Conversion of p56(lck) to p60(lck) in human peripheral blood T lymphocytes is dependent on co- stimulation through accessory receptors: involvement of phospholipase C, protein kinase C and MAP-kinases in vivo

Activation of T cells requires co-stimulation of the TCR and accessory receptors like CD2, CD4, CD8, CD11a or CD28. Engagement of the TCR without co-stimulation results in anergy / apoptosis. Here we show that induction of the shift of the tyrosine kinase p56lck from 56 kDa to apparent 60 kDa in resting human peripheral blood T cells (PBT) is strictly dependent on co-stimulation through both TCR and accessory receptors. In contrast, triggering of the TCR alone is only sufficient to induce the lck shift in preactivated cells like T cell clones or the T lymphoma line Jurkat. Our studies predict an involvement of a phospholipase C isoform which surprisingly acts downstream of a phorbolester-sensitive, H7-insensitive protein kinase C. Inhibition of the lck shift in vivo by U73122, a specific inhibitor of phospholipase C, correlates with reduced activation of the MAP-kinases ERK1 / 2. Moreover, the MEK1-specific inhibitor PD98059 blocks the lck shift in vivo. These findings demonstrate that activation of the MEK1-ERK1 / 2 pathway is required for lck conversion in vivo. The lck shift is not inducible by co-stimulation through acidic sphingomyelinase or ceramides which even prevent ERK2 activation in PBT. Moreover, it is resistant to treatment with W7, KN62 and cyclosporin A.     

Both T cell receptor (TcR)-CD3 complex and CD2 increase the tyrosine kinase activity of p56lck. CD2 can mediate TcR-CD3-independent and CD45-dependent activation of p56lck.

T cell activation by triggering the T cell receptor (TcR)-CD3 complex leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins. To date, there has been no direct evidence on the identity of the tyrosine kinase activity implicated in this signaling pathway. In this study, we demonstrate that activation of human T cells with anti-CD3 monoclonal antibody increases tyrosine kinase activity of p56lck. This extends our previous findings which demonstrated the involvement of p56lck kinase activity in the CD2 signal transduction pathway. The results from peripheral blood lymphocytes and Jurkat cell line showed in both cases an early and transient change in the specific activity of p56lck, followed by a shift to a higher apparent molecular mass. Therefore, to test directly the role of TcR-CD3 in CD2-induced activation of p56lck, we utilized mutant variants of the Jurkat cell line lacking in cell surface TcR-CD3. We found that cell surface expression of TcR-CD3 is not required for the activation of p56lck via CD2 but is necessary for the appearance of the reduced-electrophoretic-mobility form of p56lck observed after CD2 triggering. By isolating CD45- mutants from Jurkat cells, we observed that surface expression of the tyrosine phosphatase CD45 is required in order to increase p56lck activity following CD2 stimulation, while CD4-induced activation of the kinase remained unchanged. These data provide evidence for a specific functional linkage between CD2 and p56lck, in which CD45 may play an essential role.     

Interaction of CD4:lck with the T cell receptor/CD3 complex induces early signaling events in the absence of CD45 tyrosine phosphatase.

Antibody-mediated ligation of the CD3/T cell antigen receptor (TcR) activates phospholipase C (PLC) via a tyrosine kinase signaling pathway that requires expression of the transmembrane tyrosine phosphatase CD45. In normal T cells, CD3-mediated PLC activation is significantly augmented by co-ligation of CD3 with the CD4 co-receptor; however, unlike CD3-associated tyrosine kinases, antibody-induced activation of the CD4-associated tyrosine kinase p56lck does not require CD45 expression. To explore the role of CD45 in the CD3 and CD4 activation pathways further, we examined the effect of CD3/CD4 cross-linking on tyrosine phosphorylation and activation of phospholipase C in CD45- mutant cells of the T cell leukemia line HPB.ALL. In accord with previous observations, anti-CD3 stimulation of the CD45-deficient cells failed to activate tyrosine kinases, or PLC as measured by mobilization of intracellular calcium. However, we show here that ligation of CD3 with CD4 leads to tyrosine phosphorylation of PLC gamma 1 and elevation in the intracellular free Ca2+ concentration in CD45- cells that is in excess of that seen in CD45+ cells. Since CD4 stimulation alone did not activate PLC, a component of the CD3 signaling pathway must be independent of CD45. Anti-CD4-induced tyrosine phosphorylation and activation of CD4-associated lck was also enhanced in CD45- cells, suggesting that increased lck activation compensates for the defect in CD3/TcR signaling, such that interaction of the CD3 signaling pathway with the CD4-associated pathway activates PLC even in the absence of CD45. The data demonstrate that the requirement for CD45 in coupling CD3/TcR to the PI-PLC activation cascade is not absolute, but rather substantiates a role for CD45 in modifying molecular interactions that control T cell activation.     

Cross-linking of CD26 by antibody induces tyrosine phosphorylation and activation of mitogen-activated protein kinase.

CD26, a T-cell activation antigen that has dipeptidyl peptidase IV activity in its extracellular domain and has also been shown to play an important role in T-cell activation. The earliest biochemical events seen in stimulated T lymphocytes activated through the engagement of the T-cell receptor (TCR) is the tyrosine phosphorylation of a panel of cellular proteins. In this study we demonstrate that antibody-induced cross-linking of CD26-in CD26-transfected Jurkat cells induced tyrosine phosphorylation of several intracellular proteins with a similar pattern to that seen after TCR/CD3 stimulation. Herbimycin A, an inhibitor of the src family protein tyrosine kinases dramatically inhibited this CD26-mediated effect on tyrosine phosphorylation. Major tyrosine phosphorylated proteins were identified by immunoblotting, and included p56lck, p59fyn, zeta associated protein-tyrosine kinase of 70,000 MW (ZAP-70), mitogen-activated protein (MAP) kinase, c-Cb1, and phospholipase C gamma. CD26-induced tyrosine phosphorylation of MAP kinase correlated with increased MAP kinase activity. In addition, CD26 was costimulatory to CD3 signal transduction since co-cross-linking of CD26 and CD3 antigens induced prolonged and increased tyrosine phosphorylation in comparison with CD3 activation alone. We therefore conclude that CD26 is a true costimulatory entity that can up-regulate the signal transducing properties of the TCR.     

Interactions between the tyrosine kinases p56lck,p59fyn and p50csk in CD4 signaling in T cells

Interaction of the CD4 co-receptor with major histocompatibility complex (MHC) class II molecules during antigen presentation results in enhancement of antigen receptor signaling. The synergism between the two receptors is believed to result from the juxtaposition of the CD4-associated tyrosine kinase p56lck with the cytoplasmic domains of CD3 complex components. Here, we report that cross-linking of CD4 on the surface of Jurkat cells using monoclonal antibodies results in activation of the CD3-associated kinase p59fyn. Co-cross-linking of CD4 and CD3 results in synergistic activation of p59fyn. The p59fyn kinase is also hyperactive in a Jurkat cell line stably transfected with a constitutively active p56lck mutant, indicating that p56lck mediates CD4 activation of p59fyn. In support of this hypothesis, expression of a dominant inhibitory mutant of p59fyn blocks CD4 signals involved in gene activation. In addition, the p59fyn dominant inhibitor mutant blocks gene-activating signals induced by expression of a constitutively active mutant of p56lck. Overexpression of the regulatory kinase p50csk, which attenuates TcR signaling by inactivation of p59fyn, inhibits signaling from the constitutively active form of p56lck. Taken together, these data suggest that CD4/p56lck enhancement of TcR signaling is, at least in part, mediated by activation of p59fyn, and may be regulated by p50csk.     

CD2 singnaling in T cells involves tyrosine phosphorylation and activation of the Tec family kinase, EMT/ITK/TSK

Ligation of the CD2 cell surface glycoprotein expressed on Tlymphocytes and NK cells induces protein tyrosine phosphorylationand activation of the Src kinases, LCK and FYN. We show herethat in Jurkat T leukemia cells and in peripheral blood T cells,CD2 stimulation also leads to tyrosine phosphorylation and activationof the Tec family kinase, EMThTKITSK. Activation of EMT by CD2was induced by mitogenic pairs of CD2 mAb, certain single CD2mAb followed by secondary antibody cross-linking, and CD58-bearingsheep red blood cells. With the use of different Jurkat cellmutants it was demonstrated that CD2-mediated activation ofEMT required expression of LCK, but did not require surfaceexpression of the CD3 chain. Receptor-mediated activation ofLCK does not in itself lead to activation of this Tec kinasesince induction of LCK by ligation of CD4 or CD5 did not resultin activation of EMT. The activation of EMT during CD2 signalingsuggests an important role for this kinase in CD2 co-stimulationof T cell responses.     

Activation of mitogen-activated protein kinases via CD40 is distinct from that stimulated by surface IgM on B cells

CD40 plays critical roles in B cell proliferation and differentiation in response to T cell-dependent antigenic stimulation. It has been suggested that CD40-mediated biological activities are transduced by a CD40 receptor-associated factor, CRAF1 and probably by protein tyrosine kinase Lyn and its substrates, phospholipase Cγ (PLCγ) and phosphatidylinositol-3 kinase (PI-3 kinase). Here, we describe the novel finding that a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) cascade is involved in CD40 signaling in mouse B cells. Analysis of ERK activities in the B cell lymphoma cell line WEHI 231, which shows an increase in DNA synthesis or arrest of the cell cycle by cross-linking of CD40 or surface IgM (sIgM) cross-linking, respectively, indicated that one of the ERK isoforms, ERK2, was preferentially and rapidly activated after CD40 cross-linking. The CD40-mediated ERK2 activation was comparable to that after sIgM stimulation, although the activity was reduced toward the basal level within several minutes after stimulation. In contrast, ERK1 and ERK2 were activated to a similar extent by sIgM cross-linking, and the activities remained stable for at least 10 min. Furthermore, similar features of differential activation of ERK isoforms were observed in normal resting B cells in CD40 and sIgM signaling. These results suggest divergent regulatory pathways for ERK1 and ERK2 activation, and they support the notion that CD40 signaling may utilize a limited set of elements in the ERK cascade. Co-stimulation of WEHI 231 cells with anti-CD40 mAb rescues the cells from anti-IgM-mediated apoptosis, whereas this co-stimulation resulted in activation of ERK isoforms comparable to that in sIgM stimulation, without a synergistic effect. This result indicates the dominance of ERK activation in sIgM signaling over that of CD40, and it suggests that ERK activation may not be linked to the biological effect that CD40 stimulation in this cell line.     

CD28 affects the earliest signaling events generated by TCR engagement

The efficiency and magnitude of T cell responses are influenced by ligation of the co-stimulatory receptor CD28 by B7 molecules expressed on antigen-presenting cells (APC). In contrast to most previous studies in which agonistic anti-TCR/CD3 and anti-CD28 antibodies were employed, here we have investigated the contribution of CD28 to T cell activation under physiological conditions of antigen presentation. Jurkat T cells and primary T cells from TCR-transgenic mice stimulated with superantigen and antigen, respectively, presented by B7-expressing APC were utilized. In both systems we show that inhibiting CD28/B7 interaction resulted in impaired TCR-induced tyrosine phosphorylation of the signal-transducing ζ chain and ZAP-70. Consistent with a blockade of TCR-proximal signaling events, Jurkat cells stimulated in the absence of CD28 ligation were found to have strongly diminished tyrosine phosphorylation of cellular substrates and downstream signaling pathways such as Ca²⁺ /calcineurin, ERK/MAPK and JNK. Our results provide evidence for a role of CD28 in enhancing TCR signaling capacity during the earliest stages of T cell : APC interaction.     

The lymphocyte-specific protein tyrosine kinase p56lck is hyperphosphorylated on serine and tyrosine residues within minutes after activation via T cell receptor or CD2

Human T cells can be activated and induced to proliferate through either the antigen-specific receptor complex (TcR-CD3) or the CD2 surface molecule. Following stimulation, both serine and tyrosine phosphorylation of cellular protein have been demonstrated to occur. p56lck, a protein tyrosine kinase associated to the inner face of the plasma membrane, is almost exclusively expressed in lymphoid cells, especially T cells. Within minutes after activation of a human T cell-derived line (Jurkat) via stimulation of either the TcR-CD3 complex or the CD2 glycoprotein, we observed a hyperphorphosylation of p56lck. A concomitant shift to a higher molecular weight in sodium dodecyl sulfate-polyacrylamide gel was also observed. Similar changes were obtained with phorbol 12-myristate 13-acetate. Tryptic phosphopeptide analysis of the hyperphosphorylated form of p56lck yielded new phosphorylated sites in serine residues and an increased tyrosine phosphorylation. These results suggest that p56lck may be intimately connected to the signaling pathway in T cell activation.     

Glycosylation-dependent interaction of Jacalin with CD45 induces T lymphocyte activation and Th1/Th2 cytokine secretion

Jacalin, an alpha-O-glycoside of the disaccharide Thomsen-Friedenreich antigen (galactose beta1-3 N-acetylgalactosamine, T-antigen)-specific lectin from jackfruit seeds, has been shown to induce mitogenic responses and to block infection by HIV-1 in CD4+ T lymphocytes. The molecular mechanism underlying Jacalin-induced T cell activation has not been elucidated completely yet. In the present study, protein tyrosine phosphatase (PTPase) CD45 was isolated from a Jurkat T cell membrane fraction as a major receptor for Jacalin through affinity chromatography and mass spectrometry. CD45, which is highly glycosylated and expressed exclusively on the surface of lymphocytes, is a key regulator of lymphocyte signaling, playing a pivotal role in activation and development. We found that the lectin induced significant IL-2 production by a CD45-positive Jurkat T cell line (JE6.1) and primary T cells. However, this effect did not occur in a CD45-negative Jurkat T cell line (J45.01) and was blocked completely by a specific CD45 PTPase inhibitor in Jurkat T (JE6.1) and primary T cells. Furthermore, we also observed that Jacalin caused a marked increase in IL-2 secretion in response to TCR ligation and CD28 costimulation and contributed to Th1/Th2 cytokine production by activating CD45. Jacalin increased CD45 tyrosine phosphatase activity, which resulted in activation of the ERK1/2 and p38 MAPK cascades. Based on these findings, we propose a new, immunoregulatory model for Jacalin, wherein glycosylation-dependent interactions of Jacalin with CD45 on T cells elevate TCR-mediated signaling, which thereby up-regulate T cell activation thresholds and Th1/Th2 cytokine secretion.     

HIV glycoprotein gp120 inhibits TCR-CD3-mediated activation of fyn and lck

HIV major glycoprotein gp120 interacts with CD4 molecules and perturbs signaling through the TCR-CD3 complex. We examined the effects of gp120 on TCR-CD3-induced phosphorylation and activation of the src-type protein tyrosine kinases (PTK), fyn and lck. gp120 caused minimal changes in lck phosphorylation or lck enzymatic activity, but preincubation of Jurkat cells with gp120 for 20 min strongly inhibited TCR-CD3-mediated phosphorylation and activation of lck and fyn, as well as phosphorylation of CD3 zeta. Inhibition of TCR-CD3 signaling in T cells preincubated with gp120 was paralleled by inhibition of T cell proliferation to the antigen tetanus toxoid. Neither surface CD4 expression nor CD4-lck association was affected by gp120. Furthermore, gp120 inhibited lck phosphorylation induced by cross-linking of TCR-CD3 and CD4 suggesting that the inhibition of lck phosphorylation could not be simply accounted for by sequestration of CD4 molecules. gp120 selectively enhanced the phosphorylation of the lck peptide containing the autoinhibitory tyrosine residue Tyr505 relative to the lck peptide containing the positive regulatory residue Tyr394, suggesting that a qualitative alteration in lck may underlie the inhibition of TCR-CD3 signaling by gp120.      

MHC class I cross-talk with CD2 and CD28 induces specific intracellular signalling and leads to growth retardation and apoptosis via a p56(lck)-dependent mechanism

Ligation of the major histocompatibility complex class I molecules (MHC-I) on human T lymphoma cells (Jurkat) initiates p56(lck)-dependent intracellular signalling events (phosphotyrosine kinase activity; [Ca(2+)](i)) and leads to augmented growth inhibition and apoptosis. MHC-I ligation in concert with ligation of CD2 or CD28 augments, changes or modifies the pattern of activation. Ligation of MHC-I and CD2 alone resulted in growth inhibition, whereas CD28 ligation alone had no effect on cell proliferation. Ligation of MHC-I together with CD2 augmented growth inhibition and enhanced the level of apoptosis. In parallel experiments with the p56(lck)-negative Jurkat mutant cell, JCaM1.6, cross-linking neither influenced cell signalling nor cellular growth functions, indicating a cardinal role of the src kinases in signal transduction via MHC-I, CD2 and CD28 molecules. The results presented here provide evidence for the involvement of MHC-I molecules in the modulation of signal transduction via the CD2 and CD28 costimulatory molecules. Copyright Copyright 1999 S. Karger AG, Basel     

Cellular stimulation via CD95 involves activation of phospho-inositide-3-kinase

 Several distinct intracellular pathways have been recently shown to be activated during CD95/Fas/APO-1-mediated apoptosis. Here, we demonstrate that CD95 ligation induces a rapid and transient tyrosine phosphorylation and activation of phosphoinositide-3-kinase (PI-3-K) in Jurkat T lymphocytes or CD95-sensitive glioma cells. Experiments using p56lck-deficient or p56lck-reconstituted Jurkat clones and the tyrosine kinase inhibitor herbimycin A revealed that tyrosine phosphorylation and activation of PI-3-K by CD95 depends on expression of Src-like tyrosine kinases, in particular p56lck. PI-3-K stimulation seems to be critical for CD95 receptor signalling since, first, inhibition of PI-3-K prevents CD95-mediated apoptosis and, second, CD95 receptor ligation fails to induce tyrosine phosphorylation or activation of PI-3-K in CD95-resistant glioma cells. Thus, PI-3-K activation may be an early signalling event during CD95-induced apoptosis, and failure to stimulate PI-3-K may predict tumor cell resistance to CD95-triggered apoptosis. Received: 14 May 1997 / Received after revision: 28 October 1997 / Accepted: 29 October 1997     

CD28 signal transduction: tyrosine phosphorylation and receptor association of phosphoinositide-3 kinase correlate with Ca2+-independent costimulatory activity

The interaction of CD28 with its counter-receptor, B7, induces a cosignal in T cells required to prevent clonal anergy and to promote antigen-dependent interleukin-2 production. The molecular basis of the CD28 cosignal is not well understood but involves the activation of protein tyrosine kinase(s) (PTK). In this report we demonstrate that CD28 cross-linking on Jurkat T leukemic cells causes the activation of at least two PTK pathways. A CD28-induced, p56^lck kinase-independent pathway causes tyrosine-phosphorylation of a 110-kDa substrate while recruitment of p56^lck kinase activity is apparently required for CD28-induced tyrosine-phosphorylation of 97- and 68-kDa substrates as well as CD28-induced increases in intracellular calcium. The tyrosine phosphorylation of p110, but not p97 or p68, correlated with CD28 calcium-independent costimulatory activity. The pp110 molecule was tentatively identified as the catalytic subunit of phosphoinositide (PI)-3 kinase based upon its coimmunoprecipitation with the p85 regulatory subunit of PI-3 kinase. PI-3 kinase protein and catalytic activity were found complexed with the CD28 receptor if the receptor was “activated” by cross-linking on the surface of intact cells prior to detergent solubilization. The kinetics of association of PI-3 kinase with the “activated” CD28 receptor was rapid, occurring within 30 s of receptor cross-linking and was stable for at least 30 min. Analysis of the CD28 cytoplasmic peptide sequence revealed a putative PI-3 kinase src homology 2 binding motif and CD28 tyrosine phosphorylation site, DYMNM. Tyrosine phosphorylation of CD28 was detected in pervanadate-treated Jurkat B2.7 cells, but not untreated cells. Pervanadate-induced tyrosine phosphorylation of CD28 correlated with receptor association of PI-3 kinase in the absence of CD28 cross-linking, suggesting that CD28 association with PI-3 kinase uses a tyrosine phosphorylation-dependent mechanism. These data provide a model for CD28 signal transduction and support a role for PI-3 kinase in mediating the CD28 calcium-independent, cyclosporin A-insensitive costimulatory signal.     

Co-stimulation of T cells with CD2 augments TCR-CD3-mediated activation of protein tyrosine kinase p72syk, resulting in increased tyrosine phosphorylation of adapter proteins, Shc and Cbl

Complete T cell activation requires not only the first signal via TCR- CD3 engagement, but also a co-stimulatory signal through accessory receptors such as CD2, LFA-1 and CD28. However, the pathway of co- stimulatory signaling through accessory receptors is incompletely understood. We report here that CD2 provides a co-stimulus for activation of CD3-mediated syk/ZAP-70 family kinase, p72Syk (Syk), in Jurkat T cells. Although cross-linking of CD2 alone or any combination of CD2 with LFA-1alpha, LFA-1beta or CD28 did not induce tyrosine phosphorylation of Syk, co-cross-linking of CD2 with CD3 enhanced CD3- mediated tyrosine phosphorylation of Syk. Enhancement of tyrosine phosphorylation of Syk by CD2 co-stimulation was CD2 antibody concentration-dependent, and time course studies showed that CD2 co- stimulation enhanced Syk tyrosine phosphorylation by 30 s and through 5 min stimulation compared with the control. In vitro kinase assay revealed that co-cross-linking of CD2 with CD3 augmented Syk kinase activity using myelin basic protein as a substrate. Furthermore, CD2 co- stimulation with CD3 resulted in enhanced tyrosine phosphorylation of adapter proteins, such as Shc and Cbl, in an antibody concentration- dependent manner. Finally, CD2 provided a co-stimulatory signals for synthesis of IL-2 in Jurkat cells and phytohemagglutinin (PHA)- activated T cells and for proliferation of PHA-activated T cells. Taken together, these results indicate that CD2 is an important co- stimulatory receptor for CD3-mediated T cell activation and functions in concert with CD3.      

Lck tyrosine kinase is important for activation of the CD11a/CD18-integrins in human T lymphocytes

CD11a/CD18 (beta2)-integrins are expressed on leukocytes and are involved in cell adhesion and signaling. Despite extensive studies the signaling pathways and molecular mechanisms involved in integrin regulation in T cells remain not completely understood. We have now studied the involvement of the tyrosine kinase Lck in the regulation of CD11a/CD18 function in Jurkat T cells. Using the Src-family kinase inhibitor PP2, we found that CD3 ligation-induced adhesion to ICAM-1 was inhibited by PP2 at the same concentration required for complete inhibition of the MAP kinase pathway, implicating a role for Lck in integrin activation. We therefore used the Lck-deficient Jurkat cell line JCaM1.6 to further examine the involvement of Lck in integrin regulation. Interestingly, JCaM1.6 cells showed dramatically reduced levels of both CD3- and phorbol ester-induced adhesion to coated ICAM-1 as compared to normal Jurkat cells. By using flow cytometry and cell surface labeling, it was found that the surface expression of the CD11a/CD18-integrins was significantly lower in Lck-deficient T cells as compared to normal Jurkat cells. CD18 was expressed as a mature and an immaturely glycosylated form in Jurkat T cell lines, and predominantly the immature form, not associated with CD11a, was found in Lck-deficient cells. Retransfection of human Lck in JCaM1.6 cells restored adhesion. Thus, Lck is involved in regulating CD11a/CD18-integrins in T cells.     

Lysophosphatidylcholine is a regulator of tyrosine kinase activity and intracellular Ca(2+) level in Jurkat T cell line

Lysophospholipids, particularly lysophosphatidylcholine (lyso-PC), have been implicated in modulating T cell functions at the sites of inflammation and atherosclerosis. Although the chemotactic and immunomodulatory effects are well documented, the exact signaling pathway of lyso-PC action is poorly defined. In this work, we studied the earliest biochemical events in T cells triggered by lyso-PC. A marked and immediate tyrosine phosphorylation was induced in the leukemic T cell line, Jurkat. Phosphorylation of cellular substrates included src family kinase, p56(lck) and syk family kinase, ZAP70. The lyso-PC induced tyrosine phosphorylation was largely dependent on the presence of functional p56(lck). Tyrosine phosphorylation was followed by the elevation of intracellular Ca(2+) concentration. The magnitude of the mobilization of the intracellular Ca(2+) was similar in the absence of the p56(lck) activity in JCaM1.6 cells as in Jurkat cells, however, it was slightly but reproducibly delayed compared to that in the wild type cells. Inhibition of the Ser/Thr kinases and tyrosine kinases with staurosporine and genistein, respectively, decreased the rise in the intracellular Ca(2+) content. Moreover, pertussis toxin completely blocked the Ca(2+) signal supporting the role of the G-protein coupled LPC receptor in this event.     

The CD45 tyrosine phosphatase regulates Campath-1H (CD52)-induced TCR-dependent signal transduction in human T cells

Campath-1H, a humanized mAb undergoing clinical trials for treatment of leukemia, transplantation and autoimmune diseases, produces substantial lymphocyte depletion in vivo.The antibody binds to CD52, a highly glycosylated molecule attached to the membrane by a glycosylphosphatidylinositol anchor. Cross-linked Campath-1H is known to activate T cells in vitro. We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45(+)TCR(+), CD45(-)TCR(+) and CD45(+)TCR(-) Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45(+)TCR(+) Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase C gamma 1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR- and CD45-independent manner, and CD52-TCR association in CD45(+)TCR(+) cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56(lck) and p59(fyn) tyrosine kinases.     

T cell receptor activation of a ribosomal S6 kinase activity.

Stimulation of the T cell receptor-CD3 complex activates multiple signal transduction pathways, including serine/threonine and tyrosine protein kinases. Stimulation of the human T cell line Jurkat via the T cell receptor-CD3 complex with anti-CD3 monoclonal antibody or incubation with the tumor promoter phorbol 12-myristate 13-acetate caused increases in S6 kinase and microtubule-associated protein 2 (MAP) kinase activities. An S6 kinase activity that was able to phosphorylate exogenous 40S ribosomal S6 protein was recovered in immunoprecipitates obtained using a 90-kDa ribosomal S6 kinase-specific antiserum and thus represents activation of a member of the 90-kDa ribosomal S6 kinase family. Stimulation of the S6 kinase activity correlated with an increase in a kinase activity able to phosphorylate exogenous 90-kDa ribosomal S6 kinase (rsk) attributed to a MAP kinase activity. These increases in S6 and MAP kinase activities further correlated with the appearance of a 42-kDa phosphoprotein detected by anti-phosphotyrosine immunoblotting. However, while the tyrosine phosphorylation of the 42-kDa protein and the MAP kinase activity are dependent on protein kinase C activity, residual S6 kinase activity can be detected following protein kinase C depletion and subsequent anti-CD3 stimulation. Thus, T cell activation through the T cell receptor-CD3 complex results in activation of a member of the 90-kDa S6 kinase family which correlates with, but can be independent of, MAP kinase activation.