TCRζ Chain Research Articles

Forced expression of the Fc receptor gamma-chain renders human T cells hyperresponsive to TCR/CD3 stimulation.

High level expression of Fc epsilon RI gamma chain replaces the deficient TCR zeta-chain and contributes to altered TCR/CD3-mediated signaling abnormalities in T cells of patients with systemic lupus erythematosus. Increased responsiveness to Ag has been considered to lead to autoimmunity. To test this concept, we studied early signaling events and IL-2 production in fresh cells transfected with a eukaryotic expression vector encoding the Fc epsilon RI gamma gene. We found that the overexpressed Fc epsilon RI gamma chain colocalizes with the CD3 epsilon chain on the surface membrane of T cells and that cross-linking of the new TCR/CD3 complex leads to a dramatic increase of intracytoplasmic calcium concentration, protein tyrosine phosphorylation, and IL-2 production. We observed that overexpression of Fc epsilon RI gamma is associated with increased phosphorylation of Syk kinase, while the endogenous TCR zeta-chain is down-regulated. We propose that altered composition of the CD3 complex leads to increased T cell responsiveness to TCR/CD3 stimulation and sets the biochemical grounds for the development of autoimmunity.

A subset of memory effector T cells in peripheral blood defined by differential expression of the TCR-ζ chain

A subset of memory effector T cells in peripheral blood defined by differential expression of the TCR-ζ chain

Comparison of the TCR zeta-chain with the FcR gamma-chain in chimeric TCR constructs for T cell activation and apoptosis.

A promising strategy for cancer treatment is adoptive gene therapy/immunotherapy by genetically modifying T cells with a chimeric T cell receptor (cTCR). When transduced T cells (T-bodies) specifically bind to tumor antigens through cTCR, they will become cytotoxic T lymphocytes (CTL) and lyse the tumor cells in a non-major histocompatibility complex (MHC)-restricted manner. Both the FcR gamma-chain and the TCR zeta-chain have been used to construct such cTCR, and both have shown specific cytolytic functions against tumor cells. However, most researchers believe that the zeta-chain generates stronger cytolytic activities against tumor than the gamma-chain and therefore would be a better candidate for cTCR construction. On the other hand, because of the lack of costimulation signaling in such constructs, the T-body might cause activation-induced T cell death (AICD) when bound to tumor antigens. Therefore, one can argue that the gamma-chain might generate less AICD than the zeta-chain because the gamma-chain has only one immunoreceptor tyrosine-based activation motif (ITAM), and the cytolytic activities can be therefore recycled. Two cTCR, GAHgamma and GAHzeta, were constructed and evaluated for cytokine production, specific cytolytic function and AICD in T-bodies after exposure to tumor cells. Using EGP-2-positive LS174T colorectal carcinoma cells as targets, there was no substantial difference observed between a gamma-chain or zeta-chain as the T-body signaling moiety in terms of specific cytolytic functions and induced cytokine production. This paper also demonstrates that, in the absence of a costimulation system, tumor antigen may not trigger apoptosis of T cells transduced with a cTCR carrying either an FcR gamma-chain or a TCR zeta-chain. These observations challenge current ideas about the role of ITAM in T cell activation.

CTLA-4 suppresses proximal TCR signaling in resting human CD4(+) T cells by inhibiting ZAP-70 Tyr(319) phosphorylation: a potential role for tyrosine phosphatases.

The balance between positive and negative signals plays a key role in determining T cell function. CTL-associated Ag-4 is a surface receptor that can inhibit T cell responses induced upon stimulation of the TCR and its CD28 coreceptor. Little is known regarding the signaling mechanisms elicited by CTLA-4. In this study we analyzed CTLA-4-mediated inhibition of TCR signaling in primary resting human CD4(+) T cells displaying low, but detectable, CTLA-4 cell surface expression. CTLA-4 coligation with the TCR resulted in reduced downstream protein tyrosine phosphorylation of signaling effectors and a striking inhibition of extracellular signal-regulated kinase 1/2 activation. Analysis of proximal TCR signaling revealed that TCR zeta-chain phosphorylation and subsequent zeta-associated protein of 70 kDa (ZAP-70) tyrosine kinase recruitment were not significantly affected by CTLA-4 engagement. However, the association of p56(lck) with ZAP-70 was inhibited following CTLA-4 ligation, correlating with reduced actions of p56(lck) in the ZAP-70 immunocomplex. Moreover, CTLA-4 ligation caused the selective inhibition of CD3-mediated phosphorylation of the positive regulatory ZAP-70 Y319 site. In addition, we demonstrate protein tyrosine phosphatase activity associated with the phosphorylated CTLA-4 cytoplasmic tail. The major phosphatase activity was attributed to Src homology protein 2 domain-containing tyrosine phosphatase 1, a protein tyrosine phosphatase that has been shown to be a negative regulator of multiple signaling pathways in hemopoietic cells. Collectively, our findings suggest that CTLA-4 can act early during the immune response to regulate the threshold of T cell activation.

Phosphorylation and O-linked glycosylation of Elf-1 leads to its translocation to the nucleus and binding to the promoter of the TCR zeta-chain.

Elf-1, a member of the E 26-specific transcription factor family with a predicted molecular mass of 68 kDa, is involved in the transcriptional regulation of several hematopoietic cell genes. We demonstrate that Elf-1 exists primarily as a 98-kDa form in the nucleus and as an 80-kDa form in the cytoplasm. Phosphorylation and O-linked glycosylation contribute to the increased posttranslational molecular mass of Elf-1. The 98-kDa Elf-1 is released from the cytoplasm tethering retinoblastoma protein and moves to the nucleus, where it binds to the promoter of the TCR zeta-chain gene. Finally, the cytoplasmic 98-kDa form enters the proteasome pathway and undergoes degradation. In conclusion, different forms of Elf-1 are the products of posttranslational modifications that determine its subcellular localization, activity, and metabolic degradation.

Tyrosine 315 determines optimal recruitment of ZAP-70 to the T cell antigen receptor.

Recruitment of ZAP-70 protein tyrosine kinase to the T cell antigen receptor (TCR) is mediated by the binding of the SH2 domains of this enzyme to phosphorylated ITAM motifs in the CD3 and TCRzeta subunits. We have previously shown that the efficiency of both positive and negative thymocyte selection was decreased in knock-in mice expressing ZAP-70 mutated at Tyr315 (ZAP-70-Y315F), a residue laying in the interdomain B of this protein. Surprisingly, in these cells the amount of phosphorylated TCRzeta chain co-precipitating with ZAP-70-Y315F was significantly reduced compared to control mice. We report now that the binding affinity of ZAP-70-Y315F to phosphorylated ITAM is reduced as compared to the wild-type protein, whereas the intrinsic catalytic activity is untouched. Consequently, phosphorylated ITAM appear to be more accessible to protein tyrosine phosphatases (PTP) and can be readily dephosphorylated. We provide evidence suggesting that the defective ITAM binding induced by Tyr315 mutation is independent of the putative role of this residue as a binding site for Vav-1. Finally, we found that the extracellular signal-regulated kinase pathway is impaired in ZAP-70-Y315F-expressing mice. Collectively, these results demonstrate that Tyr315 has an unsuspected structural role in ZAP-70 and may allosterically regulate the function of the nearby SH2 domains.

SIGNALLING SCAFFOLDS AND ADAPTORS IN T‐CELL IMMUNITY

SIGNALLING SCAFFOLDS AND ADAPTORS IN T‐CELL IMMUNITY

Analysis of the Individual Role of the TCRζ Chain in Transgenic Mice after Conditional Activation with Chemical Inducers of Dimerization

Signaling through the TCR/CD3 complex plays a critical role in T-cell development and activation. Gene-targeted mice lacking particular components of this complex show arrested T-cell development in the thymus. As all TCR/CD3 components are required for efficient surface expression of the complex, it is difficult to assess the specific signaling role of each receptor component. To overcome this problem, we designed a strategy to examine the specific role(s) of individual receptor chains. A chimeric protein, containing binding domains for chemical inducers of dimerization fused to the cytoplasmic tail of TCRζ, was generated. Activation of the chimeric receptor after stimulation with chemical dimerizers in Jurkat cells showed tyrosine phosphorylation of the TCRζ chain chimera, recruitment of phosphorylated Zap70, and generation of NFAT in a reporter assay. Analysis of thymocytes from transgenic mice expressing this chimeric receptor showed that intracytoplasmic crosslinking of the chimera induced tyrosine phosphorylation of the protein, as well as a slow and very weak calcium mobilization response. However, this signaling did not lead to increased expression of activation markers, T-cell proliferation, or apoptosis. In addition, stimulation of thymocytes in suspension or in fetal thymic organ cultures with chemical inducers of dimerization did not lead to alterations in positive or negative selection. We conclude that signaling through the TCRζ chain alone is not sufficient to generate downstream events leading to full T-cell activation or thymocyte selection; instead, additional CD3 components must be required to induce a functional response in primary thymocytes and peripheral T cells.

Rudimentary TCR signaling triggers default IL-10 secretion by human Th1 cells.

Understanding the process of inducing T cell activation has been hampered by the complex interactions between APC and inflammatory Th1 cells. To dissociate Ag-specific signaling through the TCR from costimulatory signaling, rTCR ligands (RTL) containing the alpha1 and beta1 domains of HLA-DR2b (DRA*0101:DRB1*1501) covalently linked with either the myelin basic protein peptide 85-99 (RTL303) or CABL-b3a2 (RTL311) peptides were constructed to provide a minimal ligand for peptide-specific TCRs. When incubated with peptide-specific Th1 cell clones in the absence of APC or costimulatory molecules, only the cognate RTL induced partial activation through the TCR. This partial activation included rapid TCR zeta-chain phosphorylation, calcium mobilization, and reduced extracellular signal-related kinase activity, as well as IL-10 production, but not proliferation or other obvious phenotypic changes. On restimulation with APC/peptide, the RTL-pretreated Th1 clones had reduced proliferation and secreted less IFN-gamma; IL-10 production persisted. These findings reveal for the first time the rudimentary signaling pattern delivered by initial engagement of the external TCR interface, which is further supplemented by coactivation molecules. Activation with RTLs provides a novel strategy for generating autoantigen-specific bystander suppression useful for treatment of complex autoimmune diseases.

Antigen-specific blockade of T cells in vivo using dimeric MHC peptide.

Ag-specific immune tolerance in clinical organ transplantation is currently an unrealized but critical goal of transplant biology. The specificity and avidity of multimerized MHC-peptide complexes suggests their potential ability to modulate T cell sensitization and effector functions. In this study, we examined the ability of MHC-peptide dimers to modulate T cell function both in vitro and in vivo. Soluble MHC dimers induced modulation of surface TCR expression and inhibited T cell cytolytic activity at nanomolar concentrations in vitro. Furthermore, engagement of TCR by soluble dimers resulted in phosphorylation of the TCR zeta-chain and recruitment and phosphorylation of zeta-associated protein-70 to the signaling complex, the latter of which increased upon dimer cross-linking. Significantly, Ag-specific inhibition of an alloreactive TCR-transgenic T cell population in vivo resulted in consequent outgrowth of an allogeneic tumor. The prolonged Ag-specific suppression of expansion and/or effector function of cognate T cells in vivo suggests that soluble MHC dimers may be a means of inducing sustained Ag-specific T cell unresponsiveness in vivo.

The transmembrane adaptor protein TRIM regulates T cell receptor (TCR) expression and TCR-mediated signaling via an association with the TCR zeta chain.

T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-zeta chains and to a lesser extent via the CD3-straightepsilon/gamma heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca(2+) mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.

Functional Uncoupling of T-cell Receptor Engagement and Lck Activation in Anergic Human Thymic CD4+T Cells

Human thymic CD1a-CD4+ T cells in the final stage of thymic maturation are susceptible to anergy induced by a superantigen, toxic shock syndrome toxin-1 (TSST-1). Thymic CD4+ T-cell blasts, established by stimulating human thymic CD1a-CD4+ T cells with TSST-1 in vitro, produce a low level of interleukin-2 after restimulation with TSST-1, whereas TSST-1-induced adult peripheral blood (APB) CD4+ T-cell blasts produce high levels of interleukin-2. The extent of tyrosine phosphorylation of the T-cell receptor zeta chain induced after restimulation with TSST-1 was 2-4-fold higher in APB CD4+ T-cell blasts than in thymic CD4+ T-cell blasts. The tyrosine kinase activity of Lck was low in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, the Lck kinase activity increased in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Surprisingly, Lck was highly tyrosine-phosphorylated in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, it was markedly dephosphorylated in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Lck from APB CD4+ T-cell blasts bound the peptide containing the phosphotyrosine at the negative regulatory site of Lck-505 indicating that the site of dephosphorylation in TSST-1-activated T-cell blasts is Tyr-505. Confocal microscopy demonstrated that colocalization of Lck and CD45 was induced after restimulation with TSST-1 in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Further, remarkable accumulation of Lck in the membrane raft was observed in restimulated APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. These data indicate that interaction between Lck and CD45 is suppressed physically in thymic CD4+ T-cell blasts and plays a critical role in sustaining an anergic state.

Prolonged exposure of T cells to TNF down-regulates TCR zeta and expression of the TCR/CD3 complex at the cell surface.

A role for TNF-alpha in the pathogenesis of chronic inflammatory disease is now firmly established. Paradoxically, TNF also has potent immunomodulatory effects on CD4(+) T lymphocytes, because Ag-specific proliferative and cytokine responses are suppressed following prolonged exposure to TNF. We explored whether TNF attenuated T cell activation by uncoupling proximal TCR signal transduction pathways using a mouse T cell hybridoma model. Chronic TNF exposure induced profound, but reversible, T cell hyporesponsiveness, with TNF-treated T cells requiring TCR engagement with higher peptide concentrations for longer periods of time for commitment to IL-2 production. Subsequent experiments revealed that chronic TNF exposure led to a reversible loss of TCRzeta chain expression, in part through a reduction in gene transcription. Down-regulation of TCRzeta expression impaired TCR/CD3 assembly and expression at the cell surface and uncoupled membrane-proximal tyrosine phosphorylation events, including phosphorylation of the TCRzeta chain itself, CD3epsilon, ZAP-70 protein tyrosine kinase, and linker for activation of T cells (LAT). Intracellular Ca(2+) mobilization was also suppressed in TNF-treated T cells. We propose that TNF may contribute to T cell hyporesponsiveness in chronic inflammatory and infectious diseases by mechanisms that include down-regulation of TCRzeta expression. We speculate that by uncoupling proximal TCR signals TNF could also interrupt mechanisms of peripheral tolerance that are dependent upon intact TCR signal transduction pathways.

Polymorphisms/Mutations of TCR-ζ-Chain Promoter and 3′ Untranslated Region and Selective Expression of TCR ζ-Chain with an Alternatively Spliced 3′ Untranslated Region in Patients with Systemic Lupus Erythematosus

A vast majority of systemic lupus erythematosus (SLE) patients display decreased expression of TCR ζ-chain mRNA, a critical signaling molecule implicated in the selection of the TCR repertoire and in the prevention of autoimmunity. To identify the molecular mechanisms involved in the downregulation of TCR ζ-chain transcripts in SLE T cells, we investigated the possibility of polymorphisms/mutations in the promoter and the 3′ untranslated region. PCR, cloning and sequence analysis of the promoter region from the genomic DNA showed significantly higher number of polymorphisms in SLE T cells compared to non-SLE control subjects (P=0.044). Promoter sequence was also analysed from granulocytes to delineate the possibility of somatic mutations in activated SLE T cells. Promoter polymorphisms were significantly higher in granulocytes of SLE patients compared to non-SLE controls (P=0.048), suggesting that these polymorphisms were of genomic origin. Nucleotide analysis of the promoter sequence revealed a −76T insertion compared to the published sequence, in all of the SLE samples and controls. RT–PCR analysis of the TCR ζ-chain 3′ untranslated region showed a 344bp product in addition to the expected 906bp product. Cloning and sequence analysis of the 344bp product indicated that it is an alternatively spliced form with both splicing donor and acceptor sites, resulting in deletion of nucleotides 672–1233 of TCR ζ-chain mRNA. Unlike the nomal TCR ζ-chain, the expression of TCR ζ-chain with the alternatively spliced 344bp 3′ untranslated region was higher in SLE T cells compared to non-SLE controls. The number of mutations/polymorphisms in the 906bp TCR ζ-chain 3′ untranslated region were significantly higher in SLE T cells compared to non-SLE subjects (P=0.032). Frequent mutations/polymorphisms and aberrant splicing of the downstream 3′ untranslated region may affect the stability and/or transport of TCR ζ-chain mRNA, leading to its downregulation in SLE T cells.

Critical relationship between TCR signaling potential and TCR affinity during thymocyte selection.

Whether a developing thymocyte becomes positively or negatively selected is thought to be determined by the affinity/avidity of its TCR for MHC/peptide ligands expressed in the thymus. Presumably, differences in affinity translate into differences in the potency of the ensuing TCR-mediated signals, and these differences in signal strength determine the outcome of thymocyte selection. However, there is little direct evidence establishing a relationship between TCR-ligand affinity and signal strength during positive and negative selection. The TCR complex contains multiple signaling motifs, known as immunoreceptor tyrosine-based activation motifs (ITAMs) that are required for T cell activation. To examine the effects of TCR signal strength on selection, the signaling potential of the TCR was modified by substituting transgenic TCR zeta-chains containing either three, one, or zero ITAMs for endogenous (3-ITAM) zeta-chain. These zeta-chain variants were then bred into different alphabetaTCR transgenic backgrounds. We report that reductions in TCR signaling potential have distinct effects on the selection of thymocytes expressing different TCRs, and that the requirement for zeta-chain ITAMs critically depends upon the specificity and apparently, affinity, of the TCR for its selecting ligand(s).

Tumor-induced apoptosis of T lymphocytes: elucidation of intracellular apoptotic events

AbstractOur recent studies suggest that human squamous cell carcinoma of the head and neck (SCCHN) is capable of activating an intrinsic mechanism of programmed-cell death in interacting lymphocytes in situ and in vitro. The current study used Jurkat T-cell line as a model to investigate intracellular apoptotic events in T cells interacting with SCCHN. Apoptosis induced in T lymphocytes by tumor cells was in part Fas-mediated, since it was partially, but significantly, inhibited in the presence of anti-Fas ligand Ab or in Fas-resistant Jurkat cells. The synthetic caspase inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) and N-benzyloxycarbonyl-Asp-glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK), effectively blocked apoptosis of Jurkat cells co-incubated with SCCHN cell lines, suggesting the involvement of caspases in tumor-induced apoptosis of lymphocytes. Overexpression of CrmA, an inhibitor of caspase-1 and caspase-8, partially inhibited tumor-induced T-cell death. Caspase-8 and caspase-3 were identified as effector molecules in the execution of tumor-induced T-cell death, since the proform enzymes were processed into active subunits during co-incubation of T cells with tumor cells. Furthermore, co-incubation with tumor cells resulted in cleavage of poly(ADP-ribose) polymerase (PARP), a common caspase-3 substrate, and in cleavage of TcR-ζ chain, shown by us to be a T-cell specific caspase-3 substrate. Overexpression of Bcl-2 did not provide protection of T cells from SCCHN-induced DNA degradation. Instead, the Bcl-2 protein was cleaved in the target T cells during their co-incubation with tumor cells. These findings demonstrate that tumor cells can trigger in T lymphocytes caspase-dependent apoptotic cascades, which are not effectively protected by Bcl-2.

Translocation of tyrosine-phosphorylated TCRzeta chain to glycolipid-enriched membrane domains upon T cell activation.

Recent studies point to glycolipid-enriched membrane (GEM) microdomains as the critical sites for TCR-mediated signal transduction. However, whether the TCR complex is localized in the GEM domain is not well-defined. In the present study, we analyzed localization of the TCR-CD3 complex in the GEM domain by isolating the GEM fraction with sucrose density gradient centrifugation. Although 10% of TCRzeta chains was localized in the GEM fraction, most of the TCR complexes were excluded from the GEM before and after T cell activation, and the amount of TCRzeta in the GEM was not increased after activation. However, the tyrosine-phosphorylated form of TCRzeta was strongly concentrated in the GEM fraction upon TCR engagement. A kinetic study revealed that tyrosine phosphorylation of TCRzeta occurred initially in the Triton X-100-soluble membrane fraction followed by the accumulation of phosphorylated TCRzeta in the GEM. Thus, these results indicate that phosphorylated TCRzeta migrates into the GEM domains on T cell activation. We speculate that the GEM microdomains may function as a reservoir of activation signals from triggered TCR.

Perturbed Regulation of ZAP-70 and Sustained Tyrosine Phosphorylation of LAT and SLP-76 in c-Cbl-Deficient Thymocytes

AbstractRecent studies indicate that c-Cbl and its oncogenic variants can modulate the activity of protein tyrosine kinases. This finding is supported by studies showing that c-Cbl interacts directly with a negative regulatory tyrosine in ZAP-70, and that the levels of tyrosine-phosphorylated ZAP-70 and numerous other proteins are increased in TCR-stimulated thymocytes from c-Cbl-deficient mice. Here, we demonstrate that this enhanced phosphorylation of ZAP-70 and that of two substrates, LAT and SLP-76, is not due to altered protein levels but is the consequence of two separate events. First, we find increased expression of tyrosine-phosphorylated TCRζ chain in c-Cbl-deficient thymocytes, which results in a higher level of ζ-chain-associated ZAP-70 that is initially accessible for activation. Thus, more ZAP-70 is activated and more of its substrates (LAT and SLP-76) become tyrosine-phosphorylated after TCR stimulation. However, an additional mechanism of ZAP-70 regulation is evident at a later time poststimulation. At this time, ZAP-70 from both normal and c-Cbl−/− thymocytes becomes hyperphosphorylated; however, only in normal thymocytes does this correlate with ZAP-70 down-regulation and a diminished ability to phosphorylate LAT and SLP-76. In contrast, c-Cbl-deficient thymocytes display altered phosphorylation kinetics, for which LAT phosphorylation is increased and SLP-76 phosphorylation is sustained. Thus, the ability to down-regulate the phosphorylation of two ZAP-70 substrates is impaired in c-Cbl−/− thymocytes. These findings provide evidence that c-Cbl is involved in the negative regulation of the phosphorylation of LAT and SLP-76 by ZAP-70.

The cytoplasmic tail of killer inhibitory receptor (KIR) associates with TCR ζ in a phosphorylation-dependent manner

Killer inhibitory receptor (KIR) inhibits cytolytic function of killer cells by specific interaction with class I MHC molecules. The inhibitory effect mediated by KIR requires co-engagement of KIR with an activating receptor, such as TCR or FcR. This implies that KIR may function in the immediate vicinity of activating molecules, and previous studies have shown that p58 KIR is associated with TCR ζ- and FcR γ-chain in NK cells. To better understand the molecular interaction between KIR and TCR ζ-chain, we generated a His-tag fusion protein of a p70 KIR cytoplasmic tail (His-CytKIR) and used this protein to coprecipitate TCR ζ-chain from Jurkat T cells. Western blots of the resolved coprecipitates showed that the cytoplasmic tail of KIR associates with TCR ζ in vitro. Interestingly, the association between the His-CytKIR and TCR ζ was dependent on the phosphorylation of the His-CytKIR. Unlike the unphosphorylated His-CytKIR, the phosphorylated form no longer associated with TCR ζ. However, the association was not affected by the tyrosine phosphorylation of TCR ζ. These results suggest that the cytoplasmic tail of KIR may couple to TCR ζ in a phosphorylation-dependent manner, so it could fine-tune the activation signals induced via the TCR.

The TCR ζ-Chain Immunoreceptor Tyrosine-Based Activation Motifs Are Sufficient for the Activation and Differentiation of Primary T Lymphocytes

AbstractThe TCR complex signals through a set of 10 intracytoplasmic motifs, termed immunoreceptor tyrosine-based activation motifs (ITAMs), contained within the γ-, δ-, ε-, and ζ-chains. The need for this number of ITAMs is uncertain. Limited and contradictory studies have examined the ability of subsets of the TCR’s ITAMs to signal into postthymic primary T lymphocytes. To study signaling by a restricted set of ITAMs, we expressed in transgenic mice a chimeric construct containing the IAs class II MHC extracellular and transmembrane domains linked to the cytoplasmic domain of the TCR ζ-chain. Tyrosine phosphorylation and receptor cocapping studies indicate that this chimeric receptor signals T cells independently of the remainder of the TCR. We show that CD4+ and CD8+ primary T cells, as well as naive and memory T cells, are fully responsive to stimulation through the IAs-ζ receptor. Further, IAs-ζ stimulation can induce primary T cell differentiation into CTL, Th1, and Th2 type cells. These results show that the ζ-chain ITAMs, in the absence of the γ, δ, and ε ITAMs, are sufficient for the activation and functional maturation of primary T lymphocytes. It also supports the isolated use of the ζ-chain ITAMs in the development of surrogate TCRs for therapeutic purposes.