repair in the chromatin of yeast GAL1-10 genes.

PubMed

Li, Shisheng; Smerdon, Michael J

2004-04-02

Transcription-coupled repair (TCR) and global genomic repair (GGR) of UV-induced cyclobutane pyrimidine dimers were investigated in the yeast GAL1-10 genes. Both Rpb9- and Rad26-mediated TCR are confined to the transcribed strands, initiating at upstream sites approximately 100 nucleotides from the upstream activating sequence shared by the two genes. However, TCR initiation sites do not correlate with either transcription start sites or TATA boxes. Rad16-mediated GGR tightly correlates with nucleosome positioning when the genes are repressed and are slow in the nucleosome core and fast in linker DNA. Induction of transcription enhanced GGR in nucleosome core DNA, especially in the nucleosomes around and upstream of the transcription start sites. Furthermore, when the genes were induced, GGR was slower in the transcribed regions than in the upstream regions. Finally, simultaneous deletion of RAD16, RAD26, and RPB9 resulted in no detectable repair in all sites along the region analyzed. Our results suggest that (a). TCR may be initiated by a transcription activator, presumably through the loading of RNA polymerase II, rather than by transcription initiation or elongation per se; (b). TCR and nucleosome disruption-enhanced GGR are the major causes of rapid repair in regions around and upstream of transcription start sites; (c). transcription machinery may hinder access of NER factors to a DNA lesion in the absence of a transcription-repair coupling factor; and (d). other than GGR mediated by Rad16 and TCR mediated by Rad26 and Rpb9, no other nucleotide excision repair pathway exists in these RNA polymerase II-transcribed genes.

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man*

PubMed Central

Macauley, Matthew S.; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M.; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C.

2015-01-01

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. PMID:26507663

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man.

PubMed

Macauley, Matthew S; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C

2015-12-11

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2-6Galβ1-4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

What Climate Sensitivity Index Is Most Useful for Projections?

NASA Astrophysics Data System (ADS)

Grose, Michael R.; Gregory, Jonathan; Colman, Robert; Andrews, Timothy

2018-02-01

Transient climate response (TCR), transient response at 140 years (T140), and equilibrium climate sensitivity (ECS) indices are intended as benchmarks for comparing the magnitude of climate response projected by climate models. It is generally assumed that TCR or T140 would explain more variability between models than ECS for temperature change over the 21st century, since this timescale is the realm of transient climate change. Here we find that TCR explains more variability across Coupled Model Intercomparison Project phase 5 than ECS for global temperature change since preindustrial, for 50 or 100 year global trends up to the present, and for projected change under representative concentration pathways in regions of delayed warming such as the Southern Ocean. However, unexpectedly, we find that ECS correlates higher than TCR for projected change from the present in the global mean and in most regions. This higher correlation does not relate to aerosol forcing, and the physical cause requires further investigation.

Quantifying Appropriate De-rating of SiC MOSFETs Subject to Cosmic Rays

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

Chatty, Kiran

Terrestrial Cosmic Radiation (TCR) is known to cause failures in high-voltage Si devices resulting in de-rating of the maximum reverse blocking voltage. In this work, a test setup was developed and unaccelerated TCR testing was performed on 1200V Si IGBTs, 1200V SiC MOSFETs and 1200V SiC Schottky diodes. Failures due to TCR were generated on 1200V Si IGBTs at reverse voltages from 900V to 1175V. Si IGBTs investigated in this work will need to be operated at a maximum voltage of 800V to achieve a Failure in Time (FIT) rate of 100. No failures were observed on 1200V SiC MOSFETsmore » and Schottky diodes after testing at 1200V for over 1.5 years demonstrating low FIT rates compared to Si IGBTs. 1200V SiC Schottky diodes were fabricated in this program and the packaged devices were used in the TCR testing.« less

Fabrication of vanadium dioxide polycrystalline films with higher temperature coefficient of resistance

NASA Astrophysics Data System (ADS)

Li, Jinhua; Yuan, Ningyi; Jiang, Meiping; Kun, Li

2011-08-01

Vanadium Dioxide Polycrystalline Films with High Temperature Coefficient of Resistance(TCR) were fabricated by modified Ion Beam Enhanced Deposition(IBED) method. The TCR of the Un-doping VO2 was about -4%/K at room temperature after appropriate thermal annealing. The XRD results clearly showed that IBED polycrystalline VO2 films had a single [002] orientation of VO2(M). The TCR of 5at.%W and 7at.% Ta doped Vanadium Dioxide Polycrystalline Films were high up to -18%/K and -12%/K at room temperature, respectively. Using 7at.% Ta and 2at.% Ti co-doping, the TCR of the co-doped vanadium oxide film was -7%/K and without hysteresis during temperature increasing and decresing from 0-80°C. It should indicate that the W-doped vanadium dioxide films colud be used for high sensing IR detect and the Ta/Ti co-doped film without hysteresis is suitable for infrarid imaging application.

Nanostructured vanadium oxide thin film with high TCR at room temperature for microbolometer

NASA Astrophysics Data System (ADS)

Wang, Bin; Lai, Jianjun; Li, Hui; Hu, Haoming; Chen, Sihai

2013-03-01

In order to obtain high quality of thermal sensitive material, VOx thin film of high temperature coefficient of resistance (TCR) of 6.5%/K at room temperature has been deposited by reactive ion beam sputtering and post annealing method. AFM and XRD measurements indicate that the VOx thin film with nanostructured crystalline is composed of VO2 and V2O3. The nanostructured VOx microbolometer has been designed and fabricated. The measurement of the film system with TiN absorbing layer indicates that it has about 92% infrared absorption in the range of 8-14 μm. The performance of this bolometer, comparing with that of bolometer with common VOx, has a better result. At 20 Hz frequency and 10 μA bias current, the bolometer with high TCR has reached detectivity of 1.0 × 109 cm Hz1/2/W. It also indicates that this nanostructured VOx thin film has not only a higher TCR but also a lower noise than common VOx thin film without annealing.

Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

NASA Technical Reports Server (NTRS)

Morrow, Maureen A.

2003-01-01

Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

Radiation leukemia virus-induced thymic lymphomas express a restricted repertoire of T-cell receptor V beta gene products.

PubMed Central

Sen-Majumdar, A; Weissman, I L; Hansteen, G; Marian, J; Waller, E K; Lieberman, M

1994-01-01

We have investigated the phenotypic changes that take place during the process of neoplastic transformation in the thymocytes of C57BL/Ka mice infected by the radiation leukemia virus (RadLV). By the combined use of antibodies against the envelope glycoprotein gp70 of RadLV, the transformation-associated cell surface marker 1C11, and the CD3-T-cell receptor (TCR) complex, we found that in the RadLV-infected thymus, the earliest expression of viral gp70 is in 1C11hi cells; a small but significant percentage of these cells also express CD3. A first wave of viral replication, manifested by the expression of high levels of gp70 in thymocytes (over 70% positive), reaches a peak at 2 weeks; during this period, no significant changes are observed in the expression of 1C11 or CD3. The population of gp70+ cells is drastically reduced at 3 to 4 weeks after infection. However, a second cohort of gp70+ cells appears after 4 weeks, and these cells express high levels of 1C11 and TCR determinants as well. RadLV-induced lymphomas differ from normal thymocytes in their CD4 CD8 phenotype, with domination by one or more subsets. Characterization of TCR gene rearrangements in RadLV-induced lymphomas shows that most of these tumors are clonal or oligoclonal with respect to the J beta 2 TCR gene, while the J beta 1 TCR gene is rearranged in a minority (4 of 11) of lymphomas. TCR V beta repertoire analysis of 12 tumors reveals that 6 (50%) express exclusively the V beta 6 gene product, 2 (17%) are V beta 5+, and 1 (8%) each are V beta 8+ and V beta 9+. In normal C57BL/Ka mice, V beta 6 is expressed on 12%, V beta 5 is expressed on 9%, V beta 8 is expressed on 22%, and V beta 9 is expressed on 4% of TCRhi thymocytes. Thus, it appears that RadLV-induced thymic lymphomas are not randomly selected with respect to expressed TCR V beta type. Images PMID:8289345

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

PubMed Central

Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I.; Lluís, Carme; Cortés, Antoni; Volkow, Nora D.; Schiffmann, Serge N.; Ferré, Sergi; Casadó, Vicent

2015-01-01

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain. PMID:26100888

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer.

PubMed

Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Cortés, Antoni; Volkow, Nora D; Schiffmann, Serge N; Ferré, Sergi; Casadó, Vicent

2015-07-07

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

A General Strategy for Targeting Drugs to Bone.

PubMed

Jahnke, Wolfgang; Bold, Guido; Marzinzik, Andreas L; Ofner, Silvio; Pellé, Xavier; Cotesta, Simona; Bourgier, Emmanuelle; Lehmann, Sylvie; Henry, Chrystelle; Hemmig, René; Stauffer, Frédéric; Hartwieg, J Constanze D; Green, Jonathan R; Rondeau, Jean-Michel

2015-11-23

Targeting drugs to their desired site of action can increase their safety and efficacy. Bisphosphonates are prototypical examples of drugs targeted to bone. However, bisphosphonate bone affinity is often considered too strong and cannot be significantly modulated without losing activity on the enzymatic target, farnesyl pyrophosphate synthase (FPPS). Furthermore, bisphosphonate bone affinity comes at the expense of very low and variable oral bioavailability. FPPS inhibitors were developed with a monophosphonate as a bone-affinity tag that confers moderate affinity to bone, which can furthermore be tuned to the desired level, and the relationship between structure and bone affinity was evaluated by using an NMR-based bone-binding assay. The concept of targeting drugs to bone with moderate affinity, while retaining oral bioavailability, has broad application to a variety of other bone-targeted drugs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Doctoral Education and Transformative Consumer Research

ERIC Educational Resources Information Center

Mari, Carlo

2008-01-01

This article examines why and how transformative consumer research (TCR) can become a relevant perspective in doctoral programs. The article draws selectively from studies published in consumer behavior, marketing, and marketing education that theoretically or empirically address this topic. It discusses the meaning and background of TCR together…

Different subsets of natural killer T cells may vary in their roles in health and disease

PubMed Central

Kumar, Vipin; Delovitch, Terry L

2014-01-01

Natural killer T cells (NKT) can regulate innate and adaptive immune responses. Type I and type II NKT cell subsets recognize different lipid antigens presented by CD1d, an MHC class-I-like molecule. Most type I NKT cells express a semi-invariant T-cell receptor (TCR), but a major subset of type II NKT cells reactive to a self antigen sulphatide use an oligoclonal TCR. Whereas TCR-α dominates CD1d-lipid recognition by type I NKT cells, TCR-α and TCR-β contribute equally to CD1d-lipid recognition by type II NKT cells. These variable modes of NKT cell recognition of lipid–CD1d complexes activate a host of cytokine-dependent responses that can either exacerbate or protect from disease. Recent studies of chronic inflammatory and autoimmune diseases have led to a hypothesis that: (i) although type I NKT cells can promote pathogenic and regulatory responses, they are more frequently pathogenic, and (ii) type II NKT cells are predominantly inhibitory and protective from such responses and diseases. This review focuses on a further test of this hypothesis by the use of recently developed techniques, intravital imaging and mass cytometry, to analyse the molecular and cellular dynamics of type I and type II NKT cell antigen-presenting cell motility, interaction, activation and immunoregulation that promote immune responses leading to health versus disease outcomes. PMID:24428389

TCR revision generates functional CD4+ T cells1

PubMed Central

Hale, J. Scott; Wubeshet, Maramawit; Fink, Pamela J.

2010-01-01

CD4+Vβ5+ peripheral T cells in B6 mice respond to encounter with a peripherally-expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process, cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes, driving surface expression of novel TCRs. While post-revision CD4+Vβ5−TCRβ+ T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire, it is unknown whether they respond to homeostatic and antigenic stimuli, and thus may benefit the host. We now demonstrate that post-revision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly, post-revision cells do not proliferate in response to the tolerizing superantigen, implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of post-revision cells is not driven by the transgene-encoded receptor. Post-revision cells proliferate extensively to commensal bacterial Ags and can generate I-Ab-restricted responses to Ag by producing IFNγ following Listeria monocytogenes challenge. These data show that rescued post-revision T cells are responsive to homeostatic signals and recognize self and foreign peptides in the context of self MHC, and are thus useful to the host. PMID:20971922

TCR revision generates functional CD4+ T cells.

PubMed

Hale, J Scott; Wubeshet, Maramawit; Fink, Pamela J

2010-12-01

CD4(+)Vβ5(+) peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process, cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes, driving surface expression of novel TCRs. Although postrevision CD4(+)Vβ5(-)TCRβ(+) T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire, it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ, and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly, postrevision cells do not proliferate in response to the tolerizing superantigen, implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal bacterial Ags and can generate I-A(b)-restricted responses to Ag by producing IFN-γ following Listeria monocytogenes challenge. These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host.

Integration of T Cell Receptor, Notch and Cytokine Signals Programs in Mouse γδ T Cell Effector Differentiation.

PubMed

Zarin, Payam; In, Tracy S H; Chen, Edward L Y; Singh, Jastaranpreet; Wong, Gladys W; Mohtashami, Mahmood; Wiest, David L; Anderson, Michele K; Zúñiga-Pflücker, Juan Carlos

2018-05-13

γδ T-cells perform a wide range of tissue and disease specific functions that are dependent on the effector cytokines produced by these cells. However, the aggregate signals required for the development of interferon-γ (IFNγ) and interleukin-17 (IL-17) producing γδ T-cells remain unknown. Here, we define the cues involved in the functional programming of γδ T-cells, by examining the roles of T-cell receptor (TCR), Notch, and cytokine-receptor signaling. KN6 γδTCR-transduced Rag2 -/- T-cell progenitors were cultured on stromal cells variably expressing TCR and Notch ligands, supplemented with different cytokines. We found that distinct combinations of these signals are required to program IFNγ versus IL-17 producing γδ T cell subsets, with Notch and weak TCR ligands optimally enabling development of γδ17 cells in the presence of IL-1β, IL-21 and IL-23. Notably, these cytokines were also shown to be required for the intrathymic development of γδ17 cells. Together, this work provides a framework of how signals downstream of TCR, Notch and cytokine receptors integrate to program the effector function of IFNγ and IL-17 producing γδ T-cell subsets. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Specific recycling receptors are targeted to the immune synapse by the intraflagellar transport system

PubMed Central

Finetti, Francesca; Patrussi, Laura; Masi, Giulia; Onnis, Anna; Galgano, Donatella; Lucherini, Orso Maria; Pazour, Gregory J.; Baldari, Cosima T.

2014-01-01

ABSTRACT T cell activation requires sustained signaling at the immune synapse, a specialized interface with the antigen-presenting cell (APC) that assembles following T cell antigen receptor (TCR) engagement by major histocompatibility complex (MHC)-bound peptide. Central to sustained signaling is the continuous recruitment of TCRs to the immune synapse. These TCRs are partly mobilized from an endosomal pool by polarized recycling. We have identified IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, as a central regulator of TCR recycling to the immune synapse. Here, we have investigated the interplay of IFT20 with the Rab GTPase network that controls recycling. We found that IFT20 forms a complex with Rab5 and the TCR on early endosomes. IFT20 knockdown (IFT20KD) resulted in a block in the recycling pathway, leading to a build-up of recycling TCRs in Rab5+ endosomes. Recycling of the transferrin receptor (TfR), but not of CXCR4, was disrupted by IFT20 deficiency. The IFT components IFT52 and IFT57 were found to act together with IFT20 to regulate TCR and TfR recycling. The results provide novel insights into the mechanisms that control TCR recycling and immune synapse assembly, and underscore the trafficking-related function of the IFT system beyond ciliogenesis. PMID:24554435

Oligoclonal T cell expansions in patients with Behçet's disease

PubMed Central

DIRESKENELI, H; EKSIOGLU-DEMIRALP, E; KIBAROGLU, A; YAVUZ, S; ERGUN, T; AKOGLU, T

1999-01-01

Behçet's disease (BD) is a multisystem disorder with oral and genital ulcers, mucocutaneous, ocular, joint, vascular and central nervous system involvement. In this study, the peripheral T cell repertoire was analysed in patients with BD with MoAbs against T cell receptor (TCR) Vβ gene products in CD4+ and CD8+ T cell compartments, and these were compared with rheumatoid arthritis (RA) patients and healthy controls (HC). In the CD4+ T cell compartment, oligoclonal TCR Vβ expression was observed in 56% of BD (10/18), 71% of RA (5/7) patients and 21% (3/14) of HC. In the CD8+ T cell group 50% of BD (9/18), 57% of RA patients and 28% of HC (4/14) had an oligoclonal TCR repertoire. An increase of TCR Vβ5.1 subset was observed in five BD patients among CD8+ T cells. Other elevations of TCR Vβ subsets were heterogeneously distributed with one to three different Vβ subsets. Our results suggest an antigen-driven oligoclonal increase of T cells in BD. There was no overall increase in any Vβ group to suggest a superantigen effect. Analysis of the responsible antigens causing the increase in T cell subsets may give insights into the aetiopathogenesis of BD and immunomodulation of these T cells may lead to new treatments. PMID:10403931

T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2- restricted and melanocyte-lineage-specific CTL clone

PubMed Central

1993-01-01

HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA- A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but < 0.19% of V beta 2+ sequences expressed in PBL. These results are consistent with the hypothesis that a clonal expansion/accumulation of a melanocyte-lineage-specific and HLA-A2-restricted T cell clone occurred in vivo at the site of tumor growth. PMID:8376931

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition*

PubMed Central

Hawse, William F.; Gloor, Brian E.; Ayres, Cory M.; Kho, Kevin; Nuter, Elizabeth; Baker, Brian M.

2013-01-01

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity. PMID:23836912

A Novel Sphingomyelinase-Like Enzyme in Ixodes scapularis Tick Saliva Drives Host CD4+ T cells to Express IL-4

PubMed Central

Alarcon-Chaidez, F. J.; Boppana, V. D.; Hagymasi, A.T.; Adler, A. J.; Wikel, S. K.

2009-01-01

Tick feeding modulates host immune responses. Tick-induced skewing of host CD4+ T cells towards a Th2 cytokine profile facilitates transmission of tick-borne pathogens that would otherwise be neutralized by Th1 cytokines. Tick-derived factors that drive this Th2 response have not previously been characterized. In the current study, we examined an I. scapularis cDNA library prepared at 18-24 hours of feeding and identified and expressed a tick gene with homology to Loxosceles spider venom proteins with sphingomyelinase activity. This I. scapularis sphingomyelinase-like (IsSMase) protein is a Mg+2-dependent, neutral (pH 7.4) form of sphingomyelinase. Significantly, in an in vivo TCR transgenic adoptive transfer assay IsSMase programmed host CD4+ T cells to express the hallmark Th2 effector cytokine IL-4. IsSMase appears to directly program host CD4 T cell IL-4 expression (as opposed to its metabolic by-products) because induced IL-4 expression was not altered when enzymatic activity was neutralized. TCR transgenic CD4 T cell proliferation (CFSE-dilution) was also significantly increased by IsSMase. Furthermore, a Th2 response is superimposed onto a virally-primed Th1 response by IsSMase. Thus, IsSMase is the first identified tick molecule capable of programming host CD4+ T cells to express IL-4. PMID:19292772

Immune modulation and microchimerism after unmodified versus leukoreduced allogeneic red blood cell transfusion in cancer patients: results of a randomized study.

PubMed

Lapierre, Valérie; Aupérin, Anne; Robinet, Eric; Ferrand, Christophe; Oubouzar, Nadia; Tramalloni, Dominique; Saas, Philippe; Debaene, Bertrand; Lasser, Philippe; Tiberghien, Pierre

2007-09-01

Transfusion of red blood cells (RBCs) has been associated with immunomodulatory effects. Persistence of donor cells in the recipient may be contributive. A randomized single-center trial was conducted to compare microchimerism and immune responses in 35 patients undergoing cancer surgery and transfused perioperatively with either unmodified RBCs (UN-RBCs, n = 18) or leukoreduced RBCs (LR-RBCs, n = 17). Biologic parameters included microchimerism assessment peripheral blood mononuclear cell (PBMNC) phenotyping, cytokine production by stimulated PBMNCs, FoxP3 gene expression, and T-cell repertoire (TCR) analysis. Microchimerism was documented in 8 of 18 patients after UN-RBC transfusion while absent after LR-RBC transfusion (0/17; p = 0.001). After UN-RBC transfusion, microchimerism was associated with increased interleukin (IL)-10 production (p = 0.02), reduced TCR alteration (p = 0.04), and reduced CD56+ cell counts (p = 0.02) when compared to recipients without evidence for microchimerism. FoxP3 gene expression did not differ significantly between both treatment groups nor with the presence or absence of microchimerism in the UN-RBC group. Finally, after an initial early decrease after surgery and transfusion, IL-12 production increased and more significantly so after UN-RBC transfusion versus LR-RBC transfusion (p = 0.05). UN-RBC-induced microchimerism is associated with specific immunomodulatory effects in cancer patients who received transfusions during surgery.

Sulfonyl-containing modulators of serotonin 5-HT6 receptors and their pharmacophore models

NASA Astrophysics Data System (ADS)

Ivachtchenko, A. V.

2014-05-01

Data published in recent years on the synthesis of serotonin 5-HT6 receptor modulators are summarized. Modulators with high affinity for 5-HT6 receptors exhibiting different degrees of selectivity — from highly selective to semiselective and multimodal — are described. Clinical trial results are reported for the most promising serotonin 5-HT6 receptor modulators attracting special attention of medicinal chemists. The bibliography includes 128 references.

Association of HLA-DR1 with the allergic response to the major mugwort pollen allergen: molecular background.

PubMed

Knapp, Bernhard; Fischer, Gottfried; Van Hemelen, Dries; Fae, Ingrid; Maillere, Bernard; Ebner, Christof; Schreiner, Wolfgang; Bohle, Barbara; Jahn-Schmid, Beatrice

2012-08-08

Mugwort pollen allergens represent the main cause of pollinosis in late summer. The major allergen, Art v 1, contains only one single immunodominant, solely HLA-DR-restricted T cell epitope (Art v 125-36). The frequency of HLA-DRB1*01 is highly increased in mugwort-allergic individuals and HLA-DR1 serves as restriction element for Art v 125-36. However, Art v 125-36 also binds to HLA-DR4 with high affinity and DR1-restricted Art v 125-36 -specific T cell receptors can be activated by HLA-DR4 molecules. To understand the predominance of HLA-DR1 in mugwort allergy in spite of the degeneracy in HLA/peptide-binding and TCR-recognition, we investigated the molecular background of Art v 125-36 /MHC/TCR interactions in the context of HLA-DR1 compared to -DR4. The majority of Art v 125-36 -specific T cell lines and clones from HLA-DR1 carrying, mugwort pollen-allergic donors reacted to synthetic and naturally processed Art v 1-peptides when presented by HLA-DR1 or HLA-DR4 expressing antigen presenting cells. However, at limiting peptide concentrations DR1 was more effective in T cell stimulation. In addition, the minimal epitope for 50% of Art v 125-36 -specific T cells was shorter for DR1 than for DR4. In vitro binding assays of Art v 125-36 mutant peptides to isolated DR1- and DR4-molecules indicated similar binding capacities and use of the same register. In silico simulation of Art v 125-36 binding to HLA-DR1 and -DR4 suggested similar binding of the central part of the peptide to either molecule, but a higher flexibility of the N- and C-terminal amino acids and detachment at the C-terminus in HLA-DR1. The predominance of HLA-DR1 in the response to Art v 125-36 may be explained by subtle conformation changes of the peptide bound to DR1 compared to DR4. Computer simulation supported our experimental data by demonstrating differences in peptide mobility within the HLA-DR complex that may influence TCR-binding. We suggest that the minor differences observed in vitro may be more relevant in the microenvironment in vivo, so that only presentation by HLA-DR1, but not -DR4 permits successful T cell activation.

Severity of Acute Infectious Mononucleosis Correlates with Cross-Reactive Influenza CD8 T-Cell Receptor Repertoires.

PubMed

Aslan, Nuray; Watkin, Levi B; Gil, Anna; Mishra, Rabinarayan; Clark, Fransenio G; Welsh, Raymond M; Ghersi, Dario; Luzuriaga, Katherine; Selin, Liisa K

2017-12-05

Fifty years after the discovery of Epstein-Barr virus (EBV), it remains unclear how primary infection with this virus leads to massive CD8 T-cell expansion and acute infectious mononucleosis (AIM) in young adults. AIM can vary greatly in severity, from a mild transient influenza-like illness to a prolonged severe syndrome. We questioned whether expansion of a unique HLA-A2.01-restricted, cross-reactive CD8 T-cell response between influenza virus A-M1 58 (IAV-M1) and EBV BMLF1 280 (EBV-BM) could modulate the immune response to EBV and play a role in determining the severity of AIM in 32 college students. Only ex vivo total IAV-M1 and IAV-M1+EBV-BM cross-reactive tetramer + frequencies directly correlated with AIM severity and were predictive of severe disease. Expansion of specific cross-reactive memory IAV-M1 T-cell receptor (TCR) Vβ repertoires correlated with levels of disease severity. There were unique profiles of qualitatively different functional responses in the cross-reactive and EBV-specific CD8 T-cell responses in each of the three groups studied, severe-AIM patients, mild-AIM patients, and seropositive persistently EBV-infected healthy donors, that may result from differences in TCR repertoire use. IAV-M1 tetramer + cells were functionally cross-reactive in short-term cultures, were associated with the highest disease severity in AIM, and displayed enhanced production of gamma interferon, a cytokine that greatly amplifies immune responses, thus frequently contributing to induction of immunopathology. Altogether, these data link heterologous immunity via CD8 T-cell cross-reactivity to CD8 T-cell repertoire selection, function, and resultant disease severity in a common and important human infection. In particular, it highlights for the first time a direct link between the TCR repertoire with pathogenesis and the diversity of outcomes upon pathogen encounter. IMPORTANCE The pathogenic impact of immune responses that by chance cross-react to unrelated viruses has not been established in human infections. Here, we demonstrate that the severity of acute infectious mononucleosis (AIM), an Epstein-Barr virus (EBV)-induced disease prevalent in young adults but not children, is associated with increased frequencies of T cells cross-reactive to EBV and the commonly acquired influenza A virus (IAV). The T-cell receptor (TCR) repertoire and functions of these cross-reactive T cells differed between mild- and severe-AIM patients, most likely because these two groups of patients had selected different memory TCR repertoires in response to IAV infections encountered earlier. This heterologous immunity may explain variability in disease outcome and why young adults with more-developed IAV-specific memory T-cell pools have more-severe disease than children, who have less-developed memory pools. This study provides a new framework for understanding the role of heterologous immunity in human health and disease and highlights an important developing field examining the role of T-cell repertoires in the mediation of immunopathology. Copyright © 2017 Aslan et al.

Interplay of DNA repair with transcription: from structures to mechanisms.

PubMed

Deaconescu, Alexandra M; Artsimovitch, Irina; Grigorieff, Nikolaus

2012-12-01

Many DNA transactions are crucial for maintaining genomic integrity and faithful transfer of genetic information but remain poorly understood. An example is the interplay between nucleotide excision repair (NER) and transcription, also known as transcription-coupled DNA repair (TCR). Discovered decades ago, the mechanisms for TCR have remained elusive, not in small part due to the scarcity of structural studies of key players. Here we summarize recent structural information on NER/TCR factors, focusing on bacterial systems, and integrate it with existing genetic, biochemical, and biophysical data to delineate the mechanisms at play. We also review emerging, alternative modalities for recruitment of NER proteins to DNA lesions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification and characterization of TCRgamma and TCRdelta chains in channel catfish, Ictalurus punctatus

USDA-ARS?s Scientific Manuscript database

Channel catfish, Ictalurus punctatus, T cell receptors (TCR) gamma and delta were identified by mining of expressed sequence tag databases and full length sequences were obtained by 5'-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), (diversity; D), joining ...

Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

PubMed Central

Joshi, Rubin N.; Binai, Nadine A.; Marabita, Francesco; Sui, Zhenhua; Altman, Amnon; Heck, Albert J. R.; Tegnér, Jesper; Schmidt, Angelika

2017-01-01

Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4+CD25− T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP3 receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer. PMID:28993769

Determinants of public T cell responses.

PubMed

Li, Hanjie; Ye, Congting; Ji, Guoli; Han, Jiahuai

2012-01-01

Historically, sharing T cell receptors (TCRs) between individuals has been speculated to be impossible, considering the dramatic discrepancy between the potential enormity of the TCR repertoire and the limited number of T cells generated in each individual. However, public T cell response, in which multiple individuals share identical TCRs in responding to a same antigenic epitope, has been extensively observed in a variety of immune responses across many species. Public T cell responses enable individuals within a population to generate similar antigen-specific TCRs against certain ubiquitous pathogens, leading to favorable biological outcomes. However, the relatively concentrated feature of TCR repertoire may limit T cell response in a population to some other pathogens. It could be a great benefit for human health if public T cell responses can be manipulated. Therefore, the mechanistic insight of public TCR generation is important to know. Recently, high-throughput DNA sequencing has revolutionized the study of immune receptor repertoires, which allows a much better understanding of the factors that determine the overlap of TCR repertoire among individuals. Here, we summarize the current knowledge on public T-cell response and discuss future challenges in this field.

Generalized Procedure for Improved Accuracy of Thermal Contact Resistance Measurements for Materials With Arbitrary Temperature-Dependent Thermal Conductivity

DOE PAGES

Sayer, Robert A.

2014-06-26

Thermal contact resistance (TCR) is most commonly measured using one-dimensional steady-state calorimetric techniques. In the experimental methods we utilized, a temperature gradient is applied across two contacting beams and the temperature drop at the interface is inferred from the temperature profiles of the rods that are measured at discrete points. During data analysis, thermal conductivity of the beams is typically taken to be an average value over the temperature range imposed during the experiment. Our generalized theory is presented and accounts for temperature-dependent changes in thermal conductivity. The procedure presented enables accurate measurement of TCR for contacting materials whose thermalmore » conductivity is any arbitrary function of temperature. For example, it is shown that the standard technique yields TCR values that are about 15% below the actual value for two specific examples of copper and silicon contacts. Conversely, the generalized technique predicts TCR values that are within 1% of the actual value. The method is exact when thermal conductivity is known exactly and no other errors are introduced to the system.« less

Using an SU-8 photoresist structure and cytochrome C thin film sensing material for a microbolometer.

PubMed

Lai, Jian-Lun; Liao, Chien-Jen; Su, Guo-Dung John

2012-11-27

There are two critical parameters for microbolometers: the temperature coefficient of resistance (TCR) of the sensing material, and the thermal conductance of the insulation structure. Cytochrome c protein, having a high TCR, is a good candidate for infrared detection. We can use SU-8 photoresist for the thermal insulation structure, given its low thermal conductance. In this study, we designed a platform structure based on a SU-8 photoresist. We fabricated an infrared sensing pixel and recorded a high TCR for this new structure. The SU-8 photoresist insulation structure was fabricated using the exposure dose method. We experimentally demonstrated high values of TCR from 22%/K to 25.7%/K, and the measured noise was 1.2 × 10(-8) V2/Hz at 60 Hz. When the bias current was 2 μA, the calculated voltage responsivity was 1.16 × 10(5) V/W. This study presents a new kind of microbolometer based on cytochrome c protein on top of an SU-8 photoresist platform that does not require expensive vacuum deposition equipment.

Using an SU-8 Photoresist Structure and Cytochrome C Thin Film Sensing Material for a Microbolometer

PubMed Central

Lai, Jian-Lun; Liao, Chien-Jen; Su, Guo-Dung John

2012-01-01

There are two critical parameters for microbolometers: the temperature coefficient of resistance (TCR) of the sensing material, and the thermal conductance of the insulation structure. Cytochrome c protein, having a high TCR, is a good candidate for infrared detection. We can use SU-8 photoresist for the thermal insulation structure, given its low thermal conductance. In this study, we designed a platform structure based on a SU-8 photoresist. We fabricated an infrared sensing pixel and recorded a high TCR for this new structure. The SU-8 photoresist insulation structure was fabricated using the exposure dose method. We experimentally demonstrated high values of TCR from 22%/K to 25.7%/K, and the measured noise was 1.2 × 10−8 V2/Hz at 60 Hz. When the bias current was 2 μA, the calculated voltage responsivity was 1.16 × 105 V/W. This study presents a new kind of microbolometer based on cytochrome c protein on top of an SU-8 photoresist platform that does not require expensive vacuum deposition equipment. PMID:23443384

Chromium–niobium co-doped vanadium dioxide films: Large temperature coefficient of resistance and practically no thermal hysteresis of the metal–insulator transition

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

Miyazaki, Kenichi, E-mail: kenichi-miyazaki@denso.co.jp, E-mail: k.shibuya@aist.go.jp; University of Tsukuba, Tsukuba 305-8571; Shibuya, Keisuke, E-mail: kenichi-miyazaki@denso.co.jp, E-mail: k.shibuya@aist.go.jp

We investigated the effects of chromium (Cr) and niobium (Nb) co-doping on the temperature coefficient of resistance (TCR) and the thermal hysteresis of the metal–insulator transition of vanadium dioxide (VO{sub 2}) films. We determined the TCR and thermal-hysteresis-width diagram of the V{sub 1−x−y}Cr{sub x}Nb{sub y}O{sub 2} films by electrical-transport measurements and we found that the doping conditions x ≳ y and x + y ≥ 0.1 are appropriate for simultaneously realizing a large TCR value and an absence of thermal hysteresis in the films. By using these findings, we developed a V{sub 0.90}Cr{sub 0.06}Nb{sub 0.04}O{sub 2} film grown on amore » TiO{sub 2}-buffered SiO{sub 2}/Si substrate that showed practically no thermal hysteresis while retaining a large TCR of 11.9%/K. This study has potential applications in the development of VO{sub 2}-based uncooled bolometers.« less

Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse

PubMed Central

Lasserre, Rémi; Charrin, Stéphanie; Cuche, Céline; Danckaert, Anne; Thoulouze, Maria-Isabel; de Chaumont, Fabrice; Duong, Tarn; Perrault, Nathalie; Varin-Blank, Nadine; Olivo-Marin, Jean-Christophe; Etienne-Manneville, Sandrine; Arpin, Monique; Di Bartolo, Vincenzo; Alcover, Andrés

2010-01-01

T-cell receptor (TCR) signalling is triggered and tuned at immunological synapses by the generation of signalling complexes that associate into dynamic microclusters. Microcluster movement is necessary to tune TCR signalling, but the molecular mechanism involved remains poorly known. We show here that the membrane-microfilament linker ezrin has an important function in microcluster dynamics and in TCR signalling through its ability to set the microtubule network organization at the immunological synapse. Importantly, ezrin and microtubules are important to down-regulate signalling events leading to Erk1/2 activation. In addition, ezrin is required for appropriate NF-AT activation through p38 MAP kinase. Our data strongly support the notion that ezrin regulates immune synapse architecture and T-cell activation through its interaction with the scaffold protein Dlg1. These results uncover a crucial function for ezrin, Dlg1 and microtubules in the organization of the immune synapse and TCR signal down-regulation. Moreover, they underscore the importance of ezrin and Dlg1 in the regulation of NF-AT activation through p38. PMID:20551903

A Mechanical Switch Couples T Cell Receptor Triggering to the Cytoplasmic Juxtamembrane Regions of CD3ζζ.

PubMed

Lee, Mark S; Glassman, Caleb R; Deshpande, Neha R; Badgandi, Hemant B; Parrish, Heather L; Uttamapinant, Chayasith; Stawski, Philipp S; Ting, Alice Y; Kuhns, Michael S

2015-08-18

The eight-subunit T cell receptor (TCR)-CD3 complex is the primary determinant for T cell fate decisions. Yet how it relays ligand-specific information across the cell membrane for conversion to chemical signals remains unresolved. We hypothesized that TCR engagement triggers a change in the spatial relationship between the associated CD3ζζ subunits at the junction where they emerge from the membrane into the cytoplasm. Using three in situ proximity assays based on ID-PRIME, FRET, and EPOR activity, we determined that the cytosolic juxtamembrane regions of the CD3ζζ subunits are spread apart upon assembly into the TCR-CD3 complex. TCR engagement then triggered their apposition. This mechanical switch resides upstream of the CD3ζζ intracellular motifs that initiate chemical signaling, as well as the polybasic stretches that regulate signal potentiation. These findings provide a framework from which to examine triggering events for activating immune receptors and other complex molecular machines. Copyright © 2015 Elsevier Inc. All rights reserved.

T Cell Allorecognition via Molecular Mimicry

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

Macdonald, Whitney A.; Chen, Zhenjun; Gras, Stephanie

T cells often alloreact with foreign human leukocyte antigens (HLA). Here we showed the LC13 T cell receptor (TCR), selected for recognition on self-HLA-B*0801 bound to a viral peptide, alloreacts with B44 allotypes (HLA-B*4402 and HLA-B*4405) bound to two different allopeptides. Despite extensive polymorphism between HLA-B*0801, HLA-B*4402, and HLA-B*4405 and the disparate sequences of the viral and allopeptides, the LC13 TCR engaged these peptide-HLA (pHLA) complexes identically, accommodating mimicry of the viral peptide by the allopeptide. The viral and allopeptides adopted similar conformations only after TCR ligation, revealing an induced-fit mechanism of molecular mimicry. The LC13 T cells did notmore » alloreact against HLA-B*4403, and the single residue polymorphism between HLA-B*4402 and HLA-B*4403 affected the plasticity of the allopeptide, revealing that molecular mimicry was associated with TCR specificity. Accordingly, molecular mimicry that is HLA and peptide dependent is a mechanism for human T cell alloreactivity between disparate cognate and allogeneic pHLA complexes.« less

Anti-inflammatory and anti-bacterial properties of tetramethylhexadecenyl succinyl cysteine (TSC): a skin-protecting cosmetic functional ingredient.

PubMed

Fernandéz, J R; Rouzard, K; Voronkov, M; Huber, K L; Stock, J B; Stock, M; Gordon, J S; Pérez, E

2015-02-01

The skin is the first line of defence against exposure to microbial, physical, environmental and chemical insults. In mobilizing a protective response, several different cell types located in our skin release and respond to pro-inflammatory cytokines ensuring skin homeostasis and health. However, chronic activation of this response eventually causes damage resulting in premature ageing. Diosodium tetramethylhexadecenyl succinyl cysteine (TSC or SIG1273), an isoprenylcysteine small molecule, down modulates these inflammatory signalling pathways in various cell types (keratinocytes, peripheral blood mononuclear cells (PBMCs) and endothelial cells) and possesses anti-bacterial properties. Thus, TSC represents a novel cosmetic functional ingredient that provides a broad spectrum of benefits for the skin. To assess the anti-inflammatory properties of TSC in several cutaneous cell types and further investigate its anti-microbial activity. Cultured normal human epidermal keratinocytes were exposed to chemical irritant phorbol 12-myrisate 13-acetate (TPA) or ultraviolet-B light (UVB) to induce pro-inflammatory cytokine (IL-6, IL-8 and TNF-α) production. T-cell receptor (TCR) activation of PBMCs and nickel (Ni(2+) ) treatments of human dermal microvascular endothelial cells (HDMECs) were performed resulting in IL-4, IL-6, IL-8 and IL-17 production. Streptococcus pyogenes were cultured to determine minimal inhibitory concentration values. In vitro studies demonstrate TSC blocks TPA and UVB-induced cytokine production in cultured keratinocytes. Similarly, TSC inhibits overproduction of IL-4 and IL-17 in T-cell receptor (TCR)-activated PBMCs as well as nickel induction of IL-6 and IL-8 in HDMECs. Lastly, TSC demonstrated anti-microbial properties, inhibiting cell growth of S. pyogenes. Tetramethylhexadecenyl succinyl cysteine represents a novel cosmetic functional ingredient that provides a dual modulating benefit of skin protection to individuals by reducing inflammation in keratinocytes, endothelial and mononuclear cell types and S. pyogenes counts. © 2014 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Endogenous vs Exogenous Allosteric Modulators in GPCRs: A dispute for shuttling CB1 among different membrane microenvironments

NASA Astrophysics Data System (ADS)

Stornaiuolo, Mariano; Bruno, Agostino; Botta, Lorenzo; Regina, Giuseppe La; Cosconati, Sandro; Silvestri, Romano; Marinelli, Luciana; Novellino, Ettore

2015-10-01

A Cannabinoid Receptor 1 (CB1) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB1 modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands.

[Propranolol beta-blocker decrease in the concentration of high-affinity binding sites for calcium ions by sarcolemma membranes of the rat heart].

PubMed

Seleznev, Iu M; Martynov, A V; Smirnov, V N

1982-05-01

In vivo administration of propranolol considerably inhibits the isoproterenol-stimulated increase in 45Ca accumulation by the myocardium and completely eliminates the potentiation of isoproterenol effect by hydrocortisone. A significant lowering of the concentration of high affinity binding sites for calcium in the sarcolemmal membranes can be produced by propranolol in vitro. Under these conditions, the glucocorticoids do not change the sarcolemmal Ca2+-binding parameters or modulate the propranolol effect. Therefore, for the manifestation of glucocorticoid action to be brought about, the integrity of the cells is apparently required, while propranolol seems to change calcium binding by direct interaction with the sarcolemmal membranes. It is suggested that in vivo propranolol inhibition of catecholamine effect on calcium ion accumulation by the myocardium depends on the interaction with the beta-receptors and direct modulation of the concentration of high affinity binding sites for calcium ions on the surface of the sarcolemma.

Activated PLC-γ1 is Catalytically Induced at LAT but Activated PLC-γ1 is Localized at both LAT- and TCR-Containing Complexes

PubMed Central

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

2014-01-01

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. PMID:24412752

Two separate defects affecting true naive or virtual memory T cell precursors combine to reduce naive T cell responses with aging.

PubMed

Renkema, Kristin R; Li, Gang; Wu, Angela; Smithey, Megan J; Nikolich-Žugich, Janko

2014-01-01

Naive T cell responses are eroded with aging. We and others have recently shown that unimmunized old mice lose ≥ 70% of Ag-specific CD8 T cell precursors and that many of the remaining precursors acquire a virtual (central) memory (VM; CD44(hi)CD62L(hi)) phenotype. In this study, we demonstrate that unimmunized TCR transgenic (TCRTg) mice also undergo massive VM conversion with age, exhibiting rapid effector function upon both TCR and cytokine triggering. Age-related VM conversion in TCRTg mice directly depended on replacement of the original TCRTg specificity by endogenous TCRα rearrangements, indicating that TCR signals must be critical in VM conversion. Importantly, we found that VM conversion had adverse functional effects in both old wild-type and old TCRTg mice; that is, old VM, but not old true naive, T cells exhibited blunted TCR-mediated, but not IL-15-mediated, proliferation. This selective proliferative senescence correlated with increased apoptosis in old VM cells in response to peptide, but decreased apoptosis in response to homeostatic cytokines IL-7 and IL-15. Our results identify TCR as the key factor in differential maintenance and function of Ag-specific precursors in unimmunized mice with aging, and they demonstrate that two separate age-related defects--drastic reduction in true naive T cell precursors and impaired proliferative capacity of their VM cousins--combine to reduce naive T cell responses with aging.

Conditional deletion reveals a cell-autonomous requirement of SLP-76 for thymocyte selection.

PubMed

Maltzman, Jonathan S; Kovoor, Lisa; Clements, James L; Koretzky, Gary A

2005-10-03

The SH2 domain containing leukocyte phosphoprotein of 76 kD (SLP-76) is critical for pre-TCR-mediated maturation to the CD4+CD8+ double positive (DP) stage in the thymus. The absolute block in SLP-76null mice at the CD4-CD8-CD44-CD25+ (double-negative 3, DN3) stage has hindered our understanding of the role of this adaptor in alphabeta TCR-mediated signal transduction in primary thymocytes and peripheral T lymphocytes. To evaluate the requirements for SLP-76 in these events, we used a cre-loxP approach to generate mice that conditionally delete SLP-76 after the DN3 checkpoint. These mice develop DP thymocytes that express the alphabeta TCR on the surface, but lack SLP-76 at the genomic DNA and protein levels. The DP compartment has reduced cellularity in young mice and fails to undergo positive selection to CD4+ or CD8+ single positive (SP) cells in vivo or activation-induced cell death in vitro. A small number of CD4+SP thymocytes are generated, but these cells fail to flux calcium in response to an alphabeta TCR-generated signal. Peripheral T cells are reduced in number, lack SLP-76 protein, and have an abnormal surface phenotype. These studies show for the first time that SLP-76 is required for signal transduction through the mature alphabeta TCR in primary cells of the T lineage.

Different subsets of natural killer T cells may vary in their roles in health and disease.

PubMed

Kumar, Vipin; Delovitch, Terry L

2014-07-01

Natural killer T cells (NKT) can regulate innate and adaptive immune responses. Type I and type II NKT cell subsets recognize different lipid antigens presented by CD1d, an MHC class-I-like molecule. Most type I NKT cells express a semi-invariant T-cell receptor (TCR), but a major subset of type II NKT cells reactive to a self antigen sulphatide use an oligoclonal TCR. Whereas TCR-α dominates CD1d-lipid recognition by type I NKT cells, TCR-α and TCR-β contribute equally to CD1d-lipid recognition by type II NKT cells. These variable modes of NKT cell recognition of lipid-CD1d complexes activate a host of cytokine-dependent responses that can either exacerbate or protect from disease. Recent studies of chronic inflammatory and autoimmune diseases have led to a hypothesis that: (i) although type I NKT cells can promote pathogenic and regulatory responses, they are more frequently pathogenic, and (ii) type II NKT cells are predominantly inhibitory and protective from such responses and diseases. This review focuses on a further test of this hypothesis by the use of recently developed techniques, intravital imaging and mass cytometry, to analyse the molecular and cellular dynamics of type I and type II NKT cell antigen-presenting cell motility, interaction, activation and immunoregulation that promote immune responses leading to health versus disease outcomes. © 2014 John Wiley & Sons Ltd.

CD8+ T cells with characteristic T cell receptor beta motif are detected in blood and expanded in synovial fluid of ankylosing spondylitis patients.

PubMed

Komech, Ekaterina A; Pogorelyy, Mikhail V; Egorov, Evgeniy S; Britanova, Olga V; Rebrikov, Denis V; Bochkova, Anna G; Shmidt, Evgeniya I; Shostak, Nadejda A; Shugay, Mikhail; Lukyanov, Sergey; Mamedov, Ilgar Z; Lebedev, Yuriy B; Chudakov, Dmitriy M; Zvyagin, Ivan V

2018-02-22

The risk of AS is associated with genomic variants related to antigen presentation and specific cytokine signalling pathways, suggesting the involvement of cellular immunity in disease initiation/progression. The aim of the present study was to explore the repertoire of TCR sequences in healthy donors and AS patients to uncover AS-linked TCR variants. Using quantitative molecular-barcoded 5'-RACE, we performed deep TCR β repertoire profiling of peripheral blood (PB) and SF samples for 25 AS patients and 108 healthy donors. AS-linked TCR variants were identified using a new computational approach that relies on a probabilistic model of the VDJ rearrangement process. Using the donor-agnostic probabilistic model, we reveal a TCR β motif characteristic for PB of AS patients, represented by eight highly homologous amino acid sequence variants. Some of these variants were previously reported in SF and PB of patients with ReA and in PB of AS patients. We demonstrate that identified AS-linked clones have a CD8+ phenotype, present at relatively low frequencies in PB, and are significantly enriched in matched SF samples of AS patients. Our results suggest the involvement of a particular antigen-specific subset of CD8+ T cells in AS pathogenesis, confirming and expanding earlier findings. The high similarity of the clonotypes with the ones found in ReA implies common mechanisms for the initiation of the diseases.

Low resistivity W{sub x}V{sub 1−x}O{sub 2}-based multilayer structure with high temperature coefficient of resistance for microbolometer applications

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

Émond, Nicolas; Hendaoui, Ali; Chaker, Mohamed, E-mail: chaker@emt.inrs.ca

2015-10-05

Materials that exhibit semiconductor-to-metal phase transition (SMT) are commonly used as sensing layers for the fabrication of uncooled microbolometers. The development of highly responsive microbolometers would benefit from using a sensing material that possesses a large thermal coefficient of resistance (TCR) close to room temperature and a resistivity low enough to compromise between noise reduction and high TCR, while it should also satisfies the requirements of current CMOS technology. Moreover, a TCR that remains constant when the IR camera surrounding temperature varies would contribute to achieve reliable temperature measurements without additional corrections steps for TCR temperature dependence. In this paper,more » the characteristics of the SMT occurring in undoped and tungsten-doped vanadium dioxide thin films deposited on LaAlO{sub 3} (100) substrates are investigated. They are further exploited to fabricate a W{sub x}V{sub 1−x}O{sub 2} (0 ≤ x ≤ 2.5) multilayer structure exhibiting a bottom-up gradient of tungsten content. This MLS displays a combination of properties that is promising for application to uncooled microbolometer, such as a large TCR of −10.4%/ °C and low resistivity values ranging from 0.012 to 0.10 Ω-cm over the temperature range 22 °C–42 °C.« less

Structural basis for the initiation of eukaryotic transcription-coupled DNA repair.

PubMed

Xu, Jun; Lahiri, Indrajit; Wang, Wei; Wier, Adam; Cianfrocco, Michael A; Chong, Jenny; Hare, Alissa A; Dervan, Peter B; DiMaio, Frank; Leschziner, Andres E; Wang, Dong

2017-11-30

Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II-CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.

Conformational analysis of the ΜΒΡ83-99 (Phe91) and ΜΒΡ83-99 (Tyr91) peptide analogues and study of their interactions with the HLA-DR2 and human TCR receptors by using Molecular Dynamics

NASA Astrophysics Data System (ADS)

Potamitis, C.; Matsoukas, M.-T.; Tselios, T.; Mavromoustakos, T.; Golič Grdadolnik, S.

2011-09-01

The two new synthetic analogues of the MBP83-99 epitope substituted at Lys91 (primary TCR contact) with Phe [MBP83-99 (Phe91)] or Tyr [MBP83-99 (Tyr91)], have been structurally elucidated using 1D and 2D high resolution NMR studies. The conformational analysis of the two altered peptide ligands (APLs) has been performed and showed that they adopt a linear and extended conformation which is in agreement with the structural requirements of the peptides that interact with the HLA-DR2 and TCR receptors. In addition, Molecular Dynamics (MD) simulations of the two analogues in complex with HLA-DR2 (DRA, DRB1*1501) and TCR were performed. Similarities and differences of the binding motif of the two analogues were observed which provide a possible explanation of their biological activity. Their differences in the binding mode in comparison with the MBP83-99 epitope may also explain their antagonistic versus agonistic activity. The obtained results clearly indicate that substitutions in crucial amino acids (TCR contacts) in combination with the specific conformational characteristics of the MBP83-99 immunodominant epitope lead to an alteration of their biological activity. These results make the rational drug design intriguing since the biological activity is very sensitive to the substitution and conformation of the mutated MBP epitopes.

Evolution and Function of the TCR Vgamma9 Chain Repertoire: It’s Good to be Public

PubMed Central

Pauza, C. David; Cairo, Cristiana

2015-01-01

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 from healthy adults and the subset can be expanded 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. PMID:25769734

The molecular bases of δ/αβ T cell-mediated antigen recognition.

PubMed

Pellicci, Daniel G; Uldrich, Adam P; Le Nours, Jérôme; Ross, Fiona; Chabrol, Eric; Eckle, Sidonia B G; de Boer, Renate; Lim, Ricky T; McPherson, Kirsty; Besra, Gurdyal; Howell, Amy R; Moretta, Lorenzo; McCluskey, James; Heemskerk, Mirjam H M; Gras, Stephanie; Rossjohn, Jamie; Godfrey, Dale I

2014-12-15

αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1(+) human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity. © 2014 Pellicci et al.

The molecular bases of δ/αβ T cell–mediated antigen recognition

PubMed Central

Pellicci, Daniel G.; Uldrich, Adam P.; Le Nours, Jérôme; Ross, Fiona; Chabrol, Eric; Eckle, Sidonia B.G.; de Boer, Renate; Lim, Ricky T.; McPherson, Kirsty; Besra, Gurdyal; Howell, Amy R.; Moretta, Lorenzo; McCluskey, James; Heemskerk, Mirjam H.M.; Gras, Stephanie

2014-01-01

αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1+ human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity. PMID:25452463

T-Cell Receptor- and CD28-induced Vav1 activity is required for the accumulation of primed T cells into antigenic tissue

PubMed Central

David, Rachel; Ma, Liang; Ivetic, Aleksandar; Takesono, Aya; Ridley, Anne J.; Chai, Jian-Guo; Tybulewicz, Victor; Marelli-Berg, Federica M.

2016-01-01

Localization of primed T cells to antigenic tissue is essential for the development of effective immunity. Together with tissue-selective homing molecules, T-cell receptor (TCR)- and CD28-mediated signals have been shown to promote transendothelial migration of specific T cells into non-lymphoid antigen-rich tissue tissue. However, the cellular and molecular requirements for T-cell accumulation to target tissue following their recruitment are largely undefined. The guanine nucleotide exchange factor (GEF) Vav1 has an integral role in coupling TCR and CD28 to signalling pathways that regulate T cell activation and migration. Here, we have investigated the contribution of TCR- and CD28-induced Vav1 activity to the trafficking and localization of primed HY-specific CD4+ T cells to antigenic sites. Severe migratory defects displayed by Vav1-/- T cells in vitro were fully compensated by a combination of shear flow and chemokines, leading to normal recruitment of Vav1-/- T cells in vivo. In contrast, Vav1-/- T-cell retention into antigen-rich tissue was severely impaired, reflecting their inability to engage in sustained TCR- and CD28-mediated interactions with tissue-resident antigen-presenting cells (APCs). This novel function of APC-induced, TCR- and CD28-mediated Vav1 activity in the regulation of effector T-cell immunity highlights its potential as a therapeutic target in T-cell-mediated tissue damage. PMID:19060239

CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway.

PubMed

Gollmer, Kathrin; Asperti-Boursin, François; Tanaka, Yoshihiko; Okkenhaug, Klaus; Vanhaesebroeck, Bart; Peterson, Jeffrey R; Fukui, Yoshinori; Donnadieu, Emmanuel; Stein, Jens V

2009-07-16

CD4(+) T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)-transgenic (tg) CD4(+) T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3Kdelta(D910A/D910A) or PI3Kgamma-deficient TCR-tg CD4(+) T cells showed similar responsiveness to CCL21 costimulation as control CD4(+) T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4(+) T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca(2+) signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

PubMed

Asakura, M; Tsukamoto, T; Imafuku, J; Matsui, H; Ino, M; Hasegawa, K

1984-10-30

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

On staggered indecomposable Virasoro modules

NASA Astrophysics Data System (ADS)

Kytölä, Kalle; Ridout, David

2009-12-01

In this article, certain indecomposable Virasoro modules are studied. Specifically, the Virasoro mode L0 is assumed to be nondiagonalizable, possessing Jordan blocks of rank 2. Moreover, the module is further assumed to have a highest weight submodule, the "left module," and that the quotient by this submodule yields another highest weight module, the "right module." Such modules, which have been called staggered, have appeared repeatedly in the logarithmic conformal field theory literature, but their theory has not been explored in full generality. Here, such a theory is developed for the Virasoro algebra using rather elementary techniques. The focus centers on two different but related questions typically encountered in practical studies: How can one identify a given staggered module, and how can one demonstrate the existence of a proposed staggered module. Given just the values of the highest weights of the left and right modules, themselves subject to simple necessary conditions, invariants are defined which together with the knowledge of the left and right modules uniquely identify a staggered module. The possible values of these invariants form a vector space of dimension 0, 1, or 2, and the structures of the left and right modules limit the isomorphism classes of the corresponding staggered modules to an affine subspace (possibly empty). The number of invariants and affine restrictions is purely determined by the structures of the left and right modules. Moreover, in order to facilitate applications, the expressions for the invariants and restrictions are given by formulas as explicit as possible (they generally rely on expressions for Virasoro singular vectors). Finally, the text is liberally peppered throughout with examples illustrating the general concepts. These have been carefully chosen for their physical relevance or for the novel features they exhibit.

Horizontal Transfer of Tetracycline Resistance Genes in the Subsurface of a Poultry Farm

NASA Astrophysics Data System (ADS)

You, Y.; Ward, M.; Hilpert, M.

2008-12-01

Concentrated animal feeding operations (CAFOs) are considered to be important man-made reservoirs of antibiotic resistant bacteria and antibiotic resistance genes. At a poultry farm, we, together with Mr.~James Doolittle from USDA, measured the apparent subsurface electrical conductivity (ECa) using a EM38 meter. The resulting ECaR) associated with the poultry farm due to the fact that tetracycline (Tc) is one of the most frequently used antibiotics in food animal production and therefore is probably used at this farm. Soil and aquifer samples were taken from the farm. TcR bacteria were detected, with higher concentrations in the top layer of soil than in the aquifer. TcR bacteria were then enriched from a soil sample, and two classes of TcR genes were detected: tet(M) genes encoding ribosomal protection proteins and tet(L) genes encoding tet efflux pumps. Sequences of the PCR products were compared to known tet(M) and tet(L) genes in GenBank using BLASTN. Phylogenetic trees were also built based on the sequence information. The tet(M) genes found in our soil sample were highly similar to those located on transposons. In a soil microcosm experiment, we used the aforementioned soil sample as incubation medium as well as genetic donor (TcR soil bacteria), and a green fluorescent strain of E. coli as a model genetic recipient to study horizontal transfer of TcR genes from soil bacteria to naïve bacteria. Concentrations of inoculated E. coli were continuously monitored for 15 days, TcR E. coli isolated, and colony PCR performed. The tet(M) genes were found to be transferred to naïve E. coli. The highest horizontal transfer ratio, 0.62 transconjugant per recipient, was observed when Tc was supplemented to a soil microcosm at a concentration of 140 μg/kg soil. Modeling is also ongoing to obtain a better understanding of this complex phenomenon.

Phosphorylation of the Grb2- and phosphatidylinositol 3-kinase p85-binding p36/38 by Syk in Lck-negative T cells.

PubMed

von Willebrand, M; Williams, S; Tailor, P; Mustelin, T

1998-06-01

Activation of the mitogen-activated protein kinase (MAPK) pathway by the T-cell antigen receptor (TCR) in T cells involves a positive role for phosphatidylinositol 3-kinase (PI3K) activity. We recently reported that over-expression of the Syk protein tyrosine kinase in the Lck-negative JCaM1 cells enabled the TCR to induce a normal activation of the Erk2 MAPK and enhanced transcription of a reporter gene driven by the nuclear factor of activated T cells and AP-1. Because this system allows us to analyse the targets for Syk in receptor-mediated signalling, we examined the role of PI3K in signalling events between the TCR-regulated Syk and the downstream activation of Erk2. We report that inhibition of PI3K by wortmannin or an inhibitory p85 construct, p85deltaiSH2, reduced the TCR-induced Syk-dependent activation of Erk2, as well as the appearance of phospho-Erk and phospho-Mek. At the same time, expression of Syk resulted in the activation-dependent phosphorylation of three proteins that bound to the src homology 2 (SH2) domains of PI3K p85. The strongest of these bands had an apparent molecular mass of 36-38 kDa on SDS gels, and it was quantitatively removed from the lysates by adsorption to a fusion protein containing the SH2 domain of Grb2. The appearance of this band was Syk dependent, and it was seen only upon triggering of the TCR complex. Thus, p36/38 was phosphorylated by Syk or a Syk-regulated kinase, and this protein may provide a link to the recruitment and activation of PI3K, as well as to the Ras-MAPK pathway, in TCR-triggered T cells.

An antigen receptor-driven, interleukin 2-independent pathway for proliferation of murine cytolytic T lymphocyte clones

PubMed Central

1986-01-01

Proliferation of T lymphocytes can be induced by IL-2, either through an autocrine pathway in which the responding cell produces its own IL-2 or through an exocrine pathway in which IL-2 secreted by Th stimulates proliferation of IL-2-dependent CTL. However, proliferation of at least some CTL clones, such as CTL L3 and CTL dB45, also can be induced by stimulation of the antigen receptor in the absence of IL-2. Stimulation of these cloned CTL with T cell-depleted allogeneic spleen cells, allogeneic tumor cells, or immobilized mAb reactive with the T cell antigen receptor (TCR) induced thymidine incorporation, entry into cell cycle, and secretion of macrophage activating factor, but these stimuli did not induce the secretion of IL-2. Several observations indicated that such proliferation of cloned CTL induced by stimulation of the TCR was independent of IL-2; IL-2 could not be detected in supernatants from stimulated CTL cells. mAbs reactive with the murine IL-2-R efficiently blocked IL-2-mediated thymidine incorporation in cloned CTL and Th, but had no inhibitory effect on TCR-driven thymidine incorporation in the CTL clones. TCR-driven thymidine incorporation in cloned Th L2 cells was profoundly inhibited by these antibodies, indicating the operation of an IL-2-mediated autocrine pathway for proliferation in this cloned Th. When antibodies to the TCR were used to stimulate cloned CTL and Th, IFN-gamma mRNA was easily shown in the cloned CTL and Th. Although IL-2 mRNA could be detected in the cloned Th, it was never observed in the cloned CTL. These findings provide evidence for the existence of a TCR-mediated, IL-2-independent pathway for induction of cellular proliferation in cloned murine CTL. PMID:3486939

Differential Responsiveness of Innate-like IL-17- and IFN-γ-Producing γδ T Cells to Homeostatic Cytokines.

PubMed

Corpuz, Theresa M; Stolp, Jessica; Kim, Hee-Ok; Pinget, Gabriela V; Gray, Daniel H D; Cho, Jae-Ho; Sprent, Jonathan; Webster, Kylie E

2016-01-15

γδ T cells respond to molecules upregulated following infection or cellular stress using both TCR and non-TCR molecules. The importance of innate signals versus TCR ligation varies greatly. Both innate-like IL-17-producing γδ T (γδT-17) and IFN-γ-producing γδ T (γδT-IFNγ) subsets tune the sensitivity of their TCR following thymic development, allowing robust responses to inflammatory cytokines in the periphery. The remaining conventional γδ T cells retain high TCR responsiveness. We determined homeostatic mechanisms that govern these various subsets in the peripheral lymphoid tissues. We found that, although innate-like γδT-17 and γδT-IFNγ cells share elements of thymic development, they diverge when it comes to homeostasis. Both exhibit acute sensitivity to cytokines compared with conventional γδ T cells, but they do not monopolize the same cytokine. γδT-17 cells rely exclusively on IL-7 for turnover and survival, aligning them with NKT17 cells; IL-7 ligation triggers proliferation, as well as promotes survival, upregulating Bcl-2 and Bcl-xL. γδT-IFNγ cells instead depend heavily on IL-15. They display traits analogous to memory CD8(+) T cells and upregulate Bcl-xL and Mcl-1 upon cytokine stimulation. The conventional γδ T cells display low sensitivity to cytokine-alone stimulation and favor IL-7 for their turnover, characteristics reminiscent of naive αβ T cells, suggesting that they may also require tonic TCR signaling for population maintenance. These survival constraints suggest that γδ T cell subsets do not directly compete with each other for cytokines, but instead fall into resource niches with other functionally similar lymphocytes. Copyright © 2016 by The American Association of Immunologists, Inc.

Spread of tetracycline resistance genes at a conventional dairy farm

PubMed Central

Kyselková, Martina; Jirout, Jiří; Vrchotová, Naděžda; Schmitt, Heike; Elhottová, Dana

2015-01-01

The use of antibiotics in animal husbandry contributes to the worldwide problem of increasing antibiotic resistance in animal and human pathogens. Intensive animal production is considered an important source of antibiotic resistance genes released to the environment, while the contribution of smaller farms remains to be evaluated. Here we monitor the spread of tetracycline resistance (TC-r) genes at a middle-size conventional dairy farm, where chlortetracycline (CTC, as intrauterine suppository) is prophylactically used after each calving. Our study has shown that animals at the farm acquired the TC-r genes in their early age (1–2 weeks), likely due to colonization with TC-resistant bacteria from their mothers and/or the farm environment. The relative abundance of the TC-r genes tet(W), tet(Q), and tet(M) in fresh excrements of calves was about 1–2 orders of magnitude higher compared to heifers and dairy cows, possibly due to the presence of antibiotic residues in milk fed to calves. The occurrence and abundance of TC-r genes in fresh excrements of heifers and adult cows remained unaffected by intrauterine CTC applications, with tet(O), tet(Q), and tet(W) representing a “core TC-resistome” of the farm, and tet(A), tet(M), tet(Y), and tet(X) occurring occasionally. The genes tet(A), tet(M), tet(Y), and tet(X) were shown to be respectively harbored by Shigella, Lactobacillus and Clostridium, Acinetobacter, and Wautersiella. Soil in the farm proximity, as well as field soil to which manure from the farm was applied, was contaminated with TC-r genes occurring in the farm, and some of the TC-r genes persisted in the field over 3 months following the manure application. Concluding, our study shows that antibiotic resistance genes may be a stable part of the intestinal metagenome of cattle even if antibiotics are not used for growth stimulation, and that smaller dairy farms may also contribute to environmental pollution with antibiotic resistance genes. PMID:26074912

T-cell receptor transfer for boosting HIV-1-specific T-cell immunity in HIV-1-infected patients.

PubMed

Mummert, Christiane; Hofmann, Christian; Hückelhoven, Angela G; Bergmann, Silke; Mueller-Schmucker, Sandra M; Harrer, Ellen G; Dörrie, Jan; Schaft, Niels; Harrer, Thomas

2016-09-10

Strategies to cure HIV-1 infection require the eradication of viral reservoirs. An innovative approach for boosting the cytotoxic T-lymphocyte response is the transfer of T-cell receptors (TCRs). Previously, we have shown that electroporation of TCR-encoding mRNA is able to reprogram CD8 T cells derived from healthy donors. So far, it is unknown whether the transfer of HIV-1-specific TCRs is capable to reprogram CD8 T cells of HIV-1-infected patients. To assess the efficiency of TCR-transfer by mRNA electroporation and the functionality of reprogramed T cells in HIV-1-infected patients, we performed an in-vitro analysis of TCR-transfer into T cells from HIV-1-infected patients in various stages of disease and from healthy controls. Peripheral blood mononuclear cells from 16 HIV-1-infected patients (nine HLA-A02-positive, seven HLA-A02-negative) and from five healthy controls were electroporated with mRNA-constructs encoding TCRs specific for the HLA-A02/HIV-1-gag p17 epitope SLYNTVATL (SL9). Functionality of the TCRs was measured by γIFN-ELISpot assays. SL9/TCR transfection into peripheral blood mononuclear cells from both HLA-A02-positive and HLA-A02-negative HIV-1-infected patients and from healthy blood donors reprogramed T cells for recognition of SL9-presenting HLA-A02-positive cells in γIFN-ELISpot assays. SL9/TCR-transfer into T cells from an immunodeficient AIDS patient could induce recognition of SL9-expressing target cells only after reversion of T-cell dysfunction by antiretroviral therapy. The transfer of HIV-1-p17-specific TCRs into T cells is functional both in HIV-1-infected patients as well as in healthy blood donors. TCR-transfer is a promising method to boost the immune system against HIV-1.

Interpreting T-Cell Cross-reactivity through Structure: Implications for TCR-Based Cancer Immunotherapy.

PubMed

Antunes, Dinler A; Rigo, Maurício M; Freitas, Martiela V; Mendes, Marcus F A; Sinigaglia, Marialva; Lizée, Gregory; Kavraki, Lydia E; Selin, Liisa K; Cornberg, Markus; Vieira, Gustavo F

2017-01-01

Immunotherapy has become one of the most promising avenues for cancer treatment, making use of the patient's own immune system to eliminate cancer cells. Clinical trials with T-cell-based immunotherapies have shown dramatic tumor regressions, being effective in multiple cancer types and for many different patients. Unfortunately, this progress was tempered by reports of serious (even fatal) side effects. Such therapies rely on the use of cytotoxic T-cell lymphocytes, an essential part of the adaptive immune system. Cytotoxic T-cells are regularly involved in surveillance and are capable of both eliminating diseased cells and generating protective immunological memory. The specificity of a given T-cell is determined through the structural interaction between the T-cell receptor (TCR) and a peptide-loaded major histocompatibility complex (MHC); i.e., an intracellular peptide-ligand displayed at the cell surface by an MHC molecule. However, a given TCR can recognize different peptide-MHC (pMHC) complexes, which can sometimes trigger an unwanted response that is referred to as T-cell cross-reactivity. This has become a major safety issue in TCR-based immunotherapies, following reports of melanoma-specific T-cells causing cytotoxic damage to healthy tissues (e.g., heart and nervous system). T-cell cross-reactivity has been extensively studied in the context of viral immunology and tissue transplantation. Growing evidence suggests that it is largely driven by structural similarities of seemingly unrelated pMHC complexes. Here, we review recent reports about the existence of pMHC "hot-spots" for cross-reactivity and propose the existence of a TCR interaction profile (i.e., a refinement of a more general TCR footprint in which some amino acid residues are more important than others in triggering T-cell cross-reactivity). We also make use of available structural data and pMHC models to interpret previously reported cross-reactivity patterns among virus-derived peptides. Our study provides further evidence that structural analyses of pMHC complexes can be used to assess the intrinsic likelihood of cross-reactivity among peptide-targets. Furthermore, we hypothesize that some apparent inconsistencies in reported cross-reactivities, such as a preferential directionality, might also be driven by particular structural features of the targeted pMHC complex. Finally, we explain why TCR-based immunotherapy provides a special context in which meaningful T-cell cross-reactivity predictions can be made.

Interpreting T-Cell Cross-reactivity through Structure: Implications for TCR-Based Cancer Immunotherapy

PubMed Central

Antunes, Dinler A.; Rigo, Maurício M.; Freitas, Martiela V.; Mendes, Marcus F. A.; Sinigaglia, Marialva; Lizée, Gregory; Kavraki, Lydia E.; Selin, Liisa K.; Cornberg, Markus; Vieira, Gustavo F.

2017-01-01

Immunotherapy has become one of the most promising avenues for cancer treatment, making use of the patient’s own immune system to eliminate cancer cells. Clinical trials with T-cell-based immunotherapies have shown dramatic tumor regressions, being effective in multiple cancer types and for many different patients. Unfortunately, this progress was tempered by reports of serious (even fatal) side effects. Such therapies rely on the use of cytotoxic T-cell lymphocytes, an essential part of the adaptive immune system. Cytotoxic T-cells are regularly involved in surveillance and are capable of both eliminating diseased cells and generating protective immunological memory. The specificity of a given T-cell is determined through the structural interaction between the T-cell receptor (TCR) and a peptide-loaded major histocompatibility complex (MHC); i.e., an intracellular peptide–ligand displayed at the cell surface by an MHC molecule. However, a given TCR can recognize different peptide–MHC (pMHC) complexes, which can sometimes trigger an unwanted response that is referred to as T-cell cross-reactivity. This has become a major safety issue in TCR-based immunotherapies, following reports of melanoma-specific T-cells causing cytotoxic damage to healthy tissues (e.g., heart and nervous system). T-cell cross-reactivity has been extensively studied in the context of viral immunology and tissue transplantation. Growing evidence suggests that it is largely driven by structural similarities of seemingly unrelated pMHC complexes. Here, we review recent reports about the existence of pMHC “hot-spots” for cross-reactivity and propose the existence of a TCR interaction profile (i.e., a refinement of a more general TCR footprint in which some amino acid residues are more important than others in triggering T-cell cross-reactivity). We also make use of available structural data and pMHC models to interpret previously reported cross-reactivity patterns among virus-derived peptides. Our study provides further evidence that structural analyses of pMHC complexes can be used to assess the intrinsic likelihood of cross-reactivity among peptide-targets. Furthermore, we hypothesize that some apparent inconsistencies in reported cross-reactivities, such as a preferential directionality, might also be driven by particular structural features of the targeted pMHC complex. Finally, we explain why TCR-based immunotherapy provides a special context in which meaningful T-cell cross-reactivity predictions can be made. PMID:29046675

NK and NK-like T-cell lymphoma in extranasal sites: a comparative clinicopathological study according to site and EBV status.

PubMed

Ko, Y H; Cho, E-Y; Kim, J-E; Lee, S-S; Huh, J-R; Chang, H-K; Yang, W-I; Kim, C-W; Kim, S-W; Ree, H J

2004-05-01

To analyse the clinicopathological findings of extranasal CD56+ cytotoxic T- or NK-cell lymphomas in different organs and to compare Epstein-Barr virus (EBV)+ and EBV- lymphoma of non-blastoid cytomorphology. Fifty-one cases of cCD3+ T-cell intracellular antigen (TIA-1)+ CD56+ lymphomas of extranodal/extranasal origin were included in the study. The primary sites of the CD56+ tumours were soft tissue (n = 10), the gastrointestinal (GI) tract (n = 13), the skin (n = 15), upper aerodigestive tract excluding nasal and nasopharyngeal regions (n = 11), the testis (n = 1), and parotid gland (n = 1). TCR gene rearrangement was detected in seven of 47 cases examined (16%). EBV was positive in 39 of 51 cases (76%). The positive rate of EBV was higher in tumours of soft tissue (80%), GI tract (92%), and skin (80%), and lowest in the upper aerodigestive tract excluding the nasal and nasopharyngeal region (50%). Tumours of the soft tissue and the upper aerodigestive tract tended to present with localized disease (P = 0.002). The 2-year survival rate was lowest for tumours of the GI tract (P = 0.0256). EBV- TCR- lymphoma showed less necrosis (P = 0.0133) and a better 2-year survival rate (P = 0.0066) than EBV+ TCR- lymphoma. Patients with EBV+ TCR+ lymphomas tended to present with localized disease, more often than EBV+ TCR- lymphoma (P = 0.0186). Significant prognostic factors in all CD56+ lymphomas were the site (P = 0.0256), EBV status (P = 0.0026), necrosis with or without perforation (P = 0.0338) and the presence of pleomorphic large tumour cells (P = 0.0428). Cox's regression analysis adjusting for other pathological parameters showed EBV status to be the only independent prognostic factor (P = 0.018). Extranodal CD56+ EBV- lymphoma at extranasal sites is a clinically less aggressive malignancy and displays less necrosis than CD56+ EBV+ lymphoma. Because CD56+ EBV+ TCR+ lymphomas show similar pathological and clinical findings to CD56+ EBV+ TCR- lymphomas, nasal-type NK/T-cell lymphomas at extranasal sites should be diagnosed as such on the basis of EBV+, cytotoxic T or NK phenotype irrespective of the genotype determined by molecular study.

3D: diversity, dynamics, differential testing - a proposed pipeline for analysis of next-generation sequencing T cell repertoire data.

PubMed

Zhang, Li; Cham, Jason; Paciorek, Alan; Trager, James; Sheikh, Nadeem; Fong, Lawrence

2017-02-27

Cancer immunotherapy has demonstrated significant clinical activity in different cancers. T cells represent a crucial component of the adaptive immune system and are thought to mediate anti-tumoral immunity. Antigen-specific recognition by T cells is via the T cell receptor (TCR) which is unique for each T cell. Next generation sequencing (NGS) of the TCRs can be used as a platform to profile the T cell repertoire. Though there are a number of software tools available for processing repertoire data by mapping antigen receptor segments to sequencing reads and assembling the clonotypes, most of them are not designed to track and examine the dynamic nature of the TCR repertoire across multiple time points or between different biologic compartments (e.g., blood and tissue samples) in a clinical context. We integrated different diversity measures to assess the T cell repertoire diversity and examined the robustness of the diversity indices. Among those tested, Clonality was identified for its robustness as a key metric for study design and the first choice to measure TCR repertoire diversity. To evaluate the dynamic nature of T cell clonotypes across time, we utilized several binary similarity measures (such as Baroni-Urbani and Buser overlap index), relative clonality and Morisita's overlap index, as well as the intraclass correlation coefficient, and performed fold change analysis, which was further extended to investigate the transition of clonotypes among different biological compartments. Furthermore, the application of differential testing enabled the detection of clonotypes which were significantly changed across time. By applying the proposed "3D" analysis pipeline to the real example of prostate cancer subjects who received sipuleucel-T, an FDA-approved immunotherapy, we were able to detect changes in TCR sequence frequency and diversity thus demonstrating that sipuleucel-T treatment affected TCR repertoire in blood and in prostate tissue. We also found that the increase in common TCR sequences between tissue and blood after sipuleucel-T treatment supported the hypothesis that treatment-induced T cell migrated into the prostate tissue. In addition, a second example of prostate cancer subjects treated with Ipilimumab and granulocyte macrophage colony stimulating factor (GM-CSF) was presented in the supplementary documents to further illustrate assessing the treatment-associated change in a clinical context by the proposed workflow. Our paper provides guidance to study the diversity and dynamics of NGS-based TCR repertoire profiling in a clinical context to ensure consistency and reproducibility of post-analysis. This analysis pipeline will provide an initial workflow for TCR sequencing data with serial time points and for comparing T cells in multiple compartments for a clinical study.

Proteins feel more than they see: fine-tuning of binding affinity by properties of the non-interacting surface.

PubMed

Kastritis, Panagiotis L; Rodrigues, João P G L M; Folkers, Gert E; Boelens, Rolf; Bonvin, Alexandre M J J

2014-07-15

Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct contributions from the interface, the non-interacting surface (NIS) also plays an important role in binding affinity, especially polar and charged residues. Their percentage on the NIS is conserved over orthologous complexes indicating an evolutionary selection pressure. Their effect on binding affinity can be explained by long-range electrostatic contributions and surface-solvent interactions that are known to determine the local frustration of the protein complex surface. Including these in a simple model significantly improves the affinity prediction of protein complexes from structural models. The impact of mutations outside the interacting surface on binding affinity is supported by experimental alanine scanning mutagenesis data. These results enable the development of more sophisticated and integrated biophysical models of binding affinity and open new directions in experimental control and modulation of biomolecular interactions. Copyright © 2014. Published by Elsevier Ltd.

T-cell receptor accessory and co-receptor molecules in channel catfish

USDA-ARS?s Scientific Manuscript database

T cell receptor (TCR) associated invariant chains CD3gamma/delta,epsilon, and zeta as well as TCR co-receptors CD8alpha and CD8beta were isolated from the channel catfish, Ictalurus punctatus, at both the gene and cDNA levels. All of catfish CD3 sequences encode for proteins that resemble their resp...

75 FR 75545 - CSX Transportation, Inc.-Corporate Family Merger Exemption-Atlanta, Knoxville & Northern Railway...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-03

... transaction. CSXT is a Class I rail carrier that directly controls and operates AKNR, CIT, and TCR. The transaction involves the merger of AKNR, CIT, and TCR with and into CSXT with CSXT being the surviving corporation. The transaction is scheduled to be consummated on or after December 19, 2010, the effective date...

Characterization of the intrinsic activity for a novel class of cannabinoid receptor ligands: Indole Quinuclidine analogues

PubMed Central

Franks, Lirit N.; Ford, Benjamin M.; Madadi, Nikhil R.; Penthala, Narsimha R.; Crooks, Peter A.; Prather, Paul L.

2014-01-01

Our laboratory recently reported that a group of novel indole quinuclidine analogues bind with nanomolar affinity to cannabinoid type-1 and type-2 receptors. This study characterized the intrinsic activity of these compounds by determining whether they exhibit agonist, antagonist, or inverse agonist activity at cannabinoid type-1 and/or type-2 receptors. Cannabinoid receptors activate Gi/Go-proteins that then proceed to inhibit activity of the downstream intracellular effector adenylyl cyclase. Therefore, intrinsic activity was quantified by measuring the ability of compounds to modulate levels of intracellular cAMP in intact cells. Concerning cannabinoid type-1 receptors endogenously expressed in Neuro2A cells, a single analogue exhibited agonist activity, while eight acted as neutral antagonists and two possessed inverse agonist activity. For cannabinoid type-2 receptors stably expressed in CHO cells, all but two analogues acted as agonists; these two exceptions exhibited inverse agonist activity. Confirming specificity at cannabinoid type-1 receptors, modulation of adenylyl cyclase activity by all proposed agonists and inverse agonists was blocked by co-incubation with the neutral cannabinoid type-1 antagonist O-2050. All proposed cannabinoid type-1 receptor antagonists attenuated adenylyl cyclase modulation by cannabinoid agonist CP-55,940. Specificity at cannabinoid type-2 receptors was confirmed by failure of all compounds to modulate adenylyl cyclase activity in CHO cells devoid of cannabinoid type-2 receptors. Further characterization of select analogues demonstrated concentration-dependent modulation of adenylyl cyclase activity with potencies similar to their respective affinities for cannabinoid receptors. Therefore, indole quinuclidines are a novel structural class of compounds exhibiting high affinity and a range of intrinsic activity at cannabinoid type-1 and type-2 receptors. PMID:24858620

Modulating the DNA affinity of Elk-1 with computationally selected mutations.

PubMed

Park, Sheldon; Boder, Eric T; Saven, Jeffery G

2005-04-22

In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main-chain root-mean-square deviations than their counterparts in SAP. In particular, a single residue, D69, may contribute to Elk's lower DNA affinity for P(c-fos) by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind P(c-fos) in a reporter assay, in which D69 of wild-type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.

Observation-based Estimate of Climate Sensitivity with a Scaling Climate Response Function

NASA Astrophysics Data System (ADS)

Hébert, Raphael; Lovejoy, Shaun

2016-04-01

To properly adress the anthropogenic impacts upon the earth system, an estimate of the climate sensitivity to radiative forcing is essential. Observation-based estimates of climate sensitivity are often limited by their ability to take into account the slower response of the climate system imparted mainly by the large thermal inertia of oceans, they are nevertheless essential to provide an alternative to estimates from global circulation models and increase our confidence in estimates of climate sensitivity by the multiplicity of approaches. It is straightforward to calculate the Effective Climate Sensitivity(EffCS) as the ratio of temperature change to the change in radiative forcing; the result is almost identical to the Transient Climate Response(TCR), but it underestimates the Equilibrium Climate Sensitivity(ECS). A study of global mean temperature is thus presented assuming a Scaling Climate Response Function to deterministic radiative forcing. This general form is justified as there exists a scaling symmetry respected by the dynamics, and boundary conditions, over a wide range of scales and it allows for long-range dependencies while retaining only 3 parameter which are estimated empirically. The range of memory is modulated by the scaling exponent H. We can calculate, analytically, a one-to-one relation between the scaling exponent H and the ratio of EffCS to TCR and EffCS to ECS. The scaling exponent of the power law is estimated by a regression of temperature as a function of forcing. We consider for the analysis 4 different datasets of historical global mean temperature and 100 scenario runs of the Coupled Model Intercomparison Project Phase 5 distributed among the 4 Representative Concentration Pathways(RCP) scenarios. We find that the error function for the estimate on historical temperature is very wide and thus, many scaling exponent can be used without meaningful changes in the fit residuals of historical temperatures; their response in the year 2100 on the other hand, is very broad, especially for a low-emission scenario such as RCP 2.6. CMIP5 scenario runs thus allow for a narrower estimate of H which can then be used to estimate the ECS and TCR from the EffCS estimated from the historical data.

The translocator protein gene is associated with symptom severity and cerebral pain processing in fibromyalgia.

PubMed

Kosek, Eva; Martinsen, Sofia; Gerdle, Björn; Mannerkorpi, Kaisa; Löfgren, Monika; Bileviciute-Ljungar, Indre; Fransson, Peter; Schalling, Martin; Ingvar, Martin; Ernberg, Malin; Jensen, Karin B

2016-11-01

The translocator protein (TSPO) is upregulated during glia activation in chronic pain patients. TSPO constitutes the rate-limiting step in neurosteroid synthesis, thus modulating synaptic transmission. Related serotonergic mechanisms influence if pro- or anti-nociceptive neurosteroids are produced. This study investigated the effects of a functional genetic polymorphism regulating the binding affinity to the TSPO, thus affecting symptom severity and cerebral pain processing in fibromyalgia patients. Gene-to-gene interactions with a functional polymorphism of the serotonin transporter gene were assessed. Fibromyalgia patients (n=126) were genotyped regarding the polymorphisms of the TSPO (rs6971) and the serotonin transporter (5-HTTLPR/rs25531). Functional magnetic resonance imaging (n=24) was used to study brain activation during individually calibrated pressure pain. Compared to mixed/low TSPO affinity binders, the high TSPO affinity binders rated more severe pain (p=0.016) and fibromyalgia symptoms (p=0.02). A significant interaction was found between the TSPO and the serotonin transporter polymorphisms regarding pain severity (p<0.0001). Functional connectivity analyses revealed that the TSPO high affinity binding group had more pronounced pain-evoked functional connectivity in the right frontoparietal network, between the dorsolateral prefrontal area and the parietal cortex. In conclusion, fibromyalgia patients with the TSPO high affinity binding genotype reported a higher pain intensity and more severe fibromyalgia symptoms compared to mixed/low affinity binders, and this was modulated by interaction with the serotonin transporter gene. To our knowledge this is the first evidence of functional genetic polymorphisms affecting pain severity in FM and our findings are in line with proposed glia-related mechanisms. Furthermore, the functional magnetic resonance findings indicated an effect of translocator protein on the affective-motivational components of pain perception. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

PubMed

Xu, Wenxuan; Liu, Yajuan; Ye, Yanxin; Liu, Meng; Han, Laichuang; Song, Andong; Liu, Liangwei

2016-10-01

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs.

PubMed

Hawse, William F; Boggess, William C; Morel, Penelope A

2017-07-15

The Akt/mTOR pathway is a key driver of murine CD4 + T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3ζ and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions. Copyright © 2017 by The American Association of Immunologists, Inc.

Targeting of HPV-16+ Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed against E6.

PubMed

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

2015-10-01

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 whether TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV(+) tumor cells. 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. 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. 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. ©2015 American Association for Cancer Research.

Activation-induced Modification in the CD3 Complex of the γδ T Cell Receptor

PubMed Central

Hayes, Sandra M.; Laky, Karen; El-Khoury, Dalal; Kappes, Dietmar J.; Fowlkes, B.J.; Love, Paul E.

2002-01-01

The T cell antigen receptor complexes expressed on αβ and γδ T cells differ not only in their respective clonotypic heterodimers but also in the subunit composition of their CD3 complexes. The γδ T cell receptors (TCRs) expressed on ex vivo γδ T cells lack CD3δ, whereas αβ TCRs contain CD3δ. While this result correlates with the phenotype of CD3δ−/− mice, in which γδ T cell development is unaffected, it is inconsistent with the results of previous studies reporting that CD3δ is a component of the γδ TCR. Since earlier studies examined the subunit composition of γδ TCRs expressed on activated and expanded peripheral γδ T cells or γδ TCR+ intestinal intraepithelial lymphocytes, we hypothesized that activation and expansion may lead to changes in the CD3 subunit composition of the γδ TCR. Here, we report that activation and expansion do in fact result in the inclusion of a protein, comparable in mass and mobility to CD3δ, in the γδ TCR. Further analyses revealed that this protein is not CD3δ, but instead is a differentially glycosylated form of CD3γ. These results provide further evidence for a major difference in the subunit composition of αβ- and γδ TCR complexes and raise the possibility that modification of CD3γ may have important functional consequences in activated γδ T cells. PMID:12438426

Structural Basis for Eukaryotic Transcription-Coupled DNA Repair Initiation

PubMed Central

Xu, Jun; Lahiri, Indrajit; Wang, Wei; Wier, Adam; Cianfrocco, Michael A.; Chong, Jenny; Hare, Alissa A.; Dervan, Peter B.; DiMaio, Frank; Leschziner, Andres E.; Wang, Dong

2017-01-01

Eukaryotic transcription-coupled repair (TCR), or transcription-coupled nucleotide excision repair (TC-NER), is an important and well-conserved sub-pathway of nucleotide excision repair (NER) that preferentially removes DNA lesions from the template strand blocking RNA polymerase II (Pol II) translocation1,2. Cockayne syndrome group B protein in humans (CSB, or ERCC6), or its yeast orthologs (Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe), is among the first proteins to be recruited to the lesion-arrested Pol II during initiation of eukaryotic TCR1,3–10. Mutations in CSB are associated with Cockayne syndrome, an autosomal-recessive neurologic disorder characterized by progeriod features, growth failure, and photosensitivity1. The molecular mechanism of eukaryotic TCR initiation remains elusive, with several long-standing questions unanswered: How do cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II? How does CSB interact with the arrested Pol II complex? What is the role of CSB in TCR initiation? The lack of structures of CSB or the Pol II-CSB complex have hindered our ability to answer those questions. Here we report the first structure of S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy (cryo-EM). The structure reveals that Rad26 binds to the DNA upstream of Pol II where it dramatically alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes forward movement of Pol II and elucidate key roles for Rad26/CSB in both TCR and transcription elongation. PMID:29168508

Design and Synthesis of Non-Peptide Mimetics Mapping the Immunodominant Myelin Basic Protein (MBP83–96) Epitope to Function as T-Cell Receptor Antagonists

PubMed Central

Yannakakis, Mary-Patricia; Simal, Carmen; Tzoupis, Haralambos; Rodi, Maria; Dargahi, Narges; Prakash, Monica; Mouzaki, Athanasia; Platts, James A.; Apostolopoulos, Vasso; Tselios, Theodore V.

2017-01-01

Encephalitogenic T cells are heavily implicated in the pathogenesis of multiple sclerosis (MS), an autoimmune demyelinating disease of the central nervous system. Their stimulation is triggered by the formation of a trimolecular complex between the human leukocyte antigen (HLA), an immunodominant myelin basic protein (MBP) epitope, and the T cell receptor (TCR). We detail herein our studies directed towards the rational design and synthesis of non-peptide mimetic molecules, based on the immunodominant MBP83–96 epitope that is recognized by the TCR in complex with HLA. We focused our attention on the inhibition of the trimolecular complex formation and consequently the inhibition of proliferation of activated T cells. A structure-based pharmacophore model was generated, in view of the interactions between the TCR and the HLA-MBP83–96 complex. As a result, new candidate molecules were designed based on lead compounds obtained through the ZINC database. Moreover, semi-empirical and density functional theory methods were applied for the prediction of the binding energy between the proposed non-peptide mimetics and the TCR. We synthesized six molecules that were further evaluated in vitro as TCR antagonists. Analogues 15 and 16 were able to inhibit to some extent the stimulation of T cells by the immunodominant MBP83–99 peptide from immunized mice. Inhibition was followed to a lesser degree by analogues 17 and 18 and then by analogue 19. These studies show that lead compounds 15 and 16 may be used for immunotherapy against MS. PMID:28594344

Design and Synthesis of Non-Peptide Mimetics Mapping the Immunodominant Myelin Basic Protein (MBP83-96) Epitope to Function as T-Cell Receptor Antagonists.

PubMed

Yannakakis, Mary-Patricia; Simal, Carmen; Tzoupis, Haralambos; Rodi, Maria; Dargahi, Narges; Prakash, Monica; Mouzaki, Athanasia; Platts, James A; Apostolopoulos, Vasso; Tselios, Theodore V

2017-06-08

Encephalitogenic T cells are heavily implicated in the pathogenesis of multiple sclerosis (MS), an autoimmune demyelinating disease of the central nervous system. Their stimulation is triggered by the formation of a trimolecular complex between the human leukocyte antigen (HLA), an immunodominant myelin basic protein (MBP) epitope, and the T cell receptor (TCR). We detail herein our studies directed towards the rational design and synthesis of non-peptide mimetic molecules, based on the immunodominant MBP 83-96 epitope that is recognized by the TCR in complex with HLA. We focused our attention on the inhibition of the trimolecular complex formation and consequently the inhibition of proliferation of activated T cells. A structure-based pharmacophore model was generated, in view of the interactions between the TCR and the HLA-MBP 83-96 complex. As a result, new candidate molecules were designed based on lead compounds obtained through the ZINC database. Moreover, semi-empirical and density functional theory methods were applied for the prediction of the binding energy between the proposed non-peptide mimetics and the TCR. We synthesized six molecules that were further evaluated in vitro as TCR antagonists. Analogues 15 and 16 were able to inhibit to some extent the stimulation of T cells by the immunodominant MBP 83-99 peptide from immunized mice. Inhibition was followed to a lesser degree by analogues 17 and 18 and then by analogue 19 . These studies show that lead compounds 15 and 16 may be used for immunotherapy against MS.

Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces

PubMed Central

Briesewitz, Roger; Ray, Gregory T.; Wandless, Thomas J.; Crabtree, Gerald R.

1999-01-01

A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein–protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds. PMID:10051576

The human Vδ2+ T-cell compartment comprises distinct innate-like Vγ9+ and adaptive Vγ9- subsets.

PubMed

Davey, Martin S; Willcox, Carrie R; Hunter, Stuart; Kasatskaya, Sofya A; Remmerswaal, Ester B M; Salim, Mahboob; Mohammed, Fiyaz; Bemelman, Frederike J; Chudakov, Dmitriy M; Oo, Ye H; Willcox, Benjamin E

2018-05-02

Vδ2 + T cells form the predominant human γδ T-cell population in peripheral blood and mediate T-cell receptor (TCR)-dependent anti-microbial and anti-tumour immunity. Here we show that the Vδ2 + compartment comprises both innate-like and adaptive subsets. Vγ9 + Vδ2 + T cells display semi-invariant TCR repertoires, featuring public Vγ9 TCR sequences equivalent in cord and adult blood. By contrast, we also identify a separate, Vγ9 - Vδ2 + T-cell subset that typically has a CD27 hi CCR7 + CD28 + IL-7Rα + naive-like phenotype and a diverse TCR repertoire, however in response to viral infection, undergoes clonal expansion and differentiation to a CD27 lo CD45RA + CX 3 CR1 + granzymeA/B + effector phenotype. Consistent with a function in solid tissue immunosurveillance, we detect human intrahepatic Vγ9 - Vδ2 + T cells featuring dominant clonal expansions and an effector phenotype. These findings redefine human γδ T-cell subsets by delineating the Vδ2 + T-cell compartment into innate-like (Vγ9 + ) and adaptive (Vγ9 - ) subsets, which have distinct functions in microbial immunosurveillance.

TCRmodel: high resolution modeling of T cell receptors from sequence.

PubMed

Gowthaman, Ragul; Pierce, Brian G

2018-05-22

T cell receptors (TCRs), along with antibodies, are responsible for specific antigen recognition in the adaptive immune response, and millions of unique TCRs are estimated to be present in each individual. Understanding the structural basis of TCR targeting has implications in vaccine design, autoimmunity, as well as T cell therapies for cancer. Given advances in deep sequencing leading to immune repertoire-level TCR sequence data, fast and accurate modeling methods are needed to elucidate shared and unique 3D structural features of these molecules which lead to their antigen targeting and cross-reactivity. We developed a new algorithm in the program Rosetta to model TCRs from sequence, and implemented this functionality in a web server, TCRmodel. This web server provides an easy to use interface, and models are generated quickly that users can investigate in the browser and download. Benchmarking of this method using a set of nonredundant recently released TCR crystal structures shows that models are accurate and compare favorably to models from another available modeling method. This server enables the community to obtain insights into TCRs of interest, and can be combined with methods to model and design TCR recognition of antigens. The TCRmodel server is available at: http://tcrmodel.ibbr.umd.edu/.

T-cell receptor repertoire of human peripheral CD161hiTRAV1-2+ MAIT cells revealed by next generation sequencing and single cell analysis.

PubMed

Held, Kathrin; Beltrán, Eduardo; Moser, Markus; Hohlfeld, Reinhard; Dornmair, Klaus

2015-09-01

Mucosal-associated invariant T (MAIT) cells are a T-cell subset that expresses a conserved TRAV1-2 (Vα7.2) T-cell receptor (TCR) chain and the surface marker CD161. They are involved in the defence against microbes as they recognise small organic molecules of microbial origin that are presented by the non-classical MHC molecule 1 (MR1). MAIT cells express a semi-restricted TCR α chain with TRAV1-2 preferentially linked to TRAJ33, TRAJ12, or TRAJ20 which pairs with a limited set of β chains. To investigate the TCR repertoire of human CD161(hi)TRAV1-2(+) T cells in depth we analysed the α and β chains of this T-cell subset by next generation sequencing. Concomitantly we analysed 132 paired α and β chains from single cells to assess the αβ pairing preferences. We found that the CD161(hi)TRAV1-2(+) TCR repertoire in addition to the typical MAIT TCRs further contains polyclonal elements reminiscent of classical αβ T cells. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

The quantum chemical causality of pMHC-TCR biological avidity: Peptide atomic coordination data and the electronic state of agonist N termini.

PubMed

Antipas, Georgios S E; Germenis, Anastasios E

2015-06-01

The quantum state of functional avidity of the synapse formed between a peptide-Major Histocompatibility Complex (pMHC) and a T cell receptor (TCR) is a subject not previously touched upon. Here we present atomic pair correlation meta-data based on crystalized tertiary structures of the Tax (HTLV-1) peptide along with three artificially altered variants, all of which were presented by the (Class I) HLA-A201 protein in complexation with the human (CD8(+)) A6TCR. The meta-data reveal the existence of a direct relationship between pMHC-TCR functional avidity (agonist/antagonist) and peptide pair distribution function (PDF). In this context, antagonist peptides are consistently under-coordinated in respect to Tax. Moreover, Density Functional Theory (DFT) datasets in the BLYP/TZ2P level of theory resulting from relaxation of the H species on peptide tertiary structures reveal that the coordination requirement of agonist peptides is also expressed as a physical observable of the protonation state of their N termini: agonistic peptides are always found to retain a stable ammonium (NH3 (+)) terminal group while antagonist peptides are not.

Protein kinase D1 phosphorylates HDAC7 and induces its nuclear export after T-cell receptor activation.

PubMed

Parra, Maribel; Kasler, Herbert; McKinsey, Timothy A; Olson, Eric N; Verdin, Eric

2005-04-08

HDAC7, a class II histone deacetylase that is highly expressed in thymocytes, inhibits both transcription of the orphan steroid nuclear receptor Nur77 and induction of apoptosis in response to activation of the T-cell receptor (TCR). Here, we report that HDAC7 is exported to the cytoplasm by a calcium-independent signaling pathway after TCR activation. Protein kinase D1 (PKD1) was activated after TCR engagement, interacted with HDAC7, and phosphorylated three serines (Ser155, Ser318, and Ser448) at its N terminus, leading to its export from the nucleus. Mutation of Ser155, Ser318, and Ser448 blocked the nucleocytoplasmic shuttling of HDAC7 in response to TCR activation, as did overexpression of a kinase-inactive form of PKD1. Consistent with the regulatory role of HDAC7 in Nur77 expression, PKD1 activation led to the transcriptional activation of Nur77 via myocyte enhancer factor 2-binding sites in its promoter. In a mouse model of negative selection, PKD1 was activated during thymocyte activation. These observations indicate that PKD1 regulates the expression of Nur77 during thymocyte activation at least in part by phosphorylating HDAC7.

Research Techniques Made Simple: High-Throughput Sequencing of the T-Cell Receptor.

PubMed

Matos, Tiago R; de Rie, Menno A; Teunissen, Marcel B M

2017-06-01

High-throughput sequencing (HTS) of the T-cell receptor (TCR) is a rapidly advancing technique that allows sensitive and accurate identification and quantification of every distinct T-cell clone present within any biological sample. The relative frequency of each individual clone within the full T-cell repertoire can also be studied. HTS is essential to expand our knowledge on the diversity of the TCR repertoire in homeostasis or under pathologic conditions, as well as to understand the kinetics of antigen-specific T-cell responses that lead to protective immunity (i.e., vaccination) or immune-related disorders (i.e., autoimmunity and cancer). HTS can be tailored for personalized medicine, having the potential to monitor individual responses to therapeutic interventions and show prognostic and diagnostic biomarkers. In this article, we briefly review the methodology, advances, and limitations of HTS of the TCR and describe emerging applications of this technique in the field of investigative dermatology. We highlight studying the pathogenesis of T cells in allergic dermatitis and the application of HTS of the TCR in diagnosing, detecting recurrence early, and monitoring responses to therapy in cutaneous T-cell lymphoma. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Coevolution of T-cell receptors with MHC and non-MHC ligands

PubMed Central

Castro, Caitlin C.; Luoma, Adrienne M.; Adams, Erin J.

2015-01-01

Summary The structure and amino acid diversity of the T-cell receptor (TCR), similar in nature to that of Fab portions of antibodies, would suggest these proteins have a nearly infinite capacity to recognize antigen. Yet all currently defined native T cells expressing an α and β chain in their TCR can only sense antigen when presented in the context of a major histocompatibility complex (MHC) molecule. This MHC molecule can be one of many that exist in vertebrates, presenting small peptide fragments, lipid molecules, or small molecule metabolites. Here we review the pattern of TCR recognition of MHC molecules throughout a broad sampling of species and T-cell lineages and also touch upon T cells that do not appear to require MHC presentation for their surveillance function. We review the diversity of MHC molecules and information on the corresponding T-cell lineages identified in divergent species. We also discuss TCRs with structural domains unlike that of conventional TCRs of mouse and human. By presenting this broad view of TCR sequence, structure, domain organization, and function, we seek to explore how this receptor has evolved across time and been selected for alternative antigen-recognition capabilities in divergent lineages. PMID:26284470

Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor

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

López-Sagaseta, Jacinto; Sibener, Leah V.; Kung, Jennifer E.

2014-10-02

Invariant Natural Killer T (iNKT) cells use highly restricted {alpha}{beta} T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted {alpha}1 helix resulting in an open A pocket. Binding of the iNKT TCR requires a 7-{angstrom} displacement of the LPC headgroup but stabilizes the CD1d-LPC complex in a closed conformation. The iNKT TCR CDR loop footprint onmore » CD1d-LPC is anchored by the conserved positioning of the CDR3{alpha} loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3{beta} and J{beta} segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells.« less

Immunotherapeutic strategies targeting Natural killer T cell responses in cancer

PubMed Central

Shissler, Susannah C.; Bollino, Dominique R.; Tiper, Irina V.; Bates, Joshua; Derakhshandeh, Roshanak; Webb, Tonya J.

2017-01-01

Natural killer T (NKT) cells are a unique subset of lymphocytes that bridge the innate and adaptive immune system. NKT cells possess a classic αβ T-cell receptor (TCR) that is able to recognize self and foreign glycolipid antigens presented by the nonclassical class I major histocompatibility complex (MHC) molecule, CD1d. Type I NKT cells (referred to as invariant NKT cells) express a semi-invariant Vα14Jα18 TCR in mice and Vα24Jα18 TCR in humans. Type II NKT cells are CD1d-restricted T cells that express a more diverse set of TCR α chains. The two types of NKT cells often exert opposing effects especially in tumor immunity, where Type II cells generally suppress tumor immunity while Type I NKT cells can enhance antitumor immune responses. In this review, we focus on the role of NKT cells in cancer. We discuss their effector and suppressive functions, as well as describe preclinical and clinical studies utilizing therapeutic strategies focused on harnessing their potent anti-tumor effector functions, and conclude with a discussion on potential next steps for the utilization of NKT cell targeted therapies for the treatment of cancer. PMID:27393665

A threshold level of NFATc1 activity facilitates thymocyte differentiation and opposes notch-driven leukaemia development

PubMed Central

Klein-Hessling, Stefan; Rudolf, Ronald; Muhammad, Khalid; Knobeloch, Klaus-Peter; Maqbool, Muhammad Ahmad; Cauchy, Pierre; Andrau, Jean-Christophe; Avots, Andris; Talora, Claudio; Ellenrieder, Volker; Screpanti, Isabella; Serfling, Edgar; Patra, Amiya Kumar

2016-01-01

NFATc1 plays a critical role in double-negative thymocyte survival and differentiation. However, the signals that regulate Nfatc1 expression are incompletely characterized. Here we show a developmental stage-specific differential expression pattern of Nfatc1 driven by the distal (P1) or proximal (P2) promoters in thymocytes. Whereas, preTCR-negative thymocytes exhibit only P2 promoter-derived Nfatc1β expression, preTCR-positive thymocytes express both Nfatc1β and P1 promoter-derived Nfatc1α transcripts. Inducing NFATc1α activity from P1 promoter in preTCR-negative thymocytes, in addition to the NFATc1β from P2 promoter impairs thymocyte development resulting in severe T-cell lymphopenia. In addition, we show that NFATc1 activity suppresses the B-lineage potential of immature thymocytes, and consolidates their differentiation to T cells. Further, in the pTCR-positive DN3 cells, a threshold level of NFATc1 activity is vital in facilitating T-cell differentiation and to prevent Notch3-induced T-acute lymphoblastic leukaemia. Altogether, our results show NFATc1 activity is crucial in determining the T-cell fate of thymocytes. PMID:27312418

Targeted suppression of autoreactive CD8+ T-cell activation using blocking anti-CD8 antibodies.

PubMed

Clement, Mathew; Pearson, James A; Gras, Stephanie; van den Berg, Hugo A; Lissina, Anya; Llewellyn-Lacey, Sian; Willis, Mark D; Dockree, Tamsin; McLaren, James E; Ekeruche-Makinde, Julia; Gostick, Emma; Robertson, Neil P; Rossjohn, Jamie; Burrows, Scott R; Price, David A; Wong, F Susan; Peakman, Mark; Skowera, Ania; Wooldridge, Linda

2016-10-17

CD8 + T-cells play a role in the pathogenesis of autoimmune diseases such as multiple sclerosis and type 1 diabetes. However, drugs that target the entire CD8 + T-cell population are not desirable because the associated lack of specificity can lead to unwanted consequences, most notably an enhanced susceptibility to infection. Here, we show that autoreactive CD8 + T-cells are highly dependent on CD8 for ligand-induced activation via the T-cell receptor (TCR). In contrast, pathogen-specific CD8 + T-cells are relatively CD8-independent. These generic differences relate to an intrinsic dichotomy that segregates self-derived and exogenous antigen-specific TCRs according to the monomeric interaction affinity with cognate peptide-major histocompatibility complex class I (pMHCI). As a consequence, "blocking" anti-CD8 antibodies can suppress autoreactive CD8 + T-cell activation in a relatively selective manner. These findings provide a rational basis for the development and in vivo assessment of novel therapeutic strategies that preferentially target disease-relevant autoimmune responses within the CD8 + T-cell compartment.

SRC-like adaptor protein regulates B cell development and function.

PubMed

Dragone, Leonard L; Myers, Margaret D; White, Carmen; Sosinowski, Tomasz; Weiss, Arthur

2006-01-01

The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.

CD1d expression on chronic lymphocytic leukemia B cells affects disease progression and induces T cell skewing in CD8 positive and CD4CD8 double negative T cells.

PubMed

Zaborsky, Nadja; Gassner, Franz Josef; Asslaber, Daniela; Reinthaler, Petra; Denk, Ursula; Flenady, Sabine; Hofbauer, Josefina Piñón; Danner, Barbara; Rebhandl, Stefan; Harrer, Andrea; Geisberger, Roland; Greil, Richard; Egle, Alexander

2016-08-02

Chronic lymphocytic leukemia develops within a complex network driven by genetic mutations and microenvironmental interactions. Among the latter a complex interplay with the immune system is established by the clone. Next to a proposed recruitment of support from T and myeloid cells, potential anti-CLL immune reactions need to be subverted. By using TCL1 mice as a CLL model, we show that TCR-Vβ7+ NK1.1+ T cells are overrepresented in this disease model and constitute a main subset of peripheral CD3+ cells with biased TCR usage, showing that these cells account for a major part for T cell skewing in TCL1 mice. Moreover, we show that overrepresentation is dependent on CD1d expression in TCL1 mice, implicating that these cells belong to a NKT-like cell fraction which are restricted to antigen presented by the MHC-like surface marker CD1d. Accordingly, we observed a high fraction of CD161+ cells within overrepresented T cells in CLL patients and we found downregulation of CD1d on the surface of CLL cells, both in TCL1 mice and patients. Finally, we show that in TCL1 mice, CD1d deficiency resulted in shortened overall survival. Our results point to an interaction between CLL and CD161+ T cells that may represent a novel therapeutic target for immune modulation.

The Functions of Type I and Type II Natural Killer T (NKT) Cells in Inflammatory Bowel Diseases

PubMed Central

Liao, Chia-Min; Zimmer, Michael I.; Wang, Chyung-Ru

2013-01-01

CD1d-restricted natural killer T (NKT) cells are a distinct subset of T cells that rapidly produce an array of cytokines upon activation and play a critical role in regulating various immune responses. NKT cells are classified into two groups based on differences in T cell receptor (TCR) usage. Type I NKT cells have an invariant TCRα-chain and are readily detectable by α-galactosylceramide (α-GalCer)-loaded CD1d tetramers. Type II NKT cells have a more diverse TCR repertoire and cannot be directly identified. Both types of NKT cells as well as multiple CD1d-expressing cell types are present in the intestine and their interactions are likely to be modulated by pathogenic and commensal microbes, which in turn contribute to the intestinal immune responses in health and disease. Indeed, in several animal models of inflammatory bowel disease (IBD), Type I NKT cells have been shown to make both protective and pathogenic contributions to disease. In contrast, in human patients suffering from ulcerative colitis (UC), and a mouse model in which both CD1d expression and the frequency of Type II NKT cells are increased, Type II NKT cells appear to promote intestinal inflammation. In this review, we summarize present knowledge on the antigen recognition, activation and function of NKT cells with a particular focus on their role in IBD, and discuss factors that may influence the functional outcome of NKT cell responses in intestinal inflammation. PMID:23518808

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

Existence of negative differential thermal conductance in one-dimensional diffusive thermal transport

NASA Astrophysics Data System (ADS)

Hu, Jiuning; Chen, Yong P.

2013-06-01

We show that in a finite one-dimensional (1D) system with diffusive thermal transport described by the Fourier's law, negative differential thermal conductance (NDTC) cannot occur when the temperature at one end is fixed and there are no abrupt junctions. We demonstrate that NDTC in this case requires the presence of junction(s) with temperature-dependent thermal contact resistance (TCR). We derive a necessary and sufficient condition for the existence of NDTC in terms of the properties of the TCR for systems with a single junction. We show that under certain circumstances we even could have infinite (negative or positive) differential thermal conductance in the presence of the TCR. Our predictions provide theoretical basis for constructing NDTC-based devices, such as thermal amplifiers, oscillators, and logic devices.

DAMGO binding to mouse brain membranes: influence of salts, guanine nucleotides, substance P, and substance P fragments.

PubMed

Krumins, S A; Kim, D C; Igwe, O J; Larson, A A

1993-01-01

Substance P (SP) appears to mediate many processes of the central nervous system, including pain. This report deals with modulation of opioid binding in the mouse brain by SP and SP fragments, as well as by salts and guanine nucleotides. Binding studies of the selective mu opioid receptor agonist [D-Ala2, MePhe4,Gly(ol)5]enkephalin (DAMGO) to mouse brain membrane preparations demonstrated that guanine nucleotide modulation of DAMGO binding affinity was modified by SP. However, SP had little or no influence on inhibition of DAMGO binding induced by salts, such as MgCl2, CaCl2, or NaCl. By replacing GTP with GppNHp, SP (0.1 nM) produced multiple affinity forms of the DAMGO receptor, while at a higher concentration (10 nM), SP lost its influence on DAMGO binding. Furthermore, 0.1 nM SP changed DAMGO binding parameters in a medium containing NaCl, CaCl2, and GppNHp such that the high- and low-affinity conformations of the receptor converted to a single site following the addition of SP to the incubation medium. While the C-terminal SP fragment SP(5-11) was without effect, the N-terminal SP fragments SP(1-9) and SP(1-7) appeared to imitate SP in modifying GppNHp-modulated DAMGO binding. These results suggest that SP functions as a modulator of opioid binding at the mu receptor and it appears that the N-terminus of SP plays a role in the modulatory process.

Modernization of the Transonic Axial Compressor Test Rig

DTIC Science & Technology

2017-12-01

13. ABSTRACT (maximum 200 words) This work presents the design and simulation process of modernizing the Naval Postgraduate School’s transonic...fabricate the materials. Stiffness tests and modal analysis were conducted via Finite Element Analysis (FEA) software. This analysis was used to design ...work presents the design and simulation process of modernizing the Naval Postgraduate School’s transonic compressor test rig (TCR). The TCR, which

Control of plant phosphate homeostasis by inositol pyrophosphates and the SPX domain.

PubMed

Jung, Ji-Yul; Ried, Martina K; Hothorn, Michael; Poirier, Yves

2018-02-01

Proteins containing a SPX domain are involved in phosphate (Pi) homeostasis, including Pi transport and adaptation to Pi deficiency. The SPX domain harbors a basic surface binding Pi at low affinity and inositol pyrophosphates (PP-InsPs) at high affinity. Genetic and biochemical studies revealed that PP-InsPs serve as ligands for the SPX domain. Residues in the PHO1 SPX domain involved in PP-InsPs binding are critical for its Pi export activity, and the interaction between SPX proteins and the PHR1 transcription factor, which results in PHR1 inactivation, is promoted by PP-InsPs. Changes in PP-InsPs levels in response to Pi deficiency may thus contribute to the adaptation of plants to stress via the modulation of the activity of SPX-containing proteins and their interactors. Modulating PP-InsP levels or the affinity/specificity of the SPX domain for PP-InsP could potentially be used to engineer crops to maintain high yield under reduced Pi fertilizer input. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genes del receptor variable beta de células T en células circulantes de pacientes con lupus eritematoso generalizado y sus familiares sanos.

PubMed

Jakez-Ocampo, Juan; Paulín-Vera, Carmen María; Rivadeneyra-Espinoza, Liliana; Gómez-Martín, Diana; Carrillo-Maravilla, Eduardo; Lima, Guadalupe; Vargas-Rojas, María Inés; Pérez-Romano, Beatriz; Calva-Cevenini, Gabriella; García-Carrasco, Mario; Ruiz-Argüelles, Alejandro; Llorente, Luis

Se investigó la proporción de la expresión génica del receptor variable beta de células T (Vβ TCR) en linfocitos periféricos CD3+ en pacientes con lupus eritematoso generalizado (LEG) familiar y no familiar. El repertorio de Vβ TCR se estudió en 14 familias que presentaban más de un miembro con LEG. El uso de Vβ TCR en pacientes con LEG (n = 27) se comparó con el de los miembros sanos de estas familias (n = 47), con 37 pacientes con LEG esporádico y con 15 controles sanos. La expresión del repertorio de Vβ TCR se estudió por citometría de flujo multiparamétrica utilizando un arreglo de 24 diferentes anticuerpos monoclonales específicos de genes familiares para Vβ TCR. Se encontró el mismo perfil de expresión en las comparaciones entre los casos de LEG esporádico y familiar, así como en los consanguíneos sanos de las familias multicasos, que incluía una expresión incrementada de Vβ 5.2, Vβ 11 y Vβ 16, y una menor expresión de Vβ 3, Vβ4, Vβ 7.1 y Vβ 7. De manera interesante, solo Vβ 17 se expresó de modo diferente entre casos familiares y esporádicos de LEG. Igualmente, la expresión incrementada de Vβ 9 fue el distintivo entre los casos de LEG familiar (casos y consanguíneos sanos) y los controles sanos. Estos resultados refuerzan la noción de que el perfil final del repertorio Vβ TCR observado en LEG familiar y no familiar parece surgir de la interacción de factores genéticos, ambientales e inmunorreguladores, además de que pueden explicar las alteraciones inmunitarias que se observan en los consanguíneos sanos de pacientes con LEG. Copyright: © 2018 SecretarÍa de Salud

Tracking global changes induced in the CD4 T-cell receptor repertoire by immunization with a complex antigen using short stretches of CDR3 protein sequence.

PubMed

Thomas, Niclas; Best, Katharine; Cinelli, Mattia; Reich-Zeliger, Shlomit; Gal, Hilah; Shifrut, Eric; Madi, Asaf; Friedman, Nir; Shawe-Taylor, John; Chain, Benny

2014-11-15

The clonal theory of adaptive immunity proposes that immunological responses are encoded by increases in the frequency of lymphocytes carrying antigen-specific receptors. In this study, we measure the frequency of different T-cell receptors (TcR) in CD4 + T cell populations of mice immunized with a complex antigen, killed Mycobacterium tuberculosis, using high throughput parallel sequencing of the TcRβ chain. Our initial hypothesis that immunization would induce repertoire convergence proved to be incorrect, and therefore an alternative approach was developed that allows accurate stratification of TcR repertoires and provides novel insights into the nature of CD4 + T-cell receptor recognition. To track the changes induced by immunization within this heterogeneous repertoire, the sequence data were classified by counting the frequency of different clusters of short (3 or 4) continuous stretches of amino acids within the antigen binding complementarity determining region 3 (CDR3) repertoire of different mice. Both unsupervised (hierarchical clustering) and supervised (support vector machine) analyses of these different distributions of sequence clusters differentiated between immunized and unimmunized mice with 100% efficiency. The CD4 + TcR repertoires of mice 5 and 14 days postimmunization were clearly different from that of unimmunized mice but were not distinguishable from each other. However, the repertoires of mice 60 days postimmunization were distinct both from naive mice and the day 5/14 animals. Our results reinforce the remarkable diversity of the TcR repertoire, resulting in many diverse private TcRs contributing to the T-cell response even in genetically identical mice responding to the same antigen. However, specific motifs defined by short stretches of amino acids within the CDR3 region may determine TcR specificity and define a new approach to TcR sequence classification. The analysis was implemented in R and Python, and source code can be found in Supplementary Data. b.chain@ucl.ac.uk Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

[Somatic mutagenesis at T-cell receptor locus in inhabitants of radiation polluted regions as a result of the Chernobyl disaster].

PubMed

Zamulaeva, I A; Smirnova, S G; Orlova, N V; Vereshchagina, O A; Chekin, S Iu; Smirnova, I A; Krikunova, L I; Parshin, V S; Ivanov, V K; Saenko, A S

2006-01-01

In the period of 2001-2004, frequency of cells bearing mutations at T-cell receptor (TCR) locus was assessed in 553 inhabitants of radiation polluted regions of the Russian Federation and 154 unexposed control persons. The inhabitants were divided into three groups according to age at the moment of the Chernobyl disaster and 137Cs pollution density: 1) in utero, 37-555 kBq/m2; 2) 0-14 years old, 20-555 kBq/m2; 3) 18 and more years old, highest 137Cs density (185 more than 555 kBq/m2). The most intense changes of the TCR-mutant cell frequency were observed in the group of persons exposed to ionizing radiation in utero. The mean frequency of the mutant cells was higher in the first group than in age-matched control group by about 1.5-fold: 4.0 x 10(-4) vs 2.7 x 10(-4) accordingly (p < 0.0001). Elevation in the mean TCR-mutant cell frequency was less expressed in group of inhabitants aged 0-14 years at the moment of irradiation start: 1.3-fold increase in comparison to age-matched control (3.8 x 10(-4) vs 2.9 x 10(-4), p = 0.0002). It was not found significant differences in mutant cell frequencies between control group and adults consisting in the third group (18 and more years old at the moment of the Chernobyl accident). The changes of the TCR-mutant cell frequency in persons exposed in pre- and postnatal periods differ not only quantitatively, but qualitatively. In the fist case all persons react to irradiation by increasing number of the TCR-mutant cells in some degree. In the second case - only a part of population. Proportion of reacting persons depends on age at the start of irradiation and, perhaps, on dose absorbed. The TCR-mutant frequency was significantly higher in persons with benign tumors of different localizations and nodules in thyroid gland than in persons without this pathology.

The air quality and health co-benefits of alternative post-2020 pathways for achieving peak carbon targets in Jiangsu, China

NASA Astrophysics Data System (ADS)

Liu, M.; Bi, J.; Huang, Y.; Kinney, P. L.

2016-12-01

Jiangsu, which has three national low-carbon pilot cities, is set to be a model province in China for achieving peak carbon targets before 2030. However, according to local planning of responding to climate change, carbon emissions are projected to keep going up before 2020 even the strictest measures are implemented. In other words, innovative measures must be in action after 2020. This work aimed at assessing the air quality and health co-benefits of alternative post-2020 measures to help remove barriers of policy implementation through tying it to local incentives for air quality improvement. To achieve the aim, we select 2010 as baseline year and develop Bussiness As Usual (BAU) and Traditional Carbon Reduction (TCR) scenarios before 2020. Under BAU, only existing climate and air pollution control policies are considered; under TCR, potential climate policies in local planning and existing air pollution control policies are considered. After 2020, integrated gasification combined cycle (IGCC) plant with carbon capture and storage (CCS) technology and large-scale substitution of renewable energy seem to be two promising pathways for achieving peak carbon targets. Therefore, two additional scenarios (TCR-IGCC and TCR-SRE) are set after 2020. Based on the projections of future energy balances and industrial productions, we estimate the pollutant emissions and simulate PM2.5 and ozone concentrations by 2017, 2020, 2030 and 2050 using CMAQ. Then using health impact assessment approach, the premature deaths are estimated and monetized. Results show that the carbon peak in Jiangsu will be achieved before 2030 only under TCR-IGCC and TCR-SRE scenarios. Under three policy scenarios, Jiangsu's carbon emission control targets would have substantial effects on primary air pollutant emissions far beyond those we estimate would be needed to meet the PM2.5 concentration targets in 2017. Compared with IGCC with CCS, large-scale substitutions of renewable energy bring comparable pollutant emission reductions but more health benefits because it reduces more emissions from traffic sources which are more harmful to health. However, large-scale substitution of renewable energy posed challenges on energy supply capacity, which need to be seriously considered in future policy decision.

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

PubMed Central

Koenig, Patrick; Lee, Chingwei V.; Walters, Benjamin T.; Janakiraman, Vasantharajan; Stinson, Jeremy; Patapoff, Thomas W.; Fuh, Germaine

2017-01-01

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. PMID:28057863

Modulation of Active Site Electronic Structure by the Protein Matrix to Control [NiFe] Hydrogenase Reactivity

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

Smith, Dayle MA; Raugei, Simone; Squier, Thomas C.

2014-09-30

Control of the reactivity of the nickel center of the [NiFe] hydrogenase and other metalloproteins commonly involves outer coordination sphere ligands that act to modify the geometry and physical properties of the active site metal centers. We carried out a combined set of classical molecular dynamics and quantum/classical mechanics calculations to provide quantitative estimates of how dynamic fluctuations of the active site within the protein matrix modulate the electronic structure at the catalytic center. Specifically we focused on the dynamics of the inner and outer coordination spheres of the cysteinate-bound Ni–Fe cluster in the catalytically active Ni-C state. There aremore » correlated movements of the cysteinate ligands and the surrounding hydrogen-bonding network, which modulate the electron affinity at the active site and the proton affinity of a terminal cysteinate. On the basis of these findings, we hypothesize a coupling between protein dynamics and electron and proton transfer reactions critical to dihydrogen production.« less

Modulation of active site electronic structure by the protein matrix to control [NiFe] hydrogenase reactivity.

PubMed

Smith, Dayle M A; Raugei, Simone; Squier, Thomas C

2014-11-21

Control of the reactivity of the nickel center of the [NiFe] hydrogenase and other metalloproteins commonly involves outer coordination sphere ligands that act to modify the geometry and physical properties of the active site metal centers. We carried out a combined set of classical molecular dynamics and quantum/classical mechanics calculations to provide quantitative estimates of how dynamic fluctuations of the active site within the protein matrix modulate the electronic structure at the catalytic center. Specifically we focused on the dynamics of the inner and outer coordination spheres of the cysteinate-bound Ni-Fe cluster in the catalytically active Ni-C state. There are correlated movements of the cysteinate ligands and the surrounding hydrogen-bonding network, which modulate the electron affinity at the active site and the proton affinity of a terminal cysteinate. On the basis of these findings, we hypothesize a coupling between protein dynamics and electron and proton transfer reactions critical to dihydrogen production.

Proven in vitro evolution of protease cathepsin E-inhibitors and -activators at pH 4.5 using a paired peptide method.

PubMed

Kitamura, Koichiro; Komatsu, Masayuki; Biyani, Madhu; Futakami, Masae; Kawakubo, Tomoyo; Yamamoto, Kenji; Nishigaki, Koichi

2012-12-01

Improving a particular function of molecules is often more difficult than identifying such molecules ab initio. Here, a method to acquire higher affinity and/or more functional peptides was developed as a progressive library selection method. The primary library selection products were utilized to build a secondary library composed of blocks of 4 amino acids, of which selection led to peptides with increased activity. These peptides were further converted to randomly generate paired peptides. Cathepsin E-inhibitors thus obtained exhibited the highest activities and affinities (pM order). This was also the case with cathepsin E-activating peptides, proving the methodological effectiveness. The primary, secondary, and tertiary library selections can be regarded as module-finding, module-shuffling, and module-pairing, respectively, which resembles the progression of the natural evolution of proteins. The mode of peptide binding to their target proteins is discussed in analogy to antibodies and epitopes of an antigen. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

PubMed Central

Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

2015-01-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-binding Module*

PubMed Central

Taylor, Courtney B.; Talib, M. Faiz; McCabe, Clare; Bu, Lintao; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2012-01-01

Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3–6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function. PMID:22147693

Impaired thymic selection in mice expressing altered levels of the SLP-76 adaptor protein.

PubMed

Ramsey, Kimberley; Luckashenak, Nancy; Koretzky, Gary A; Clements, James L

2008-02-01

Intracellular signaling initiated by ligation of the TCR influences cell fate at multiple points during the lifespan of a T cell. This is especially evident during thymic selection, where the nature of TCR-dependent signaling helps to establish a MHC-restricted, self-tolerant T cell repertoire. The Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) adaptor protein is a required intermediate in multiple signaling pathways triggered by TCR engagement, several of which have been implicated in dictating the outcome of thymic selection (e.g., intracellular calcium flux and activation of ERK family MAPKs). To determine if thymocyte maturation and selection at later stages of development are sensitive to perturbations in SLP-76 levels, we analyzed these crucial events using several transgenic (Tg) lines of mice expressing altered levels of SLP-76 in the thymus. In Tg mice expressing low levels of SLP-76 in preselection thymocytes, the CD4:CD8 ratio in the thymus and spleen was skewed in a manner consistent with impaired selection and/or maturation of CD4+ thymocytes. Low SLP-76 expression also correlated with reduced CD5 expression on immature thymocytes, consistent with reduced TCR signaling potential. In contrast, reconstitution of SLP-76 at higher levels resulted in normal thymic CD5 expression and CD4:CD8 ratios in the thymus and periphery. It is curious that thymic deletion of TCR-Tg (HY) thymocytes was markedly impaired in both lines of Tg-reconstituted SLP-76-/- mice. Studies using chimeric mice indicate that the defect in deletion of HY+ thymocytes is intrinsic to the developing thymocyte, suggesting that maintenance of sufficient SLP-76 expression from the endogenous locus is a key element in the selection process.

Interplay between membrane elasticity and active cytoskeleton forces regulates the aggregation dynamics of the immunological synapse

NASA Astrophysics Data System (ADS)

Dharan, Nadiv; Farago, Oded

Adhesion between a T cell and an antigen presenting cell is achieved by TCR-pMHC and LFA1-ICAM1 protein complexes. These segregate to form a special pattern, known as the immunological synapse (IS), consisting of a central quasi-circular domain of TCR-pMHC bonds surrounded by a peripheral domain of LFA1-ICAM1 complexes. Insights gained from imaging studies had led to the conclusion that the formation of the central adhesion domain in the IS is driven by active (ATP-driven) mechanisms. Recent studies, however, suggested that passive (thermodynamic) mechanisms may also play an important role in this process. Here, we present a simple physical model, taking into account the membrane-mediated thermodynamic attraction between the TCR-pMHC bonds and the effective forces that they experience due to ATP-driven actin retrograde flow and transport by dynein motor proteins. Monte Carlo simulations of the model exhibit a good spatio-temporal agreement with the experimentally observed pattern evolution of the TCR-pMHC microclusters. The agreement is lost when one of the aggregation mechanisms is "muted", which helps to identify the respective roles in the process. We conclude that actin retrograde flow drives the centripetal motion of TCR-pMHC bonds, while the membrane-mediated interactions facilitate microcluster formation and growth. In the absence of dynein motors, the system evolves into a ring-shaped pattern, which highlights the role of dynein motors in the formation of the final concentric pattern. The interplay between the passive and active mechanisms regulates the rate of the accumulation process, which in the absence of one them proceeds either too quickly or slowly.

The effect of inhibitory signals on the priming of drug-hapten-specific T-cells that express distinct Vβ receptors

PubMed Central

Gibson, Andrew; Faulkner, Lee; Lichtenfels, Maike; Ogese, Monday; Al-Attar, Zaid; Alfirevic, Ana; Esser, Philipp R.; Martin, Stefan F.; Pirmohamed, Munir; Park, B. Kevin; Naisbitt, Dean J.

2017-01-01

Drug hypersensitivity involves the activation of T-cells in an HLA allele-restricted manner. Since the majority of individuals who carry HLA risk alleles do not develop hypersensitivity, other parameters must control development of the drug-specific T-cell response. Thus, we have utilized a T-cell priming assay and nitroso sulfamethoxazole (SMX-NO) as a model antigen to investigate (1) the activation of specific T-cell receptor (TCR)Vβ subtypes, (2) the impact of PD-1, CTLA4 and TIM-3 co-inhibitory signalling on activation of naïve and memory T-cells and (3) the ability of Tregs to prevent responses. An expansion of the TCR repertoire was observed for nine different Vβ subtypes, while spectratyping revealed that SMX-NO-specific T-cell responses are controlled by public TCRs present in all individuals alongside private TCR repertoires specific to each individual. We proceeded to evaluate the extent to which the activation of these TCR Vβ-restricted antigen-specific T-cell responses is governed by regulatory signals. Blockade of PDL-1/CTLA4 signalling dampened activation of SMX-NO-specific naïve and memory T-cells, while blockade of TIM-3 produced no effect. PD-1, CTLA4, and TIM-3 displayed discrete expression profiles during drug-induced T-cell activation and expression of each receptor was enhanced on dividing T-cells. As these receptors are also expressed on Tregs, Treg-mediated suppression of SMX-NO-induced T-cell activation was investigated. Tregs significantly dampened the priming of T-cells. In conclusion, our findings demonstrate that distinct TCR Vβ subtypes, dysregulation of co-inhibitory signalling pathways and dysfunctional Tregs may influence predisposition to hypersensitivity. PMID:28687658

64Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

PubMed Central

Griessinger, Christoph M.; Maurer, Andreas; Kesenheimer, Christian; Kehlbach, Rainer; Reischl, Gerald; Ehrlichmann, Walter; Bukala, Daniel; Harant, Maren; Cay, Funda; Brück, Jürgen; Nordin, Renate; Kohlhofer, Ursula; Rammensee, Hans-Georg; Quintanilla-Martinez, Leticia; Schaller, Martin; Röcken, Martin; Pichler, Bernd J.; Kneilling, Manfred

2015-01-01

T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific 64Cu-monoclonal antibody (mAb)–TCR complex enables a stable labeling of T cells. The TCR–mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied 64Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover. PMID:25587131

The Calcineurin Pathway Inhibitor Tacrolimus Enhances the In Vitro Activity of Azoles against Mucorales via Apoptosis

PubMed Central

Shirazi, F.

2013-01-01

The calcineurin pathway regulates antifungal drug resistance and the virulence of several major human-pathogenic fungi, including the recalcitrant Mucorales. We hypothesized that the fungistatic triazoles posaconazole (PCZ) and itraconazole (ICZ) become fungicidal in the setting of the calcineurin inhibitor tacrolimus (TCR) and that such an effect is mediated through apoptosis. Fungicidal activity and apoptosis were studied using standard microbiological techniques and hyphal metabolic and vital dye reduction assays at 37°C in RPMI 1640. Apoptosis was characterized by detecting intracellular Ca2+, phosphatidylserine (PS) externalization, DNA fragmentation, plasma membrane integrity, chromatin condensation, reactive oxygen species (ROS) generation, caspase-like activity, ATP, and cytochrome c release. MICs for PCZ and ICZ alone were significantly higher (8 to 128 μg/ml) than those of PCZ or ICZ plus TCR (0.25 to 4 μg/ml) for Rhizopus oryzae, Cunninghamella bertholletiae, and Mucor circinelloides. Both PCZ and ICZ in combination with TCR became fungicidal, and their activity was mediated through increased apoptotic cell death of R. oryzae (10 to 50%), C. bertholletiae (5 to 50%), and M. circinelloides (5 to 55%) germlings, with morphological apoptotic changes characterized by externalization of PS, nuclear condensation, and DNA fragmentation. Moreover, activation of the caspase-like activity was correlated with cell death induced by TCR plus PCZ or ICZ. These changes correlated with elevated intracellular Ca2+ and ROS levels and disturbance of mitochondrial potential. We found that PCZ or ICZ in combination with TCR renders Mucorales sensitive to triazoles via apoptotic death. These observations could serve as a new paradigm for the development of new therapeutic strategies. PMID:23851337

The calcineurin pathway inhibitor tacrolimus enhances the in vitro activity of azoles against Mucorales via apoptosis.

PubMed

Shirazi, F; Kontoyiannis, D P

2013-09-01

The calcineurin pathway regulates antifungal drug resistance and the virulence of several major human-pathogenic fungi, including the recalcitrant Mucorales. We hypothesized that the fungistatic triazoles posaconazole (PCZ) and itraconazole (ICZ) become fungicidal in the setting of the calcineurin inhibitor tacrolimus (TCR) and that such an effect is mediated through apoptosis. Fungicidal activity and apoptosis were studied using standard microbiological techniques and hyphal metabolic and vital dye reduction assays at 37°C in RPMI 1640. Apoptosis was characterized by detecting intracellular Ca(2+), phosphatidylserine (PS) externalization, DNA fragmentation, plasma membrane integrity, chromatin condensation, reactive oxygen species (ROS) generation, caspase-like activity, ATP, and cytochrome c release. MICs for PCZ and ICZ alone were significantly higher (8 to 128 μg/ml) than those of PCZ or ICZ plus TCR (0.25 to 4 μg/ml) for Rhizopus oryzae, Cunninghamella bertholletiae, and Mucor circinelloides. Both PCZ and ICZ in combination with TCR became fungicidal, and their activity was mediated through increased apoptotic cell death of R. oryzae (10 to 50%), C. bertholletiae (5 to 50%), and M. circinelloides (5 to 55%) germlings, with morphological apoptotic changes characterized by externalization of PS, nuclear condensation, and DNA fragmentation. Moreover, activation of the caspase-like activity was correlated with cell death induced by TCR plus PCZ or ICZ. These changes correlated with elevated intracellular Ca(2+) and ROS levels and disturbance of mitochondrial potential. We found that PCZ or ICZ in combination with TCR renders Mucorales sensitive to triazoles via apoptotic death. These observations could serve as a new paradigm for the development of new therapeutic strategies.

The link between CD8⁺ T-cell antigen-sensitivity and HIV-suppressive capacity depends on HLA restriction, target epitope and viral isolate.

PubMed

Lissina, Anna; Fastenackels, Solène; Inglesias, Maria C; Ladell, Kristin; McLaren, James E; Briceño, Olivia; Gostick, Emma; Papagno, Laura; Autran, Brigitte; Sauce, Delphine; Price, David A; Saez-Cirion, Asier; Appay, Victor

2014-02-20

Although it is established that CD8 T-cell immunity is critical for the control of HIV replication in vivo, the key factors that determine antiviral efficacy are yet to be fully elucidated. Antigen-sensitivity and T-cell receptor (TCR) avidity have been identified as potential determinants of CD8⁺ T-cell efficacy. However, there is no general consensus in this regard because the relationship between these parameters and the control of HIV infection has been established primarily in the context of immunodominant CD8⁺ T-cell responses against the Gag₂₆₃₋₂₇₂ KK10 epitope restricted by human leukocyte antigen (HLA)-B27. To investigate the relationship between antigen-sensitivity, TCR avidity and HIV-suppressive capacity in vitro across epitope specificities and HLA class I restriction elements, we used a variety of techniques to study CD8⁺ T-cell clones specific for Nef₇₃₋₈₂ QK10 and Gag₂₀₋₂₉ RY10, both restricted by HLA-A3, alongside CD8⁺ T-cell clones specific for Gag₂₆₃₋₂₇₂ KK10. For each targeted epitope, the linked parameters of antigen-sensitivity and TCR avidity correlated directly with antiviral efficacy. However, marked differences in HIV-suppressive capacity were observed between epitope specificities, HLA class I restriction elements and viral isolates. Collectively, these data emphasize the central role of the TCR as a determinant of CD8⁺ T-cell efficacy and demonstrate that the complexities of antigen recognition across epitope and HLA class I boundaries can confound simple relationships between TCR engagement and HIV suppression.

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

PubMed

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

2016-07-15

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

Human common acute lymphoblastic leukemia-derived cell lines are competent to recombine their T-cell receptor delta/alpha regions along a hierarchically ordered pathway.

PubMed

Hansen-Hagge, T E; Yokota, S; Reuter, H J; Schwarz, K; Bartram, C R

1992-11-01

Rearrangements of the T-cell receptor (TCR) delta locus are observed in the majority of human B-cell precursor acute lymphoblastic leukemias (ALL) with a striking predominance of V delta 2(D)D delta 3 recombinations in common ALL (cALL) patients. Recently, we and others showed that almost 20% of cALL cases are characterized by further recombination of V delta 2(D)D delta 3 segments to J alpha elements, thereby deleting the TCR delta locus in analogy to the delta Rec/psi J alpha pathway in differentiating alpha/beta-positive T cells. We report here that two human cALL-derived cell lines, REH and Nalm-6, are competent to recombine the TCR delta/alpha locus under standard tissue culture conditions. Analysis of different REH subclones obtained by limiting dilution of the initial culture showed a biased recombination of V delta 2D delta 3 to distinct J alpha elements. During prolonged tissue culture, a subclone acquired growth advantage and displaced parental cells as well as other subclones. Frequently, the DJ junctions of REH subclones contained extended stretches of palindromic sequences derived from modified D delta 3 coding elements. The other cell line, Nalm-6, started the TCR delta/alpha recombination with an unusual signal joint of a cryptic recombinase signal sequence (RSS) upstream of D delta 3 to the 3' RSS of D delta 3. The RSS dimer was subsequently rearranged in all investigated subclones to an identical J alpha element. Both cell lines might become valuable tools to unravel the complex regulation of TCR delta/alpha recombination pathways in malignant and normal lymphopoiesis.

Analysis of the T-cell receptor repertoire of human T-cell leukemia virus type 1 (HTLV-1) Tax-specific CD8+ cytotoxic T lymphocytes from patients with HTLV-1-associated disease: evidence for oligoclonal expansion.

PubMed

Utz, U; Banks, D; Jacobson, S; Biddison, W E

1996-02-01

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurological disease characterized by marked degeneration of the spinal cord and the presence of antibodies against HTLV-1. Patients with HAM/TSP, but not asymptomatic carriers, show very high precursor frequencies of HTLV-1-specific CD8+ T cells in peripheral blood and cerebrospinal fluid, suggestive of a role of these T cells in the pathogenesis of the disease. In HLA-A2+ HAM/TSP patients, HTLV-1-specific T cells were demonstrated to be directed predominantly against one HTLV-1 epitope, namely, Tax11-19. In the present study, we analyzed HLA-A2-restricted HTLV-1 Tax11-19-specific cytotoxic T cells from three patients with HAM/TSP. An analysis of the T-cell receptor (TCR) repertoire of these cells revealed an absence of restricted variable (V) region usage. Different combinations of TCR V alpha and V beta genes were utilized between, but also within, the individual patients for the recognition of Tax11-19. Sequence analysis of the TCR showed evidence for an oligoclonal expansion of few founder T cells in each patient. Apparent structural motifs were identified for the CDR3 regions of the TCR beta chains. One T-cell clone could be detected within the same patient over a period of 3 years. We suggest that these in vivo clonally expanded T cells might play a role in the pathogenesis of HAM/TSP and provide information on HTLV-1-specific TCR which may elucidate the nature of the T cells that infiltrate the central nervous system in HAM/TSP patients.

Toward Fast and Accurate Binding Affinity Prediction with pmemdGTI: An Efficient Implementation of GPU-Accelerated Thermodynamic Integration.

PubMed

Lee, Tai-Sung; Hu, Yuan; Sherborne, Brad; Guo, Zhuyan; York, Darrin M

2017-07-11

We report the implementation of the thermodynamic integration method on the pmemd module of the AMBER 16 package on GPUs (pmemdGTI). The pmemdGTI code typically delivers over 2 orders of magnitude of speed-up relative to a single CPU core for the calculation of ligand-protein binding affinities with no statistically significant numerical differences and thus provides a powerful new tool for drug discovery applications.

Regulation of TCR Signaling to NF-kB

DTIC Science & Technology

2013-03-20

applications and benefits to basic research are discussed in detail in Chapter 4.      16   CHAPTER 2: Selective autophagy of the...To confirm the specificity of the chemical inhibitors of autophagy, we employed a genetic approach. Specifically, we in vitro differentiated Th2...MG132 treatment. These data provide strong genetic evidence that TCR-mediated degradation of endogenous Bcl10 is controlled by autophagy, with a

SPAK kinase is a substrate and target of PKCθ in T-cell receptor-induced AP-1 activation pathway

PubMed Central

Li, Yingqiu; Hu, Junru; Vita, Randi; Sun, Binggang; Tabata, Hiroki; Altman, Amnon

2004-01-01

Protein kinase C-θ (PKCθ) plays an important role in T-cell activation via stimulation of AP-1 and NF-κB. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKCθ-interacting kinase. SPAK interacted with PKCθ (but not with PKCα) via its 99 COOH-terminal residues. TCR/CD28 costimulation enhanced this association and stimulated the catalytic activity of SPAK. Recombinant SPAK was phosphorylated on Ser-311 in its kinase domain by PKCθ, but not by PKCα. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCθ-deficient T cells. Transfected SPAK synergized with constitutively active PKCθ to activate AP-1, but not NF-κB. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKCθ-interacting region of SPAK, and Ser-311 mutation greatly reduced these activities of SPAK. Conversely, a SPAK-specific RNAi or a dominant-negative SPAK mutant inhibited PKCθ- and TCR/CD28-induced AP-1, but not NF-κB, activation. These results define SPAK as a substrate and target of PKCθ in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-κB) activation. PMID:14988727

Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

PubMed

Davenport, A J; Cross, R S; Watson, K A; Liao, Y; Shi, W; Prince, H M; Beavis, P A; Trapani, J A; Kershaw, M H; Ritchie, D S; Darcy, P K; Neeson, P J; Jenkins, M R

2018-02-27

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. Copyright © 2018 the Author(s). Published by PNAS.

Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity

PubMed Central

Davenport, A. J.; Cross, R. S.; Watson, K. A.; Liao, Y.; Shi, W.; Prince, H. M.; Beavis, P. A.; Trapani, J. A.; Kershaw, M. H.; Ritchie, D. S.; Darcy, P. K.; Jenkins, M. R.

2018-01-01

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. PMID:29440406

Climate system properties determining the social cost of carbon

NASA Astrophysics Data System (ADS)

Otto, Alexander; Todd, Benjamin J.; Bowerman, Niel; Frame, David J.; Allen, Myles R.

2013-06-01

The choice of an appropriate scientific target to guide global mitigation efforts is complicated by uncertainties in the temperature response to greenhouse gas emissions. Much climate policy discourse has been based on the equilibrium global mean temperature increase following a concentration stabilization scenario. This is determined by the equilibrium climate sensitivity (ECS) which, in many studies, shows persistent, fat-tailed uncertainty. However, for many purposes, the equilibrium response is less relevant than the transient response. Here, we show that one prominent policy variable, the social cost of carbon (SCC), is generally better constrained by the transient climate response (TCR) than by the ECS. Simple analytic expressions show the SCC to be directly proportional to the TCR under idealized assumptions when the rate at which we discount future damage equals 2.8%. Using ensemble simulations of a simple climate model we find that knowing the true value of the TCR can reduce the relative uncertainty in the SCC substantially more, up to a factor of 3, than knowing the ECS under typical discounting assumptions. We conclude that the TCR, which is better constrained by observations, less subject to fat-tailed uncertainty and more directly related to the SCC, is generally preferable to the ECS as a single proxy for the climate response in SCC calculations.

Wide-scale quantitative phosphoproteomic analysis reveals that cold treatment of T cells closely mimics soluble antibody stimulation

PubMed Central

Ji, Qinqin; Salomon, Arthur R.

2015-01-01

The activation of T-lymphocytes through antigen-mediated T-cell receptor (TCR) clustering is vital in regulating the adaptive-immune response. Although T cell receptor signaling has been extensively studied, the fundamental mechanisms for signal initiation are not fully understood. Reduced temperature initiated some of the hallmarks of TCR signaling such as increased phosphorylation and activation on ERK and calcium release from the endoplasmic reticulum as well as coalesce T-cell membrane microdomains. The precise mechanism of TCR signaling initiation due to temperature change remains obscure. One critical question is whether signaling initiated by cold treatment of T cells differs from signaling initiated by crosslinking of the T cell receptor. To address this uncertainty, a wide-scale, quantitative mass spectrometry-based phosphoproteomic analysis was performed on T cells stimulated either by temperature shift or through crosslinking of the TCR. Careful statistical comparison between the two stimulations revealed a striking level of identity between the subset of 339 sites that changed significantly with both stimulations. This study demonstrates for the first time, at unprecedented detail, that T cell cold treatment was sufficient to initiate signaling patterns nearly identical to soluble antibody stimulation, shedding new light on the mechanism of activation of these critically important immune cells. PMID:25839225

Generation of TCR-Expressing Innate Lymphoid-like Helper Cells that Induce Cytotoxic T Cell-Mediated Anti-leukemic Cell Response.

PubMed

Ueda, Norihiro; Uemura, Yasushi; Zhang, Rong; Kitayama, Shuichi; Iriguchi, Shoichi; Kawai, Yohei; Yasui, Yutaka; Tatsumi, Minako; Ueda, Tatsuki; Liu, Tian-Yi; Mizoro, Yasutaka; Okada, Chihiro; Watanabe, Akira; Nakanishi, Mahito; Senju, Satoru; Nishimura, Yasuharu; Kuzushima, Kiyotaka; Kiyoi, Hitoshi; Naoe, Tomoki; Kaneko, Shin

2018-06-05

CD4 + T helper (Th) cell activation is essential for inducing cytotoxic T lymphocyte (CTL) responses against malignancy. We reprogrammed a Th clone specific for chronic myelogenous leukemia (CML)-derived b3a2 peptide to pluripotency and re-differentiated the cells into original TCR-expressing T-lineage cells (iPS-T cells) with gene expression patterns resembling those of group 1 innate lymphoid cells. CD4 gene transduction into iPS-T cells enhanced b3a2 peptide-specific responses via b3a2 peptide-specific TCR. iPS-T cells upregulated CD40 ligand (CD40L) expression in response to interleukin-2 and interleukin-15. In the presence of Wilms tumor 1 (WT1) peptide, antigen-specific dendritic cells (DCs) conditioned by CD4-modified CD40L high iPS-T cells stimulated WT1-specific CTL priming, which eliminated WT1 peptide-expressing CML cells in vitro and in vivo. Thus, CD4 modification of CD40L high iPS-T cells generates innate lymphoid helper-like cells inducing bcr-abl-specific TCR signaling that mediates effectiveanti-leukemic CTL responses via DC maturation, showing potential for adjuvant immunotherapy against leukemia. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

IMGT, the International ImMunoGeneTics database.

PubMed Central

Lefranc, M P; Giudicelli, V; Busin, C; Bodmer, J; Müller, W; Bontrop, R; Lemaitre, M; Malik, A; Chaume, D

1998-01-01

IMGT, the international ImMunoGeneTics database, is an integrated database specialising in Immunoglobulins (Ig), T cell Receptors (TcR) and Major Histocompatibility Complex (MHC) of all vertebrate species, created by Marie-Paule Lefranc, CNRS, Montpellier II University, Montpellier, France (lefranc@ligm.crbm.cnrs-mop.fr). IMGT includes three databases: LIGM-DB (for Ig and TcR), MHC/HLA-DB and PRIMER-DB (the last two in development). IMGT comprises expertly annotated sequences and alignment tables. LIGM-DB contains more than 23 000 Immunoglobulin and T cell Receptor sequences from 78 species. MHC/HLA-DB contains Class I and Class II Human Leucocyte Antigen alignment tables. An IMGT tool, DNAPLOT, developed for Ig, TcR and MHC sequence alignments, is also available. IMGT works in close collaboration with the EMBL database. IMGT goals are to establish a common data access to all immunogenetics data, including nucleotide and protein sequences, oligonucleotide primers, gene maps and other genetic data of Ig, TcR and MHC molecules, and to provide a graphical user friendly data access. IMGT has important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutical approaches (antibody engineering), genome diversity and genome evolution studies. IMGT is freely available at http://imgt.cnusc.fr:8104 PMID:9399859

[Immunoglobulin genes and T-cell receptors as molecular markers in children with acute lymphoblastic leukaemia].

PubMed

Lazić, Jelena; Dokmanović, Lidija; Krstovski, Nada; Predojević, Jelica; Tosić, Natasa; Pavlović, Sonja; Janić, Dragana

2009-01-01

Acute lymphoblastic leukaemia (ALL) is a malignant clonal disease, one of the most common malignancies in childhood. Contemporary protocols ensure high remission rate and long term free survival. The ability of molecular genetic methods help to establish submicroscopic classification and minimal residual disease (MRD) follow up, in major percent responsible for relapse. The aim of the study was to detect the frequency of IgH and TCR gene rearrangements and their correlation with clinical parameters. Forty-one children with ALL were enrolled in the study group, with initial diagnosis of IgH and TCR gene rearrangements by polimerase chain reaction (PCR). MRD follow-up was performed in induction phase when morphological remission was expected, and after intensive chemiotherapy. In the study group IgH rearrangement was detected in 82.9% of children at the diagnosis, while TCR rearrangement was seen in 56.1%. On induction day 33, clonal IgH rearrangements persisted in 39% and TCR rearrangements in 36.5% of children. Molecular analysis of genetic alterations and their correlation with standard prognostic parameters show the importance of risk stratification revision which leads to new therapy intensification approach. MRD stands out as a precise predictive factor for the relapse of disease.

TCR-independent CD28-mediated gene expression in peripheral blood lymphocytes from donors chronically infected with HIV-1.

PubMed Central

Wong, J G; Smithgall, M D; Haffar, O K

1997-01-01

Complete activation of peripheral blood T cells requires both T-cell receptor (TCR) stimulation and CD28 costimulation. Signalling pathways associated specifically with CD28 are not well understood, however, because ligation of CD28 in the absence of TCR stimulation does not give rise to cellular responses in normal cells. In peripheral blood lymphocytes (PBL) from donors chronically infected with human immunodeficiency virus-1 (HIV-1), CD28 can induce viral replication through an alternative pathway that does not require TCR ligation. We have exploited this observation to study CD28-mediated signal transduction using reverse transcriptase-mediated polymerase chain reaction (RT-PCR) to amplify viral RNA. Independent ligation of CD28 on donor PBL induced expression of the HIV-1 tat gene but not the interleukin-2 (IL-2) gene. Viral induction did not occur following pretreatment of cells with actinomycin D, suggesting it was mediated through transcriptional activation of the viral long terminal repeat (LTR). tat was induced in the presence of the protein kinase C inhibitor H-7, but was inhibited by cyclosporin A. Our results demonstrate that CD28 is linked directly to specific signalling pathways leading to de novo induction of genes in PBL. Images Figure 1 Figure 2 Figure 3 PMID:9135558

Improved sake metabolic profile during fermentation due to increased mitochondrial pyruvate dissimilation.

PubMed

Agrimi, Gennaro; Mena, Maria C; Izumi, Kazuki; Pisano, Isabella; Germinario, Lucrezia; Fukuzaki, Hisashi; Palmieri, Luigi; Blank, Lars M; Kitagaki, Hiroshi

2014-03-01

Although the decrease in pyruvate secretion by brewer's yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and (13) C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer's yeasts. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

T-cell Receptor Signaling Activates an ITK/NF-κB/GATA-3 axis in T-cell Lymphomas Facilitating Resistance to Chemotherapy.

PubMed

Wang, Tianjiao; Lu, Ye; Polk, Avery; Chowdhury, Pinki; Zamalloa, Carlos Murga; Fujiwara, Hiroshi; Suemori, Koichiro; Beyersdorf, Niklas; Hristov, Alexandra C; Lim, Megan S; Bailey, Nathanael G; Wilcox, Ryan A

2017-05-15

Purpose: T-cell lymphomas are a molecularly heterogeneous group of non-Hodgkin lymphomas (NHL) that account for a disproportionate number of NHL disease-related deaths due to their inherent and acquired resistance to standard multiagent chemotherapy regimens. Despite their molecular heterogeneity and frequent loss of various T cell-specific receptors, the T-cell antigen receptor is retained in the majority of these lymphomas. As T-cell receptor (TCR) engagement activates a number of signaling pathways and transcription factors that regulate T-cell growth and survival, we examined the TCR's role in mediating resistance to chemotherapy. Experimental Design: Genetic and pharmacologic strategies were utilized to determine the contribution of tyrosine kinases and transcription factors activated in conventional T cells following TCR engagement in acquired chemotherapy resistance in primary T-cell lymphoma cells and patient-derived cell lines. Results: Here, we report that TCR signaling activates a signaling axis that includes ITK, NF-κB, and GATA-3 and promotes chemotherapy resistance. Conclusions: These observations have significant therapeutic implications, as pharmacologic inhibition of ITK prevented the activation of this signaling axis and overcame chemotherapy resistance. Clin Cancer Res; 23(10); 2506-15. ©2016 AACR . ©2016 American Association for Cancer Research.

Lineage determination of CD7+ CD5- CD2- and CD7+ CD5+ CD2- lymphoblasts: studies on phenotype, genotype, and gene expression of myeloperoxidase, CD3 epsilon, and CD3 delta.

PubMed

Yoneda, N; Tatsumi, E; Teshigawara, K; Nagata, S; Nagano, T; Kishimoto, Y; Kimura, T; Yasunaga, K; Yamaguchi, N

1994-04-01

The gene expression of myeloperoxidase (MPO), CD3 epsilon, and CD3 delta molecules, the gene rearrangement of T-cell receptor (TCR) delta, gamma, and beta and immunoglobulin heavy (IgH) chain, and the expression of cell-surface antigens were investigated in seven cases of CD7+ CD5- CD2- and four cases of CD7+ CD5+ CD2- acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LBL) blasts, which were negative for cytochemical myeloperoxidase (cyMPO). More mature T-lineage blasts were also investigated in a comparative manner. In conclusion, the CD7+ CD5- CD2- blasts included four categories: undifferentiated blasts without lineage commitment, T-lineage blasts, T-/myeloid lineage blasts, and cyMPO-negative myeloblasts. The CD7+ CD5+ CD2- blasts included two categories; T-lineage and T-/myeloid lineage blasts. The 11 cases were of the germ-line gene (G) for TCR beta and IgH. Four cases were G for TCR delta and TCR gamma. The others were of the monoclonally rearranged gene (R) for TCR delta and G for TCR gamma or R for both TCR delta and TCR gamma. The expression or in vitro induction of CD13 and/or CD33 antigens correlated with the immaturity of these neoplastic T cells, since it was observed in all 11 CD7+ CD5- CD2- and CD7+ CD5+ CD2-, and some CD7+ CD5+ CD2+ (CD3- CD4- CD8-) cases, but not in CD3 +/- CD4+ CD8+ or CD3+ CD4+ CD8- cases. CD3 epsilon mRNA, but not CD3 delta mRNA, was detected in two CD7+ CD5- CD2- cases, while mRNA of neither of the two CD3 molecules was detected in the other tested CD7+ CD5- CD2- cases. In contrast, mRNA of both CD3 epsilon and CD3 delta were detected in all CD7+ CD5+ CD2- cases, indicating that CD7+ CD5- CD2- blasts at least belong to T-lineage. The blasts of two CD7+ CD5- CD2- cases with entire germ-line genes and without mRNA of the three molecules (MPO, CD3 epsilon, and CD3 delta) were regarded as being at an undifferentiated stage prior to their commitment to either T- or myeloid-lineage. The co-expression of the genes of MPO and CD3 epsilon in a CD7+ CD5- CD2- case MPO, CD3 epsilon, and CD3 delta in a CD7+ CD5+ CD2- case suggested the presence of some overlapping phase for T- and myeloid-lineage commitment during immature stages of differentiation. This helps understand the conversion of some T-ALL/LBL cases to acute myeloblastic leukemia (AML).(ABSTRACT TRUNCATED AT 400 WORDS)

A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding

PubMed Central

Kostrhon, Sebastian; Kontaxis, Georg; Kaufmann, Tanja; Schirghuber, Erika; Kubicek, Stefan; Konrat, Robert

2017-01-01

N-terminal histone tails are subject to many posttranslational modifications that are recognized by and interact with designated reader domains in histone-binding proteins. BROMO domain adjacent to zinc finger 2B (BAZ2B) is a multidomain histone-binding protein that contains two histone reader modules, a plant homeodomain (PHD) and a bromodomain (BRD), linked by a largely disordered linker. Although previous studies have reported specificity of the PHD domain for the unmodified N terminus of histone H3 and of the BRD domain for H3 acetylated at Lys14 (H3K14ac), the exact mode of H3 binding by BAZ2B and its regulation are underexplored. Here, using isothermal titration calorimetry and NMR spectroscopy, we report that acidic residues in the BAZ2B PHD domain are essential for H3 binding and that BAZ2B PHD–BRD establishes a polyvalent interaction with H3K14ac. Furthermore, we provide evidence that the disordered interdomain linker modulates the histone-binding affinity by interacting with the PHD domain. In particular, lysine-rich stretches in the linker, which resemble the positively charged N terminus of histone H3, reduce the binding affinity of the PHD finger toward the histone substrate. Phosphorylation, acetylation, or poly(ADP-ribosyl)ation of the linker residues may therefore act as a cellular mechanism to transiently tune BAZ2B histone-binding affinity. Our findings further support the concept of interdomain linkers serving a dual role in substrate binding by appropriately positioning the adjacent domains and by electrostatically modulating substrate binding. Moreover, inhibition of histone binding by a histone-mimicking interdomain linker represents another example of regulation of protein–protein interactions by intramolecular mimicry. PMID:28864776

Enhancing the Breadth and Efficacy of Therapeutic Vaccines for Breast Cancer

DTIC Science & Technology

2014-10-01

sequence data produced by the Slansky team following their single-cell emulsion RT-PCR technique; however, it can be packaged and shared for use...cell emulsion RT-PCR. Additional modifications were made to our epitope discovery workflow to increase efficacy of transcript and neoantigen candidate...the MiTCR [8] open source software package developed by MiLaboratory. MiTCR is a highly efficient and fast approach to CDR3 extraction, clonotype

Characteristics of the Energetic Igniters Through Integrating B/Ti Nano-Multilayers on TaN Film Bridge

NASA Astrophysics Data System (ADS)

Yan, YiChao; Shi, Wei; Jiang, HongChuan; Cai, XianYao; Deng, XinWu; Xiong, Jie; Zhang, WanLi

2015-05-01

The energetic igniters through integrating B/Ti nano-multilayers on tantalum nitride (TaN) ignition bridge are designed and fabricated. The X-ray diffraction (XRD) and temperature coefficient of resistance (TCR) results show that nitrogen content has a great influence on the crystalline structure and TCR. TaN films under nitrogen ratio of 0.99 % exhibit a near-zero TCR value of approximately 10 ppm/°C. The scanning electron microscopy demonstrates that the layered structure of the B/Ti multilayer films is clearly visible with sharp and smooth interfaces. The electrical explosion characteristics employing a capacitor discharge firing set at the optimized charging voltage of 45 V reveal an excellent explosion performance by (B/Ti) n /TaN integration film bridge with small ignition delay time, high explosion temperature, much more bright flash of light, and much large quantities of the ejected product particles than TaN film bridge.

Increased T-cell receptor mutation frequency in radiation-exposed residents living near the Semipalatinsk nuclear test site.

PubMed

Taooka, Yasuyuki; Takeichi, Nobuo; Noso, Yoshihiro; Kawano, Noriyuki; Apsalikov, Kazbek N; Hoshi, Masaharu

2006-02-01

From 1949 to 1989, 488 nuclear explosions were carried out in Semipalatinsk, and the cancer risk is increased in this region. Measuring somatic-cell mutation frequencies may be a useful tool for evaluating cancer risk within radiation-exposed populations. Here, we report the first evidence of increased T-cell receptor (TCR) mutations in peripheral blood from radiation-exposed residents of Semipalatinsk. The TCR mutation frequency in the highly exposed residents (Dolon and Sarzhal) was significantly higher than in the control group (Kokpekti). There was no statistically significant difference between the control group and the weakly exposed group (Kaynar and Semipalatinsk-city). The TCR mutation assay appeared to be a useful biological dosimeter even after a period of 40 years since radiation exposure. This may be the result of specific conditions, such as the presence of internal exposure.

THEMIS, a new T cell specific protein important for late thymocyte development

PubMed Central

Lesourne, Renaud; Uehara, Shoji; Lee, Jan; Song, Ki-Duk; Li, LiQi; Pinkhasov, Julia; Zhang, Yongqing; Weng, Nan-Ping; Wildt, Kathryn F.; Wang, Lie; Bosselut, Remy; Love, Paul E.

2010-01-01

During positive selection, thymocytes transition through a stage during which T cell receptor (TCR) signaling controls CD4 versus CD8 lineage choice and subsequent maturation. Here, we describe a new T cell specific protein, THEMIS, that performs a distinct function during this stage. In Themis-/- mice, thymocyte selection was impaired and the number of transitional CD4+CD8int thymocytes as well as CD4 and CD8 single positive thymocytes was decreased. Remarkably, although no overt TCR-proximal signaling deficiencies were detected, Themis-/-CD4+CD8int thymocytes exhibited developmental defects consistent with attenuated signaling that were reversible by increased TCR stimulation. These results identify THEMIS as a critical component of the T cell developmental program and suggest that THEMIS functions to sustain and/or integrate signals required for proper lineage commitment and maturation. PMID:19597498

The J beta segment of the T cell receptor contributes to the V beta-specific T cell expansion caused by staphylococcal enterotoxin B and Urtica dioica superantigens.

PubMed

Musette, P; Galelli, A; Truffa-Bachi, P; Peumans, W; Kourilsky, P; Gachelin, G

1996-03-01

We have used a new polymerase chain reaction-based technique to analyze at the clonal level the CDR3 diversity and the J beta usage associated with the V beta-dependent T cell receptor (TCR) recognition of two superantigens: the staphylococcal enterotoxin B and the Urtica dioica agglutinin. Our results show that subset of J beta elements is preferentially expanded in a given V beta family, independently of the nature of the superantigen. By contrast, the CDR3 loop does not contribute significantly to the T cell expansion induced by the superantigens. We conclude that the J beta segment of the TCR beta chain, but not the CDR3 region, participates in superantigen binding, presumably by influencing the quaternary structure of the TCR beta chain.

Platypus TCRμ provides insight into the origins and evolution of a uniquely mammalian TCR locus1

PubMed Central

Wang, Xinxin; Parra, Zuly E.; Miller, Robert D.

2011-01-01

TCRμ is an unconventional TCR that was first discovered in marsupials and appears to be absent from placental mammals and non-mammals. Here we show that TCRμ is also present in the duckbill platypus, an egg-laying monotreme, consistent with TCRμ being ancient and present in the last common ancestor of all extant mammals. As in marsupials, platypus TCRμ is expressed in a form containing double V domains. These V domains more closely resemble antibody V than that of conventional TCR. Platypus TCRμ differs from its marsupial homologue by requiring two rounds of somatic DNA recombination to assemble both V exons and has a genomic organization resembling the likely ancestral form of the receptor genes. These results demonstrate that the ancestors of placental mammals would have had TCRμ but it has been lost from this lineage. PMID:21976776

Platypus TCRμ provides insight into the origins and evolution of a uniquely mammalian TCR locus.

PubMed

Wang, Xinxin; Parra, Zuly E; Miller, Robert D

2011-11-15

TCRμ is an unconventional TCR that was first discovered in marsupials and appears to be absent from placental mammals and nonmammals. In this study, we show that TCRμ is also present in the duckbill platypus, an egg-laying monotreme, consistent with TCRμ being ancient and present in the last common ancestor of all extant mammals. As in marsupials, platypus TCRμ is expressed in a form containing double V domains. These V domains more closely resemble Ab V than that of conventional TCR. Platypus TCRμ differs from its marsupial homolog by requiring two rounds of somatic DNA recombination to assemble both V exons and has a genomic organization resembling the likely ancestral form of the receptor genes. These results demonstrate that the ancestors of placental mammals would have had TCRμ but it has been lost from this lineage.

Normal T lymphocytes can express two different T cell receptor beta chains: implications for the mechanism of allelic exclusion

PubMed Central

1995-01-01

We have examined the extent of allelic exclusion at the T cell receptor (TCR) beta locus using monoclonal antibodies specific for V beta products. A small proportion (approximately 1%) of human peripheral blood T cells express two V beta as determined by flow cytometric analysis, isolation of representative clones, and sequencing of the corresponding V beta chains. Dual beta T cells are present in both the CD45R0+ and CD45R0- subset. These results indicate that dual beta expression is compatible with both central and peripheral selection. They also suggest that the substantial degree of TCR beta allelic exclusion is dependent only on asynchronous rearrangements at the beta locus, whereas the role of the pre-TCR is limited to signaling the presence of at least one functional beta protein. PMID:7699339

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist.

PubMed

Sanchez, Elisa; Huse, Morgan

2018-04-25

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla

PubMed Central

McCaughtry, Tom M.; Baldwin, Troy A.; Wilken, Matthew S.; Hogquist, Kristin A.

2008-01-01

The thymic medulla is generally held to be a specialized environment for negative selection. However, many self-reactive thymocytes first encounter ubiquitous self-antigens in the cortex. Cortical epithelial cells are vital for positive selection, but whether such cells can also promote negative selection is controversial. We used the HYcd4 model, where T cell receptor for antigen (TCR) expression is appropriately timed and a ubiquitous self-antigen drives clonal deletion in male mice. We demonstrated unambiguously that this deletion event occurs in the thymic cortex. However, the kinetics in vivo indicated that apoptosis was activated asynchronously relative to TCR activation. We found that radioresistant antigen-presenting cells and, specifically, cortical epithelial cells do not efficiently induce apoptosis, although they do cause TCR activation. Rather, thymocytes undergoing clonal deletion were preferentially associated with rare CD11c+ cortical dendritic cells, and elimination of such cells impaired deletion. PMID:18936237

TCR and IL-7 Signaling Are Altered in the Absence of Functional GTPase of the Immune Associated Nucleotide Binding Protein 5 (GIMAP5)

PubMed Central

Chen, Xi-Lin; Serrano, Daniel; Ghobadi, Farnaz; Mayhue, Marian; Hoebe, Kasper; Ilangumaran, Subburaj; Ramanathan, Sheela

2016-01-01

GTPase of the immune associated nucleotide binding protein (GIMAP) family of proteins are expressed essentially in cells of the hematopoietic system. Mutation in the founding member of this gene family, Gimap5, results in the lymphopenic phenotype in Bio-Breeding diabetes prone rats. In mice, deletion of functional Gimap5 gene affects the survival and renewal of hematopoietic stem cells in addition to the defects observed in T cells. Here we show that T cells from OTII TCR-transgenic Gimap5sph/sph mice do not proliferate in response to its cognate antigen. Furthermore, T cells from Gimap5 mutant rats and mice show decreased phosphorylation of STAT5 following stimulation with IL-7. Our results suggest that functional Gimap5 is required for optimal signaling through TCR and IL-7R in T cells. PMID:27023180

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

PubMed Central

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

2016-01-01

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

Alpha-actinin binding kinetics modulate cellular dynamics and force generation

PubMed Central

Ehrlicher, Allen J.; Krishnan, Ramaswamy; Guo, Ming; Bidan, Cécile M.; Weitz, David A.; Pollak, Martin R.

2015-01-01

The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease. PMID:25918384