Intracellular Phospho-Flow cytometry reveals novel insights into TCR proximal signaling events. A comparison with Western blot.

Phospho-site specific antibodies become increasingly available, enabling the study of signaling events by Western blotting (WB) or intracellular flow cytometry (Phospho-Flow). Here we compared data generated by WB or Phospho-Flow regarding the kinetics and degree of phosphorylation of membrane proximal TCR signaling molecules. Phosphorylation events in Jurkat T cells were triggered by anti-CD3 stimulation (OKT3) or by oxidative stress (H(2)O(2)) and were analyzed by Phospho-Flow or WB. Both techniques showed that OKT3- or H(2)O(2)-induced, transient phosphorylation of ZAP70 or LAT was dependent on functional Lck. Phospho-Flow data revealed differences in the kinetics and the degree of H(2)O(2)- or OKT3-mediated protein phosphorylation compared with WB data. In addition, using Phospho-Flow we discovered that H(2)O(2)-induced phosphorylation of TCR signaling proteins was inhibited by small molecular weight kinase inhibitors far more potently than OKT3-triggered protein phosphorylation, despite a superior induction of phosphorylation by H(2)O(2). This finding was confirmed by WB. Interestingly, we identified by Phospho-Flow that, in P116 Jurkat cells lacking ZAP70 protein expression, H(2)O(2) potently triggered the phosphorylation of ZAP70 residues Y493 and Y292 but not Y319. The phosphorylation of these ZAP70 tyrosine residues cells was blocked by an Lck inhibitor, suggesting the existence of an Lck-coupled truncated ZAP70 protein or a novel isoform of ZAP70 in P116 cells. Phospho-Flow is a largely quantitative technology with excellent throughput, highly suited in studying the function or inhibition of TCR signaling pathways and allowing the detection of novel pathway insights. It can serve as a good complement to Western blot analysis.

Profound inhibition of antigen-specific T-cell effector functions by dasatinib.

PURPOSE: The dual BCR-ABL/SRC kinase inhibitor dasatinib entered the clinic for the treatment of chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia. Because SRC kinases are known to play an important role in physiologic T-cell activation, we analyzed the immunobiological effects of dasatinib on T-cell function. The effect of dasatinib on multiple T-cell effector functions was examined at clinically relevant doses (1-100 nmol/L); the promiscuous tyrosine kinase inhibitor staurosporine was used as a comparator. EXPERIMENTAL DESIGN: Purified human CD3+ cells and virus-specific CD8+ T cells from healthy blood donors were studied directly ex vivo; antigen-specific effects were confirmed in defined T-cell clones. Functional outcomes included cytokine production (interleukin-2, IFN gamma, and tumor necrosis factor alpha), degranulation (CD107a/b mobilization), activation (CD69 up-regulation), proliferation (carboxyfluorescein diacetate succinimidyl ester dilution), apoptosis/necrosis induction, and signal transduction. RESULTS: Both dasatinib and staurosporine inhibited T-cell activation, proliferation, cytokine production, and degranulation in a dose-dependent manner. Mechanistically, this was mediated by the blockade of early signal transduction events and was not due to loss of T-cell viability. Overall, CD4+ T cells seemed to be more sensitive to these effects than CD8+ T cells, and naïve T cells more sensitive than memory T-cell subsets. The inhibitory effects of dasatinib were so profound that all T-cell effector functions were shut down at therapeutically relevant concentrations. CONCLUSION: These findings indicate that caution is warranted with use of this drug in the clinical setting and provide a rationale to explore the potential of dasatinib as an immunosuppressant in the fields of transplantation and T-cell-driven autoimmune diseases.

Localization of Src homology 2 domain-containing phosphatase 1 (SHP-1) to lipid rafts in T lymphocytes: functional implications and a role for the SHP-1 carboxyl terminus.

The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) has previously been shown to be a negative regulator of signaling mediated via the TCR. A growing body of evidence indicates that the regulated localization of proteins within certain membrane subdomains, referred to as lipid rafts, is important for the successful transduction of signaling events downstream of the TCR. However, considerably less is known about the localization of negative regulators during these lipid raft-dependent signaling events. In this study we have investigated the subcellular localization of SHP-1 and its role in regulation of TCR-mediated signaling. Our studies demonstrate that in a murine T cell hybridoma as well as in primary murine thymocytes, a fraction of SHP-1 localizes to the lipid rafts, both basally and after TCR stimulation. Interestingly, although SHP-1 localized in the nonraft fractions is tyrosine phosphorylated, the SHP-1 isolated from the lipid rafts lacks the TCR-induced tyrosine phosphorylation, suggesting physical and/or functional differences between these two subpopulations. We identify a requirement for the C-terminal residues of SHP-1 in optimal localization to the lipid rafts. Although expression of SHP-1 that localizes to lipid rafts potently inhibits TCR-mediated early signaling events and IL-2 production, the expression of lipid raft-excluded SHP-1 mutants fails to elicit any of the inhibitory effects. Taken together these studies reveal a key role for lipid raft localization of SHP-1 in mediating the inhibitory effects on T cell signaling events.

Control of TCR-mediated activation of beta 1 integrins by the ZAP-70 tyrosine kinase interdomain B region and the linker for activation of T cells adapter protein.

One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. In this report, we use human ZAP-70-deficient Jurkat T cells to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain.

Architectural changes in the TCR:CD3 complex induced by MHC:peptide ligation.

A hallmark of T cell activation is the ligation-induced down-modulation of the TCR:CD3 complex. However, little is known about the molecular events that drive this process. The CD3 zeta-chain has been shown to play a unique role in regulating the assembly, transport, and cell surface expression of the TCR:CD3 complex. In this study we have investigated the relationship between CD3zeta and the TCRalphabetaCD3epsilondeltagamma complex after ligation by MHC:peptide complexes. Our results show that there is a significant increase in free surface CD3zeta, which is not associated with the TCR:CD3 complex, after T cell stimulation. This may reflect dissociation of CD3zeta from the TCRalphabetaCD3epsilondeltagamma complex or transport of intracellular CD3zeta directly to the cell surface. We also show that MHC:peptide ligation also results in exposure of the TCR-associated CD3zeta NH2 terminus, which is ordinarily buried in the complex. These observations appears to be dependent on Src family protein tyrosine kinases, which are known to be critical for efficient T cell activation. These data suggest a mechanism by which ligated TCR may be differentiated from unligated TCR and selectively down-modulated.

A regulatory role for CD37 in T cell proliferation.

CD37 is a leukocyte-specific protein belonging to the tetraspanin superfamily. Previously thought to be predominantly a B cell molecule, CD37 is shown in this study to regulate T cell proliferation. CD37-deficient (CD37(-/-)) T cells were notably hyperproliferative in MLR, in response to Con A, or CD3-TCR engagement particularly in the absence of CD28 costimulation. Hyperproliferation was not due to differences in memory to naive T cell ratios in CD37(-/-) mice, apoptosis, or TCR down-modulation. Division cycle analyses revealed CD37(-/-) T cells to enter first division earlier than wild-type T cells. Importantly, proliferation of CD37(-/-) T cells was preceded by enhanced early IL-2 production. We hypothesized CD37 to be involved in TCR signaling and this was supported by the observation that CD4/CD8-associated p56(Lck) kinase activity was increased in CD37(-/-) T cells. Remarkably, CD37 cross-linking on human T cells transduced signals that led to complete inhibition of CD3-induced proliferation. In the presence of CD28 costimulation, CD37 engagement still significantly reduced proliferation. Taken together, these results demonstrate a regulatory role for CD37 in T cell proliferation by influencing early events of TCR signaling.

TCR zeta mRNA with an alternatively spliced 3'-untranslated region detected in systemic lupus erythematosus patients leads to the down-regulation of TCR zeta and TCR/CD3 complex.

The reduction or absence of TCR zeta-chain (zeta) expression in systemic lupus erythematosus (SLE) patients is thought to be related to the pathogenesis of SLE. Recently, we reported the predominant expression of zeta mRNA containing an alternatively spliced 3'-untranslated region (3'UTR; zetamRNA/as-3'UTR) and a reduction in the expression of zeta mRNA containing the wild-type 3'UTR (zetamRNA/w-3'UTR) in T cells from SLE patients. Here we show that AS3'UTR mutants (MA5.8 cells deficient in zeta protein that have been transfected with zetamRNA/as-3'UTR) exhibit a reduction in the expression of TCR/CD3 complex and zeta protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab compared with that in wild-type 3'UTR mutants (MA5.8 cells transfected with zetamRNA/w-3'UTR). Furthermore, the real-time PCR analyses demonstrated that the half-life of zetamRNA/as-3'UTR in AS3'UTR mutants (3 h) was much shorter than that of zetamRNA/w-3'UTR in wild-type 3'UTR mutants (15 h). Thus, the lower stability of zetamRNA/as-3'UTR, which is predominant in SLE T cells, may be responsible for the reduced expression of the TCR/CD3 complex, including zeta protein, in SLE T cells.

Selective inhibition of T cell activation via CD147 through novel modulation of lipid rafts.

The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor alpha-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.

TCR dynamics in human mature T lymphocytes lacking CD3 gamma.

The contribution of CD3gamma to the surface expression, internalization, and intracellular trafficking of the TCR/CD3 complex (TCR) has not been completely defined. However, CD3gamma is believed to be crucial for constitutive as well as for phorbol ester-induced internalization. We have explored TCR dynamics in resting and stimulated mature T lymphocytes derived from two unrelated human congenital CD3gamma-deficient (gamma(-)) individuals. In contrast to gamma(-) mutants of the human T cell line Jurkat, which were selected for their lack of membrane TCR and are therefore constitutively surface TCR negative, these natural gamma(-) T cells constitutively expressed surface TCR, mainly through biosynthesis of new chains other than CD3gamma. However, surface (but not intracellular) TCR expression in these cells was less than wild-type cells, and normal surface expression was clearly CD3gamma-dependent, as it was restored by retroviral transduction of CD3gamma. The reduced surface TCR expression was likely caused by an impaired assembly or membrane transport step during recycling, whereas constitutive internalization and degradation were apparently normal. Ab binding to the mutant TCR, but not phorbol ester treatment, caused its down-modulation from the cell surface, albeit at a slower rate than in normal controls. Kinetic confocal analysis indicated that early ligand-induced endocytosis was impaired. After its complete down-modulation, TCR re-expression was also delayed. The results suggest that CD3gamma contributes to, but is not absolutely required for, the regulation of TCR trafficking in resting and Ag-stimulated mature T lymphocytes. The results also indicate that TCR internalization is regulated differently in each case.

Inducible T cell tyrosine kinase regulates actin-dependent cytoskeletal events induced by the T cell antigen receptor.

The tec family kinase, inducible T cell tyrosine kinase (Itk), is critical for both development and activation of T lymphocytes. We have found that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events. Expression of Src homology (SH) 2 domain mutant Itk transgenes into Jurkat T cells inhibits these events. Furthermore, Itk(-/-) murine T cells display significant defects in TCR/CD3-induced actin polymerization. In addition, Jurkat cells deficient in linker for activation of T cells expression, an adaptor critical for Itk activation, display impaired cytoskeletal events and expression of SH3 mutant Itk transgenes reconstitutes this impairment. Interestingly, expression of an Itk kinase-dead mutant transgene into Jurkat cells has no effect on cytoskeletal events. Collectively, these data suggest that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events, possibly in a kinase-independent fashion.

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes.

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.

The alternatively spliced domain TnFnIII A1A2 of the extracellular matrix protein tenascin-C suppresses activation-induced T lymphocyte proliferation and cytokine production.

Several lines of evidences have suggested that T cell activation could be impaired in the tumor environment, a condition referred to as tumor-induced immunosuppression. We have previously shown that tenascin-C, an extracellular matrix protein highly expressed in the tumor stroma, inhibits T lymphocyte activation in vitro, raising the possibility that this molecule might contribute to tumor-induced immunosuppression in vivo. However, the region of the protein mediating this effect has remained elusive. Here we report the identification of the minimal region of tenascin-C that can inhibit T cell activation. Recombinant fragments corresponding to defined regions of the molecule were tested for their ability to inhibit in vitro activation of human peripheral blood T cells induced by anti-CD3 mAbs in combination with fibronectin or IL-2. A recombinant protein encompassing the alternatively spliced fibronectin type III domains of tenascin-C (TnFnIII A-D) vigorously inhibited both early and late lymphocyte activation events including activation-induced TCR/CD8 down-modulation, cytokine production, and DNA synthesis. In agreement with this, full length recombinant tenascin-C containing the alternatively spliced region suppressed T cell activation, whereas tenascin-C lacking this region did not. Using a series of smaller fragments and deletion mutants issued from this region, we have identified the TnFnIII A1A2 domain as the minimal region suppressing T cell activation. Single TnFnIII A1 or A2 domains were no longer inhibitory, while maximal inhibition required the presence of the TnFnIII A3 domain. Altogether, these data demonstrate that the TnFnIII A1A2 domain mediate the ability of tenascin-C to inhibit in vitro T cell activation and provide insights into the immunosuppressive activity of tenascin-C in vivo.

LAP, a lymphocyte activation gene-3 (LAG-3)-associated protein that binds to a repeated EP motif in the intracellular region of LAG-3, may participate in the down-regulation of the CD3/TCR activation pathway.

The threshold, extent and termination of TCR activation is controlled in part by inhibitory co-receptors expressed on activated T cells. The lymphocyte activation gene product (LAG-3), a ligand for MHC class II molecules co-caps with the CD3/TCR complex and inhibits cell proliferation and cytokine secretion in response to CD3 signaling. We first investigated whether LAG-3 is localized in activated T cells in detergent-resistant membrane rafts enriched in glycosphingolipids and cholesterol. We showed that both LAG-3 and MHC class II are present in the cell fraction of glycosphingolipid-rich complexes (GSL complexes) before the assembly of the immunological synapse by CD3/TCR complex cross-linking. Using the LAG-3 intracytoplasmic region as bait in the yeast two-hybrid cloning system, we next identified a novel protein termed LAP for LAG-3-associated protein. LAP is encoded by a 1.8-kb RNA message in lymphocytes and encodes a 45-kDa protein that is expressed in most tissues. We showed that LAP binds specifically in vitro and in vivo to the Glu-Pro (EP) repeated motif present in the LAG-3 intracytoplasmic region. LAP also binds to the EP motif of another functionally important receptor, the PDGFR. Thus, LAP is a candidate molecule for a new type of signal transduction and/or coupling of clustered rafts to the microtubule networks that could explain how negative signaling of co-receptors may occur through molecules devoid of any immunoreceptor tyrosine-based inhibitory motif consensus sequence.

Production of TNF-alpha by human V gamma 9V delta 2 T cells via engagement of Fc gamma RIIIA, the low affinity type 3 receptor for the Fc portion of IgG, expressed upon TCR activation by nonpeptidic antigen.

Human lymphocytes expressing the gammadelta TCR represent a minor T cell subpopulation found in blood. The majority of these cells express Vgamma9Vdelta2 determinants and respond to nonpeptidic phosphoantigens. Several studies have shown that, in vivo, the percentage of Vgamma9Vdelta2 T cells dramatically increases during pathological infection, leading to the hypothesis that they play an important role in the defense against pathogens. However, the specific mechanisms involved in this response remain poorly understood. It has been established that Vgamma9Vdelta2 T cells display potent cytotoxic activity against virus-infected and tumor cells, thereby resembling NK cells. In this study, we show that, upon stimulation by nonpeptidic Ags, Vgamma9Vdelta2 T cells express FcgammaRIIIA (CD16), a receptor that is constitutively expressed on NK cells. CD16 appears to be an activation Ag for Vgamma9Vdelta2 T cells. Indeed, ligation of CD16 on Vgamma9Vdelta2 T cells leads to TNF-alpha production. This TNF-alpha production, which is dependent (like that induced via the TCR-CD3 complex) on the activation of the p38 and extracellular signal-regulated kinase-2 mitogen-activated protein kinases, can be modulated by CD94 NK receptors. Therefore, it appears that Vgamma9Vdelta2 T cells can be physiologically activated by two sequential steps via two different cell surface Ags: the TCR-CD3 complex and the FcgammaRIIIA receptor, which are specific cell surface Ags for T lymphocytes and NK cells, respectively. This strongly suggests that, in the general scheme of the immune response, Vgamma9Vdelta2 T cells represent an important subpopulation of cells that play a key role in the defense against invading pathogens.

Cutting edge: Ly49A inhibits TCR/CD3-induced apoptosis and IL-2 secretion.

To evaluate the importance of Ly49A on TCR-induced cellular events, we established clones of the 1F2 T cell hybridoma expressing either Ly49A or a chimeric version, Ly49A/H, where the Ly49A cytoplasmic domain has been replaced by the Ly49H cytoplasmic domain. Ligation of Ly49A, but not Ly49A/H, with its ligand H-2D(d) or anti-Ly49A mAbs caused a specific inhibition of TCR/CD3-induced IL-2 secretion. Moreover, flow cytometry analysis of hypodiploid DNA and annexin V binding revealed that ligation of Ly49A protected cells from apoptosis induced by anti-CD3 mAbs or Ag. In contrast, ligation of the Ly49A/H chimeric receptor had no antiapoptotic effect. In addition, engagement of Ly49A selectively inhibited TCR-induced Fas ligand expression whereas TCR-induced Fas expression was not significantly affected. Expression of Ly49 inhibitory receptors on T cells may represent an important mechanism for the regulation of T cell survival in vivo by inhibiting TCR-induced apoptosis and IL-2 secretion.

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.

SLP-76 binding to p56lck: a role for SLP-76 in CD4-induced desensitization of the TCR/CD3 signaling complex.

Nonreceptor protein tyrosine kinases and associated substrates play a pivotal role in Ag receptor stimulation of resting cells and in the initiation of activation-induced cell death (AICD) of preactivated T cells. CD4-associated p56lck has been implicated not only in the activation of primary T cells, but also in the inhibition of T cell responses. We have previously shown that CD4+ T cell clones can be rescued from AICD when surface CD4 is engaged before the TCR stimulus. In this study, we show that prevention of AICD is associated with a CD4-dependent inhibition of TCR-triggered tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) and Vav. We provide evidence for a SLP-76 interaction with Src homology 3 domains of p56lck and identify amino acids 185-194 of SLP-76 as relevant docking site. In view of the multiple functions of p56lck and SLP-76/Vav in the initiation of TCR/CD3/CD4 signaling, we propose a model for the CD4-dependent inhibition of TCR signaling and AICD of preactivated T cells. Our data suggest that preformed activation complexes of adapter proteins and enzymes in the vicinity of the CD4/p56lck complex are no longer available for the TCR signal when CD4 receptors are engaged before TCR stimulation.

CD2 rescues T cells from T-cell receptor/CD3 apoptosis: a role for the Fas/Fas-L system.

Anti-CD3 monoclonal antibodies (MoAbs) and glucocorticoid hormones induce apoptosis in immature thymocytes and peripheral T lymphocytes. This process is inhibited by a number of growth factors, including interleukin-2 (IL-2), IL-3, and IL-4, as well as by triggering of the adhesion molecule CD44, which would indicate that signals generated by membrane receptors can modulate the survival of lymphoid cells. To investigate whether triggering of CD2 may also affect apoptosis in lymphoid cells, we analyzed the effect of stimulation with anti-CD2 MoAbs on T-cell apoptosis induced by two stimuli, anti-CD3 MoAbs and dexamethasone (DEX), using a hybridoma T-cell line and a T-helper cell clone. The results show that CD2 engagement decreased anti-CD3 MoAb-induced apoptosis, but did not influence DEX-induced cell death. Furthermore, the decrease appeared to be related to the expression of Fas/APO-1 (CD95) and Fas-ligand (Fas-L). In fact, we show that CD2 stimulation inhibits apoptosis by preventing the CD3-induced upregulation of Fas and Fas-L in a Fas-dependent experimental system. These data suggest that a costimulatory molecule may control a deletion pathway and may therefore contribute to the regulation of peripheral tolerance.

The integrin-triggered rescue of T lymphocyte apoptosis is blocked in HIV-1-infected individuals.

HIV infection is associated with a disease status-dependent impairment of Ag-specific T cell responses, resulting in anergy or unchecked apoptotic cell death. beta1 integrins play an important role in the induction of T lymphocyte responses to antigenic challenge by providing a T cell costimulatory signal, and have been shown to rescue various cell types from undergoing apoptosis. We examined the integrin-triggered cell survival signal and associated pathways in CD3+ T cells derived from 69 HIV-1-infected individuals in comparison with healthy controls. We found beta1 integrin-mediated costimulation of TCR-induced T cell proliferation and protection from aberrant cell death to be absent in the majority of patients with AIDS, but intact in asymptomatic, infected individuals. The lack of integrin-mediated rescue may be partly due to an early impairment of TCR/integrin-costimulated secretion of IFN-gamma, a type 1 lymphokine that protects against TCR-induced apoptosis of T cells from HIV-seropositive donors, but not loss of integrin expression. The mechanism of integrin hyporesponsiveness appeared to correlate with a failure of the integrin-generated signal to induce pp125FAK mRNA and protein expression. Protein kinase C activation in CD3+ T cells following integrin stimulation was also impaired in HIV-infected individuals, mostly among the symptomatic/AIDS patients. Protein kinase C inactivation in T cells was shown to have a destabilizing effect in vitro on pp125FAK mRNA that contains an AUUUA motif in the 3'-untranslated region, a consensus sequence for the AU-rich elements responsible for mRNA destabilization. These aberrant changes in pp125FAK expression may have direct significance to the overall immunopathogenesis during infection with HIV-1.