TCR Zeta-chain Research Articles

The phosphotyrosine phosphatase SHP-2 participates in a multimeric signaling complex and regulates T cell receptor (TCR) coupling to the Ras/mitogen-activated protein kinase (MAPK) pathway in Jurkat T cells.

Src homology 2 (SH2) domain-containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase-mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-zeta chain tyrosine phosphorylation or TCR-elicited Ca2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2-associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3'-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3'-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process.

Altered pattern of TCR/CD3-mediated protein-tyrosyl phosphorylation in T cells from patients with systemic lupus erythematosus. Deficient expression of the T cell receptor zeta chain.

Cellular immunity aberrations in patients with SLE are underscored by the abnormal early Ag receptor-mediated lymphocyte signal transduction pathway. To further characterize the T cell receptor (TCR)/CD3-initiated signaling defects, we studied 22 patients with SLE, 12 patients with other systemic rheumatic diseases, and 14 normal donors. The early (1 min) TCR/CD3-mediated tyrosine phosphorylation of cellular proteins with a molecular size between 36 and 64 kD was increased in 15 of 21 SLE patients, compared to normal or disease control subjects. The deficiency or absence of a band with a molecular size of approximately 16 kD in the immunoblots of SLE patients led us to investigate the expression of the TCRzeta chain. In immunoblots using anti-zeta antibodies we found that 10 of 22 lupus patients tested lacked the expression of TCRzeta, which was always present in control subjects (P < 0.001). Flow cytometric studies using permeabilized cells confirmed the deficiency or absence of the TCRzeta chain in lupus T cells. Using Northern blots we found that for eight patients tested, the TCRzeta mRNA was missing in three, decreased in three, and apparently normal in two patients (P < 0.003), but was always present in control subjects. Reverse transcriptase-PCR verified Northern blot results. We conclude that TCRzeta chain expression is either decreased or absent in the majority of patients with SLE, but not in patients with other systemic rheumatic diseases, regardless of disease activity, treatment status, or clinical manifestations. The previously described increases in TCR-initiated Ca2+ responses and the herein described increases in TCR-induced protein tyrosine phosphorylation and deficient TCRzeta expression may represent intrinsic defects modulating lupus T cell function.

Anti-peptide Antibodies Detect Conformational Changes of the Inter-SH2 Domain of ZAP-70 Due to Binding to the ζ Chain and to Intramolecular Interactions

T cell receptor (TCR) triggering induces association of the protein tyrosine kinase ZAP-70, via its two src-homology 2 (SH2) domains, to di-phosphorylated Immunoreceptor Tyrosine-based Activation Motifs (2pY-ITAMs) present in the intracellular tail of the TCR-zeta chain. The crystal structure of the SH2 domains complexed with a 2pY-ITAM peptide suggests that the 60-amino acid-long inter-SH2 spacer helps the SH2 domains to interact with each other to create the binding site for the 2pY-ITAM. To investigate whether the inter-SH2 spacer has additional roles in the whole ZAP-70, we raised antibodies against two peptides of this region and probed ZAP-70 structure under various conditions. We show that the reactivity of antibodies directed at both sequences was dramatically augmented toward the tandem SH2 domains alone compared with that of the entire ZAP-70. This indicates that the conformation of the inter-SH2 spacer is not maintained autonomously but is controlled by sequences C-terminal to the SH2 domains, namely, the linker region and/or the kinase domain. Moreover, antibody binding to the same two determinants was also inhibited when ZAP-70 or the SH2 domains bound to the zeta chain or to a 2pY-ITAM. Together, these two observations suggest a model in which intramolecular contacts keep ZAP-70 in a closed configuration with the two SH2 domains near to each other.

Pharmacological control of antigen responsiveness in genetically modified T lymphocytes.

A chimeric TCR gene, comprising an anti-hapten single-chain Ab variable fragment fused to the transmembrane and cytoplasmic regions of the human TCR zeta-chain, was used to determine whether the tetracycline-regulatable system could be used to regulate gene expression in T cells. Jurkat T cells were stably transfected with a single vector encoding the tetracycline trans-activator protein, controlled by a constitutive promoter, and the chimeric TCR, under the control of a trans-activator protein-responsive promoter. In the absence of tetracyclines, the transfected T cells were shown to express the chimeric receptor on the cell surface and could be activated by its cognate Ag, leading to the secretion of IL-2. When the cells were exposed to increasing concentrations of tetracyclines, surface expression of the chimeric receptor was suppressed in a dose-dependent manner, and this suppression was sufficient to result in complete loss of responsiveness to the targeted Ag. Prolonged suppression of trans-gene expression for up to 7 days was observed after doxycycline was removed from the cultures, but eventual recovery of surface expression was complete, and the absolute time to recovery was directly proportional to the initial concentration of the drug. Pharmacologic control of trans-gene expression in gene-modified T cells will not only facilitate new approaches to the study of different aspects of T cell biology, but will also provide the basis for new gene therapy strategies.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) interferes with extracellular signal-regulated kinase (ERK) and Jun NH2-terminal kinase (JNK) activation, but does not affect phosphorylation of T cell receptor zeta and ZAP70.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an important regulator of T cell homeostasis. Ligation of this receptor leads to prominent downregulation of T cell proliferation, mainly as a consequence of interference with IL-2 production. We here report that CTLA-4 engagement strikingly selectively shuts off activation of downstream T cell receptor (TCR)/CD28 signaling events, i.e., activation of the microtubule-associated protein kinase (MAPKs) ERK and JNK. In sharp contrast, proximal TCR signaling events such as ZAP70 and TCR-zeta chain phosphorylation are not affected by CTLA-4 engagement on activated T cells. Since activation of the ERK and JNK kinases is required for stimulation of interleukin (IL)-2 transcription, these data provide a molecular explanation for the block in IL-2 production imposed by CTLA-4.

Defective TCR-mediated signaling in synovial T cells in rheumatoid arthritis.

In rheumatoid arthritis (RA), the functional status of T cells is incompletely understood. Synovial T cells display phenotypic evidence of former activation, but there is poor production of T cell-derived cytokines in the synovium. In addition, synovial T cell proliferation upon mitogenic and antigenic stimulation was decreased compared with that in peripheral blood T cells. Moreover, previous reports revealed that early Ca2+ rises induced by TCR/CD3 stimulation were decreased in RA T cells compared with those in healthy controls. To investigate the molecular mechanisms of RA synovial T cell hyporesponsiveness, we analyzed the TCR/CD3-mediated protein tyrosine phosphorylation in RA peripheral blood and synovial fluid (SF) T cells. SF T cells exhibited a decreased overall tyrosine phosphorylation pattern upon stimulation. Most notably, the induction of phosphorylation of p38 was virtually absent. Moreover, we found that tyrosine phosphorylation of the TCR zeta-chain, one of the most proximal events in TCR signaling, is clearly diminished in RA SF T cells. The decrease in tyrosine phosphorylation was accompanied by a decrease in detectable levels of zeta-protein within synovial T cells. These results suggest that a defective TCR signaling underlies the hyporesponsiveness of synovial T cells in RA.

Diminished TCR signaling in cutaneous T cell lymphoma is associated with decreased activities of Zap70, Syk and membrane-associated Csk.

Malignant cells of patients with cutaneous T cell lymphoma (CTCL) are of monoclonal origin and of the CD4+/CD45RO+ subset. Since unlike their normal counterparts, triggering of their TCR/CD3 in vitro elicits only a weak mitogenic response, we set out to determine which of the signal transduction molecules initiated by anti-CD3E antibodies are affected in neoplastic cells. The results obtained from analysis of tumor cells from four patients show a general reduction in basal and induced tyrosine phosphorylation of a wide range of signaling proteins. Furthermore, the function of members from distinct families of protein tyrosine kinases was altered in neoplastic cells. The enzymatic activity of the membrane-bound fraction of Csk was suppressed, and its association with other cellular proteins was altered. There was a decline in the amount and activity of Syk, and a slight decrease in the specific activity of Lck kinases. Zap70 tyrosyl phosphorylation was reduced or undetectable and the kinase associated weakly, or not at all, with the TCR zeta chain. We propose that dampened TCR-triggered responses in CTCL are caused by suppression of an array of effector molecules required for coupling cell surface receptors to early and late signaling events.

Fc epsilonRI gamma can support T cell development and function in mice lacking endogenous TCR zeta-chain.

Fc epsilonRI gamma (Fc gamma) is a member of the zeta family of signal transducing molecules that function as components of both the TCR and Fc receptors (FcR). While the majority of thymocytes and T cells express TCRs containing zeta-chain homodimers, certain unique populations of T cells express TCRs that contain both zeta and Fc gamma. To examine the ability of Fc gamma to substitute for zeta-chain in T cell development and function, we introduced a transgene encoding Fc gamma into mice made genetically deficient for zeta-chain (zeta(e)-/-). Analysis of thymocyte development in zeta(e)-/-;Fc gamma Tg mice demonstrated that Fc gamma was able to support the maturation of both gammadelta TCR+ and alphabeta TCR+ T cells. However, positive selection of alphabeta TCR+ thymocytes was less efficient in zeta(e)-/-;Fc gamma Tg mice than in zeta(e)-/- mice reconstituted with zeta-chain. This difference may be due to the fact that Fc gamma contains a single immunoreceptor tyrosine-based activation motif (ITAM) whereas zeta-chain contains three ITAMs. Interestingly, the peripheral T cells that develop in zeta(e)-/- mice reconstituted with Fc gamma are functional and respond to TCR-specific stimuli. These data suggest that Fc gamma and zeta are interchangeable in their ability to mediate T cell development and function, however zeta-chain is more efficient at promoting positive selection and T cell maturation. The difference in efficiency between zeta and Fc gamma may be responsible in part for the unusual developmental and functional properties of T cells that constitutively express Fc gamma as a signaling component of their TCRs.

Aberrant TCR-mediated signaling in CD45-null thymocytes involves dysfunctional regulation of Lck, Fyn, TCR-zeta, and ZAP-70.

CD45 is a transmembrane phosphotyrosine phosphatase expressed on all nucleated hemopoietic cells. Targeting of CD45 exon 9 has generated a mouse line completely lacking CD45 expression (CD45-null) in which there are severe abnormalities in T cell development. Defects in TCR-mediated signals underlying these abnormalities have now been investigated using CD45-null T cells. No T cell proliferation was detected in response to a CD3 mAb. In thymocytes the p56(lck) and p59(fyn) tyrosine kinases were hyperphosphorylated, and p56(lck) was in its inactive conformation. Both basal and TCR-stimulated tyrosine phosphorylation of TCR-zeta and CD3-epsilon were much reduced, and TCR stimulation induced an abnormal p18 phosphoisomer of TCR-zeta previously noted in T cells stimulated by altered peptide ligands. These defects were associated with the failure of ZAP-70 kinase recruitment to the TCR-zeta chain. TCR coupling to the tyrosine phosphorylation of several proteins, including HS1 and p120(cbl), was also much reduced. However, TCR-induced signaling was not ablated, and significant inositol phosphate and calcium signals were observed in CD45-null thymocytes. Our molecular analysis suggests that the threshold for TCR signal transduction is greatly increased in CD45-null T cells, thus explaining the profound defects in thymic development.

Phosphatidylinositol-based glycolipid-anchored proteins enhance proximal TCR signaling events.

Previous reports suggested that T lymphocyte activation through phosphatidylinositol-based glycolipid (GPI)-anchored molecules is dependent on surface expression of the TCR. Here we show that stimulation of the TCR in five mutant cell lines with deficiencies in GPI biosynthesis fails to induce tyrosine phosphorylation of the TCR zeta-chain and ZAP-70, indicating that early events in TCR-mediated signal transduction are affected in these mutants. The Src kinases Fyn and Lck coprecipitating with activated TCR complexes are significantly less kinase active in the GPI-processing mutants than in wild-type cells. These data suggest that GPI-anchored proteins may play an important role in the initiation of TCR signal transduction by contributing to the accumulation of Src kinase activity in the TCR complex.

Sequential development of structural and functional alterations in T cells from tumor-bearing mice.

The TCR alpha beta or -gamma delta chains bind the peptide ligand, whereas the associated CD3 delta epsilon gamma and TCR zeta subunits couple the TCR to intracellular signal transduction components. Recently, several groups have described marked alterations in signal transduction elements in T cells from cancer patients or in mice bearing tumor for a few weeks (>26 days). The sequence in which these alterations develop is unknown. The aim of this study was to explore the kinetics of the development of alterations in signal transduction molecules (TCR zeta chain, NF kappaB family proteins, and tyrosine kinase p56(lck)) in mice bearing MC38 colon adenocarcinoma. The results demonstrate that alterations in NF kappaB family proteins, specifically the failure of p65 translocation to the nucleus, occur earlier and more frequently than the decrease in zeta-chain. These defects are paralleled by an impaired ability to produce Th1 cytokines (IL-2 and IFN-gamma). These initial changes are followed by the eventual loss of TCR zeta chain and p56(lck) and a marked decrease in cytotoxic function. An increased rate of lysosomal degradation is one of the mechanisms responsible for the loss of zeta-chain.

Defective TCR-mediated signaling in synovial T cells in rheumatoid arthritis

In rheumatoid arthritis (RA), the functional status of T cells is incompletely understood. Synovial T cells display phenotypic evidence of former activation, but there is poor production of T cell-derived cytokines in the synovium. In addition, synovial T cell proliferation upon mitogenic and antigenic stimulation was decreased compared with that in peripheral blood T cells. Moreover, previous reports revealed that early Ca2+ rises induced by TCR/CD3 stimulation were decreased in RA T cells compared with those in healthy controls. To investigate the molecular mechanisms of RA synovial T cell hyporesponsiveness, we analyzed the TCR/CD3-mediated protein tyrosine phosphorylation in RA peripheral blood and synovial fluid (SF) T cells. SF T cells exhibited a decreased overall tyrosine phosphorylation pattern upon stimulation. Most notably, the induction of phosphorylation of p38 was virtually absent. Moreover, we found that tyrosine phosphorylation of the TCR zeta-chain, one of the most proximal events in TCR signaling, is clearly diminished in RA SF T cells. The decrease in tyrosine phosphorylation was accompanied by a decrease in detectable levels of zeta-protein within synovial T cells. These results suggest that a defective TCR signaling underlies the hyporesponsiveness of synovial T cells in RA.

Defective TCR-mediated signaling in synovial T cells in rheumatoid arthritis

In rheumatoid arthritis (RA), the functional status of T cells is incompletely understood. Synovial T cells display phenotypic evidence of former activation, but there is poor production of T cell-derived cytokines in the synovium. In addition, synovial T cell proliferation upon mitogenic and antigenic stimulation was decreased compared with that in peripheral blood T cells. Moreover, previous reports revealed that early Ca2+ rises induced by TCR/CD3 stimulation were decreased in RA T cells compared with those in healthy controls. To investigate the molecular mechanisms of RA synovial T cell hyporesponsiveness, we analyzed the TCR/CD3-mediated protein tyrosine phosphorylation in RA peripheral blood and synovial fluid (SF) T cells. SF T cells exhibited a decreased overall tyrosine phosphorylation pattern upon stimulation. Most notably, the induction of phosphorylation of p38 was virtually absent. Moreover, we found that tyrosine phosphorylation of the TCR zeta-chain, one of the most proximal events in TCR signaling, is clearly diminished in RA SF T cells. The decrease in tyrosine phosphorylation was accompanied by a decrease in detectable levels of zeta-protein within synovial T cells. These results suggest that a defective TCR signaling underlies the hyporesponsiveness of synovial T cells in RA.

Purification and Characterization of Human ZAP-70 Protein-tyrosine Kinase from a Baculovirus Expression System

The ZAP-70 protein tyrosine kinase is essential for T cell antigen receptor (TCR)-mediated signaling. The absence of ZAP-70 results in impaired differentiation of T cells and a lack of responsiveness to antigenic stimulation. In order to study the characteristics of ZAP-70 in vitro, we overexpressed an epitopically tagged human ZAP-70 in a recombinant baculovirus expression system and purified it by column chromatography. The kinase activity of purified, recombinant ZAP-70 required cation and exhibited a strong preference for Mn2+ over Mg2+. The apparent Km of ZAP-70 for ATP was approximately 3.0 microM. The activity of the recombinant ZAP-70, unlike that of the homologous protein tyrosine kinase, Syk, was not affected by binding of TCR-derived tyrosine phosphorylated immunoreceptor tyrosine-based activation motif peptides. Several proteins were tested as potential in vitro substrates of ZAP-70. Only alpha-tubulin and the cytoplasmic fragment of human erythrocyte band 3 (cfb3), which have a region of sequence identity at the phosphorylation site, proved to be good substrates, exhibiting Kmvalues of approximately 3.3 and approximately 2.5 microM, respectively ([ATP] = 50 microM). alpha- and beta-Casein were poor substrates for ZAP-70, and no activity toward enolase, myelin basic protein, calmodulin, histone proteins, or angiotensin could be detected. In contrast to the T cell protein tyrosine kinase, Lck, ZAP-70 did not phosphorylate the cytoplasmic portion of the TCRzeta chain or short peptides corresponding to the CD3epsilon or the TCRzeta immunoreceptor tyrosine-based activation motifs. Our studies suggest that ZAP-70 exhibits a high degree of substrate specificity.

Qualitative and quantitative contributions of the T cell receptor zeta chain to mature T cell apoptosis.

Engagement of the T cell receptor (TCR) of mature T lymphocytes can lead either to activation/proliferation responses or programmed cell death. To understand the molecular regulation of these two fundamentally different outcomes of TCR signaling, we investigated the participation of various components of the TCR-CD3 complex. We found that the TCR-zeta chain, while not absolutely required, was especially effective at promoting mature T cell apoptosis compared with the CD3 epsilon, gamma, or delta chains. We also carried out mutagenesis to address the role of the immunoreceptor tyrosine-based activation motifs (ITAMs) that are the principal signaling components found three times in the TCR-zeta chain and once in each of the CD3 epsilon, gamma, or delta chains. We found that the ability of the TCR-zeta chain to promote apoptosis results both from a quantitative effect of the presence of multiple ITAMs as well as qualitatively different contributions made by individual ITAMs. Apoptosis induced by single chain chimeras revealed that the first zeta ITAM stimulated greater apoptosis than the third zeta ITAM, and the second zeta ITAM was unable to trigger apoptosis. Because microheterogeneity in the amino acid sequence of the various ITAM motifs found in the TCR-zeta and CD3 chains predicts interactions with distinct src-homology-2-domain signaling proteins, our results suggest the possibility that individual ITAM motifs might play unique roles in TCR responses by engaging specific signaling pathways.

Defective TCR-mediated signaling in anergic T cells.

Extrathymic T cell tolerance to Ags can be achieved through clonal deletion by activation-induced programmed cell death as well as by functional unresponsiveness (anergy) of Ag reactive-T cells. Previous studies demonstrated that in vivo administration of staphylococcal enterotoxin B (SEB) induces SEB-specific T cell anergy. To investigate the molecular mechanisms of T cell anergy, we analyzed the TCR-mediated signal transduction in normal and anergic T cells. Anergic T cells exhibited impaired protein tyrosine phosphorylation on TCR-mediated stimulation. The altered tyrosine phosphorylation in anergic T cells may be caused by a defect in tyrosine phosphorylation of TCR zeta-chain and its subsequent association with ZAP-70 tyrosine kinase. Our data demonstrated that TCR zeta-chain phosphorylation and its sequential interaction with ZAP-70 are essential for the initiation of TCR-mediated signal transduction.

Physical dissociation of the TCR-CD3 complex accompanies receptor ligation.

Recent studies indicate that there may be functional uncoupling of the TCR-CD3 complex and suggest that the TCR-CD3 complex is composed of two parallel signal-transducing units, one made of gamma delta epsilon chains and the other of zeta chains. To elucidate the molecular mechanisms that may explain the functional uncoupling of TCR and CD3, we have analyzed their expression by using flow cytometry as well as immunochemical means both before and after stimulation with anti-TCR-beta, anti-CD3 epsilon, anti-CD2, staphylococcal enterotoxin B, and ionomycin. We present evidence that TCR physically dissociates from CD3 after stimulation of the TCR-CD3 complex. Stimulation with anti-CD3 resulted in down-modulation of TCR within 45 min whereas CD3 epsilon was still expressed on the cell surface as detected by flow cytometry. However, the cell surface expression of TCR and CD3 was not affected when cells were stimulated with anti-TCR-beta under the same conditions. In the case of anti-CD3 treatment of T cells, the TCR down-modulation appeared to be due to the internalization of TCR, as determined by immunoelectron microscopy. Immunochemical analysis of cells after stimulation with either anti-TCR or anti-CD3 mAbs revealed that the overall protein levels of TCR and CD3 were similar. More interestingly, the dissociation of the TCR-CD3 complex was observed with both treatments and occurred in a manner that the TCR and the associated TCR-zeta chain dissociated as a unit from CD3. These results provide the first report of physical dissociation of TCR and CD3 after stimulation through the TCR-CD3 complex. The results also suggest that the signal transduction pathway triggered by TCR may differ from that induced by CD3.

The pre-T cell receptor (TCR) complex is functionally coupled to the TCR-zeta subunit.

The pre-T cell receptor (TCR) complex regulates early T cell development and consists of a heterodimer of the TCR-beta subunit in association with the pre-TCR-alpha chain. Notably, in contrast to alpha/beta-expressing T cells, several studies suggested that the TCR-zeta chain is not stably associated with this pre-TCR complex. To examine the proximal signaling processes mediated by the pre-TCR complex and the role of the TCR-zeta chain in these processes, we stimulated pre-TCR-expressing cells and analyzed the interactions of the TCR/CD3 invariant chains with the Syk/ZAP-70 family of protein tyrosine kinases. Stimulation of the pre-TCR complex led to the tyrosine phosphorylation of the CD3 epsilon and TCR-zeta chains, as well as the phosphorylation and association of ZAP-70 and Syk with phosphorylated CD3 epsilon and TCR-zeta. These results demonstrate that the pre-TCR complex is functionally coupled to the TCR-zeta subunit and to the ZAP-70 and Syk protein tyrosine kinases.

Transcriptional regulation of the murine TCR gene

The TCR zeta chain plays a significant role in the assembly of the receptor complex and in coupling antigen recognition to the intracellular signal transduction apparatus. Since the zeta protein level is considered the limiting factor for receptor assembly in mature T cells, aberrant expression of the zeta chain affects both receptor structure and function. To understand the regulatory mechanisms controlling zeta gene expression, we characterized the 5' flanking region of the gene. Our analysis reveals the existence of at least three regions within the -784 to +121 fragment involved in the transcription of the zeta gene in T cells: (i) the fragment from -216 to +121 contains the basal promoter; (ii) the sequence between -561 and -216 includes positive elements which confer strong transcriptional activity; and (iii) the region between -784 and -561 which contains negative element(s) that down-regulate zeta gene transcription. The entire 5' flanking region of the zeta gene is functional in both T and fibroblast cell lines, although the transcriptional levels and the specific regions required for maximal activity differ between the two cell types. Maximal transcriptional activity is achieved when T cells are stimulated simultaneously via the TCR and with PMA. The transcriptional activity of the zeta gene can be induced by PMA alone in T cells but not in fibroblasts, suggesting that this effect is mediated by T cell-specific factors. We also demonstrate that upregulation of the transcriptional activity induced by the different stimuli is consistent with increased expression of zeta mRNAm, pointing to the possibility that signal transduction events initiated during T cell activation may be involved in controlling zeta gene expression.

T cells infiltrating non-Hodgkin's B cell lymphomas show altered tyrosine phosphorylation pattern even though T cell receptor/CD3-associated kinases are present.

Although tumor infiltrating lymphocytes (T-TIL) from B cell non-Hodgkins lymphoma patients contain tumor-reactive T cells, they display poor proliferation and IFN-gamma production when stimulated through the TCR-CD3. To determine if there was altered signaling linked to TCR-CD3 ligation, tyrosine phosphorylation was examined in T-TIL because it represents an early and critical event in T cell activation. After stimulation with anti-CD3 Ab, Western blotting with anti-phosphotyrosine showed reduced phosphorylation in T-TIL when compared with peripheral blood-derived T cells from normal individuals. The altered phosphorylation was not due to the reduced expression of signaling elements linked to the TCR-CD3 complex. T-TIL expressed normal levels of CD3 epsilon, TCR zeta chain, and the three tyrosine kinases, p56lck (Lck), p59fyn, and ZAP-70. However, in T-TIL, anti-Lck Ab reacted with a 60-kDa protein, which appears to be the phosphorylated form of Lck. Binding of anti-Lck Ab to the 60-kDa protein was blocked by Lck peptide. In addition, anti-Lck Ab immunoprecipitated a phosphorylated 60-kDa protein from gamma-32P-labeled T-TIL that was not seen in normal resting T cells. In vitro kinase assay studies also demonstrated that TCR-CD3 engagement increased the kinase activity of Lck in normal T cells but not in T-TIL. These results suggest that although T-TIL from B cell non-Hodgkins lymphoma patients contain the signal transduction molecules associated with TCR-CD3 activation pathway, they are impaired in tyrosine phosphorylation and Lck activity, which may contribute to the functional defects of these cells.