CD4 co-receptor dependent signaling promotes competency for re-stimulation induced cell death of effector T cells

The elimination of activated T cells by FAS-mediated signaling is an important immunoregulatory mechanism used to maintain homeostasis and prevent tissue damage. T cell receptor-dependent signals are required to confer sensitivity to FAS-mediated re-stimulation-induced cell death (RICD), however, the nature of these signals is not well understood. In this report, we show, using T cells from CD4-deficient mice reconstituted with a tail-less CD4 transgene, that CD4-dependent signaling events are a critical part of the competency signal required for RICD. This is in part due to defects in FAS receptor signaling complex formation as shown by decreased recruitment of FAS and caspase 8 into lipid rafts following antigen re-stimulation in the absence of CD4-dependent signals. In addition, in the absence of CD4-dependent signals, effector T cells have a selective defect in IL-2 secretion following peptide re-stimulation, while provision of exogenous IL-2 during re-stimulation partially restores susceptibility to RICD. Importantly, IL-2 production and proliferation after primary peptide stimulation is comparable between wild type and CD4-deficient T cells indicating that the requirement for CD4-dependent signaling events for IL-2 production is developmentally regulated and is particularly critical in previously activated effector T cells. In total, our results indicate that CD4 co-receptor dependent signaling events specifically regulate effector T cell survival and function. Further, these data suggest that CD4-dependent signaling events may protect against the decreased IL-2 production and resistance to cell death seen during chronic immune stimulation.     

Cutting edge: Rac GTPases sensitize activated T cells to die via Fas

In activated CD4(+) T cells, TCR restimulation triggers apoptosis that depends on interactions between the death receptor Fas and its ligand, FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but what pathways mediate this process are not known. In this study we identify the Rho GTPases Rac1 and Rac2 as essential components in restimulation-induced cell death. RNA interference-mediated knockdown of Rac GTPases greatly reduced Fas-dependent, TCR-induced apoptosis. The ability of Rac1 to sensitize T cells to Fas-induced apoptosis correlated with Rac-mediated cytoskeletal reorganization, dephosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal linker proteins, and the translocation of Fas to lipid raft microdomains. In primary activated CD4(+) T cells, Rac1 and Rac2 were independently required for maximal TCR-induced apoptosis. Activating Rac signaling may be a novel way to sensitize chronically stimulated lymphocytes to Fas-induced apoptosis, an important goal in the treatment of autoimmune diseases.     

The CD3-zeta chimeric antigen receptor overcomes TCR Hypo-responsiveness of human terminal late-stage T cells

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

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

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

T cell, Ig domain, mucin domain-2 gene-deficient mice reveal a novel mechanism for the regulation of Th2 immune responses and airway inflammation

The development of asthma and other atopic diseases is influenced by cytokines produced by Th2 effector T cells. How effector T cell responses are regulated once these cell populations are established remains unclear. The recently described T cell and airway phenotype regulator locus, containing the T cell, Ig domain, mucin domain (TIM) genes, is genetically associated with Th2 cytokine production and Th2-dependent immune responses. In this study, we report the phenotype of the TIM-2 gene-deficient mouse, and demonstrate exacerbated lung inflammation in an airway atopic response model. Immune responses in the TIM-2-deficient mouse reveal disregulated expression of Th2 cytokines, and adoptive transfer experiments show that the T cell compartment is responsible for the heightened inflammatory phenotype. These studies show that TIM-2 is a novel and critical regulator of effector T cell activity.     

Modulation of T cell activation by stomatin-like protein 2

T cell activation through the Ag receptor (TCR) requires sustained signaling from signalosomes within lipid raft microdomains in the plasma membrane. In a proteomic analysis of lipid rafts from human T cells, we identified stomatin-like protein (SLP)-2 as a candidate molecule involved in T cell activation through the Ag receptor. In this study, we show that SLP-2 expression in human primary lymphocytes is up-regulated following in vivo and ex vivo activation. In activated T cells, SLP-2 interacts with components of TCR signalosomes and with polymerized actin. More importantly, up-regulation of SLP-2 expression in human T cell lines and primary peripheral blood T cells increases effector responses, whereas down-regulation of SLP-2 expression correlates with loss of sustained TCR signaling and decreased T cell activation. Our data suggest that SLP-2 is an important player in T cell activation by ensuring sustained TCR signaling, which is required for full effector T cell differentiation, and point to SLP-2 as a potential target for immunomodulation.     

Carcinoembryonic antigen-related cell adhesion molecule 1 inhibits proximal TCR signaling by targeting ZAP-70

The long cytoplasmic tail (CT) isoforms of carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1) are expressed on activated human T cells and possess two ITIM motifs in the CT. These isoforms of CEACAM1 are inhibitory for T cell responses initiated by the TCR/CD3 complex with the inhibition dependent upon the ITIMs of CEACAM1 and Src homology 2 domain-containing phosphatase 1 (SHP-1). However, the mechanism by which this inhibition occurs in T cells is unknown. We demonstrate here that the Src family kinase, Lck, and the ability of CEACAM1 to bind homophilically are required for the ITIM phosphorylation of CEACAM1 that is a prerequisite for CEACAM1 association with SHP-1. We further show that CEACAM1 associates with and recruits SHP-1 to the TCR/CD3 complex leading to decreased phosphorylation of CD3-zeta and ZAP-70 and consequently decreased activation of the elements downstream of ZAP-70. This is physiologically relevant because extinction of SHP-1 expression or blockade of homophilic binding by CEACAM1 using a Fab that specifically recognizes the homophilic binding region of human CEACAM1 increases the cytolytic function initiated by the TCR/CD3 complex. These studies show that long CT isoforms of CEACAM1 orchestrate an inhibitory program that abrogates extremely proximal events downstream of the TCR/CD3 complex by focusing on the activation of ZAP-70.     

TIM-3: a novel regulatory molecule of alloimmune activation

T cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4(+)FoxP3(-) and CD8(+) T cells in acutely rejecting graft recipients. A blocking anti-TIM-3 mAb accelerated allograft rejection only in the presence of host CD4(+) T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ-, IL-6-, and IL-17-producing splenocytes, enhanced CD8(+) cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4(+) T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4(+) T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response.     

Human TIM-1 associates with the TCR complex and up-regulates T cell activation signals

The T cell, Ig domain, and mucin domain-1 (TIM-1) gene is associated with Th2 T cell responses and human atopic diseases. The mechanism by which TIM-1 influences T cell responses remains unknown. We demonstrate that TIM-1 is recruited to the TCR-signaling complex via association with CD3. TIM-1 up-regulates TCR-associated signaling events, including phosphorylation of Zap70 and IL-2-inducible T cell kinase. This activity requires TIM-1 tyrosine phosphorylation. TIM-1 expression induces formation of a novel complex that includes PI3K and ITK. Finally, the consequences of TIM-1 activation include increased expression of effector cytokines. These results demonstrate that TIM-1 is a critical component of the human T cell response and provide a mechanistic hypothesis for the role of TIM-1 in disease.     

T cell-signaling network analysis reveals distinct differences between CD28 and CD2 costimulation responses in various subsets and in the MAPK pathway between resting and activated regulatory T cells

To uncover signaling system differences between T cell stimuli and T cell subsets, phosphorylation status of 18 signaling proteins at six different time points following TCR triggering and CD28/CD2 costimulation was examined in human T cell subsets by phospho-epitope-specific flow cytometry of fluorescent cell barcoded samples, thereby providing a high-resolution signaling map. Compared with effector/memory T cells, naive T cells displayed stronger activation of proximal signaling molecules after TCR triggering alone. Conversely, distal phosphorylation events, like pErk and pS6-ribosomal protein, were stronger in effector/memory subsets. CD28 costimulation specifically induced signaling necessary for proper NF-κB activation, whereas CD2 signaled more strongly to S6-ribosomal protein. Analysis of resting regulatory T cells (rTregs; CD4(+)CD45RA(+)FOXP3(+)) and activated regulatory T cells (actTregs; CD4(+)CD45RA(-)FOXP3(++)) revealed that, although rTregs had low basal, but inducible, Erk activity, actTregs displayed high basal Erk phosphorylation and little or no Akt activation. Interestingly, the use of Mek inhibitors to block Erk activation inhibited activation-dependent FOXP3 upregulation in rTregs, their transition to actTregs, and the resulting increase in suppressive capacity. In summary, our systems approach unraveled distinct differences in signaling elicited by CD28 and CD2 costimulation and between rTregs and actTregs. Blocking rTreg transition to highly suppressive actTregs by Mek inhibitors might have future therapeutic applications.     

Mutual antagonism of TGF-beta and Interleukin-2 in cell survival and lineage commitment of induced regulatory T cells

Transforming growth factor beta (TGF-β)- and Interleukin-2 (IL-2)-mediated signaling enables the generation and expansion of induced regulatory T (iTreg) cells that carry high hopes for the treatment of chronic inflammatory and autoimmune diseases. Knowledge about factors stabilizing their lineage commitment and lifespan, however, is limited. Here, we investigated the behavior of iTreg cells, derived from apoptosis-defective mouse mutants, during activated cell autonomous cell death, triggered by cytokine-deprivation, or activation-induced cell death (AICD) after restimulation of the T-cell receptor, and compared these responses with those of effector T cells. We observed that iTreg cells were much more sensitive to IL-2-deprivation but poorly susceptible to AICD. In fact, when apoptosis was compromised, T-cell receptor (TCR)-religation resulted in methylation-independent, ERK- and PI3K/mTOR-mediated loss of Foxp3 expression, impaired suppressive capacity and effector cytokine production. Although iTreg cells prevented colitis induction they rapidly lost Foxp3-GFP expression and gained ability to produce effector cytokines thereby imposing Th1 cell fate on resident effector cells. Surprisingly, iTreg cell conversion itself was limited by TGF-β-mediated Bim/Bcl2L11-dependent apoptosis. Hence, the very same cytokine that drives the generation of iTreg cells can trigger their demise. Our results provide novel insights in iTreg cell biology that will assist optimization of iTreg-based therapy.     

Cbl-b regulates antigen-induced TCR down-regulation and IFN-gamma production by effector CD8 T cells without affecting functional avidity

The E3 ubiquitin ligase Cbl-b is a negative regulator of TCR signaling that: 1) sets the activation threshold for T cells; 2) is induced in anergic T cells; and 3) protects against autoimmunity. However, the role of Cbl-b in regulating CD8 T cell activation and functions during physiological T cell responses has not been systematically examined. Using the lymphocytic choriomeningitis virus infection model, we show that Cbl-b deficiency did not significantly affect the clonal expansion of virus-specific CD8 T cells. However, Cbl-b deficiency not only increased the steady-state cell surface expression levels of TCR and CD8 but also reduced Ag-induced down-modulation of cell surface TCR expression by effector CD8 T cells. Diminished Ag-stimulated TCR down-modulation and sustained Ag receptor signaling induced by Cbl-b deficiency markedly augmented IFN-gamma production, which is known to require substantial TCR occupancy. By contrast, Cbl-b deficiency minimally affected cell-mediated cytotoxicity, which requires limited engagement of TCRs. Surprisingly, despite elevated expression of CD8 and reduced Ag-induced TCR down-modulation, the functional avidity of Cbl-b-deficient effector CD8 T cells was comparable to that of wild-type effectors. Collectively, these data not only show that Cbl-b-imposed constraint on TCR signaling has differential effects on various facets of CD8 T cell response but also suggest that Cbl-b might mitigate tissue injury induced by the overproduction of IFN-gamma by CD8 T cells. These findings have implications in the development of therapies to bolster CD8 T cell function during viral infections or suppress T cell-mediated immunopathology.     

Distinct Rho GTPase activities regulate epithelial cell localization of the adhesion molecule CEACAM1: involvement of the CEACAM1 transmembrane domain

CEACAM1 is an intercellular adhesion glycoprotein. As CEACAM1 plays an important role in epithelial cell signaling and functions, we have examined its localization in epithelial cells. We have observed that distribution at cell contacts is not always seen in these cells, suggesting that CEACAM1 localization might be regulated. In Swiss 3T3 cells, the targeting of CEACAM1 at cell-cell boundaries is regulated by the Rho GTPases. In the present study, we have used the MDCK epithelial cells to characterize the effects of the Rho GTPases and their effectors on CEACAM1 intercellular targeting. Activated Cdc42 and Rac1 or their downstream effector PAK1 targeted CEACAM1 to sites of cell-cell contacts. On the other hand, neither activated RhoA nor activated Rho kinase directed CEACAM1 to cell boundaries, resulting in a condensed distribution of CEACAM1 at the cell surface. Interestingly, inhibition of this pathway resulted in CEACAM1 intercellular localization suggesting that a tightly regulated balance of Rho GTPase activities is necessary to target CEACAM1 at cell-cell boundaries. In addition, using CEACAM1 mutants and chimeric fusion constructs containing domains of the colony-stimulating factor receptor, we have shown that the transmembrane domain of CEACAM1 is responsible for the Cdc42-induced targeting at cell-cell contacts.     

Carcinoembryonic antigen-related cell adhesion molecule 1 on murine dendritic cells is a potent regulator of T cell stimulation

Dendritic cells (DC) are important APCs that play a key role in the induction of an immune response. The signaling molecules that govern early events in DC activation are not well understood. We therefore investigated whether DC express carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1, also known as BGP or CD66a), a well-characterized signal-regulating cell-cell adhesion molecule that is expressed on granulocytes, monocytes, and activated T cells and B cells. We found that murine DC express in vitro as well as in vivo both major isoforms of CEACAM1, CEACAM1-L (having a long cytoplasmic domain with immunoreceptor tyrosine-based inhibitory motifs) and CEACAM1-S (having a short cytoplasmic domain lacking phosphorylatable tyrosine residues). Ligation of surface-expressed CEACAM1 on DC with the specific mAb AgB10 triggered release of the chemokines macrophage inflammatory protein 1alpha, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 and induced migration of granulocytes, monocytes, T cells, and immature DC. Furthermore, the surface expression of the costimulatory molecules CD40, CD54, CD80, and CD86 was increased, indicating that CEACAM1-induced signaling regulates early maturation and activation of dendritic cells. In addition, signaling via CEACAM1 induced release of the cytokines IL-6, IL-12 p40, and IL-12 p70 and facilitated priming of naive MHC II-restricted CD4(+) T cells with a Th1-like effector phenotype. Hence, our results show that CEACAM1 is a signal-transducing receptor that can regulate early maturation and activation of DC, thereby facilitating priming and polarization of T cell responses.     

The FcR gamma subunit and Syk kinase replace the CD3 zeta-chain and ZAP-70 kinase in the TCR signaling complex of human effector CD4 T cells

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.     

TIM-1 signaling in B cells regulates antibody production

Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3⁺ anti-CD28-stimulated CD4⁺ T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.     

Primed T cells are more resistant to Fas-mediated activation-induced cell death than naive T cells.

Memory T cells respond in several functionally different ways from naive T cells and thus function as efficient effector cells. In this study we showed that primed T cells were more resistant to Fas-mediated activation-induced cell death (AICD) than naive T cells using OVA-specific TCR transgenic DO10 mice and Fas-deficient DO10 lpr/lpr mice. We found that apoptosis was efficiently induced in activated naive T cells at 48 and 72 h after Ag restimulation (OVA peptide; 0.3 and 3 microM), whereas apoptosis was not significantly increased in activated primed T cells at 24-72 h after Ag restimulation. We further showed that the resistance to AICD in primed T cells was due to the decreased sensitivity to apoptosis induced by Fas-mediated signals, but TCR-mediated signaling equally activated both naive and primed T cells to induce Fas and Fas ligand expressions. Furthermore, we demonstrated that primed T cells expressed higher levels of Fas-associated death domain-like IL-1beta-converting enzyme inhibitory protein (FLIP), an inhibitor of Fas-mediated apoptosis, at 24-48 h after Ag restimulation than naive T cells. In addition, Bcl-2 expression was equally observed between activated naive and primed T cells after Ag restimulation. Thus, these results indicate that naive T cells are sensitive to Fas-mediated AICD and are easily deleted by Ag restimulation, while primed/memory T cells express higher levels of FLIP after Ag restimulation, are resistant to Fas-mediated AICD, and thus function as efficient effector cells for a longer period.     

TGF-beta inhibits IL-2 production and promotes cell cycle arrest in TCR-activated effector/memory T cells in the presence of sustained TCR signal transduction

TGF-beta signaling is critical for controlling naive T cell homeostasis and differentiation; however, the biological and biochemical changes induced by TGF-beta in effector/memory T cells are poorly defined. We show that although TGF-beta inhibits effector/memory peripheral blood T lymphoblast proliferation and IL-2 production, the intensity and kinetics for TCR-induced global tyrosine phosphorylation are markedly increased compared with that in untreated cells or naive T cells. After TCR ligation, tyrosine phosphorylation of proximal tyrosine kinases and docking proteins like linker for activation of T cells is maintained for >30 min in TGF-beta-primed cells compared with untreated cells where phosphorylation of these targets returned to basal levels by 10 min. Extended phosphorylation of linker for activation of T cells in treated peripheral blood T selectively prolongs ERK 1/2 signaling and phospholipase C-gamma1 activation leading to increased Ca(2+) flux. A kinase/phosphatase imbalance could not account for extended phosphorylation as CD45R, SHP-1, and SHP-2 expression remains unaltered. The contradiction between prolonged signal transduction and inhibition of proliferation is partially explained by the observation that TGF-beta priming results in ERK 1/2-independent p21 induction and decreased cyclin D1 expression leading to accumulation of T cells in G(0)/G(1) phases of the cell cycle and cell cycle arrest. Despite inhibition of T cell function by TGF-beta priming, TCR and cytokine signaling pathways are intact and selectively extended, suggesting that suppression in the effector/memory T cell is mediated by reprogramming signal transduction, rather than its inhibition as in the naive T cell.     

The Src-homology domain 2-bearing protein tyrosine phosphatase-1 inhibits antigen receptor-induced apoptosis of activated peripheral T cells.

Restimulation of Ag receptors on peripheral T lymphocytes induces tyrosine phosphorylation-based signaling cascades that evoke Fas ligand expression and induction of Fas-mediated programmed cell death. In view of the role for the Src homology domain 2-bearing protein tyrosine phosphatase-1 (SHP-1) in modulating TCR signaling, we investigated the influence of SHP-1 on TCR-mediated apoptosis by assaying the sensitivity of peripheral T cells from SHP-1-deficient viable motheaten (mev) mice to cell death following TCR restimulation. The results of these studies revealed mev peripheral T cells to be markedly more sensitive than wild-type cells to induction of cell death following TCR stimulation. By contrast, PMA/ionophore and anti-Fas Ab-induced apoptotic responses were no different in mev compared with wild-type activated cells. Enhanced apoptosis of TCR-restimulated mev lymphocytes was associated with marked increases in Fas ligand expression as compared with wild-type cells, but was almost abrogated in both mev and wild-type cells by Fas-Fc treatment. Thus, the increased sensitivity of mev T cells to apoptosis following TCR restimulation appears to reflect a TCR-driven phenomenon mediated through up-regulation of Fas-Fas ligand interaction and induction of the Fas signaling cascade. These findings, together with the hyperproliferative responses of mev peripheral T cells to initial TCR stimulation, indicate that SHP-1 modulation of TCR signaling translates to the inhibition of both T cell proliferation and activation and, as such, is likely to play a pivotal role in regulating the expansion of Ag-stimulated T cells during an immune response.