Regulation of the alternative pathway of T cell activation by anti-T3 monoclonal antibody

T cell activation may be triggered either through the T3-Ti antigen receptor complex or via an alternative macrophage-independent pathway involving the 50KD T11 sheep erythrocyte-binding glycoprotein. Monoclonal antibodies anti-T11(2) and anti-T11(3), directed at distinct epitopes of the T11 molecule, trigger mature T cells to proliferate and express their functional programs, and induce expression of IL 2 receptors on both T3+ and T3- thymocytes. We now show that a non-mitogenic anti-T3 antibody blocks activation via the T11 pathway of not only peripheral blood T cells, but also T3+ thymocytes. Anti-T3 does not affect surface expression of T11 or the rapid augmentation of T11(3) expression after incubation of cells with anti-T11(2). However, anti-T3 inhibits generation of IL 2 receptors and production of IL 2 by T lineage cells cultured with anti-T11(2) plus anti-T11(3). In contrast, modulation of the T11 molecule by a non-mitogenic anti-T11 antibody does not inhibit activation of T cells by a mitogenic anti-T3 antibody. The ability of anti-T3 to block expression of IL 2 receptors on both thymocytes and mature T cells activated by the T11 pathway suggests that a regulatory interaction may be important during T cell ontogeny to provide a mechanism for inhibiting expansion of autoreactive clones.     

Sheep erythrocyte rosetting induces multiple alterations in T lymphocyte function: inhibition of T cell receptor activity and stimulation of T11/CD2

When T lymphocytes from human blood or lymphoid organs are prepared by the sheep red blood cell (SRBC) rosetting procedure, glycoproteins of the SRBC membrane interact intimately with the CD2 (T11) molecule on the T cell surface. We now show that rosette formation has measurable short- and long-term effects upon the T cells. First, for a period of 24-48 hr after rosetting, the signal transducing and activation functions of the T3/Ti T cell antigen receptor complex is paralyzed for anti-T3-induced calcium mobilization, with a concomitant decrease in proliferative response to mitogens or stimulatory anti-T3 antibodies. Calcium mobilization through the alternate pathway of T cell activation, the T11/CD2 SRBC receptor, was also inhibited by rosetting. Second, rosetting appears to confer a partial stimulatory signal through the T11/CD2 pathway. Thus, 72 hr or more after rosetting, there was increased expression of the T11(3) activation epitope, and rosetted T cells were stimulated to proliferate in the presence of anti-T11(3) antibodies alone. These results provide further details on the effects of SRBC-T cell interactions, with important methodological implications. Moreover, they suggest a hitherto unrecognized down-regulatory effect of engaging the CD2 molecule, and provide further evidence that the T cell receptor is functionally interconnected to the CD2 activation pathway.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.     

Activation of human thymocytes via the 50KD T11 sheep erythrocyte binding protein induces the expression of interleukin 2 receptors on both T3+ and T3- populations

Recent studies have demonstrated that the 50KD T11 molecule is a surface component of a macrophage-independent alternative pathway of human T cell activation that is unrelated to the T3/Ti antigen-MHC receptor complex. Given the expression of T11 on all human thymocytes, it was of interest to determine whether they could be activated via this pathway. The triggering of T11 by monoclonal antibodies anti-T112 and anti-T113, directed at two unique epitopes on the molecule, induced IL 2 receptor expression on both T3+ and T3- thymocytes but did not induce IL 2 production. Consequently, in contrast to peripheral blood T cells, thymocytes did not proliferate in response to anti-T112 and anti-T113 in the absence of exogenous IL 2. These studies suggest that IL 2 receptor gene activation precedes IL 2 gene activation in T cell development. The ability of the alternative pathway of T cell activation to induce IL 2 receptor expression on T3- thymocytes implies that the T11 molecule may have an important role in early thymocyte ontogeny.     

Mechanisms of immune memory. T cell activation and CD3 phosphorylation correlates with Ta1 (CDw26) expression

The Ta1 (CDw26) Ag distinguishes a subset of circulating T lymphocytes that is the major population proliferating to recall Ag challenge. Unlike receptors for growth factors such as IL-2 and transferrin, the Ta1 Ag is present on T cell lines and clones irrespective of cell cycle. The appearance of Ta1 on T cells that respond to recall Ag allowed us to investigate activation requirements that may be associated with T cell immune memory. Ta1+ peripheral blood T cells were induced to proliferate by mAb recognizing either the invariant chains of the TCR, or by pairs of mitogenic antibodies directed to the CD2 molecule. In contrast, Ta1- cells were not stimulated by these antibodies. In addition, Ta1-cells did not proliferate maximally after addition of the phorbol ester PMA in combination with the calcium ionophore Ionomycin, suggesting that the intracellular targets of these agents may not be fully active. Anti-CD3-induced elevation of intracellular calcium levels was equivalent in the two subpopulations, suggesting that calcium mobilization mechanisms were intact. In contrast, PMA-induced phosphorylation of TCR CD3 chains was significantly greater in Ta1+ cells as compared to Ta1- T cells. Taken together, our results indicate that Ta1 expression, which is associated with T cell activation and memory, may be causally related to TCR and CD2-mediated activation mechanisms. The PMA inducible TCR phosphorylation in Ta1+ memory cells associated with their increased ability to proliferate after CD3/TCR or CD2 stimulation suggests that intracellular phosphorylation events may be causally associated with T cell immune memory.     

Human T cells can be directed to lyse tumor targets through the alternative activation/T11-E rosette receptor pathway

We investigated the ability of human T cells to be directed to lyse murine and human tumor targets by antibodies (Ab) to the T11-E rosette (CD2) receptor. We found that the human cytotoxic T lymphocyte clone TBI-6, which is specific for the Epstein-Barr virus-transformed cell line, CM-EBV, could be directed to lyse the Fc receptor-positive murine tumor P388D1, by the combination of anti-T11(2) plus anti-T11(3) Ab. This activation and lysis was demonstrable only with an Fc receptor expressing tumor target and only with those Ab or with anti-T3 (CD3) Ab but not with other anti-T11 Ab or other Ab directed against surface structures on the clone. We therefore constructed heterodimeric Ab consisting of anti-T11(2) or anti-T11(3) Ab and the J5 anti-common acute lymphoblastic leukemic antigen (anti-CALLA) Ab. The purity and retained functional properties of the dimers were demonstrated by sodium dodecyl sulfate-polyacrylamide gels, fluorescence-activated cell sorter analysis on relevant cells, and by the ability of these conjugates to activate human peripheral blood lymphocytes to proliferate. These heterodimeric Ab conjugates were shown to be able to direct the lysis of CALLA+ targets by TBI-6. The specificity of this lysis was demonstrated by the inability of these heterodimers to direct the lysis of CALLA- targets by the cytotoxic T lymphocyte clone, and by the ability of excess free J5, but not an irrelevant Ab of the same isotype, to block this type of lysis. The potential clinical significance of these reagents is discussed.     

Cross-linking of T3 (CD3) with T4 (CD4) enhances the proliferation of resting T lymphocytes

Monoclonal antibodies reactive with defined T lymphocyte surface antigens were covalently coupled to protein A-Sepharose beads using the bifunctional imidoester, dimethyl pimelimidate. Sepharose-immobilized antibody reactive with T3 induced the proliferation of resting T lymphocytes in the presence of either recombinant interleukin 2 or phorbol myristate acetate. When monoclonal antibodies reactive with T3 and T4 were coupled to the same Sepharose bead (hereafter designated Sepharose (T3:T4)), proliferation was enhanced an average of three-fold. Similarly prepared Sepharose beads coupled to anti-T3 and anti-T8 also enhanced proliferation over that observed with anti-T3 alone. Sepharose (T3:T4) similarly increased the proliferation of T4+ lymphocytes and a T4+ clone but failed to enhance the proliferation of T8+ lymphocytes. The increased proliferation of T4+ lymphocytes resulted from a preferential activation of the T4+2H4- helper population over the T4+2H4+ suppressor-inducer population. The enhanced proliferation induced by Sepharose (T3:T4) could be completely inhibited by soluble anti-T4. These results suggest that perturbation of T3 may be a minimal signal for T cell activation and that the assembly of a multimeric complex including T3 and T4 may be required for optimal T cell activation.     

Signaling from LFA-1 contributes signal transduction through CD2 alternative pathway in T cell activation.

LFA-1, a member of the integrin family of molecules, is involved in mediating cellular adhesion in all phases of the immune response, playing a role in the interaction of helper T cells as well as in killing of target cells by both cytotoxic T cells and natural killer cells. We have developed a monoclonal antibody, anti-HVS6B6, which recognizes a functionally unique epitope of the LFA-1 molecule. Although this mAb itself was not mitogenic against T cells, it induced a strong proliferative response when added to T cells with submitogenic concentrations of anti-CD2 (anti-T11(2) and anti-T11(3)) mAbs. In contrast, other anti-LFA-1 mAbs (CD11a and CD18) suppressed this anti-CD2 mAb-induced T cell proliferation. Kinetic studies showed that anti-HVS6B6 acts on an early event in CD2-mediated T cell activation. Although T11(3)-epitope expression induced by anti-T11(2) mAb was not affected by treatment of cells with anti-HVS6B6, both Ca2+ influx and phosphatidylinositol turnover induced by anti-CD2 mAbs were markedly enhanced by the pretreatment of T cells with anti-HVS6B6 mAb. These results indicate that the LFA-1 mediating signal contributes to a very early phase of signal transduction during CD2-mediated T cell activation.     

Specific CD45 isoforms differentially regulate T cell receptor signaling.

Multiple isoforms of T cell CD45 tyrosine phosphatase are expressed as a result of alternative RNA splicing among extracellular exons. To discern the presence and identity of distinct functions among CD45 isoforms, we compared thymic T cell activation responses by elevating expression of two CD45 isoforms normally found on quiescent T cells. We report that CD45RABC significantly increased CD4+ thymic T cell proliferation in both a mixed lymphocyte reaction and following anti-T cell receptor (TCR) antibody stimulation. Additionally, CD45RABC enhanced Ca2+ mobilization and phosphotyrosine accumulation, and suppressed the inhibitory effect of anti-CD4 antibodies. By contrast, CD45R0 did not enhance TCR signaling or phosphotyrosine levels in CD4+ thymic T cells and required a TCR co-stimulus to augment cellular proliferation. These studies provide genetic evidence that alternative CD45 isoforms are functionally distinct and disclose a unique mechanism by which T cell immunologic responsiveness can be modified.     

Dynamic recruitment of human CD2 into lipid rafts. Linkage to T cell signal transduction

CD2 mediates T cell adhesion via its ectodomain and signal transduction utilizing its 117-amino acid cytoplasmic tail. Here we show that a significant fraction of human CD2 molecules is inducibly recruited into lipid rafts upon CD2 cross-linking by a specific pair of mitogenic anti-CD2 monoclonal antibodies (anti-T11(2) + anti-T11(3)) or during cellular conjugate formation by CD58, the physiologic ligand expressed on antigen-presenting cells. Translocation to lipid microdomains is independent of the T cell receptor (TCR) and, unlike inducible TCR-raft association, requires no tyrosine phosphorylation. Structural integrity of rafts is necessary for CD2-stimulated elevation of intracellular free calcium and tyrosine phosphorylation of cellular substrates. Whereas murine CD2 contains two membrane-proximal intracellular cysteines, partitioning CD2 into cholesterol-rich lipid rafts constitutively, human CD2 has no cytoplasmic cysteines. Mapping studies using CD2 point mutation, deletion, and chimeric molecules suggest that conformational change in the CD2 ectodomain participates in inducible raft association and excludes the membrane-proximal N-linked glycans, the transmembrane segment, and the CD2 cytoplasmic region (residues 8-117) as necessary for translocation. Translocation of CD2 into lipid rafts may reorganize the membrane into an activation-ready state prior to TCR engagement by a peptide associated with a major histocompatibility complex molecule, accounting for synergistic T cell stimulation by CD2 and the TCR.     

Characterization and expression of the human alpha beta T cell receptor by using a framework monoclonal antibody

A monoclonal antibody (mAb) with framework reactivity against the T cell receptor (TCR) alpha beta complex is characterized. The mAb, beta Framework 1 (beta F1) is capable of immunoprecipitating the TCR alpha beta complex from 125I-labeled human T cell tumors, immunocompetent T cell clones, and peripheral blood lymphocytes (PBL). beta F1 recognizes the separated TCR beta subunit in Western blotting. Because it does not bind to the surface of viable T cells but does react with the plasma membrane form of the TCR after treatment with membrane solubilizing agents, the beta F1 mAb reacts with a "hidden" determinant on the TCR beta subunit. After solubilization with 70% ethanol, the TCR alpha beta complex is shown to exist on greater than 92% of T3+ human PBL, whereas 2 to 8% of T3+ PBL do not react with the mAb. The beta F1 mAb demonstrates the existence of differently glycosylated surface 125I-labeled TCR alpha-chains (alpha, alpha', alpha") in association with a common TCR beta-chain on the HPB-MLT T cell leukemia. Reactivity of the beta F1 mAb on thymus tissue sections is similar to that of anti-Leu-4 (anti-T3). The beta F1 mAb should prove useful as a research tool for both the immunochemical characterization and isolation of virtually any alpha beta T cell receptor, whether from individual T cell clones or polyclonal populations of T lymphocytes. Recognition of T cell receptors in histologic tissue sections suggests that the beta F1 mAb may be useful in the clinical diagnosis of T cell lineage neoplasms. In failing to recognize all T3+ lymphocytes, it allows the identification of novel populations of T3+ lymphocytes that may express non-alpha, non-beta T cell receptors.     

Growth inhibition of human T cells by antibodies recognizing the T cell antigen receptor complex

Monoclonal antibodies that bind to the T cell MHC-antigen recognition complex (anti-T3 or anti-Ti) are known to either mimic ligand binding and activate T cells or block ligand binding, leading to an inhibition of T cell activation. In the present experiments, we demonstrate a direct inhibitory effect on the growth of human T cells by anti-T3 or anti-Ti antibodies. The proliferation of human peripheral blood T cells preactivated by exposure to PHA was inhibited in a specific manner by anti-T3. Colony formation in soft agar by REX cells, a leukemic cell line of early T cell phenotype, was completely inhibited by anti-T3 or anti-Ti antibodies, whereas isotype-matched antibodies to a variety of other T cell markers had no effect. Growth of REX cells in suspension culture was not affected by anti-T3 or anti-Ti. A cell line, T3.N1, was established from an agar colony of anti-T3-resistant REX cells. T3.N1 was phenotypically identical to REX except for failure to express any detectable T3 or Ti surface antigen. T3.N1 colony formation in soft agar was not inhibited by anti-T3 or anti-Ti. There was no rise in [Ca2+]i of T3.N1 cells after anti-T3 or anti-Ti exposure. These results indicate that in addition to the well-known positive regulatory effects of ligand binding to the T3/Ti complex, T3/Ti binding can also result in a down-regulatory signal for human T cell growth.     

Inefficient phospholipase C activation and reduced Lck expression characterize the signaling defect of umbilical cord T lymphocytes.

Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the immune response of cord blood-derived T lymphocytes compared with that of adult T lymphocytes. Our findings demonstrate that phospholipase C (PLC) isozymes, which play a pivotal role in the control of protein kinase C activation and Ca2+ mobilization, are differently expressed in cord and adult T lymphocytes. PLCbeta1 and delta1 are expressed at higher levels in cord T cells, while PLCbeta2 and gamma1 expression is higher in adult T lymphocytes. PLCdelta2 and gamma2 appear to be equally expressed in both cell types. In addition, a functional defect in PLC activation via CD3 ligation or pervanadate treatment, stimuli that activate tyrosine kinases, was observed in cord blood T cells, whereas treatment with aluminum tetrafluoride (AlF4-), a G protein activator, demonstrated a similar degree of PLC activation in cord and adult T cells. The impaired PLC activation of cord blood-derived T cells was associated with a a very low expression of the Src kinase, Lck, along with a reduced level of ZAP70. No mitogenic response to CD3 ligation was observed in cord T cells. However, no signaling defect was apparent downstream of PLC activation, as demonstrated by the mitogenic response of cord T cells to the pharmacologic activation of protein kinase C and Ca2+ by treatment with PMA and ionomycin. Thus, neonatal cord blood-derived T cells show a signaling immaturity associated with inadequate PLCgamma activation and decreased Lck expression.     

Anti-Qa-2-induced T cell activation. The parameters of activation, the definition of mitogenic and nonmitogenic antibodies, and the differential effects on CD4+ vs CD8+ T cells

The MHC Ag Qa-2 is a glycolipid anchored class I molecule expressed at high levels on all peripheral T lymphocytes. In this study we found that anti-Qa-2 antibodies could stimulate the proliferation of murine T cells in vitro. Anti-Qa-2-induced proliferation required secondary cross-linking with anti-Ig antibody and the presence of PMA. Only Qa-2+ strains could be induced to proliferate by anti-Qa-2 antibody, but under the conditions employed, anti-CD3 could induce proliferation in Qa-2+ and Qa-2-strains. Interestingly, only anti-Qa-2 reagents directed against the alpha 3 domain of the Qa-2 class I molecule were effective in inducing proliferation. Furthermore, unlike purified CD4+ cells, purified CD8+ cells were unable to be stimulated by the anti-Qa-2 antibodies. These results lead to the inclusion of Qa-2 in a group of physiologically relevant, glycolipid-anchored, cell-surface molecules, mobilization of which can generate signals that initiate the proliferation of T cells. Such molecules may play a secondary role in cellular activation after the primary engagement of the TCR.     

Distinct epitopes on the T cell antigen receptor of HPB-ALL tumor cells identified by monoclonal antibodies

The T cell antigen receptor is a approximately 90,000 dalton disulfide linked heterodimer that is non-covalently associated with the CD3 complex. Prior studies have demonstrated that anti-CD3 or -Ti antibodies can mimic antigen and induce cellular proliferation and the secretion of lymphokines. An early event in activation via CD3/Ti is a rapid increase in concentration of intracellular Ca2+ levels. In the present studies, we have produced a panel of monoclonal antibodies (MAb) against the Ti expressed on HPB-ALL tumor cells. All MAb immunoprecipitate a approximately 90,000 dalton disulfide linked heterodimer and induced co-modulation of Ti and CD3. On the basis of competitive binding studies, four distinct epitopes on the Ti of HPB-ALL were identified with MAb L38, L39, L41, and L42. These epitopes were additionally discriminated on the basis of reactivity with normal polyclonal T cell populations and functional effects on HPB-ALL. L39 reacted with a monomorphic epitope present on approximately 2 to 5% of peripheral blood T lymphocytes from all donors examined and was specifically mitogenic for peripheral blood T cells expressing this epitope. L39+ T cells in blood included both CD4+ and CD8+ lymphocytes. In contrast, L38, L41, and L42 failed to react with peripheral blood T cells and were not mitogenic for peripheral blood lymphocytes. Anti-Leu-4, L38, L39, and L41 MAb all induced a rapid increase in (Ca2+)i in HPB-ALL tumor cells, similar to previous findings with anti-CD3 and anti-Ti MAb against various tumor cells and peripheral blood T cells. In contrast, L42 MAb did not induce a substantial increase in (Ca2+)i. Failure of L42 to induce a substantial increased (Ca2+)i could not be attributed to the apparent titer, avidity, or isotype of the antibody. These findings suggest that induction of increased (Ca2+)i upon binding of Ti is epitope dependent. Furthermore, these data demonstrate that several distinct public and private epitopes can be identified on the T cell antigen receptor.     

Antigen recognition by human T lymphocytes is linked to surface expression of the T3 molecular complex

Four distinct surface molecules on human T cells are defined by the monoclonal antibodies anti-T1, anti-T3 (anti-T3_A), anti-T11 and anti-T12. Following cell binding, anti-T3 (anti-T3_A) and anti-T1 induce independent modulation of their respective ligands, whereas anti-T11 and anti-T12 do not. To explore the biological consequences of this modulation, we used cloned populations of T4 and T8 cytotoxic T lymphocytes. Anti-T3 (anti-T3_A), but not anti-T1, inhibits cytotoxic T lymphocyte effector function by T4 and T8 clones as well as antigen-specific T cell recognition. The latter is not secondary to a generalized inhibitory effect since responsiveness to interleukin 2 is maintained. Moreover, after modulation, cytotoxic T lymphocytes recover cytolytic function in parallel with reexpression of surface T3 molecules. We provide evidence for a direct linkage between antigen recognition by T lymphocytes and surface expression of the T3 molecular complex.     

CD1d-specific NK1.1+ T cells with a transgenic variant TCR

The majority of T lymphocytes carrying the NK cell marker NK1.1 (NKT cells) depend on the CD1d molecule for their development and are distinguished by their potent capacity to rapidly secrete cytokines upon activation. A substantial fraction of NKT cells express a restricted TCR repertiore using an invariant TCR Valpha14-Jalpha281 rearrangement and a limited set of TCR Vbeta segments, implying recognition of a limited set of CD1d-associated ligands. A second group of CD1d-reactive T cells use diverse TCR potentially recognizing a larger diversity of ligands presented on CD1d. In TCR-transgenic mice carrying rearranged TCR genes from a CD1d-reactive T cell with the diverse type receptor (using Valpha3. 2/Vbeta9 rearrangements), the majority of T cells expressing the transgenic TCR had the typical phenotype of NKT cells. They expressed NK1.1, CD122, intermediate TCR levels, and markers indicating previous activation and were CD4/CD8 double negative or CD4+. Upon activation in vitro, the cells secreted large amounts of IL-4 and IFN-gamma, a characteristic of NKT cells. In mice lacking CD1d, TCR-transgenic cells with the NKT phenotype were absent. This demonstrates that a CD1d-reactive TCR of the "non-Valpha 14" diverse type can, in a ligand-dependent way, direct development of NK1.1+ T cells expressing expected functional and cell-surface phenotype characteristics.     

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.     

Differential roles of PKC-theta in the regulation of intracellular calcium concentration in primary T cells

Activation of T lymphocytes requires protein kinase C theta (PKC-theta) and an appropriately elevated free intracellular Ca2+ concentration ([Ca2+]i). Here, we show that phorbol 12 myristate 13-acetate (PMA) inhibited Ca2+ influx in wild-type but not PKC-theta-/- T cells, suggesting that PKC-theta plays a role in PMA-mediated inhibition of Ca2+ influx. In contrast, T cell receptor (TCR) crosslinking in the same PKC-theta-/- T cells did result in significantly decreased [Ca2+]i compared to wild-type T cells, suggesting a positive role for PKC-theta in TCR-mediated Ca2+ mobilization. In PKC-theta-/- mice, peripheral mature T cells, but not developing thymocytes, displayed significantly decreased TCR-induced Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation upon sub-optimal TCR crosslinking. The decreased intracellular free Ca2+ was due to changes in Ca2+ influx but not efflux, as observed in extracellular and intracellular Ca2+ mobilization studies. However, these differences in Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation disappeared with increasing intensity of TCR crosslinking. The enhancing effect of PKC-theta on Ca2+ influx is not only dependent on the strength of TCR crosslinking but also on the developmental stage of T cells. The underlying mechanism involved phospholipase Cgamma1 activation and inositol triphosphate production. Furthermore, knockdown of endogenous PKC-theta expression in Jurkat cells resulted in significant inhibition of TCR-induced activation of NFAT, as evidenced from NFAT reporter studies. Forced expression of a constitutively active form of calcineurin in PKC-theta-/- Jurkat cells could readily overcome the above inhibition. Thus, PKC-theta can both positively and negatively regulate the Ca2+ influx that is critical for NFAT activity.