Activation of human T lymphocytes via the CD2 antigen results in tyrosine phosphorylation of T cell antigen receptor zeta-chains.

The phosphorylation of the invariant chains associated with the human TCR has been investigated after the stimulation of T lymphocytes with CD2 mAb T11(2) and T11(3), PHA, or phorbol 12,13-dibutyrate. As described previously, stimulation of T cells with either CD2 mAb or phorbol 12,13-dibutyrate resulted in the phosphorylation of the CD3 gamma-chain. The combination of T11(2) and T11(3) mAb also induced phosphorylation of the TCR zeta-chain. The phosphorylated zeta-polypeptide of CD2-activated cells was immunoprecipitated with antiphosphotyrosine antibodies and migrated to a 21- to 23-kDa position during SDS/PAGE. These results indicate that stimulation of human T cells via the CD2 Ag with the T11(2) and T11(3) mAb activates not only protein kinase C but also tyrosine kinase(s), resulting in the phosphorylation of the CD3 gamma-chain and the tyrosine phosphorylation of the zeta-chain, respectively.     

Immunosuppression induced by Salmonella involves inhibition of tyrosine phosphorylation in murine T lymphocytes

Abstract It is well known that facultative intracellular pathogens such as Salmonella suppress the host immune system. In the present study we attempted to clarify the mechanism responsible for the suppression of T-cell proliferation in mice infected with Salmonella typhimurium . The proliferation of murine spleen cells stimulated with a T-cell mitogen such as phytohemagglutinin (PHA) or concanavalin A (ConA) was significantly suppressed when the mice were infected with S. typhimurium , but not with Eschirichia coli . The suppression of T-cell proliferation did not necessarily parallel the level of interleukin-2 (IL-2) secretion, and was not restored by treatment with a calcium ionophore, indomethacin or IL-2. Only phorbol 12-myristate-13 acetate (PMA), an activator of protein kinase C (PKC), caused a slight recovery of cell proliferation with an augmentation of IL-2 secretion. Furthermore, Western blotting using anti-phosphotyrosine antibodies showed that the mitogen-induced tyrosine phosphorylation of 120-, 106-, 94-, 68- and 57-kDa proteins in murine splenic T-cells was inhibited by S. typhimurium infection. Also, the inhibition of tyrosine phosphorylation was not restored by treatment with PMA. These results suggest that the suppression of T-cell proliferation induced by Salmonella infection may be regulated by inhibition of tyrosine phosphorylation in T-cells, although the inhibition is not associated with PKC activation and subsequent IL-2 secretion of T cells.     

CD45-protein tyrosine phosphatase cross-linking inhibits T cell receptor CD3-mediated activation in human T cells

Activation of peripheral blood T cells, and the leukemic T cell line Jurkat, as measured by mobilization of intracellular calcium, by an anti-TCR antibody is blocked by mAb (T191) to the leukocyte common Ag (CD45). T191 also blocked down-regulation of the CD3-TCR complex induced by an anti-CD3 mAb. Vanadate, a phosphotyrosine phosphatase inhibitor, partially blocks the effect of T191 and restored mobilization of intracellular calcium. Assays of the immunoprecipitates of T191 and CD45 from both Jurkat-BM1 and peripheral T cells showed that the immune complexes had intrinsic phosphatase activity. A parallel immunoprecipitate using a mAb (4-10) against HLA class I showed no such activity. Further analysis of the T191 immunocomplex revealed activity against phosphotyrosine, p-nitrophenylphosphate, and [32P-poly-glu-tyr, but not against phosphoserine. Phosphatase activity was inhibited by Vanadate, but not by Zn2+ or F-. These results show that CD45 is a phosphotyrosine phosphatase, and strongly suggest that tyrosine phosphorylation/dephosphorylation is critically involved in activation of T cells through the TCR-CD3 complex.     

CD45 cross-linking regulates phospholipase C activation and tyrosine phosphorylation of specific substrates in CD3/Ti-stimulated T cells.

In lymphocytes, CD45 regulates the increase in cytoplasmic calcium concentration that occurs after receptor cross-linking. Here we show that T cell receptor complex (CD3/Ti)-mediated inositol phosphate production was inhibited by CD45 ligation in Jurkat cells. CD3/Ti signaling in normal T cells was also inhibited by CD45 ligation, but coupling of CD4 with CD3/Ti gave augmented calcium signals that were entirely resistant to the inhibitory effect of CD45. In contrast, CD3-induced T cell proliferation was suppressed by immobilized CD45 mAb even in the presence of CD4 mAb. The effect of CD45 and CD4 ligation on tyrosine phosphorylation during T cell activation was directly examined by immunoblotting with anti-phosphotyrosine. Using immobilized mAb, CD45 ligation suppressed the tyrosine phosphorylation of specific substrates induced by CD3/Ti stimulation, including almost complete suppression of 150-, 36-, and 35-kDa proteins and partial suppression of 76- and 80-kDa proteins. Other tyrosine-phosphorylated proteins induced by CD3/Ti stimulation, including 135- and 21-kDa proteins, were not suppressed by simultaneous ligation of CD3/Ti and CD45. Simultaneous ligation of CD3 and CD4 enhanced tyrosine phosphorylation of all substrates, but did not overcome the CD45-mediated suppression of tyrosine phosphorylation of the 35- and 36-kDa proteins. The CD45-mediated suppression of phospholipase C activation is therefore modulated by association with CD4 without altering the specific inhibition of tyrosine phosphorylation and T cell proliferation after co-ligation of CD45 and CD3/Ti.     

Regulation of basal tyrosine phosphorylation of the B cell antigen receptor complex by the protein tyrosine phosphatase, CD45.

Signal transduction via the B cell AgR complex has recently been shown to be dependent on the activation of one or more protein tyrosine kinases. Similarly, it has been found that signal transduction requires the expression of the protein tyrosine phosphatase CD45. Thus, transduction of a signal after AgR cross-linking must involve the coordinate interaction of these two enzymatic activities. It is therefore logical to hypothesize that the competence of the B cell to respond to ligands that bind the AgR may be dependent on the maintenance of an equilibrium between the tyrosine phosphorylation and dephosphorylation of specific signal transduction components. We have demonstrated in the present study that in resting B cells, the basal level of AgR complex tyrosine phosphorylation is regulated by cellular protein tyrosine phosphatases. Treatment of cells with the protein tyrosine phosphatase inhibitor, Na3VO4, resulted in rapid hyperphosphorylation of the receptor complex. Based on this observation, experiments were designed to examine the role of CD45 in regulation of AgR complex phosphorylation. Treatment of B cells with anti-CD45 mAb alone was found to have no effect on cytoskeletal association of CD45 or on its distribution within the membrane. Addition of a secondary cross-linking reagent, however, induced the association of CD45 with the cytoskeleton and caused capping. Subsequent studies demonstrated that increased tyrosine phosphorylation of the mIg-associated proteins MB-1 and B29 could be induced after incubating cells with anti-CD45 mAb and a secondary cross-linker, but not after the addition of anti-CD45 mAb alone. Changes in tyrosine phosphorylation of MB-1 and B29 were found to correlate with the cytoskeletal association of CD45. Interestingly, although cross-linking CD45 induced alterations in its association with the cytoskeleton and in its distribution within the membrane, no significant change in the level of protein tyrosine phosphatase activity could be detected under these conditions. These findings support the possibility that ligand binding to CD45 can induce biochemical and/or physical alterations in the molecule that presumably inhibit its ability to interact with specific substrates in the cell, thereby shifting the established equilibrium between tyrosine-specific phosphorylation and dephosphorylation.     

Activation of tyrosine phosphorylation in human T cells via the CD2 pathway. Regulation by the CD45 tyrosine phosphatase

In this study we compare the effect of CD3 and CD2 ligation on tyrosine kinase activation in human peripheral blood T cells. Using antiphosphotyrosine antibody to detect tyrosine phosphorylation of cellular substrates, we demonstrate that mAb stimulation of either CD3 or CD2 results in tyrosine phosphorylation of the TCR-zeta chain and 135- and 100-kDa proteins. However, differences are observed between CD3 and CD2 ligation; only the former results in rapid tyrosine phosphorylation of 72-, 65-, and 40-kDa substrates. Co-aggregation of CD2 and CD45, a tyrosine phosphatase, results in inhibition of intracellular calcium elevation and T cell proliferation. We demonstrate in this study that this manipulation also inhibits polyphosphoinositide hydrolysis and tyrosine phosphorylation of the 100-kDa substrate. The failure of tyrosine phosphorylation of the 100-kDa substrate is specific in that phosphorylation of the 135-kDa protein is not inhibited. Similar results are observed when CD2 and CD45 are independently cross-linked rather than co-aggregated. The observation that CD45 cross-linking alters tyrosine phosphorylation of T cell substrates and effects polyphosphoinositide hydrolysis is further evidence that tyrosine phosphorylation regulates early events in T cell activation including, perhaps, phospholipase C activity.     

Evidence for GTP-binding protein involvement in the tyrosine phosphorylation of the T cell receptor zeta chain.

The zeta subunit of the T cell receptor (TCR) is a prominent substrate for a TCR-activated tyrosine kinase. Tyrosine phosphorylation of the zeta subunit in response to antibody-mediated receptor cross-linking was synergized in permeabilized T cells by either of two non-hydrolyzable GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) or guanosine 5'-[beta, gamma-imido]triphosphate Gpp(NH)p. ATP analogues did not significantly affect antibody-induced tyrosine phosphorylation. Unlike the GTP analogues, the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP beta S) did not enhance phosphorylation of zeta. The effect induced by the GTP analogues required TCR occupancy and was independent of protein kinase C. Taken together these observations implicate a GTP-binding protein in the modulation of TCR-induced tyrosine phosphorylation.     

Down-regulation of cell surface CD4 molecule expression induced by anti-CD4 antibodies in human T lymphocytes.

Antigenic modulation was defined as the down-regulation of a cell surface antigen expression induced by exposure to specific antibody. We investigated the modulation of CD4 surface expression in human peripheral blood lymphocytes incubated in vitro with anti-CD4 monoclonal antibodies (mAbs). Modulation of surface CD4 was achieved at 37 degrees C, but not at 4 degrees C, with five different murine anti-CD4 mAbs of IgG1 and IgG2a subclasses, with different epitope specificities. Modulation was dose dependent with a maximum at nonsaturating mAb concentration. It was reversible upon culture in mAb-free medium. It was accelerated and amplified in the presence of monocytes or after cross-linking of anti-CD4 mAbs. It could be induced with solid phase anti-CD4 mAbs, but not with soluble F(ab')2 fragments. Its magnitude was identical on all CD4+ lymphocytes. It was associated with a moderate down-regulation of CD2 and CD3 but not of CD8 and HLA class I surface expression. Modulation was slightly augmented by addition of inhibitors of the endosome/lysosome pathway but not by protein synthesis inhibitors. The anti-CD4 mAb initially bound to cell surface was no longer detectable after 24 hr of culture. Most of surface CD4 proteins complexed with antibody were rapidly internalized and transiently replaced by CD4 from an intracytoplasmic pool and then no longer were expressed. CD4 mRNA was moderately decreased in cells incubated with anti-CD4 mAb while beta-actin and beta 2-microglobulin mRNAs remained at stable levels. It was concluded that down-regulation of CD4 surface expression induced by anti-CD4 mAb concerned only a part of CD4 molecules and was associated with a decreased synthesis. The delay required to achieve maximal modulation is likely to reflect exhaustion of the intracytoplasmic recycling pool of CD4 molecules.     

4-1BB cross-linking enhances the survival and cell cycle progression of CD4 T lymphocytes

4-1BB, a T cell co-stimulatory receptor, prolongs the survival and multiplication of CD4 T cells. Cross-linking 4-1BB stimulated expression of the anti-apoptotic genes bcl-XL and bcl-2, as well as of cyclins D2 and E, and inhibited expression of the cyclin-dependent kinase (cdk) inhibitor p27kip1. Ova-activated CD4 T cells of 4-1BB-deficient/DO11.10 TCR transgenic mice survived less well and underwent less expansion than cells of wild type DO11.10 TCR transgenic mice. These findings demonstrate that 4-1BB is a co-stimulatory molecule for CD4 T cell survival and expansion in vivo.     

Induction of the tyrosine phosphorylation of a 66 kd soluble protein by DMSO in human peripheral blood T lymphocytes

Previous studies from our laboratory have demonstrated that a 66 KD cytoplasmic protein (TPP 66) is newly phosphorylated on tyrosine when human peripheral blood T lymphocytes are incubated with a variety of agents that activate these cells or augment activation by known mitogens. Since DMSO has been shown to activate tyrosine specific protein kinases we have examined the role of this agent on the phosphorylation of TPP 66. 5 to 40% DMSO induced the phosphorylation of TPP 66 with the maximal increase in phosphorylation seen at 20%. Concentrations greater than 40% were inhibitory. Phosphorylation of TPP 66 in DMSO treated cells could be detected as early as 2 min following the addition of DMSO, with increased [32P]O4 incorporation over the next 60 min. The phosphorylation was on tyrosine residues demonstrated by base hydrolysis of TPP 66 extracted from the gels followed by single dimension high voltage electrophoresis. Since DMSO augments activation of T lymphocytes by lectins, this data provides further support for a critical role for the tyrosine phosphorylation of TPP 66 in the mediation or modulation of T lymphocyte activation.     

Characterization of anti-CD8-resistant cytolytic T lymphocytes induced by multivalent cross-linking of CD8 on precursor cells

The lytic activity of most CD8+ MHC class I allospecific CTL generated in vitro can be inhibited by anti-CD8 antibodies. Such inhibition has led to hypotheses that CD8/class I interactions normally contribute to the triggering of CTL with low or moderate avidity Ag-specific TCR by providing those CTL with auxiliary binding avidity. However, CD8 has also been proposed to play an active signaling role in T cell activation. We have recently reported that multivalent cross-linking of CD8 on CTL precursors in MLC does appear to mediate activation signals, and induces the generation of CD8+ MHC class I allospecific CTL whose lytic activity cannot be blocked by anti-CD8 antibodies. In our present study, we have further characterized such anti-CD8 uninhibitable effector cells. These CTL are resistant to blocking of their lytic function by anti-Lyt-3 mAb as well as anti-Lyt-2 mAb, but remain sensitive to blocking by anti-LFA-1 mAb, indicating that they do use non-CD8 cell adhesion molecules during target cell recognition and lysis. As a consequence of mAb-induced multivalent CD8 cross-linking during their generation, anti-CD8 uninhibitable CTL significantly reduce their cell surface expression of CD8, which permits their identification and facilitates their purification from heterogeneous MLC populations. Such anti-CD8 uninhibitable effector cells can be maintained as stable CTL lines, in the absence of anti-CD8 mAb after the initial induction period. The in vitro generation of anti-CD8 uninhibitable CTL, which may be highly enriched for cells bearing high affinity TCR, could represent a new experimental approach to studies of TCR gene usage and repertoire, as well as a potentially important strategy for the deliberate generation of high affinity effector cells for adoptive immunotherapy.     

Tyrosine phosphorylation of a c-Src-like protein is increased in membranes of CD4- CD8- T lymphocytes from lpr/lpr mice.

Mice homozygous for the autosomal recessive lpr gene have a disorder that results in autoimmunity and massive accumulation of T lymphocytes lacking CD4 and CD8 surface markers. These abnormal T cells exhibit constitutive tyrosine phosphorylation of a component of the CD3-T-cell receptor complex. We compared membrane tyrosine phosphorylation in lpr/lpr CD4- CD8- T cells and control T cells, lpr membranes exhibited a 7.3-fold increase (n = 16) in tyrosine phosphorylation of a 60-kilodalton protein. The increase was correlated with the Lpr but not the CD4- CD8- phenotype in that p60 phosphorylation was not increased in membranes from normal CD4- CD8- thymocytes. To identify the p60 in lpr cells, we examined the activity of several T-cell tyrosine-specific protein kinases. p56lck phosphorylation was only slightly increased in lpr membranes (2.2-fold; n = 16). Phorbol ester treatment of intact T cells before membrane isolation caused p56lck to migrate as pp60lck; however, pp60lck could be clearly distinguished from the pp60 in lpr cells by two-dimensional gel electrophoresis. The pp60 from lpr cells exhibited several isoforms at pH approximately 6.3 to 6.5. Although on two-dimensional gels pp60c-src had a pI (6.4 to 6.8) within a similar region, p60c-src mRNA, protein, and kinase activities were not increased in lpr cells. In addition, staphylococcal V8 proteolytic cleavage of the lpr pp60 isolated on two-dimensional gels yielded two major fragments, a pattern distinct from that of pp60c-src. However, by using an antiserum against the C-terminal sequence of c-Src and other related kinases, including p59fyn, the pp60 could be immunoprecipitated in greater amounts from lpr than from control T cells. When pp59(fyn) was selectively immunoprecipitated from T-cell membranes with specific antisera, its molecular weight, proteolytic cleavage pattern, and behavior on two-dimensional gels were identical to those of the pp60 from lpr cells. We conclude that p59(fyn) phosphorylation is increased in membranes from lpr/lpr CD4(-) CD8(-) T cells and that the increase is correlated with constitutive tyrosine phosphorylation and perhaps with the expansion of this unusual T-cell population.     

Expression and tyrosine phosphorylation of the T cell receptor zeta-subunit in human thymocytes.

Recent evidence suggests that the zeta-subunit of the TCR complex plays a critical role in transducing signals initiated by the Ag receptor heterodimer. Because thymic maturation involves specific interactions between the TCR complex and thymic stromal cells, the zeta-subunit has been postulated to also play a role in this process. To assess the potential for zeta to contribute to thymocyte maturation, we have used an anti-zeta mAb (TIA-2) to quantitate its expression in mature (CD3bright) and immature (CD3dim and CD3-) populations of human thymocytes. Using both flow cytometric and immunoblotting analysis, we found that the relative expression of TCR-zeta varied directly with the surface expression of CD3. Importantly, TCR-zeta was detected in the majority of CD3- thymocytes, indicating that its expression precedes the surface appearance of CD3:TCR. In thymocytes, TCR-zeta was found to be constitutively phosphorylated on tyrosine residues. The relative expression of phospho-zeta varied directly with the maturational stage of the thymocyte, with the mature (CD3bright), single positive cells accounting for most of the phospho-zeta found in the human thymus. The expression of phospho-zeta could be significantly increased by activating thymocytes with mAb reactive with either CD3 or CD2. These results suggest that TCR-zeta is functionally linked to the major thymocyte activation receptors.     

A protein kinase C pseudosubstrate peptide inhibits phosphorylation of the CD3 antigen in streptolysin-O-permeabilized human T lymphocytes.

Activation of human T lymphocytes leads to the phosphorylation of the CD3-antigen gamma polypeptide. We have investigated a possible role for protein kinase C (PKC) in mediating this phosphorylation event by using T cells permeabilized with streptolysin-O in the presence of 120 mM-K+ buffers containing Ca2+-EGTA. The gamma-chain was phosphorylated by [gamma-32P]ATP in permeabilized T lymphoblasts in the presence of phorbol 12,13-dibutyrate (Pdbu) or phytohaemagglutinin (PHA). Ca2+ alone in the range 0.5-1.0 microM also induced gamma-chain phosphorylation in some T-lymphoblast preparations; that in Jurkat-6 cells occurred at lower concentrations (50-500 nM). Two experimental approaches were used to investigate the possible involvement of PKC. Firstly, when permeabilization was carried out in buffer lacking free Ca2+, PKC was lost from the cells, and gamma-chain phosphorylation could then no longer be induced on subsequent addition of Pdbu or PHA in 400 nM-Ca2+, or 800 nM-Ca2+ alone, to permeabilized cells. However, when permeabilization was carried out in the presence of these three agents, PKC was translocated to intracellular membranes, and subsequent addition of [gamma-32P]ATP to these cells then resulted in gamma-chain phosphorylation. In the second approach, induction of gamma-chain phosphorylation by Pdbu, 1-oleoyl-2-acetylglycerol, 1,2-diolein, PHA or Ca2+ alone was effectively blocked by permeabilizing T cells in the presence of a PKC pseudosubstrate peptide (50 microM). Pseudosubstrate concentrations in the range 7-20 microM inhibited gamma-chain phosphorylation by 50%. In contrast, addition of four other 'irrelevant' basic peptides (50 microM) did not result in detectable inhibition, and 50 microM-pseudosubstrate did not inhibit the phosphorylation of 17 other polypeptides isolated from permeabilized T cells. These data suggest that Pdbu-, 1,2-diacylglycerol-, PHA- and Ca2+-induced phosphorylation of the CD3-antigen gamma chain in permeabilized T cells is mediated by PKC.     

Regulation of T cell receptor- and CD28-induced tyrosine phosphorylation of the focal adhesion tyrosine kinases Pyk2 and Fak by protein kinase C. A role for protein tyrosine phosphatases

The T cell receptor (TCR)-CD3 complex and the costimulatory molecule CD28 are critical for T cell function. Both receptors utilize protein tyrosine kinases (PTKs) for the phosphorylation of various signaling molecules, a process that is critical for the function of both receptors. The PTKs of the focal adhesion family, Pyk2 and Fak, have been implicated in the signaling of TCR and CD28. We show here evidence for the regulation of TCR- and CD28-induced tyrosine phosphorylation of the focal adhesion PTKs by protein kinase C (PKC). Thus, treating Jurkat T cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) rapidly and strongly reversed receptor-induced tyrosine phosphorylation of the focal adhesion PTKs. In contrast, PMA did not affect TCR-induced tyrosine phosphorylation of CD3zeta or the PTKs Fyn and Zap-70. However, PMA induced a strong and rapid dephosphorylation of the linker molecule for activation of T cells. PMA failed to induce the dephosphorylation of proteins in PKC-depleted cells or in cells pretreated with the PKC inhibitor Ro-31-8220, confirming the role of PKC in mediating the PMA effect on receptor-induced protein tyrosine phosphorylation. The involvement of protein tyrosine phosphatases (PTPases) in mediating the dephosphorylation of the focal adhesion PTKs was confirmed by the failure of PMA to dephosphorylate Pyk2 in cells pretreated with the PTPase inhibitor orthovanadate. These results implicate PKC in the regulation of receptor-induced tyrosine phosphorylation of the focal adhesion PTKs in T cells. The data also suggest a role for PTPases in the PKC action.     

Activation of tyrosine kinase p60fyn following T cell antigen receptor cross-linking.

Activation of T cells by specific antigens in the context of major histocompatibility complex encoded proteins is mediated by the T cell antigen receptor (TcR), consisting of a variable (Ti) and an invariant (CD3) subunits. Tyrosine phosphorylation is considered to be one of the earliest steps in TcR-mediated signal transduction. There are indications that the p60fyn protein tyrosine kinase is involved in signaling via TcR. However, enzymatic activation of the Src-related tyrosine kinases upon TcR triggering has not been shown yet, therefore the identity of TcR-activated tyrosine kinase(s) remains unclear. We demonstrate that cross-linking of CD3 activates p60fyn and induces tyrosine phosphorylation of cellular proteins in human T cells (resting peripheral T cells, a helper T cell clone, a helper T cell clone immortalized with Herpesvirus saimiri, and a leukemic T cell line). Activation of p60fyn was fast, and its maximum (2-4-fold activation as compared with the basal activity) was followed by a decline. The amount of p60fyn in the cells remained unchanged. None of the other T cell Src-related tyrosine kinases was activated after cross-linking of CD3. Activation of p60fyn was induced by anti-CD3, but not by anti-CD4, anti-CD2, or anti-CD28. The activation was correlated with an increase of the phosphotyrosine content of p60fyn. These studies provide direct proof for the functional association between p60fyn and the TcR.     

Biochemical differences in the alphabeta T cell receptor.CD3 surface complex between CD8+ and CD4+ human mature T lymphocytes

We have reported the existence of biochemical and conformational differences in the alphabeta T cell receptor (TCR) complex between CD4(+) and CD8(+) CD3gamma-deficient (gamma(-)) mature T cells. In the present study, we have furthered our understanding and extended the observations to primary T lymphocytes from normal (gamma(+)) individuals. Surface TCR.CD3 components from CD4(+) gamma(-) T cells, other than CD3gamma, were detectable and similar in size to CD4(+) gamma(+) controls. Their native TCR.CD3 complex was also similar to CD4(+) gamma(+) controls, except for an alphabeta(deltaepsilon)(2)zeta(2) instead of an alphabetagammaepsilondeltaepsilonzeta(2) stoichiometry. In contrast, the surface TCRalpha, TCRbeta, and CD3delta chains of CD8(+) gamma(-) T cells did not possess their usual sizes. Using confocal immunofluorescence, TCRalpha was hardly detectable in CD8(+) gamma(-) T cells. Blue native gels (BN-PAGE) demonstrated the existence of a heterogeneous population of TCR.CD3 in these cells. Using primary peripheral blood T lymphocytes from normal (gamma(+)) donors, we performed a broad epitopic scan. In contrast to all other TCR.CD3-specific monoclonal antibodies, RW2-8C8 stained CD8(+) better than it did CD4(+) T cells, and the difference was dependent on glycosylation of the TCR.CD3 complex but independent of T cell activation or differentiation. RW2-8C8 staining of CD8(+) T cells was shown to be more dependent on lipid raft integrity than that of CD4(+) T cells. Finally, immunoprecipitation studies on purified primary CD4(+) and CD8(+) T cells revealed the existence of TCR glycosylation differences between the two. Collectively, these results are consistent with the existence of conformational or topological lineage-specific differences in the TCR.CD3 from CD4(+) and CD8(+) wild type T cells. The differences may be relevant for cis interactions during antigen recognition and signal transduction.     

Evidence that a GTP binding protein regulates phosphorylation of the CD3 antigen in human T lymphocytes

The role of guanine nucleotide binding regulatory proteins (G proteins) in the regulation of phosphorylation of the gamma subunit of the CD3 antigen has been examined. CD3 gamma chain phosphorylation in isolated T cell microsomes was stimulated by the G protein activator guanosine 5'-0 thiotriphosphate (GTP gamma S), but cyclic adenosine monophosphate and guanosine 5'-diphosphate were ineffective at inducing gamma chain phosphorylation. The effect of GTP gamma S was rapid and transient; a half maximal effect was observed with 50 microM of the nucleotide. gamma polypeptide phosphorylated in vitro in GTP gamma S stimulated microsomes incorporated phosphate on Serines 123 and 126. These data are consistent with the involvement of a G protein in the signalling mechanisms that regulate the phosphorylation of the CD3 gamma chain.     

Investigation of the kinetics and order of tyrosine phosphorylation in the T-cell receptor zeta chain by the protein tyrosine kinase Lck.

We report experiments to investigate the role of the physiologically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptor zeta chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the zeta chain. Preliminary 1H-NMR studies of recombinant zeta chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines. Phosphorylation kinetics were determined for each tyrosine by assaying the transfer of 32P by recombinant Lck on to each of the peptides. The rates of phosphorylation were found to depend on the location of the tyrosine, leading to the proposal that Lck phosphorylates the six zeta chain ITAM tyrosines in the order 1N (first) > 3N > 3C > 2N > 1C > 2C (last) as a result of differences in the amino-acid sequence surrounding each tyrosine. This proposal was then tested on cytosolic, recombinant T-cell receptor zeta chain. After in vitro phosphorylation by Lck, the partially phosphorylated zeta chain was digested with trypsin. Separation and identification of the zeta chain fragments using LC-MS showed, as predicted by the peptide phosphorylation studies, that tyrosine 1N is indeed the first to be phosphorylated by Lck. We conclude that differences in the amino-acid context of the six zeta chain ITAM tyrosines affect the efficiency of their phosphorylation by the kinase Lck, which probably contributes to the distinct patterns of phosphorylation observed in vivo.