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Loss of ζ-associated protein 70 (Zap70) results in severe immunodeficiency in humans and mice because of the critical role of Zap70 in T-cell receptor (TCR) signalling. Here we describe a novel mouse strain generated by N-ethyl-N-nitrosourea mutagenesis, with the reduced protein stability (rps) mutation in Zap70. The A243V rps mutation resulted in decreased Zap70 protein and a reduced duration of TCR-induced calcium responses, equivalent to that induced by a 50% decrease in catalytically active Zap70. The reduction of signalling through Zap70 was insufficient to substantially perturb thymic differentiation of conventional CD4 and CD8 T cells, although Foxp3(+) regulatory T cells demonstrated altered thymic production and peripheral homeostasis. Despite the mild phenotype, the Zap70(A243V) variant lies just above the functional threshold for TCR signalling competence, as T cells relying on only a single copy of the Zap70(rps) allele for TCR signalling demonstrated no intracellular calcium response to TCR stimulation. This addition to the Zap70 allelic series indicates that a rate-limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null., (© 2013 John Wiley & Sons Ltd.)

Many autoimmune diseases exhibit familial aggregation, indicating that they have genetic determinants. Single nucleotide polymorphisms in PTPN2, which encodes T cell protein tyrosine phosphatase (TCPTP), have been linked with the development of several autoimmune diseases, including type 1 diabetes and Crohn's disease. In this study, we have identified TCPTP as a key negative regulator of TCR signaling, which might explain the association of PTPN2 SNPs with autoimmune disease. We found that TCPTP dephosphorylates and inactivates Src family kinases to regulate T cell responses. Using T cell-specific TCPTP-deficient mice, we established that TCPTP attenuates T cell activation and proliferation in vitro and blunts antigen-induced responses in vivo. TCPTP deficiency lowered the in vivo threshold for TCR-dependent CD8(+) T cell proliferation. Consistent with this, T cell-specific TCPTP-deficient mice developed widespread inflammation and autoimmunity that was transferable to wild-type recipient mice by CD8(+) T cells alone. This autoimmunity was associated with increased serum levels of proinflammatory cytokines and anti-nuclear antibodies, T cell infiltrates in non-lymphoid tissues, and liver disease. These data indicate that TCPTP is a critical negative regulator of TCR signaling that sets the threshold for TCR-induced naive T cell responses to prevent autoimmune and inflammatory disorders arising.

Genome-wide association studies (GWAS) have identified a large number of SNPs that are linked to human autoimmune diseases. However, the functional consequences of most of these genetic variations remain undefined. T cell protein tyrosine phosphatase (TCPTP, which is encoded by PTPN2) is a JAK/STAT and growth factor receptor phosphatase that has been linked to the pathogenesis of type 1 diabetes, rheumatoid arthritis, and Crohn's disease by GWAS. In this issue of the JCI, Wiede and colleagues have generated a T cell-specific deletion of TCPTP and identified a novel role for this phosphatase as a negative regulator of TCR signaling. These data provide new insight as to how noncoding PTPN2 SNPs identified in GWAS could drive human autoimmune diseases.

The CXCR4 chemokine receptor is a G protein-coupled receptor that signals in T lymphocytes by forming a heterodimer with the 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 MAPK and downstream AP-1-dependent cytokine transcription in response to stromal cell-derived factor-1 (SDF-1), the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate G protein-coupled receptor-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. In this study, 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 calcium ion concentrations, whereas 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 to distinctly regulate migration versus other signaling functions.

CD45 is the most abundant protein tyrosine phosphatase in the plasma membrane of T cells and serves a critical role in TCR signaling. Different CD45 isoforms are made by alternative mRNA splicing depending on the stage of T cell development and activation, yet their role remains unclear. Expression of CD45RA and RC isoforms is increased 20- to 200-fold on T cells from thunder mice with a loss-of-function mutation in the RNA-binding protein, heterogeneous nuclear ribonucleoprotein L-like (hnRNPLL), although total CD45 expression is unaltered. In this study, we test the hypothesis that this shift in CD45 isoform expression alters TCR signaling, thymic selection, and accumulation of peripheral T cells. There was no discernable effect of the change in CD45 isoform expression upon Lck phosphorylation or T cell positive and negative selection, whereas these indices were strongly affected by a decrease in the overall amount of CD45 in Ptprc mutant animals. The one exception to this conclusion was in thymocytes from Ptprc(loc/loc) animals with 4% of normal CD45 protein levels, where Lck505 phosphorylation was increased 25% in Hnrpll mutant cells, suggesting that high m.w. CD45 isoforms had lower Lck505 phosphatase activity in this context. In T cells with no CD45 protein, hnRNPLL mutation still diminished peripheral T cell accumulation, demonstrating that hnRNPLL regulates T cell longevity independently from its effects on CD45 splicing.

Human Vgamma9Vdelta2 T cells, a major innate-like peripheral T cell subset, are thought to play in vivo an important role in innate and adaptive immune responses to infection agents and tumors. However, the mechanisms regulating their broad effector functions, such as cytotoxicity and cytokine responses, remain poorly understood. In this study, we used single-cell calcium video imaging to analyze the early intracellular events associated with TCR-induced Vgamma9Vdelta2 T cell functional responses. When compared with other human T cell subsets, including NKT and Vdelta2(neg) gammadelta T cells, TCR/CD3-activated Vgamma9Vdelta2 T cells displayed an unusually delayed and sustained intracellular calcium mobilization, which was dramatically quickened and shortened on costimulation by NKG2D, a main activating NKR regulating gammadelta T cell tumor cytolysis. Importantly, the protein kinase C transduction pathway was identified as a main regulator of the NKG2D-mediated costimulation of antitumor Vgamma9Vdelta2 cytolytic responses. Therefore, this study identifies a new mechanism regulating Vgamma9Vdelta2 T cell functional plasticity through fine-tuning of early signal transduction events.

Immune responses to citrullinated neoantigens and clinical efficacy of costimulation blockade indicate a general defect in maintaining T cell tolerance in rheumatoid arthritis (RA). To examine whether TCR threshold calibration contributes to disease pathogenesis, signaling in RA T cells was quantified. RA patients had a selective increase in ERK phosphorylation compared with demographically matched controls due to a mechanism distal of Ras activation. Increased ERK responses included naive and memory CD4 and CD8 T cells and did not correlate with disease activity. The augmented ERK activity delayed SHP-1 recruitment to the TCR synapse and sustained TCR-induced Zap70 and NF-kappaB signaling, facilitating responses to suboptimal stimulation. Increased responsiveness of the ERK pathway was also a characteristic finding in the SKG mouse model of RA where it preceded clinical symptoms. Treatment with subtherapeutic doses of a MEK-1/2 inhibitor delayed arthritis onset and reduced severity, suggesting that increased ERK phosphorylation predisposes for autoimmunity and can be targeted to prevent disease.

MHC class II expression identifies an effector subset of human CD4(+)CD25(high)FoxP3(high) natural regulatory T cells (DR(+) Tregs) that induces more rapid suppression and exhibits higher FoxP3 expression than the remaining Treg population. Although Tregs are known to be highly sensitive to apoptosis, in this study we demonstrate that this sensitivity is primarily a feature of DR(+) Tregs. Granzyme B (GzmB) is strongly expressed by nonregulatory responder CD4 T cells, whereas effector DR(+) Tregs express little GzmB. Strong TCR stimulation markedly increases the expression of GzmB in all dividing responder CD4 T cells and mitigates the suppression by DR(+) Tregs. DR(+) Treg suppressive activity reemerges if GzmB is neutralized. We show that responder cells actively kill effector Tregs by producing GzmB in response to strong TCR stimulation. Thus, the production of GzmB by strongly activated CD4 T cells represents a mechanism by which CD4 T cells resist Treg suppression.

The addition of calcineurin inhibitors, including cyclosporine A (CsA) and FK-506 (tacrolimus), to transplant protocols has markedly reduced acute allograft rejection and prolonged patient survival. Although monitoring of serum drug levels has been shown to be a poor indicator of efficacy, there is little data on calcineurin enzymatic activity in humans. Therefore, we measured calcineurin in isolated CD3(+)/4(+) T cells from 81 non-transplant controls and 39 renal allograft patients by using a (32)PO(4)-labeled calcineurin-specific substrate. A gender difference was observed in the control cohort, with activity in males significantly higher than that in females (1073 +/- 134 versus 758 +/- 75 fmol/microg/min, respectively). Activity of both groups was comparably inhibited by 5 ng/ml tacrolimus (27 +/- 4 versus 30 +/- 4%). Calcineurin is a downstream target of the T-cell receptor (TCR). Therefore, activity was measured in isolated T cells after incubation with anti-CD3/CD28 antibodies to stimulate the TCR. Calcineurin activity increased significantly from 1214 +/- 111 to 1652 +/- 138 fmol/microg/min; addition of either tacrolimus or CsA (500 ng/ml) blocked CD3/CD28 stimulation. Despite therapeutic levels of tacrolimus and CsA (mean 11.4 and 172 ng/ml), basal calcineurin activity was significantly higher among renal transplant recipients than controls (1776 +/- 175 versus 914 +/- 78 fmol/microg/min). In contrast, anti-CD3/CD28 antibodies failed to stimulate calcineurin activity in transplant subjects. Finally, we found that basal and stimulated calcineurin activities are inversely related. Consistent with this finding, basal activity in resting T cells rose over time after transplant but stimulation fell (r(2) = 0.785, p < 0.05). These data suggest that examination of TCR-stimulated calcineurin activity after renal transplantation may be useful for monitoring immunosuppression of individual patients.

Short-lived TCR microclusters and a longer-lived protein kinase Ctheta-focusing central supramolecular activation cluster (cSMAC) have been defined in model immunological synapses (IS). In different model systems, CD28-mediated costimulatory interactions have been detected in microclusters, the cSMAC, or segregated from the TCR forming multiple distinct foci. The relationship between TCR and costimulatory molecules in the physiological IS of T cell-dendritic cell (DC) is obscure. To study the dynamic relationship of CD28-CD80 and TCR interactions in the T cell-DC IS during Ag-specific T cell activation, we generated CD80-eCFP mice using bacterial artificial chromosome transgenic technology. In splenic DCs, endogenous CD80 and CD80-eCFP localized to plasma membrane and Golgi apparatus, and CD80-eCFP was functional in vivo. In the OT-II T cell-DC IS, multiple segregated TCR, CD80, and LFA-1 clusters were detected. In the T cell-DC synapse CD80 clusters were colocalized with CD28 and PKCtheta, a characteristic of the cSMAC. Acute blockade of TCR signaling with anti-MHC Ab resulted in a rapid reduction in Ca(2+) signaling and the number and size of the CD80 clusters, a characteristic of TCR microclusters. Thus, the T cell-DC interface contains dynamic costimulatory foci that share characteristics of microclusters and cSMACs.

The CD8 coreceptor plays a crucial role in thymocyte and T cell sensitivity by binding to class I MHC and recruiting downstream signaling molecules to the TCR. Previous studies reported considerable changes in TCR-independent CD8/class I MHC binding (i.e., CD8 noncognate interactions) during T cell development, changes that correlated with altered glycosylation of surface molecules. In particular, expression of the sialyltransferase ST3Gal-I has been proposed as a critical factor regulating the attenuation of CD8 avidity during the double-positive to CD8 single-positive progression. This hypothesis is strengthened by the fact that ST3Gal-I(-/-) animals show a profound disregulation of CD8 T cell homeostasis. In contrast to this model, however, we report in this study that ST3Gal-I deficiency had no detectable impact on CD8 noncognate binding to multimeric peptide/MHC class I ligands at any stage of thymocyte development. We also found that the susceptibility to CD8-induced cell death is not markedly influenced by ST3Gal-I deficiency. Thus, the profound effects of ST3Gal-I on CD8 T cell survival evidently do not involve a role for this enzyme in controlling CD8-class I binding.

Mucosal immune tolerance in the healthy intestine is typified by lamina propria T cell (LPT) functional hyporesponsiveness after TCR engagement when compared with peripheral blood T cell (PBT). When LPT from an inflamed intestine are activated through TCR cross-linking, their responsiveness is stronger. LPT are thus capable of switching from a tolerant to a reactive state, toggling between high and low thresholds of activation. We demonstrate that in normal LPT global tyrosine phosphorylation upon TCR cross-linking or an increase in intracellular H2O2, an inhibitor of protein tyrosine phosphatases, is muted. Thus, we propose that LPT have a greater reducing capacity than PBT, shifting the balance between kinases and protein tyrosine phosphatases in favor of the latter. Surface gamma-glutamyl transpeptidase, an indirect indicator of redox potential, and glutathione are significantly elevated in LPT compared with PBT, suggesting that elevated glutathione detoxifies TCR-induced reactive oxygen species. When glutathione is depleted, TCR-induced LPT tyrosine phosphorylation rises to PBT levels. Conversely, increasing glutathione in PBT attenuates tyrosine phosphorylation. In LPT isolated from inflamed mucosa, TCR cross-linking induces greater phosphorylation, and gamma-glutamyl transpeptidase levels are reduced compared with those from autologous noninflamed tissue. We conclude that the high TCR signaling threshold of mucosal T cells is tuned by intracellular redox equilibrium, whose dysregulation may mediate intestinal inflammation.

T cell development is regulated at two critical checkpoints that involve signaling events through the TCR. These signals are propagated by kinases of the Src and Syk families, which activate several adaptor molecules to trigger Ca(2+) release and, in turn, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. In this study, we show that a constitutively active form of CaMKII antagonizes TCR signaling and impairs positive selection of thymocytes in mice. Following TCR engagement, active CaMKII decreases TCR-mediated CD3zeta chain phosphorylation and ZAP70 recruitment, preventing further downstream events. Therefore, we propose that CaMKII belongs to a negative-feedback loop that modulates the strength of the TCR signal through the tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP-2).

Signaling through the T cell antigen receptor leading to elimination (negative selection) or differentiation (positive selection) of developing thymocytes generates a self-tolerant T cell repertoire. Here we report that the serine-threonine kinase MINK selectively connects the T cell receptor to a signaling pathway that mediates negative but not positive selection. Analysis of this pathway suggested that the essential function of MINK in the elimination of self-reactive thymocytes may be associated with 'downstream' activation of Jun kinase and enhancement of expression of the proapoptotic molecule Bim.

Among the three isoenzymes of neuraminidase (Neu) or sialidase, Neu-1 has been suggested to be induced by cell activation and to be involved in IL (interleukin)-4 biosynthesis in murine T-cells. In the present study, we found that antigen-induced airway eosinophilia, a typical response dependent on Th2 (T-helper cell type 2) cytokines, as well as mRNA expression of Th2 cytokines, including IL-4, are suppressed in Neu-1-deficient mice, thereby demonstrating the in vivo role of murine Neu-1 in regulation of Th2 cytokines. To elucidate the roles of various sialidases in human T-cell activation, we investigated their tissue distribution, gene induction and function. Neu-1 is the predominant isoenzyme at the mRNA level in most tissues and cells in both mice and humans, including T-cells. T-cells also have significant levels of Neu-3 mRNAs, albeit much lower than those of Neu-1, whereas the levels of Neu-2 mRNAs are minimal. In human T-cells, both Neu-1 and Neu-3 mRNAs are significantly induced by T-cell-receptor stimulation, as is sialidase activity against 4-methylumbelliferyl- N -acetylneuramic acid (a substrate for both Neu-1 and Neu-3) and the ganglioside G(D1a) [NeuAcalpha2-3Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-cer] (a substrate for Neu-3, but not for Neu-1). The expression of the two sialidase genes may be under differential regulation. Western blot analysis and enzymic comparison with recombinant sialidases have revealed that Neu-3 is induced as a major isoform in activated cells. The induction of Neu-1 and Neu-3 in T-cells is unique. In human monocytes and neutrophils stimulated with various agents, the only observation of sialidase induction has been by IL-1 in neutrophils. Functionally, a major difference has been observed in Jurkat T-cell lines over-expressing Neu-1- and Neu-3. Upon T-cell receptor stimulation, IL-2, interferon-gamma, IL-4 and IL-13 are induced in the Neu-1 line, whereas in the Neu-3 line the same cytokines are induced, with the exception of IL-4. Taken together, these results suggest an important immunoregulatory role for both Neu-1 and Neu-3 in humans.

T cell activation is associated with the partitioning of TCRs and other signaling proteins, forming an immunological synapse. This study demonstrates a novel function for the CD4 coreceptor in regulating molecular clustering at the immunological synapse site. We show using transgenic mouse and retroviral reconstitution studies that CD4 is required for TCR/protein kinase C (PKC) theta clustering. Specifically, we demonstrate that CD4 palmitoylation sequences are required for TCR/PKCtheta raft association and subsequent clustering, indicating a particular role for raft-associated CD4 molecules in regulating immune synapse organization. Although raft association of CD4 is necessary, it is not sufficient to mediate clustering, as cytoplasmic tail deletion mutants are able to localize to rafts, but are unable to mediate TCR/PKCtheta clustering, indicating an additional requirement for CD4 signaling. These studies suggest that CD4 coreceptor function is regulated not only through its known signaling function, but also by posttranslational lipid modifications which regulate localization of CD4 in lipid rafts.

TCR engagement can induce either T cell proliferation and differentiation or activation-induced T cell death (AICD) through apoptosis. The intracellular signaling pathways that dictate such a disparate fate after TCR engagement have only been partially elucidated. Non-FcR-binding anti-CD3 mAbs induce a partial agonist TCR signaling pattern and cause AICD on Ag-activated, cycling T cells. In this study, we examined TCR signaling during the induction of AICD by anti-CD3 fos, a non-FcR-binding anti-CD3 mAb. This mAb activates Fyn, Lck, and extracellular signal-regulated kinase, and induces phosphorylation of Src-like adapter protein, despite the inability to cause calcium mobilization or TCR polarization. Anti-CD3 fos also fails to effectively activate zeta-associated protein of 70 kDa or NF-kappaB. Using Ag-specific T cells deficient for Fyn or Lck, we provide compelling evidence that activation of Lck is required for the induction of AICD. Our data indicate that a selective and distinct TCR signaling pattern is required for AICD by TCR partial agonist ligands.

CD24, also referred to as the heat-stable Ag, is a T cell differentiation Ag that is highly expressed on both CD4-CD8- double negative and CD4+CD8+ double positive thymocytes. Here, we report that CD24 ligation by a new anti-CD24 Ab, mT-20, induced the apoptosis of both double negative and double positive thymocytes, as well as the Scid.adh thymic lymphoma cell line, in the absence of TCR/CD3 engagement. CD24-mediated apoptosis of mouse thymocytes and its signaling pathway appeared not to be associated with p53, CD95, TNFR, or caspases. Furthermore, we found that cell death was blocked by the addition of scavengers of reactive oxygen species or by Bcl-2 overexpression, implying the role of CD24 signaling in the mitochondrial regulation. In this study, we suggest that CD24 ligation induced the apoptosis of immature thymocytes independently of both caspase and TCR.

Chimeric receptors that include CD28 signaling in series with TCRzeta in the same receptor have been demonstrated to activate prestimulated human primary T cells more efficiently than a receptor providing TCRzeta signaling alone. We examined whether this type of receptor can also activate resting human primary T cells, and whether molecules other than CD28 could be included in a single chimeric receptor in series with TCRzeta to mediate the activation of resting human primary T cells. Human CD33-specific chimeric receptors were generated with CD28, inducible costimulator, CD134, or CD137 signaling regions in series with TCRzeta signaling region and transfected by electroporation into resting human primary T cells. Their ability to mediate Ag-specific activation was analyzed in comparison with a receptor providing TCRzeta signaling alone. Inclusion of any of the costimulatory signaling regions in series with TCRzeta enhanced the level of specific Ag-induced IL-2, IFN-gamma, TNF-alpha, and GM-CSF cytokine production and enabled resting primary T cells to survive and proliferate in response to Ag in the absence of any exogenous factors. Inclusion of CD28, inducible costimulator, or CD134 enhanced TCRzeta-mediated, Ag-specific target cell lysis. Chimeric receptors providing B7 and TNFR family costimulatory signals in series with TCRzeta in the same receptor can confer self-sufficient clonal expansion and enhanced effector function to resting human T cells. This type of chimeric receptor may now be used to discover the most potent combination of costimulatory signals that will improve current immunotherapeutic strategies.

Notch genes encode membrane receptors that regulate cell fate decisions in metazoa. Notch receptors and ligands are expressed in developing lymphoid tissue and mature lymphocytes and the role of Notch signaling in early T and B cell development has been studied extensively. However, its contribution to mature T cell function is unknown. TCR-mediated T cell activation is a fundamental process of the adaptive immune system that has been studied for decades; however, the details of this process are incompletely understood. In this study, we present evidence that Notch is required for TCR-mediated activation of peripheral T cells. Inhibition of Notch activation dramatically decreases T cell proliferation in both CD4 and CD8 cells and blocks both NF-kappaB activity and IFN-gamma production in peripheral T cells. Our data reveal a new, nondevelopmental function of Notch as a previously unknown key link in peripheral T cell activation and cytokine secretion.

Negative selection is the process whereby immature thymocytes expressing TCRs with high affinity for self-peptide:MHC complexes are induced to undergo apoptosis. The transcriptional events that occur as a result of TCR signaling during negative selection are not well-characterized. Using oligonucleotide arrays, we have identified 33 genes that exhibit changes in RNA levels in CD4(+)CD8(+) thymocytes during negative selection in vivo. Of 18 genes that have been further characterized, 13 are regulated in response to stimulation with Ag or anti-CD3 and anti-CD28 Abs ex vivo, indicating that these genes are regulated independently of activation of the peripheral immune system. These data also support the idea that anti-CD3/CD28-mediated thymocyte apoptosis is a valid model for negative selection in vivo. A detailed examination of the regulation of many of the identified genes in response to treatment with dexamethasone or gamma-radiation or in response to anti-CD3/anti-CD28 stimulation in the presence of pharmacological inhibitors of mitogen-activated protein kinase kinase kinase 1, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, calcineurin, and cyclin-dependent kinase 2 has facilitated the elucidation of a map of the transcriptional events that occur downstream of the TCR. These studies support a model whereby similar signal transduction pathways are activated by stimuli that induce positive and negative selection and are consistent with the idea that the balance between opposing proapoptotic and antiapoptotic pathways determines cell fate. The data presented in this study also suggest that calcineurin functions to amplify TCR signals by promoting sustained increases in the levels of specific transcripts.

Altered peptide ligands (APL) induce T cell responses different from those induced by the original agonistic peptide. As shown for CD4(+) T cells, partial agonists induce partial T cell activation without proliferation because of lower affinities and higher off rates to TCR than those of agonists. To determine whether overexpression of partially agonistic TCR ligands on antigen-presenting cells provides high-avidity TCR ligands, we generated L cell transfectants expressing various numbers of HLA-DR4 covalently linked with APL derived from a streptococcal peptide and observed responses of the cognate T cells. Some overexpressed HLA-DR4/partially agonistic APL complexes induced T cell proliferation in a density-dependent manner. However, tyrosine phosphorylation of zeta-associated protein-70 (ZAP-70) and linker for activation of T cells and kinase activity of ZAP-70 were not detectable. T cell proliferation stimulated with L cell transfectants was sensitive to the PKC inhibitor Gö6976, but to a lesser extent to Gö6983, suggesting the involvement of mu isotype of PKC (PKCmu). In vitro kinase assays revealed that PKCmu activity was up-regulated only in T cells stimulated with L cell transfectants that induced T cell proliferation. Our data suggest the presence of a unique signaling pathway coupling TCR ligation with T cell proliferation associated with PKCmu activation and impaired ZAP-70 activation.

The TCR-mediated signals required to activate resting T cells have been well characterized; however, it is not known how TCR-coupled signals are transduced in differentiated effector T cells that coordinate ongoing immune responses. Here we demonstrate that human effector CD4 T cells up-regulate the expression of the CD3zeta-related FcRgamma signaling subunit that becomes part of an altered TCR/CD3 signaling complex containing CD3epsilon, but not CD3zeta. The TCR/CD3/FcRgamma complex in effector cells recruits and activates the Syk, but not the ZAP-70, tyrosine kinase. This physiologic switch in TCR signaling occurs exclusively in effector, and not naive or memory T cells, suggesting a potential target for manipulation of effector responses in autoimmune, malignant, and infectious diseases.

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

TCR and cytokine signals induce naive T cells to undergo spontaneous divisions as part of a homeostatic response to conditions of T cell deficiency. The conditions under which these signals evoke the homeostatic response and their interaction with each other are poorly understood, and yet are very important clinically in considering strategies for immune reconstitution. Here, we show that p56(lck) (lck)-mediated TCR signals and IL-7R signals are each able to stimulate T cell proliferation in lymphopenic hosts independently of one another, but can also synergize to facilitate proliferation. Furthermore, the relative contribution to the homeostatic response by TCR and cytokine signals is not fixed and critically depends on both the degree of lymphopenia and specific characteristics of individual T cell clones. Finally, we show that only lck and not fyn can mediate the TCR-driven proliferation, while neither lck nor fyn is required for IL-7R-induced proliferation.

The role of TCR signals triggered by recognition of self MHCs in maintaining the survival of naive peripheral T cells remains controversial. Here we examine the role of the Src family kinases, p56(lck) (Lck) and p59(fyn) (Fyn), in the survival of naive T cells. We show that long term survival requires a combination of signals transduced by Src family kinases and signals through the IL-7R. In the absence of either one, naive T cells die slowly, but if both signals are removed, cell loss is greatly accelerated. The TCR signal can be mediated by either Fyn or Lck at wild-type levels of expression, but not by Lck alone if expressed suboptimally. The disappearance of T cells in the absence of Fyn and Lck was associated with a complete loss of TCRzeta-chain phosphorylation and down-regulation of CD5, both of which are also MHC contact dependent, indicating that the Src family kinases are critical for transducing a TCR-MHC survival signal.

Studies in Jurkat cells have shown that combined stimulation through the TCR and CD28 is required for activation of c-Jun N-terminal kinase (JNK), suggesting that JNK activity may mediate the costimulatory function of CD28. To examine the role of JNK signaling in CD28 costimulation in normal T cells, murine T cell clones and CD28(+/+) or CD28(-/-) TCR transgenic T cells were used. Although ligation with anti-CD28 mAb augmented JNK activation in Th1 and Th2 clones stimulated with low concentrations of anti-CD3 mAb, higher concentrations of anti-CD3 mAb alone were sufficient for JNK activation even in the absence of anti-CD28. JNK activity was comparably induced in both CD28(+/+) and CD28(-/-) 2C/recombinase-activating gene 2(RAG2)(-/-) T cells stimulated with anti-CD3 mAb alone, and with L(d)/peptide dimers, a direct alphabeta TCR ligand. Moreover, JNK activation was also detected in 2C/RAG2(-/-) T cells stimulated with P815 cells that express the relevant alloantigen L(d) whether or not B7-1 was coexpressed. However, IL-2 production by both Th1 clones and CD28(+/+) 2C/RAG2(-/-) T cells was detected only upon TCR and CD28 coengagement. Thus, CD28 coligation is not necessary, and stimulation through the TCR is sufficient, for JNK activation in normal murine T cells. The concept that JNK mediates the costimulatory function of CD28 needs to be reconsidered.

Positive selection is a process that ensures that peripheral T cells express TCR that are self-MHC restricted. This process occurs in the thymus and requires both self-MHC and self-peptides. We have recently established a TCR transgenic (TCR(trans)(+)) mouse model using the C10.4 TCR restricted to the MHC class Ib molecule, H2-M3. Having defined H2-M3 as the positively selecting MHC molecule, the severely limited number of H2-M3 binding peptides allowed us to characterize a mitochondrial NADH dehydrogenase subunit 1-derived 9-mer peptide as the physiological ligand of positive selection. Here, we demonstrate that the NADH dehydrogenase subunit 1 self-peptide is seen by mature C10.4 TCR(trans)(+) T cells as a weak agonist and induces positive selection at a defined concentration range. We also found that the full-length cognate peptide, a strong agonist for mature C10.4 TCR(trans)(+) T cells, initiated positive selection, albeit at significantly lower concentrations. At increased peptide concentrations, and thus increased epitope densities, either peptide only induced the development of partially functional T cells. We conclude that successful positive selection only proceeded at a defined, yet fairly narrow window of avidity.

The CD45 protein tyrosine phosphatase regulates Ag receptor signaling in T and B cells. In the absence of CD45, TCR coupling to downstream signaling cascades is profoundly reduced. Moreover, in CD45-null mice, the maturation of CD4+CD8+ thymocytes into CD4+CD8- or CD4-CD8+ thymocytes is severely impaired. These findings suggest that thymic selection may not proceed normally in CD45-null mice, and may be biased in favor of thymocytes expressing TCRs with strong reactivity toward self-MHC-peptide ligands to compensate for debilitated TCR signaling. To test this possibility, we purified peripheral T cells from CD45-null mice and fused them with the BWalpha-beta- thymoma to generate hybridomas expressing normal levels of TCR and CD45. The reactivity of these hybridomas to self or foreign MHC-peptide complexes was assessed by measuring the amount of IL-2 secreted upon stimulation with syngeneic or allogeneic splenocytes. A very high proportion (55%) of the hybridomas tested reacted against syngeneic APCs, indicating that the majority of T cells in CD45-null mice express TCRs with high avidity for self-MHC-peptide ligands, and are thus potentially autoreactive. Furthermore, a large proportion of TCRs selected in CD45-null mice (H-2b) were also shown to display reactivity toward closely related MHC-peptide complexes, such as H-2bm12. These results support the notion that modulating the strength of TCR-mediated signals can alter the outcome of thymic selection, and demonstrate that CD45, by molding the window of affinity/avidity for positive and negative selection, directly participates in the shaping of the T cell repertoire.

In this work, we have tried to establish whether human memory T cells may be protected from Fas (CD95)-induced apoptosis when correctly activated by Ag, and not protected when nonspecifically or incorrectly activated. In particular, we wanted to investigate the molecular mechanisms that regulate the fate of memory T cells following an antigenic challenge. To address this issue, we chose an experimental system that closely mimics physiological T cell activation such as human T cell lines and clones specific for viral peptides or alloantigens. We demonstrate that memory T cells acquire an activation-induced cell death (AICD)-resistant phenotype when TCRs are properly engaged by specific Ag bound to MHC molecules. Ag concentration and costimulation are critical parameters in regulating the protective effect. The analysis of the mechanisms involved in the block of CD95 signal transduction pathways revealed that the crucial events are the inhibition of CD95-associated IL-1beta-converting enzyme (ICE)-like protease (FLICE) activation and poly(ADP)-ribose polymerase cleavage, and the mRNA expression of FLICE-like inhibitory protein. Furthermore, we have observed that TCR-mediated neosynthesis of FLICE-like inhibitory protein mRNA is suppressed either by protein tyrosine kinase inhibitors or cyclosporin A. In conclusion, the present analysis of the effects of TCR triggering on the regulation of AICD suggests that AICD could be inhibited in human memory T cells activated in vivo by a foreign Ag, but may become operative when the Ag has been cleared.

Galectin-1 is an endogenous lectin expressed by thymic and lymph node stromal cells at sites of Ag presentation and T cell death during normal development. It is known to have immunomodulatory activity in vivo and can induce apoptosis in thymocytes and activated T cells (1-3). Here we demonstrate that galectin-1 stimulation cooperates with TCR engagement to induce apoptosis, but antagonizes TCR-induced IL-2 production and proliferation in a murine T cell hybridoma and freshly isolated mouse thymocytes, respectively. Although CD4+ CD8+ double positive cells are the primary thymic subpopulation susceptible to galectin-1 treatment alone, concomitant CD3 engagement and galectin-1 stimulation broaden susceptible thymocyte subpopulations to include a subset of each CD4- CD8-, CD4+ CD8+, CD4- CD8+, and CD4+ CD8- subpopulations. Furthermore, CD3 engagement cooperates with suboptimal galectin-1 stimulation to enhance cell death in the CD4+ CD8+ subpopulation. Galectin-1 stimulation is shown to synergize with TCR engagement to dramatically and specifically enhance extracellular signal-regulated kinase-2 (ERK-2) activation, though it does not uniformly enhance TCR-induced tyrosine phosphorylation. Unlike TCR-induced IL-2 production, TCR/galectin-1-induced apoptosis is not modulated by the expression of kinase inactive or constitutively activated Lck. These data support a role for galectin-1 as a potent modulator of TCR signals and functions and indicate that individual TCR-induced signals can be independently modulated to specifically affect distinct TCR functions.

Signaling from the TCR involves the protein tyrosine kinase p59fyn (Fyn). Previous studies have shown that T cell development occurs normally in Fyn-/- mice. In this study, we investigated the requirement for Fyn in the development and function of T cells expressing either the transgenic 2C TCR, with high affinity for its Ag ligand, or the transgenic H-Y TCR, representative of a low affinity TCR. Although Fyn was not essential for positive selection of thymocytes expressing either the 2C or the H-Y TCR, it facilitated the down-regulation of the heat-stable Ag in positively selected CD4-CD8+ thymocytes in both 2C and H-Y mice. Negative selection of thymocytes expressing the H-Y TCR also occurs efficiently in Fyn-/- mice. However, in Fyn-/- mice, there was a preferential survival of thymocytes that expressed higher levels of the CD8 coreceptor and the transgenic TCR. Positively selected CD4-CD8+ thymocytes and peripheral T cells expressing either the 2C or the H-Y TCR differed in their requirement of Fyn for optimal proliferation responses to stimulation by antigenic ligands. Whereas 2C Fyn-/- or 2C Fyn+/+ thymocytes and peripheral T cells responded optimally to stimulation by the specific Ag, H-Y Fyn-/- thymocytes and peripheral T cells were hyporesponsive compared with Fyn+/+ cells. Significantly, in response to a defined low affinity ligand, both 2C Fyn-/- thymocytes and peripheral T cells required Fyn for optimal response to Ag stimulation. Thus, Fyn plays a role during thymocyte development and is required for optimal responses to low affinity/avidity ligands.

CD5 is a 67-kDa T cell surface Ag that can be found physically associated with the CD3-TCR molecular complex. In different experimental models it has been shown to act as a costimulatory receptor for T cell activation. Unexpectedly, studies in CD5-deficient mice suggested a negative role for the CD5 Ag in CD3-TCR signaling in the thymus. In this report we assessed the constitutive interactions of CD5 in freshly isolated human thymocytes with signaling elements of the CD3-TCR complex. We determined that the ZAP-70 protein tyrosine kinase was present in CD5 immunoprecipitates. The two molecules were constitutively tyrosine phosphorylated in a complex also associating with unphosphorylated as well as phosphorylated zeta-chains. Although both p21 and p23 tyrosine-phosphorylated forms of zeta as well as phospho-CD3 epsilon molecules were constitutively present in human thymocytes and could be immunoprecipitated with ZAP-70- or CD3 epsilon-specific Abs, the p21 species of zeta was predominant in CD5 immune complexes. The interaction between CD5 and ZAP-70 was not observed in CD3-negative thymocytes, where the constitutive tyrosine phosphorylation of ZAP-70 was very low. We conclude that CD5 may affect in vivo the signaling capacity of TCRs expressed by human thymocytes by altering the phosphorylation state of CD3 and/or by retaining ZAP-70 with the p21 species of zeta.

The Ag receptors on mature B and T cells are not coupled to the activation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Moreover, phorbol esters such as PMA, which can activate cPLA2 via mitogen-activated protein (MAP) kinase in most cell types, also failed to induce the release of arachidonate from mature cells, suggesting that the cPLA2 pathway may not be functional in mature lymphocytes. Interestingly, Western blot analysis revealed that cPLA2, which had previously been thought to be expressed ubiquitously, is not expressed in mature B or T cells and that cytosolic phospholipase A2 expression could not be up-regulated in lymphocytes following culture with a range of cytokines most likely to be involved in an immune response such as IL-1 alpha, IL-3, or TNF-alpha. In contrast, cPLA2 was shown to be expressed and activated in thymocytes and immature B cells under conditions in which ligation of the Ag receptors led to growth arrest and/or apoptosis. Taken together, these data suggest that cPLA2 does not play a role in Ag receptor-mediated lymphocyte activation, but may be involved in the molecular mechanisms underlying lymphocyte maturation and/or self tolerance by clonal deletion.

Ca2+/calmodulin-dependent protein kinase type Gr (CaM kinase-Gr) is a Ca2+/calmodulin-dependent protein kinase which is enriched in the brain and thymus. In this study, we examined the expression of CaM kinase-Gr in human lymphocytes and the regulation of its catalytic activity by antigen receptor signaling. CaM kinase-Gr was found selectively expressed in T lymphocytes in a developmentally regulated manner. It was present at severalfold higher levels in immature thymocytes (CD3low, CD4+CD8+) relative to mature thymocytes (CD3high, CD4+CD8-/CD8+CD4-) or to circulating T lymphocytes. The kinase was preferentially expressed in CD4+ T lymphocytes, but was not detected in B lymphocytes or in monocytes. The impact of T cell antigen receptor-CD3 complex (TCR.CD3) signaling on kinase activity was examined using Jurkat human leukemic T lymphocytes as a model. Treatment of Jurkat cells with anti TCR.CD3 monoclonal antibody induced rapid autophosphorylation of the kinase on serine residues and a dramatic, autophosphorylation-dependent enhancement of both Ca2+/calmodulin-dependent and autonomous kinase activity. Enzyme autophosphorylation and activation were dependent on the influx of extracellular Ca2+ following receptor signaling but could not be induced by an influx of extra-cellular Ca2+ triggered by ionophores, indicating that additional signals delivered via TCR.CD3 contribute to the activation of CaM kinase-Gr. These findings suggest a role for CaM kinase-Gr in T lymphocyte development and activation and indicate the presence of stringent regulatory mechanisms governing the activity of this kinase in situ.

The expression of the V(D)J [variable (diversity) joining elements] recombination activating genes, RAG-1 and RAG-2, has been examined during T cell development in the thymus. In situ hybridization to intact thymus and RNA blot analysis of isolated thymic subpopulations separated on the basis of T cell receptor (TCR) expression demonstrated that both TCR- and TCR+ cortical thymocytes express RAG-1 and RAG-2 messenger RNA's. Within the TCR+ population, RAG expression was observed in immature CD4+CD8+ (double positive) cells, but not in the more mature CD4+CD8- or CD4-CD8+ (single positive) subpopulations. Thus, although cortical thymocytes that bear TCR on their surface continue to express RAG-1 and RAG-2, it appears that the expression of both genes is normally terminated during subsequent thymic maturation. Since thymocyte maturation in vivo is thought to be regulated through the interaction of the TCR complex with self major histocompatibility complex (MHC) antigens, these data suggest that signals transduced by the TCR complex might result in the termination of RAG expression. Consistent with this hypothesis, thymocyte TCR cross-linking in vitro led to rapid termination of RAG-1 and RAG-2 expression, whereas cross-linking of other T cell surface antigens such as CD4, CD8, or HLA class I had no effect.