Negative regulation of TCR signaling by linker for activation of X cells via phosphotyrosine-dependent and -independent mechanisms

The activation of T cells and the initiation of an immune response is tightly controlled through the crosstalk of both positive and negative regulators. Two adaptors that function as negative regulators of T cell activation are adaptor in lymphocytes of unknown function X (ALX) and linker for activation of X cell (LAX). Previously, we showed that T cells from mice deficient in ALX and LAX display similar hyperresponsiveness, with increased IL-2 production and proliferation upon TCR/CD28 stimulation, and that these adaptors physically associate. In this study, we analyze the nature of the association between ALX and LAX. We demonstrate that this association occurs in the absence of TCR/CD28 signaling via a mechanism independent of both tyrosine phosphorylation of LAX and the SH2 domain of ALX. Cotransfection of ALX with LAX resulted in LAX tyrosine phosphorylation in the absence of TCR/CD28 stimulation. ALX-mediated LAX phosphorylation depends upon the ALX SH2 domain, which functions to recruit Lck to LAX. We also show that LAX, like ALX, can inhibit RE/AP reporter activation. However, in contrast to its inhibition of NFAT, the inhibition of RE/AP by LAX is independent of its tyrosine phosphorylation. Therefore, it can be concluded that inhibition of signaling events involved in T cell activation by LAX occurs through mechanisms both dependent on and independent of its tyrosine phosphorylation.     

Negative regulation of interleukin-2 and p38 mitogen-activated protein kinase during T-cell activation by the adaptor ALX

Activation of na?ve T cells requires synergistic signals produced by the T-cell receptor (TCR) and by CD28. We previously identified the novel adaptor ALX, which, upon overexpression in Jurkat T cells, inhibited activation of the interleukin-2 (IL-2) promoter by TCR/CD28, suggesting that it is a negative regulator of T-cell activation. To further understand the physiological role of ALX, ALX-deficient mice were generated. Purified T cells from ALX-deficient mice demonstrated increased IL-2 production, CD25 expression, and proliferation in response to TCR/CD28 stimulation. Enhanced IL-2 production and proliferation were also observed when ALX-deficient mice were primed in vivo with ovalbumin-complete Freund's adjuvant and then restimulated ex vivo. Consistent with our initial overexpression studies, these data demonstrate that ALX is a negative regulator of T-cell activation. While TCR/CD28-mediated activations of phosphotyrosine induction, extracellular signal-regulated kinase 1/2, Jun N-terminal protein kinase, IkappaB kinase alpha/beta, and Akt were unaltered, constitutive activation of p38 mitogen-activated protein kinase and its upstream regulators MKK3/6 were observed for ALX-deficient splenocytes. The phenotype of ALX-deficient mice resembled the phenotype of those deficient in the transmembrane adaptor LAX, and an association between ALX and LAX proteins was demonstrated. These results suggest that ALX, in association with LAX, negatively regulates T-cell activation through inhibition of p38.     

Negative regulation of lymphocyte activation by the adaptor protein LAX

The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.     

The related adaptors, adaptor in lymphocytes of unknown function X and Rlk/Itk-binding protein, have nonredundant functions in lymphocytes

Adaptors play a critical role in regulating signaling pathways that control lymphocyte development and activation. Adaptor in lymphocytes of unknown function X (ALX) and Rlk/Itk-binding protein (RIBP) are adaptors related by structure and sequence, coexpressed in T cells. Mice deficient for each adaptor demonstrated that ALX and RIBP, respectively, negatively and positively regulate T cell activation in response to TCR/CD28 stimulation. However, these results did not preclude that they may function redundantly in other cell populations, or in response to other stimuli. Therefore, to understand the relationship between these related adaptors, ALX/RIBP-deficient mice were generated. We demonstrate that although ALX and RIBP are expressed throughout T cell development, T cell development occurs normally in these mice. Using the H-Y TCR transgenic model, positive and negative selection were found to proceed unimpeded in the absence of ALX and RIBP. We demonstrate that RIBP is also expressed in B cells; however, RIBP- and ALX/RIBP-deficient mice had normal B cell development, and responded equivalently to wild type in response to IgM, CD40, B cell-activating factor/B lymphocyte stimulator, CpG, and LPS. Interestingly, T cells deficient in both ALX and RIBP behaved similarly to those deficient in ALX alone during T cell activation in response to TCR/CD28, exhibiting increased IL-2 production, CD25 expression, and proliferation, thus showing that ALX deficiency masked the effect of RIBP deficiency. ALX/RIBP-deficient T cells did not have any alterations in either activation-induced cell death or Th1/2 polarization. Therefore, we did not find any functional redundancy or synergy during lymphocyte development, selection, activation, or survival in ALX/RIBP-deficient mice, demonstrating that these molecules function independently.     

Differential Src family kinase activity requirements for CD3 zeta phosphorylation/ZAP70 recruitment and CD3 epsilon phosphorylation

The current model of T cell activation is that TCR engagement stimulates Src family tyrosine kinases (SFK) to phosphorylate CD3zeta. CD3zeta phosphorylation allows for the recruitment of the tyrosine kinase ZAP70, which is phosphorylated and activated by SFK, leading to the phosphorylation of downstream targets. We stimulated mouse CTLs with plate-bound anti-CD3 and, after cell lysis, recovered proteins that associated with the CD3 complex. The protein complexes were not preformed, and a number of tyrosine-phosphorylated proteins were inducibly and specifically associated with the TCR/CD3 complex. These results suggest that complex formation only occurs at the site of TCR engagement. The recruitment and tyrosine phosphorylation of most proteins were abolished when T cells were stimulated in the presence of the SFK inhibitor PP2. Surprisingly, CD3zeta, but not CD3epsilon, was inducibly tyrosine phosphorylated in the presence of PP2. Furthermore, ZAP70 was recruited, but not phosphorylated, after TCR stimulation in the presence of PP2, thus confirming the phosphorylation status of CD3zeta. These data suggest that there is a differential requirement for SFK activity in phosphorylation of CD3zeta vs CD3epsilon. Consistent with this possibility, ZAP70 recruitment was also detected with anti-CD3-stimulated, Lck-deficient human Jurkat T cells. We conclude that TCR/CD3-induced CD3zeta phosphorylation and ZAP70 recruitment do not absolutely require Lck or other PP2-inhibitable SFK activity, but that SFK activity is absolutely required for CD3epsilon and ZAP70 phosphorylation. These data reveal the potential for regulation of signaling through the TCR complex by the differential recruitment or activation of SFK.     

A serine phosphatase is involved in CD2-mediated activation of human T lymphocytes and natural killer cells.

We investigated early activation events after T cell triggering via the Ag receptor (TCR/CD3) complex as compared to activation via the CD2 surface molecule. To this end, resting peripheral human T lymphocytes were preincubated with 32P-orthophosphate and subsequently exposed to mitogenic mAb directed at either TCR/CD3 or CD2 for varying time periods. Cells were lysed and postnuclear lysates subjected to two-dimensional-gel electrophoresis (IEF and SDS-PAGE). As early as 10 min after stimulation through CD2, dephosphorylation of a cytosolic 19-kDa protein was observed. In contrast, this protein remained phosphorylated in unstimulated as well as CD3 activated T cells. Phosphoprotein (pp) 19 dephosphorylation was transient because, at later time points (2-4 h) after CD2 triggering, this protein was phosphorylated again. Phosphoaminoacid analysis indicated that pp19 is dephosphorylated on serine residues. Identical results were obtained using a CD2+ but TCR/CD3- human NK cell clone indicating that pp19 dephosphorylation occurs independent of surface expression of a TCR/CD3 complex. These data show that, in addition to protein phosphorylation events, serine dephosphorylation is involved in T cell triggering. More important, a selective signaling mechanism appears to be linked to T cell activation through the CD2 pathway.     

Critical role of TNF receptor type-2 (p75) as a costimulator for IL-2 induction and T cell survival: a functional link to CD28

CD28 provides important signals that lower the threshold of T cell activation, augment the production of IL-2, and promote T cell survival. The recent identification of a second family of costimulatory molecules within the TNFR family has reshaped the "two-signal" model of T cell activation. In this study the role of p75 as a T cell costimulatory molecule in controlling cell fate during TCR/CD28-mediated stimulation was examined. We found that p75-deficient T cells possess a profound defect in IL-2 production in response to TCR/CD28-mediated stimulation. Examination of key signaling intermediates revealed that TCR proximal events such as global tyrosine phosphorylation and ZAP70 phosphorylation, as well as downstream MAPK cascades are unperturbed in p75-deficient T cells. In contrast, p75 is nonredundantly coupled to sustained AKT activity and NF-kappaB activation in response to TCR/CD28-mediated stimulation. Moreover, p75-deficient T cells possess a defect in survival during the early phase of T cell activation that is correlated with a striking defect in Bcl-x(L) expression. These data indicate discrete effects of p75 on the intracellular signaling milieu during T cell activation, and reveal the synergistic requirement of TCR, CD28, and p75 toward optimal IL-2 induction and T cell survival. We propose that p75 acts as one of the earliest of the identified costimulatory members of the TNFR family, and is functionally linked to CD28 for initiating and determining T cell fate during activation.     

Tyrosine phosphorylation of a novel 100-kDa protein coupled to CD28 in resting human T cells is enhanced by a signal through TCR/CD3 complex

For T cell activation, two signals are required, i.e., a T cell receptor (TCR)/CD3-mediated main signal and a CD28-mediated costimulatory signal. CD28 binds to its ligand (CD80 or CD86) and transduces the most important costimulatory signal. The cytoplasmic domain of the CD28 molecule, composed of 41 amino acids, does not contain any intrinsic enzyme activity. The cytoplasmic domain of CD28 is remarkably conserved among species and is associated with a number of signaling molecules that affect the main signal. We report here that a tyrosine phosphorylated 100-kDa protein (ppl00) was coupled to the CD28 cytoplasmic domain in Jurkat and human peripheral T cells. The pp100 was distinguished from other CD28 associated molecules such as Vav, STAT5, PI 3-kinase, Valosin-containing protein (VCP), Nucleolin, Gab2 (Grb2-associated binding protein 2), and STAT6. The tyrosine phosphorylation of pp100 coprecipitated with CD28 was enhanced by CD3 stimulation by the specific antibody, tyrosine phosphatase inhibitor and PKC activator. Tyrosine phosphorylation of pp100 was attenuated by the prior addition of PKC inhibitor. These findings indicate that pp100 is a novel tyrosine phosphorylated protein coupled to CD28 under continuous control of tyrosine phosphatases and might play a role in T cell activation augmented by a TCR/CD3-mediated main signal.     

Abnormal T cell receptor signal transduction of CD4 Th cells in X-linked lymphoproliferative syndrome

The molecular basis of X-linked lymphoproliferative (XLP) disease has been attributed to mutations in the signaling lymphocytic activation molecule-associated protein (SAP), an src homology 2 domain-containing intracellular signaling molecule known to interact with the lymphocyte-activating surface receptors signaling lymphocytic activation molecule and 2B4. To investigate the effect of SAP defects on TCR signal transduction, herpesvirus saimiri-immortalized CD4 Th cells from XLP patients and normal healthy individuals were examined for their response to TCR stimulation. CD4 T cells of XLP patients displayed elevated levels of tyrosine phosphorylation compared with CD4 T cells from healthy individuals. In addition, downstream serine/threonine kinases are constitutively active in CD4 T cells of XLP patients. In contrast, TCR-mediated activation of Akt, c-Jun-NH(2)-terminal kinases, and extracellular signal-regulated kinases in XLP CD4 T cells was transient and rapidly diminished when compared with that in control CD4 T cells. Consequently, XLP CD4 T cells exhibited severe defects in up-regulation of IL-2 and IFN-gamma cytokine production upon TCR stimulation and in MLRs. Finally, SAP specifically interacted with a 75-kDa tyrosine-phosphorylated protein upon TCR stimulation. These results demonstrate that CD4 T cells from XLP patients exhibit aberrant TCR signal transduction and that the defect in SAP function is likely responsible for this phenotype.     

Involvement of hematopoietic progenitor kinase 1 in T cell receptor signaling

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related serine/threonine protein kinase, is a hematopoietic-specific upstream activator of the c-Jun N-terminal kinase. Here, we provide evidence to demonstrate the involvement of HPK1 in T cell receptor (TCR) signaling. HPK1 was activated and tyrosine-phosphorylated with similar kinetics following TCR/CD3 or pervanadate stimulation. Co-expression of protein-tyrosine kinases, Lck and Zap70, with HPK1 led to HPK1 activation and tyrosine phosphorylation in transfected mammalian cells. Upon TCR/CD3 stimulation, HPK1 formed inducible complexes with the adapters Nck and Crk with different kinetics, whereas it constitutively interacted with the adapters Grb2 and CrkL in Jurkat T cells. Interestingly, HPK1 also inducibly associated with linker for activation of T cells (LAT) through its proline-rich motif and translocated into glycolipid-enriched microdomains (also called lipid rafts) following TCR/CD3 stimulation, suggesting a critical role for LAT in the regulation of HPK1. Together, these results identify HPK1 as a new component of TCR signaling. T cell-specific signaling molecules Lck, Zap70, and LAT play roles in the regulation of HPK1 during TCR signaling. Differential complex formation between HPK1 and adapters highlights the possible involvement of HPK1 in multiple signaling pathways in T cells.     

An adenoviral vector encoding dominant negative Cbl lowers the threshold for T cell activation in post-thymic T cells

Cbl family ubiquitin ligases act as key negative regulators of TCR signaling. Knockout mice lacking Cbl-b and c-Cbl show augmented T cell activation and CD28-independent IL-2 production. In order to study Cbl function directly in post-thymic T cells, a DN Cbl adenovirus was generated for transduction of T cells from Coxsackie/adenovirus receptor (CAR) transgenic (Tg) mice. We show that dominant negative (DN) Cbl-transduced CD4⁺ T cells exhibited enhanced IL-2 production upon TCR/CD28 engagement compared with empty adenoviral vector-transduced cells. This augmentation was reflected at both IL-2 mRNA and protein level, and correlated with increased protein phosphorylation of Vav, Akt, ERK, and p38MAPK. Our results indicate that introduction of dominant negative Cbl can potentiate activation of post-thymic CD4⁺ T cells, which argues for development of strategies to interfere with Cbl function as a method of immunopotentiation.     

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

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

Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation.

Antiphosphotyrosine immunoblots were used to characterize tyrosine phosphorylated proteins after stimulation of the human TCR. Increased tyrosine phosphorylation was evident on at least 12 substrates within 2 min after ligation of the TCR with mAb. Analysis of the time course for increased tyrosine phosphorylation revealed distinct patterns. Increased phosphorylation of 135-kDa and 100-kDa substrates was evident within 5 s, whereas increased phosphorylation of the TCR-zeta-chain required several minutes after treatment with anti-CD3 mAb. This rapid cellular tyrosine phosphorylation occurred independent of the cell cycle, as it occurred after stimulation of resting T cells, T cell blasts, and the Jurkat T cell leukemia line. When the TCR complex was cross-linked together with the CD4 receptor by heteroconjugate anti-CD3/CD4 mAb, an increased magnitude of tyrosine phosphorylation occurred, although no new substrates could be detected. The increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates was specific in that anti-HLA class I, anti-CD6, anti-CD7, and anti-CD28 antibodies did not cause increased tyrosine phosphorylation. Anti-CD4 stimulation of resting T cells did not cause increased tyrosine phosphorylation of pp100 and pp135, suggesting that the CD4-associated kinase, lck, does not account for the tyrosine phosphorylation observed after TCR stimulation. Similarly, pharmacologic treatment of cells with phorbol ester and calcium ionophore did not cause increased tyrosine phosphorylation of these substrates, indicating that activation of protein kinase C or phospholipase C does not account for these early increases in tyrosine phosphorylation. The time of onset of pp100 phosphorylation, and the magnitude of phosphorylation correlated with the magnitude of calcium mobilization when cells were stimulated with different forms of TCR stimulation. When cells were labeled with [3H]myoinositol and analyzed after stimulation by anti-CD3 mAb, increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates preceded the activation of phospholipase C, as measured by the appearance of inositol 1,4,5-trisphosphate. This occurred in both T cell blasts and in the Jurkat T cell line. Thus, these findings show that increased tyrosine phosphorylation is the earliest yet detected signal observed after ligation of the TCR complex, and furthermore suggest that tyrosine phosphorylation might link the TCR to the phosphatidylinositolbisphosphate hydrolysis signaling pathway.     

Structural and functional evidence that Nck interaction with CD3epsilon regulates T-cell receptor activity

Recruitment of signaling molecules to the cytoplasmic domains of the CD3 subunits of the T-cell receptor (TCR) is crucial for early T-cell activation. These transient associations either do or do not require tyrosine phosphorylation of CD3 immune tyrosine activation motifs (ITAMs). Here we show that the non-ITAM-requiring adaptor protein Nck forms a complex with an atypical PxxDY motif of the CD3ε tail, which encompasses Tyr166 within the ITAM and a TCR endocytosis signal. As suggested by the structure of the complex, we find that Nck binding inhibits phosphorylation of the CD3ε ITAM by Fyn and Lck kinases in vitro. Moreover, the CD3ε-Nck interaction downregulates TCR surface expression upon physiological stimulation in mouse primary lymph node cells. This indicates that Nck performs an important regulatory function in T lymphocytes by inhibiting ITAM phosphorylation and/or removing cell surface TCR via CD3ε interaction.     

Cutting edge: TCR stimulation by antibody and bacterial superantigen induces Stat3 activation in human T cells.

Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.