T-cell Receptor Subunits Research Articles

E proteins control the development of NKγδT cells through their invariant T cell receptor

T cell receptor (TCR) signaling regulates important developmental transitions, partly through induction of the E protein antagonist, Id3. Although normal γδ T cell development depends on Id3, Id3 deficiency produces different phenotypes in distinct γδ T cell subsets. Here, we show that Id3 deficiency impairs development of the Vγ3+ subset, while markedly enhancing development of NKγδT cells expressing the invariant Vγ1Vδ6.3 TCR. These effects result from Id3 regulating both the generation of the Vγ1Vδ6.3 TCR and its capacity to support development. Indeed, the Trav15 segment, which encodes the Vδ6.3 TCR subunit, is directly bound by E proteins that control its expression. Once expressed, the Vγ1Vδ6.3 TCR specifies the innate-like NKγδT cell fate, even in progenitors beyond the normally permissive perinatal window, and this is enhanced by Id3-deficiency. These data indicate that the paradoxical behavior of NKγδT cells in Id3-deficient mice is determined by its stereotypic Vγ1Vδ6.3 TCR complex.

Allosteric inhibition of the T cell receptor by a designed membrane ligand.

The T cell receptor (TCR) is a complex molecular machine that directs the activation of T cells, allowing the immune system to fight pathogens and cancer cells. Despite decades of investigation, the molecular mechanism of TCR activation is still controversial. One of the leading activation hypotheses is the allosteric model. This model posits that binding of pMHC at the extracellular domain triggers a dynamic change in the transmembrane (TM) domain of the TCR subunits, which leads to signaling at the cytoplasmic side. We sought to test this hypothesis by creating a TM ligand for TCR. Previously we described a method to create a soluble peptide capable of inserting into membranes and binding to the TM domain of the receptor tyrosine kinase EphA2 (Alves et al., eLife, 2018). Here, we show that the approach is generalizable to complex membrane receptors, by designing a TM ligand for TCR. We observed that the designed peptide caused a reduction of Lck phosphorylation of TCR at the CD3ζ subunit in T cells. As a result, in the presence of this peptide inhibitor of TCR (PITCR), the proximal signaling cascade downstream of TCR activation was significantly dampened. Co-localization and co-immunoprecipitation in diisobutylene maleic acid (DIBMA) native nanodiscs confirmed that PITCR was able to bind to the TCR. AlphaFold-Multimer predicted that PITCR binds to the TM region of TCR, where it interacts with the two CD3ζ subunits. Our results additionally indicate that PITCR disrupts the allosteric changes in the compactness of the TM bundle that occur upon TCR activation, lending support to the allosteric TCR activation model. The TCR inhibition achieved by PITCR might be useful to treat inflammatory and autoimmune diseases and to prevent organ transplant rejection, as in these conditions aberrant activation of TCR contributes to disease.

Next Generation Genomic Analyses in T-ALL Patients Identify Recurrent and Novel Genomic Abnormalities

Background and Objective 15% of pediatric and 40% of adult T-cell Acute Lymphoblastic Leukemia (T-ALL) patients fail conventional therapy highlighting the need for novel therapeutic strategies based on genomic alterations of individuals. We interrogated genomic alterations in Australian T-ALL patients for patterns of mutation and druggable targets. A subset of samples were used to establish patient-derived xenograft (PDX) models to evaluate novel therapies. Methods T-ALL patients' samples underwent next generation genomic analyses (128 total samples including diagnosis, refractory and relapse timepoints from 118 patients of all age groups). mRNA sequencing (mRNAseq) identified gene fusions and structural variants and assessed gene expression (n=101 patients). Fusions were called when identified by 2/3 predictors (FusionCatcher, SOAPfuse, JAFFA). Variant calling utilized GATK HaplotypeCaller and underwent several filtering steps to eliminate possible germline alterations and common SNPs. DNA copy number variations (CNVs) were detected via Multiplex Ligation-dependent Probe Amplification (MLPA: P202, P335, P383; n=64 patients). Establishment of PDX models from patient material (bone marrow or peripheral blood) is ongoing. Recapitulation of human disease was confirmed by mRNAseq in a subset of xenografts. Results Genomic fusion genes were identified in 46/101 samples (46%) by mRNAseq; the most common fusion identified was STIL-TAL1 (n=6). Increased expression of LCK and/or LAT (encoded proteins are involved in T-cell receptor (TCR) signal transduction) was observed in 100% of patients with the STIL-TAL1 gene fusion indicating TCR signaling pathways may be perturbed in this sub-group. Other common gene fusions were MLLT10-DDX3X (n=5) and KMT2A-MLLT4/AFDN (n=4). We also observed the previously reported fusions SET-NUP214, KMT2A-MLLT1, PICALM-MLLT10, NUP214-ABL1, several fusions involving TCR subunits as well as novel fusions involving KMT2A, NOTCH1, LMO1, ZEB2. Numerous nonsynonymous mutations were identified in 81/101 patients (80%) with mRNAseq data available (Figure 1). Broadly, the mutated genes encode proteins in the following categories: oncoproteins (NRAS, KRAS); tumour suppressors (TP53, CHEK2, BRCA1, BRCA2, PTEN), epigenetic regulators (EZH2, SETD5, DNMT3A); regulators of NOTCH signalling (NOTCH1, NOTCH2, FBXW7); transcription factors and regulators (IKZF1, KMT2A, EP400, SMARCD1, RUNX1, AFF1, AFF3); kinase and cytokine signal regulators (ATM, JAK2, JAK3, TYK2, FLT3, PTPN11). INDELS in clinically relevant genes were identified in 37/101 patients (37%) including alterations to: NOTCH1, PHF6, PTEN, STAT1, IL7R, CDKN2A, LYL1, WT1, JAK3, LEF1. The most common copy number alterations identified in our patient cohort were CDKN2A/B deletions (30/64 patients, 47%), PHF6 duplication (20/64 patients, 31%) and MLLT3 deletion (13/64 patients, 20%). In patients with CDKN2A/B deletions and additional CNVs, PHF6 duplication (n=6) and MYB duplication (n=4) were mutually exclusive. However, one patient without CDKN2A/B deletions harbored both MYB and PHF6 duplications. MLLT3 deletion always co-occurred with CDKN2A/B deletions (13/13 patients with MLLT3 deletion), but was never observed with PTEN deletion (0/7 patients with PTEN deletion). Patients with either CDKN2A/B deletions or PHF6 duplications frequently harbored NOTCH1 abnormalities: 13/32 patients (41%) and 8/21 patients (38%), respectively. PDX primagrafts investigated the engraftment latency and peripheral organ infiltration. Primagrafts established from patients harboring NUP214-SET1 or NUP214-ABL1 fusions engrafted at a slower rate (82 d and 91 d, respectively) than primagrafts from patients harboring a STIL-TAL1 fusion (45 d) or CDKN2A/B deletions (31 d and 48 d). Conclusions In our T-ALL cohort we demonstrate that the majority of cases harbor rearrangements, structural variations and duplication/deletion of genes associated with malignant transformation. We identified several co-occurring lesions as well as mutually exclusive genomic abnormalities. The top 20 mutated genes in our patient cohort differ to those reported for a pediatric cohort (Roberts et al 2019 Blood 134:649), indicating an association between patient age and genomic alteration. Secondary PDX models investigating novel targeted treatment strategies are ongoing. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. White:Bristol-Myers Squibb: Honoraria, Research Funding; Amgen: Honoraria.

Abstract 2190: Engineering off-the-shelf T cell receptor fusion construct (TRuC) T cells

Abstract The T-Cell Receptor (TCR) can be functionally split into antigen recognition mediated by the TCRα/β heterodimer and signal transduction triggered by the CD3 complex comprising CD3ϵ/γ, CD3ϵ/δ and CD3ζ/ζ dimers. Unlike antibodies, the TCR recognizes its cognate peptide antigen only when presented on human leukocyte antigen (HLA) molecules. Recently, we reported that tethering an antibody-derived binder to one of the TCR subunits redirects T cells to specifically kill tumor cells independent of HLA. Different from CAR-T cells, the T cell receptor fusion constructs (TRuC™) are integrated into the natural TCR and require all TCR subunits for receptor translocation to the cell surface. The development of off-the-shelf TRuC-T cells is desirable to shorten the vein-to-vein production time and reduce manufacturing costs. Here, we describe the generation of TRuC-T cells expressing a fully functional TRuC TCR without alloreactivity. Inactivation of the endogenous TRAC gene employing CRISPR/Cas9 endonuclease disrupts natural TCR formation. Yet, replacing the endogenous TCRα subunit with a human TCRα constant region without variable domain to avoid alloreactivity is insufficient for TRuC TCR expression. A functional TRuC TCR, however, can be created by substituting the following constructs for the inactivated TRAC gene: (i) murine TCRα and β constant domains without variable domains, (ii) chimeric human/murine TCRα and β constant regions or (iii) TCRγ and δ constant domains. Off-the-shelf TRuC-T cells upregulate activation markers, secrete cytokines, and kill tumor cells in an antigen-specific manner. Importantly, the genome-engineered TRuC-T cells lack alloreactivity as demonstrated in mixed lymphocyte reactions and clear tumors in NSG xenograft models without signs of Graft versus host disease (GvHD). Our findings warrant further development of allogeneic TRuC-T cells for cancer therapy. Citation Format: Julie Donaghey, Philippe Kieffer-Kwon, Troy Patterson, Tiffany Chan, Holly Horton, Robert Tighe, Dario A. Gutierrez, Daniel Getts, Robert Hofmeister. Engineering off-the-shelf T cell receptor fusion construct (TRuC) T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2190.

Abstract A21: Engineering antigen-specific natural killer cells against the melanoma-associated antigen tyrosinase via TCR gene transfer

Abstract Introduction of chimeric antigen receptors (CARs) to natural killer (NK) cells has so far been the only practical method for specific targeting of NK cells against surface antigens. In contrast, T-cell receptor (TCR) gene therapy can supply large populations of cytotoxic T-lymphocytes (CTL) genetically modified to express a TCR that can also target intracellular antigens. However, the mispairing of endogenous and genetically transferred TCR subunits constitutes a bottleneck in the development of safe therapies as it often leads to formation of TCRs with unknown specificity. In order to overcome this obstacle and enable intracellular antigen targeting, we propose the use of NK cells for TCR gene therapy. In this study, we approach the obstacles associated with TCR gene therapy from a unique perspective that results in MHC-I-restricted epitope-specific targeting of tumors cells through expression of a functional TCR complex on NK cells. Our results show that the ectopic expression of CD3δ, CD3γ, and CD3ϵ chains along with TCR α/β gene delivery to NK cells enables the functional expression of a TCR specific to the HLA-A2-restiricted tyrosinase-derived melanoma epitope, Tyr368-379. NK cells expressing a functional TCR exhibit the capacity to degranulate in an antigen-specific manner in response to engagement of the cognate peptide/MHC ligand on target cells. In addition, upon engagement of their TCR, NK cells are fully capable of producing proinflammatory cytokines IFNγ and TNF-α, a signature mark of NK cell activation and immune cell recruitment. Finally, NK-TCR cells exhibit MHC-I-restricted antigen detection and antigen-specific lysis of tumor cells both in vitro and in vivo. Antigen-specific targeting of NK cells via TCR gene delivery stands out as a unique discovery providing a novel tool in the field of adoptive immunotherapy that can also overcome the major hurdle of “mispairing” in TCR gene therapy. Clinical trials using NK cells, including genetically modified NK cells expressing activating receptors or CARs, have clearly demonstrated a significant benefit in patients with various malignancies. The overall safety profile and promising clinical benefits of NK cells combined with the exclusive antigen specificity of the TCR, all together provide a novel approach in the design of efficient antigen-specific adoptive immunotherapy. Citation Format: Ayhan Parlar, Ece C. Sayitoglu, Cevriye Pamukcu, Anna-Maria Georgoudaki, Didem Ozkazanc, Mertkaya Aras, Benjamin Josey, Michael Chrobok, Suzanne Branecki, Pegah Zahedimaram, Lolai Ikromzoda, Evren Alici, Batu Erman, Tolga Sutlu, Adil D. Duru. Engineering antigen-specific natural killer cells against the melanoma-associated antigen tyrosinase via TCR gene transfer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A21.

Engineering antigen-specific NK cell lines against the melanoma-associated antigen tyrosinase via TCR gene transfer.

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL-TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off-target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen-specific TCR complex on NK cell lines NK-92 and YTS, demonstrated by using a TCR against the HLA-A2-restricted tyrosinase-derived melanoma epitope, Tyr368-377 . Most importantly, the introduction of a TCR complex to NK cell lines enables MHC-restricted, antigen-specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of "mispairing" in TCR gene therapy.

The Quest for Psoriasis Autoantigens: Genetics Meets Immunology in the Melanocyte

The Quest for Psoriasis Autoantigens: Genetics Meets Immunology in the Melanocyte

Defining an allogeneic CAR-T approach by shRNA-mediated knockdown of the T-cell receptor.

e14551 Background: An allogeneic approach for CAR T-Cell therapy can significantly streamline manufacturing, provide more accessible options to patients as well as enhance safety by reducing the possibility of graft-versus-host-disease. The T-Cell Receptor (TCR) is comprised of multiple subunits and functions to activate T-cells by a signal transduction cascade that is initiated upon antigen binding. Eliminating expression of the endogenous TCR on modified CAR-T-Cells may eliminate the ability to recognize major and minor histocompatibility antigens in the recipient. This study assessed if simultaneous expression of multiple short hairpin RNAs that knockdown levels of individual TCR subunits could result in the loss of TCR expression and TCR-mediated T-Cell activation. Methods: Recombinant DNA producing short hairpin RNAs against the TCR complex was transfected into T-cells. Cell surface TCR was analyzed by FACS. Following CD3 activation or co-culture with B-cells, T-Cell activation was quantified by measuring the levels of IL-2 by ELISA and QPCR. Results: When expressed individually, each shRNA inhibited it’s cognate TCR subunit by up to 93% of the endogenous levels. However, upon simultaneous expression of the shRNAs against the different subunits from the same vector, we observed a near complete depletion of the TCR complex from the cell surface ( > 99%) as measured by FACS analyses. Furthermore, TCR functionality was inhibited when treated cells were stimulated with either CD3 or in B cell co-cultures with Staphylococcal enterotoxins. IL-2 secretion was inhibited to undetectable levels by ELISA by the multi-shRNA treatment and > 98% by qPCR. Conclusions: Though knockdown of any single component of TCR never exceeded 93%, simultaneous knockdown of several TCR subunits abrogated surface TCR and downstream activation suggesting that disruption of stoichiometric expression the subunits prevented TCR formation. The small size of the shRNA expression cassette ( < 2 Kb) permits co-expression from the same lentiviral vector as the CAR. Altogether, these data point to a viable strategy towards generating a single vector approach for the production of allogeneic T-Cells for immunotherapies against certain cancers.

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

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

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

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.

Loss of T cell-mediated antitumor immunity after construct-specific downregulation of retrovirally encoded T-cell receptor expression in vivo.

Adoptive T-cell therapy is clinically efficacious in the treatment of select cancers. However, it is often difficult to obtain adequate numbers of tumor-specific T cells for therapy. One method for overcoming this limitation is to generate tumor-specific T cells by retrovirally mediated T-cell-receptor (TCR) gene transfer. However, despite instances of therapeutic success, major obstacles remain, including attaining the survival of retrovirally modified T cells in vivo as well as inducing long-term and multi-gene retroviral expression. Using a murine model of adoptively transferred retrovirally modified CD8(+) T cells, where antitumor immunity was dependent on sustained, multigene expression, we found that in vitro assays are poor indicators of in vivo efficacy. Despite persisting for over 9 months in a nonlymphopenic environment, genetically modified T cells exhibited discordant retrovirally mediated gene expression in vivo not readily evident from initial in vitro assays. In particular, one of the two TCR subunit genes necessary for antigen specificity was selectively lost in vivo. As this discordant gene expression was associated with the loss of antitumor immunity, consideration of these findings may provide guidance in the design, evaluation and application of retroviral vectors for use in the treatment of cancer and other human disease.

The Cyclophilin-like Domain of Ran-binding Protein-2 Modulates Selectively the Activity of the Ubiquitin-Proteasome System and Protein Biogenesis

The ubiquitin-proteasome system (UPS) plays a critical role in protein degradation. The 19S regulatory particle (RP) of the 26S proteasome mediates the recognition, deubiquitylation, unfolding, and channeling of ubiquitylated substrates to the 20S proteasome. Several subunits of the 19S RP interact with a growing number of factors. The cyclophilin-like domain (CLD) of Ran-binding protein-2 (RanBP2/Nup358) associates specifically with at least one subunit, S1, of the base subcomplex of the 19S RP, but the functional implications of this interaction on the UPS activity are elusive. This study shows the CLD of RanBP2 promotes selectively the accumulation of a subset of reporter substrates of the UPS, such as the ubiquitin (Ub)-fusion yellow fluorescent protein (YFP) degradation substrate, Ub(G76V)-YFP, and the N-end rule substrate, Ub-R-YFP. Conversely, the degradation of endoplasmic reticulum and misfolded proteins, and of those linked to UPS-independent degradation, is not affected by CLD. The selective effect of CLD on the UPS in vivo is independent of, and synergistic with, proteasome inhibitors, and CLD does not affect the intrinsic proteolytic activity of the 20S proteasome. The inhibitory activity of CLD on the UPS resides in a purported SUMO binding motif. We also found two RanBP2 substrates, RanGTPase-activating protein and retinitis pigmentosa GTPase regulator interacting protein-1alpha1, whose steady-state levels are selectively modulated by CLD. Hence, the CLD of RanBP2 acts as a novel auxiliary modulator of the UPS activity; it may contribute to the molecular and subcellular compartmentation of the turnover of properly folded proteins and modulation of the expressivity of several neurological diseases.

A novel function of VCP (valosin-containing protein; p97) in the control of N-glycosylation of proteins in the endoplasmic reticulum

α-Chain of T-cell receptor (TCR) is a typical ERAD (ER-associated degradation) substrate degraded in the absence of other TCR subunits. Depletion of derlin 1 fails to induce accumulation of αTCR despite inducing accumulation of α1-antitrypsin, another ERAD substrate. Furthermore, while depletion of VCP does not affect levels of α1-antitrypsin, it induces an increase in levels of αTCR. RNAi of VCP induces preferential accumulation of αTCR with less mannose residues, suggesting its retention within the ER. Mass spectrometric analysis of cellular N-linked glycans revealed that depletion of VCP decreases the level of high-mannose glycoproteins, increases the levels of truncated low-mannose glycoproteins and induces changes in the abundance of complex glycans assembled in post-ER compartments. Since proteasome inhibition was unable to mimic those changes, they cannot be regarded as a simple consequence of inhibited ERAD but represent a complex effect of VCP on the function of the ER.

Enhanced Antitumor Activity of T Cells Engineered to Express T-Cell Receptors with a Second Disulfide Bond

Adoptive transfer of genetically T-cell receptor (TCR)-modified lymphocytes has been recently reported to cause objective cancer regression. However, a major limitation to this approach is the mispairing of the introduced chains with the endogenous TCR subunits, which leads to reduced TCR surface expression and, subsequently, to their lower biological activity. We here show that it is possible to improve TCR gene transfer by adding a single cysteine on each receptor chain to promote the formation of an additional interchain disulfide bond. We show that cysteine-modified receptors were more highly expressed on the surface of human lymphocytes compared with their wild-type counterparts and able to mediate higher levels of cytokine secretion and specific lysis when cocultured with specific tumor cell lines. Furthermore, cysteine-modified receptors retained their enhanced function in CD4(+) lymphocytes. We also show that this approach can be employed to enhance the function of humanized and native murine receptors in human cells. Preferential pairing of cysteine-modified receptor chains accounts for these observations, which could have significant implications for the improvement of TCR gene therapy.

Distribution of lipid raft markers in live cells

GPI (glycosylphosphatidylinositol)-anchored proteins are characteristic components of biochemically defined lipid rafts. Rafts may be involved in T-cell stimulation, but it is not clear whether molecules involved in TCR (T-cell receptor) signalling are partitioned to T-cell synapses through raft microdomains or through specific protein-protein interactions. We have used FRET (fluorescence resonance energy transfer) analysis to study the distribution of GPI-anchored fluorescent proteins in the plasma membrane of live cells. Multiple criteria suggested that FRET between different GPI-anchored fluorescent proteins in COS-7 or unstimulated Jurkat T-cells is generated by a random, unclustered distribution. Stimulation of TCR signalling in Jurkat T-cells by beads coated with antibodies against TCR subunits resulted in localized increases in fluorescence of raft markers. However, measurements of FRET and ratio imaging showed that there was no detectable clustering and no overall enrichment of raft markers in these regions.

TCR zeta-chain downregulation: curtailing an excessive inflammatory immune response.

The T-cell receptor (TCR) functions in both antigen recognition and signal transduction, which are crucial initial steps of antigen-specific immune responses. TCR integrity is vital for the induction of optimal and efficient immune responses, including the routine elimination of invading pathogens and the elimination of modified cells and molecules. Of the TCR subunits, the zeta-chain has a key role in receptor assembly, expression and signalling. Downregulation of TCR zeta-chain expression and impairment of T-cell function have been shown for T cells isolated from hosts with various chronic pathologies, including cancer, and autoimmune and infectious diseases. This review summarizes studies of the various pathologies that show this phenomenon and provides new insights into the mechanism responsible for downregulation of zeta-chain expression, its relevance and its clinical implications.

Masking of the CD3 gamma di-leucine-based motif by zeta is required for efficient T-cell receptor expression.

The T-cell receptor (TCR) is a multimeric receptor composed of the Ti alpha beta heterodimer and the noncovalently associated CD3 gamma delta epsilon and zeta(2) chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated zeta chains, indicating that the zeta chain masks the endocytosis motif in CD3 gamma and thereby stabilizes TCR cell surface expression.

TCR subunit specificity of CTLA-4-mediated signaling.

Cytotoxic T-lymphocyte-associated antigen (CTLA)-4 is an activation-induced receptor that down-regulates T cell responses by antagonizing B7-dependent costimulation and/or by transducing a negative signal. The mechanism of CTLA-4-mediated negative signaling is unknown. Recently, it has been postulated that CTLA-4 inhibits T cell activation by causing specific dephosphorylation of the T cell receptor (TCR)-zeta chain of the antigen-receptor complex through an lck-dependent recruitment of the Src homology-2-containing tyrosine phosphatase-2. To test this hypothesis, we generated stably transfected T cell clones expressing doxycycline-inducible CTLA-4 with CD25:TCR-zeta (CD25-zeta) or CD25:CD3-epsilon (CD25-epsilon) fusion proteins. In these clones, ligation of CD25-zeta or of CD25-epsilon with antibodies against CD25 induced full T cell activation, as illustrated by extracellular signal-regulated kinase (ERK) activation and interleukin (IL)-2 production. More importantly, coligation of CTLA-4 with CD25-zeta or of CTLA-4 with CD25-epsilon in the respectively transfected clones inhibited ERK activation and IL-2 production, demonstrating that CTLA-4 does not specifically inhibit signals from TCR-zeta but can also inhibit signals from CD3-epsilon. Our results suggest that the target specificity of CTLA-4 is determined by its coligation with any given transmembrane receptor rather than by its intracellular mediators.

Sequence Variation and Linkage Disequilibrium in the Human T-Cell Receptor β ( TCRB) Locus

The T-cell receptor (TCR) plays a central role in the immune system, and > 90% of human T cells present a receptor that consists of the alpha TCR subunit (TCRA) and the beta subunit (TCRB). Here we report an analysis of 63 variable genes (BV), spanning 553 kb of TCRB that yielded 279 single-nucleotide polymorphisms (SNPs). Samples were drawn from 10 individuals and represent four populations-African American, Chinese, Mexican, and Northern European. We found nine variants that produce nonfunctional BV segments, removing those genes from the TCRB genomic repertoire. There was significant heterogeneity among population samples in SNP frequency (including the BV-inactivating sites), indicating the need for multiple-population samples for adequate variant discovery. In addition, we observed considerable linkage disequilibrium (LD) (r(2) > 0.1) over distances of approximately 30 kb in TCRB, and, in general, the distribution of r(2) as a function of physical distance was in close agreement with neutral coalescent simulations. LD in TCRB showed considerable spatial variation across the locus, being concentrated in "blocks" of LD; however, coalescent simulations of the locus illustrated that the heterogeneity of LD we observed in TCRB did not differ markedly from that expected from neutral processes. Finally, examination of the extended genotypes for each subject demonstrated homozygous stretches of >100 kb in the locus of several individuals. These results provide the basis for optimization of locuswide SNP typing in TCRB for studies of genotype-phenotype association.

Dissociation of intracellular signaling pathways in response to partial agonist ligands of the T cell receptor.

The T cell receptor (TCR) is a versatile receptor able to generate different signals that result in distinct T cell responses. The pattern of early signals is determined by the TCR binding kinetics that control the ability of the ligand to coengage TCR and coreceptor. Coengagement of TCR and CD4 results in an agonist signaling pattern with complete tyrosine phosphorylation of TCR subunits, and recruitment and activation of ZAP-70. In contrast, TCR engagement without CD4 coengagement causes a partial agonist type of signaling, characterized by distinct phosphorylation of TCR subunits and recruitment but no activation of ZAP-70. The pathways triggered by partial agonist signaling are unknown. Here, we show that agonists cause association of active lck and active ZAP-70 with p120-GTPase-activating protein (p120-GAP). These associations follow engagement of CD4 or CD3, respectively. In contrast, partial agonists do not activate lck or ZAP-70, but induce association of p120-GAP with inactive ZAP-70. Despite these differences, both agonist and partial agonist signals activate the mitogen-activated protein kinase (MAPK) pathway. However, MAPK activation by partial agonists is transient, supporting a kinetic, CD4-dependent model for the mechanism of action of variant TCR ligands. Transient MAPK activation may explain some of the responses to TCR partial agonists and antagonists.