Lck is required for activation-induced T cell death after TCR ligation with partial agonists

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.     

Non-Fc receptor-binding humanized anti-CD3 antibodies induce apoptosis of activated human T cells

Human trials in organ allografts have demonstrated that murine anti-CD3 mAbs are immunosuppressive. By mimicking Ag, anti-CD3 can produce T cell activation, anergy, or death. Activation of resting T cells in vivo results in dose-limiting cytokine release and is caused by Ab-mediated cross-linking of T cells and Fcgamma receptor (FcR)-bearing cells. With the goal of minimizing cytokine-induced toxicity, anti-CD3 have been engineered to lower Fc binding avidity. Preclinical murine studies have indicated that non-FcR-binding anti-CD3 can induce apoptosis of Ag-activated T cells. Since induction of T cell apoptosis may be an important mechanism of immunosuppression by anti-CD3, we tested whether Fc mutations affect the ability of anti-human CD3 to induce apoptosis of activated T cells. We compared wild-type murine anti-CD3, M291, and OKT3 and their humanized, FcR- and non-FcR-binding structural variants in quantitative assays of T cell apoptosis. Non-FcR-binding variants produced more sustainable phosphorylation of extracellular signal-regulated kinase-2, greater release of IFN-gamma, and more effectively caused activation-dependent T cell apoptosis. Non-FcR-binding variants dissociated more quickly from the T cell surface and caused less internalization of the TCR, which then remained available in greater abundance on the cell surface for signaling. Cross-linking of non-FcR-binding variants by antiglobulin enhanced TCR internalization and minimized induction of T cell apoptosis. We conclude that non-FcR-binding, humanized anti-CD3 have improved ability to induce apoptosis of activated T cells, presumably by allowing durable expression of the TCR and sustained signaling.     

Naive T cells are resistant to anergy induction by anti-CD3 antibodies

Anti-CD3 mAbs are potent immunosuppressive agents used in clinical transplantation. It has been generally assumed that one of the anti-CD3 mAb-mediated tolerance mechanisms is through the induction of naive T cell unresponsiveness, often referred to as anergy. We demonstrate in this study that naive T cells stimulated by anti-CD3 mAbs both in vivo and in vitro do not respond to the superantigen staphylococcal enterotoxin B nor to soluble forms of anti-CD3 mAbs and APC, but express increased reactivity to plastic-coated forms of the same anti-CD3 mAbs and to their nominal Ag/class II MHC, a finding that is difficult to rationalize with the concept of anergy. Phenotypic and detailed kinetic studies further suggest that a strong signal 1 delivered by anti-CD3 mAbs in the absence of costimulatory molecules does not lead to anergy, but rather induces naive T cells to change their mitogen responsiveness and acquire features of memory T cells. In marked contrast, Ag-experienced T cells are sensitive to anergy induction under the same experimental settings. Collectively, these studies demonstrate that exposure of naive T cells in vivo and in vitro to a strong TCR stimulus does not induce Ag unresponsiveness, indicating that sensitivity to negative signaling through TCR/CD3 triggering is developmentally regulated in CD4(+) T cells.     

Defective T cell receptor-mediated signal transduction in memory CD4 T lymphocytes exposed to superantigen or anti-T cell receptor antibodies

Lymphocytes must promote protective immune responses while still maintaining self-tolerance. Stimulation through the T cell receptor (TCR) can lead to distinct responses in naive and memory CD4 T cells. Whereas peptide antigen stimulates both naive and memory T cells, soluble anti-CD3 antibodies and bacterial superantigens stimulate only naive T cells to proliferate and secrete cytokines. Further, superantigens, like staphylococcal enterotoxin B (SEB), cause memory T cells to become anergic while soluble anti-CD3 does not. In the present report, we show that signal transduction through the TCR is impaired in memory cells exposed to either anti-CD3 or SEB. A block in signaling leads to impaired activation of the kinase ZAP-70 so that downstream signals and cell proliferation do not occur. We further show that the signaling defect is unique to each agent. In anti-CD3-treated memory T cells, the src kinase Lck is only transiently activated and does not phosphorylate and activate ZAP-70. In SEB-treated memory T cells, ZAP-70 does not interact with the TCR/CD3 complex to become accessible to Lck. Finally, we provide evidence that alternative signaling pathways are initiated in SEB-treated memory cells. Altered signaling, indicated by an elevation in activity of the src kinase Fyn, may be responsible for memory cell anergy caused by SEB. Thus, differentiation of naive T cells into memory cells is accompanied by alterations in TCR-mediated signaling that can promote heightened recall immunity or specific tolerance.     

SKAP55 recruits to lipid rafts and positively mediates the MAPK pathway upon T cell receptor activation

T cell receptor (TCR) engagement triggers a series of events including protein tyrosine kinase activation, tyrosine phosphorylation of adapter proteins, and multiple protein-protein interactions. We observed that adapter protein SKAP55, the Src kinase-associated phosphoprotein, formed homodimers through its SH3 domain and SK region. SKAP55 as a substrate interacted with Fyn kinase in vivo. In Jurkat cells, interaction between SKAP55 and Fyn kinase depended on TCR activation. Stable overexpression of SKAP55 in Jurkat cells caused mitogen-activated protein kinase activation following TCR engagement. Anti-CD3 stimulation also promoted the interaction of SKAP55 with Grb-2 in T cells. Mutational analysis revealed that tyrosine 271 in SKAP55 played a pivotal role for interaction with both Fyn kinase and adapter protein Grb-2, indicating that the Fyn-phosphorylated SKAP55 transiently associates with adapter Grb-2 to mediate mitogen-activated protein kinase activation. Intriguingly, T cell receptor engagement dramatically induced the translocation of endogenous SKAP55 to lipid rafts where SKAP55 was found to interact with Fyn kinase, suggesting that the positive function of SKAP55 via its association with Fyn and other signaling components may have been involved in raft-mediated T cell activation.     

Superantigen-induced CD4 memory T cell anergy. I. Staphylococcal enterotoxin B induces Fyn-mediated negative signaling

Memory CD4 T cells must provide robust protection for an organism while still maintaining self-tolerance. Superantigens reveal a memory cell-specific regulatory pathway, by which signaling through the TCR can lead to clonal tolerance (anergy). Here we show that the src kinase Fyn is a critical regulator of anergy in murine memory CD4 T cells induced by the bacterial superantigen staphylococcal enterotoxin B (SEB). Exposure to SEB results in impaired TCR signaling due to failed CD3/ZAP-70 complex formation. Further, signal transduction through the TCR remains similarly blocked when anergic memory cells are subsequently exposed to agonist peptide antigen. Pharmacological inhibition or genetic elimination of Fyn kinase reverses memory cell anergy, resulting in SEB-induced cell proliferation. The mechanism underlying impaired TCR signaling and subsequent memory cell anergy must involve a Fyn signaling pathway given that the suppression of Fyn activity restores CD3/ZAP-70 complex formation and TCR proximal signaling.     

In vitro and in vivo activation of murine gamma/delta T cells induces the expression of IgA, IgM, and IgG Fc receptors.

The present studies examined resting and activated murine gamma/delta T lymphocytes, in vitro and in vivo, for surface expression of FcR. Polyclonal gamma/delta TCR+ lymphocytes selectively grown from the spleen and intestine of normal mice did not express FcR when the cells were in a resting state, but when cells were activated with anti-CD3 antibody virtually all of the splenic gamma/delta lymphocytes and a large subpopulation of the intestinal gamma/delta lymphocytes expressed IgA and IgM FcR. This was confirmed by using transgenic mice. Resting gamma/delta TCR+ lymphocytes from the spleen, thymus, lymph node, and blood of gamma/delta TCR transgenic mice did not express FcR for any of the five major classes of Ig H chains. Activation of the gamma/delta TCR+ cells via the CD3/TCR complex induced high levels of IgM and IgA FcR and low levels of IgG FcR. Finally, in hepatic granulomas of schistosome-infected mice, activated gamma/delta TCR+ cells are present and express high levels of IgA and IgM FcR and low levels of IgG FcR. These investigations establish that transition of gamma/delta TCR+ lymphocytes from a resting to an activated state (triggered via the T3Ti TCR complex) is accompanied by the induction of surface membrane receptors specific for Ig H chain isotypes. The activation-linked expression of FcR on gamma/delta TCR+ lymphocytes provides potential mechanisms for coupling the functional activities of gamma/delta T lymphocytes with immune mechanisms that involve Ig molecules, such as antibody-dependent cellular cytotoxicity.     

Lck和Fyn对T细胞发育过程的影响

胸腺中T细胞的发育及次级淋巴组织中成熟T细胞的活化均需要细胞能够对环境信号分子做出适应性的反应。在共刺激分子及细胞因子受体介导的信号参与下通过TCR(T cell receptor)及其辅助受体CD4和CD8与MHC/抗原肽复合物相互作用,可以诱导TCR信号通路激活并最终导致T细胞免疫反应的发生。Src家族激酶Lck(Lymphocyte-specific protein tyrosine kinase)和Fyn(Proto-oncogene tyrosine-protein kinase)的激活是启动TCR信号通路的关键因素。在T细胞的发育、阳性选择、初始T细胞的外周存活及由淋巴细胞缺失诱导的细胞增殖中都起着关键性的作用。研究显示,虽然这两种信号分子紧密相关,但在某些条件下Lck发挥着比Fyn更重要的作用,并且Fyn仅可以补充Lck的部分功能。文章针对这两个激酶在T细胞发育的整个过程中的作用机制进行了论述。     

Lck和Fyn对T细胞发育过程的影响

胸腺中T细胞的发育及次级淋巴组织中成熟T细胞的活化均需要细胞能够对环境信号分子做出适应性的反应。在共刺激分子及细胞因子受体介导的信号参与下通过TCR(T cell receptor )及其辅助受体CD4和CD8与MHC/抗原肽复合物相互作用, 可以诱导TCR信号通路激活并最终导致T细胞免疫反应的发生。Src家族激酶Lck(Lymphocyte-specific protein tyrosine kinase)和Fyn (Proto-oncogene tyrosine-protein kinase)的激活是启动TCR信号通路的关键因素。在T细胞的发育、阳性选择、初始T细胞的外周存活及由淋巴细胞缺失诱导的细胞增殖中都起着关键性的作用。研究显示, 虽然这两种信号分子紧密相关, 但在某些条件下Lck发挥着比Fyn更重要的作用, 并且Fyn仅可以补充Lck的部分功能。文章针对这两个激酶在T细胞发育的整个过程中的作用机制进行了论述。     

Cell surface comodulation of CD4 and T cell receptor by anti-CD4 monoclonal antibody

In our study we have used anti-CD4 mAb to investigate the cell surface association between CD4 and the Ag-specific TCR complex on mature peripheral T cells. Anti-CD4 mAb was administered in vivo and in vitro and its effects on CD4 and CD3 cell surface expression were determined. In vivo, anti-CD4 mAb reduced cell surface expression of its ligand, CD4, and secondarily also reduced cell surface expression of CD3/TCR on CD4+ splenic T cells. In vitro, multivalent cross-linking of CD4 by anti-CD4 mAb and either FcR+ cells or anti-Ig mAb also resulted in decreased surface expression of CD4 and specific comodulation of CD3/TCR. The secondary reduction in cell surface CD3/TCR expression induced by CD4 cross-linking could be pharmacologically disrupted by high doses of PMA, indicating that the comodulation of CD3 with CD4 was dependent upon intracellular mediators, possibly including protein kinase C. These results demonstrate that, in the presence of anti-CD4 mAb, CD4 is functionally associated with the CD3/TCR complex, and that this association is dependent upon the activity of intracellular mediators. Such intracellular mediators might induce the coordinate down-modulation of physically unassociated CD4 and CD3/TCR molecules, or, alternatively, might promote a physical interaction between CD4 and CD3/TCR molecules.     

Ligation of the T cell receptor complex results in phosphorylation of Smad2 in T lymphocytes

TGF-beta modulates immune responses by regulating T cell function. The Smad family of proteins has been recently shown to transduce signals for the TGF-beta superfamily and Smad2 mediates TGF-beta signaling. Here, we showed that TGF-beta phosphorylated Smad2 and induced interaction between Smad2 and Smad4 in primary T cells and the Jurkat T cell line. Interestingly, ligation of the T cell receptor (TCR)/CD3 complex with anti-CD3 mAb also phosphorylated Smad2, but failed to induce interaction between Smad2 and Smad4 in the Jurkat T cell line. Phosphorylation of Smad2 via the TCR/CD3 complex was not abrogated by treatment with neutralizing antibody against TGF-beta. Furthermore, PD98059, a MEK inhibitor, suppressed Smad2 phosphorylation by stimulation with anti-CD3 mAb in Jurkat T cell line. These findings indicated that not only TGF-beta but also stimulation via the TCR/CD3 complex phosphorylated Smad2 through mitogen-activated protein (MAP) kinase cascades, suggesting that Smad2 may function in both TGF-beta- and TCR/CD3 complex-mediated signaling pathways in T cells.     

Human T cell activation induced by a monoclonal mouse IgG3 anti-CD3 antibody (RIV9) requires binding of the Fc part of the antibody to the monocytic 72-kDa high-affinity Fc receptor (FcRI)

The mitogenic activity of anti-CD3 mouse monoclonal antibodies (mAb) in cultures of human peripheral blood mononuclear cells (PBMC) depends on the ability of the mAb to interact with CD3 molecules on the T cells, and with Fc receptors (FcR) on monocytes. Two types of FcR with distinct specificity for murine (m) IgG subclasses are involved: a 72-kDa receptor (FcRI) binds mIgG2a and a 40-kDa receptor (FcRII) binds mIgG1. In this study we examined the mitogenic activity of mIgG3 anti-CD3 mAb RIV9. In cultures of human PBMC, the mAb induced T cell proliferation and interleukin 2 production. We found that subjects, unresponsive to mIgG2a anti-CD3 (e.g., OKT3), were also RIV9 nonresponders. In contrast, nonresponders to mIgG1 anti-CD3 (e.g., anti-Leu4) had a normal response to RIV9. Our results therefore suggested that anti-CD3 mAb of the mIgG2a and mIgG3 subclass bind to the same monocytic FcR. Human monomeric IgG, which has been shown to bind to FcRI only, blocked T cell proliferation induced by mIgG2a and mIgG3 anti-CD3, but had no effect on T cell proliferation induced by mIgG1 anti-CD3. In contrast, a mAb (IV.3) to FcRII, which blocks ligand binding of the receptor, blocked the mitogenic activity of mIgG1 anti-CD3 antibodies, but had no effect on T cell proliferation induced by mIgG3 anti-CD3 or by mIgG2a anti-CD3. Binding of RIV9 to FcR of responder monocytes could be demonstrated in immunofluorescence. Monocytes from the RIV9 nonresponder subjects however were unable to bind the Fc portion of this antibody. The binding of fluorescein (FITC)-conjugated mIgG3 or FITC-conjugated mIgG2a to responder monocytes could be inhibited by human monomeric IgG and by mIgG2a and mIgG3, but not by the mAb to FcRII. The results demonstrate that mIgG3 binds to FcRI on human monocytes and that this binding is needed for the mitogenic activity of mIgG3 anti-CD3.     

Molecular basis for T cell response induced by altered peptide ligand of type II collagen

Mounting evidence from animal models has demonstrated that alterations in peptide-MHC interactions with the T cell receptor (TCR) can lead to dramatically different T cell outcomes. We have developed an altered peptide ligand of type II collagen, referred to as A9, which differentially regulates TCR signaling in murine T cells leading to suppression of arthritis in the experimental model of collagen-induced arthritis. This study delineates the T cell signaling pathway used by T cells stimulated by the A9·I-A(q) complex. We have found that T cells activated by A9 bypass the requirement for Zap-70 and CD3-ζ and signal via FcRγ and Syk. Using collagen-specific T cell hybridomas engineered to overexpress either Syk, Zap-70, TCR-FcRγ, or CD3-ζ, we demonstrate that A9·I-A(q) preferentially activates FcRγ/Syk but not CD3-ζ/Zap-70. Moreover, a genetic absence of Syk or FcRγ significantly reduces the altered peptide ligand induction of the nuclear factor GATA3. By dissecting the molecular mechanism of A9-induced T cell signaling we have defined a new alternate pathway that is dependent upon FcRγ and Syk to secrete immunoregulatory cytokines. Given the interest in using Syk inhibitors to treat patients with rheumatoid arthritis, understanding this pathway may be critical for the proper application of this therapy.     

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.     

JNK2 is required for efficient T-cell activation and apoptosis but not for normal lymphocyte development

BACKGROUND: The Jun N-terminal kinase (JNK) signaling pathway has been implicated in cell proliferation and apoptosis, but its function seems to depend on the cell type and inducing signal. In T cells, JNK has been implicated in both antigen-induced activation and apoptosis. RESULTS: We generated mice lacking the JNK2 isozymes. The mutant mice were healthy and fertile but defective in peripheral T-cell activation induced by antibody to the CD3 component of the T-cell receptor (TCR) complex - proliferation and production of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) were reduced. The proliferation defect was restored by exogenous IL-2. B-cell activation was normal in the absence of JNK2. Activation-induced peripheral T-cell apoptosis was comparable between mutant and wild-type mice, but immature (CD4(+) CD8(+)) thymocytes lacking JNK2 were resistant to apoptosis induced by administration of anti-CD3 antibody in vivo. The lack of JNK2 also resulted in partial resistance of thymocytes to anti-CD3 antibody in vitro, but had little or no effect on apoptosis induced by anti-Fas antibody, dexamethasone or ultraviolet-C (UVC) radiation. CONCLUSIONS: JNK2 is essential for efficient activation of peripheral T cells but not B cells. Peripheral T-cell activation is probably required indirectly for induction of thymocyte apoptosis resulting from administration of anti-CD3 antibody in vivo. JNK2 functions in a cell-type-specific and stimulus-dependent manner, being required for apoptosis of immature thymocytes induced by anti-CD3 antibody but not for apoptosis induced by anti-Fas antibody, UVC or dexamethasone. JNK2 is not required for activation-induced cell death of mature T cells.     

CD28 costimulation induces delta opioid receptor expression during anti-CD3 activation of T cells

Previous studies have demonstrated that naive splenic mouse T cells express no or only very low levels of the delta-type opioid receptor (delta OR), but stimulation of mouse splenocytes with Con A results in induction of delta OR mRNA and protein. In this report we have shown that stimulation of highly purified populations of naive mouse T cells with anti-CD3 mAb alone results in T cell activation, as evidenced by sustained IL-2 secretion and cell proliferation, but fails to elicit delta OR expression. However, delta OR expression is induced by costimulation of these very pure T cells with anti-CD3 and anti-CD28 mAbs. The delta OR induction by anti-CD3 and anti-CD28 costimulation was completely blocked by inhibition of phosphatidylinositol 3-kinase with wortmannin. Because phosphatidylinositol 3-kinase activation in T cells is linked to costimulation, these results suggest that induction of delta OR expression during T cell activation is strictly dependent on costimulation. It also appears that costimulatory receptors other than CD28 can provide the signaling required for delta OR expression because delta OR mRNA was induced by Con A stimulation of splenocytes from CD28-deficient mice.     

Lipid raft assembly and Lck recruitment in TRAIL costimulation mediates NF-κB activation and T cell proliferation

The TNF-related apoptosis-inducing ligand was shown to provide a costimulatory signal that cooperates with the TCR/CD3 complex to induce T cell proliferation and cytokine production. Although a number of signaling pathways were linked to the TCR/CD3 complex, it is not known how these two receptors cooperate to induce T cell activation. In this study, we show that TRAIL-induced costimulation of T cells depends on activation of the NF-κB pathway. TRAIL induced the NF-κB pathway by phosphorylation of inhibitor of κB factor kinase and protein kinase C in conjunction with anti-CD3. Furthermore, we demonstrated that TRAIL costimulation induced phosphorylation of the upstream TCR-proximal tyrosine kinases, Lck and ZAP70. Ligation of the TRAIL by its soluble receptor, DR4-Fc, alone was able to induce the phosphorylation of Lck and ZAP70 and to activate the NF-κB pathway; however, it was insufficient to fully activate T cells to support T cell proliferation. In contrast, TRAIL engagement in conjunction with anti-CD3, but not TRAIL ligation alone, induced lipid raft assembly and recruitment of Lck and PKC. These results demonstrate that TRAIL costimulation mediates NF-κB activation and T cell proliferation by lipid raft assembly and recruitment of Lck. Our results suggest that in TRAIL costimulation, lipid raft recruitment of Lck integrates mitogenic NF-κB-dependent signals from the TCR and TRAIL in T lymphocytes.     

p59fyn (Fyn) promotes the survival of anergic CD4-CD8- alpha beta TCR+ cells but negatively regulates their proliferative response to antigen stimulation

T cell anergy is characterized by alterations in TCR signaling that may play a role in controlling the unresponsiveness of the anergic cell. We have addressed questions regarding the importance of the Src kinase p59(fyn) (Fyn) in this process by using Fyn null mice. We demonstrate that a mature population of CD4(-)CD8(-) alphabeta TCR(+) anergic T cells lacking Fyn have a substantial recovery of their proliferation defect in response to Ag stimulation. This recovery cannot be explained by ameliorated production of IL-2, and the improved proliferation correlates with an enhanced ability of the Fyn(-/-) anergic T cells to up-regulate the high affinity IL-2 receptor. We also observe that anergic CD4(-)CD8(-) alphabeta TCR(+) T cells have a heightened survival ability that is partially dependent on the elevated levels of Fyn and IL-2 receptor beta-chain expressed by these cells. The enhanced survival correlates with an increased capacity of the anergic cells to respond to IL-15. We conclude that Fyn plays an important role in aspects of T cell anergy pertaining to TCR signaling and to cell survival.     

T cell-specific adapter protein inhibits T cell activation by modulating Lck activity

We previously reported the isolation of a cDNA encoding a T cell-specific adapter protein (TSAd). Its amino acid sequence contains an SH2 domain, tyrosines in protein binding motifs, and proline-rich regions. In this report we show that expression of TSAd is induced in normal peripheral blood T cells stimulated with anti-CD3 mAbs or anti-CD3 plus anti-CD28 mAbs. Overexpression of TSAd in Jurkat T cells interfered with TCR-mediated signaling by down-modulating anti-CD3/PMA-induced IL-2 promoter activity and anti-CD3 induced Ca2+ mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-gamma1, SH2-domain-containing leukocyte-specific phosphoprotein of 76kDa, and linker for activation of T cells was also reduced. Furthermore, TSAd inhibited Zap-70 recruitment to the CD3zeta-chains in a dose-dependent manner. Consistent with this, Lck kinase activity was reduced 3- to 4-fold in COS-7 cells transfected with both TSAd and Lck, indicating a regulatory effect of TSAd on Lck. In conclusion, our data strongly suggest an inhibitory role for TSAd in proximal T cell activation.