CD2 can mediate TCR/CD3-independent T cell activation.

T lymphocytes can be activated clonotypically through TCR/CD3 complex or polyclonally via the CD2 molecule. Whether CD2-mediated activation is dependent on TCR/CD3 expression or signaling is controversial. We have re-explored this issue by using a series of CD2-transfected, TCR/CD3 surface membrane-negative human and mouse T cells. Our results clearly show that such T cells can be triggered for IL-2 secretion and increases in intracellular Ca2+ through the CD2 molecule in the absence of surface expression of TCR/CD3 complexes. These responses are only observed when cells express high levels of CD2 and there is a critical threshold of CD2 expression necessary for such activation in the absence of CD3. Concomitant expression of TCR/CD3 complex markedly lowers the level of CD2 required for activation via the latter pathway. These results provide a clear resolution of the controversy concerning the requirement for surface CD3 expression in T cell activation through CD2 and further suggest a possible role for CD2 in activation of TCR/CD3-negative cells.     

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.     

The T11 (CD2) molecule is functionally linked to the T3/Ti T cell receptor in the majority of T cells

Most mature human T lymphocytes express both the multichain T3 (CD3)/Ti T cell receptor for antigen (TCR), and the biochemically distinct 55-kDa T11 (CD2) glycoprotein. Stimulating the T11 molecule causes profound T cell proliferation and functional activation in vitro, but the relationship of T11-mediated activation to antigenic stimulation of T lymphocytes in vivo remains unknown. We now present evidence that T11 function is directly linked to TCR components in T3/Ti+ T11+ human T cells. First, we found that stimulating peripheral blood T cells with the mitogenic combination of anti-T11(2) cells with the mitogenic combination of anti-T11(2) plus anti-T11(3) monoclonal antibodies caused the phosphorylation of TCR T3 chains. The predominance of T3-gamma-phosphorylation that occurred in anti-T11(2) plus anti-T11(3)-treated T cells is similar to the pattern previously observed in antigen-stimulated T cell clones. Second, T11 function depended upon concurrent cell-surface expression of the TCR. Thus, when peripheral blood T cells were deprived of cell surface T3/Ti by anti-T3 modulation, anti-T11(2) plus anti-T11(3)-induced mitogenesis and transmembrane signal generation in the form of calcium mobilization were inhibited. The mechanism of TCR-T11 interdependence was investigated in a series of TCR-deficient variants of a T cell lymphoblastoid cell line. T3/Ti negative variants expressed cell surface T11, but anti-T11(2) plus anti-T11(3) failed to cause detectable calcium mobilization. The TCR-deficient variants also failed to express T11(3) activation epitopes after incubation with anti-T11(2) antibodies, suggesting that T11(3) expression is an essential and TCR-dependent intermediate in the T11 activation mechanism in these cells. Taken together, our results suggest that T11 function depends upon cell-surface expression of TCR in many T3/Ti+ T11+ T lymphocytes, and T11-mediated activation is intimately interconnected with TCR activation mechanisms. A model in which stimulating signals delivered via T11 may be a part of antigenic activation of T lymphocytes is presented.     

Differential role of tyrosine phosphorylation in the induction of apoptosis in T cell clones via CD95 or the TCR/CD3-complex

Activated T cells undergo apoptosis when the Fas-antigen (APO-1, CD95) is ligated by Fas Ligand (FasL) or agonistic anti-Fas antibodies. Repeated stimulation of T lymphocytes via the TCR/CD3-complex induces activation-induced cell death (AICD) associated with FasL surface expression. FasL binding to Fas molecules triggers the Fas-dependent death signaling cascade. Since it is still controversial whether Fas-induced cell death is associated with tyrosine kinase activity, we investigated the tyrosine kinase activation requirements in anti-Fas antibody-induced cell death and AICD in human T cell clones. We report that cell death triggered by anti-Fas antibody is not accompanied by an increase in tyrosine phosphorylation and cannot be blocked by inhibitors of protein tyrosine kinases (PTK). Under the same conditions, AICD of T cell clones is clearly associated with tyrosine kinase activation. In fact, semiquantitative RT-PCR analysis of FasL mRNA expression triggered in T cell clones via the TCR/CD3-complex revealed that tyrosine phosphorylation is required for functional FasL mRNA and surface expression.     

Signal transduction via leukocyte antigen CD43 (sialophorin). Feedback regulation by protein kinase C

CD43 is a constitutively phosphorylated 115-kDa sialoglycoprotein expressed on a variety of blood cells including lymphocytes and monocytes. L10, a mAb directed against CD43, triggers T cell activation and enhances hydrogen peroxide production in monocytes. Activation of mononuclear cells by L10 initiates phosphoinositides hydrolysis, C2+ mobilization, and protein kinase C (PKC) activation. In turn, activated PKC hyperphosphorylates CD43, suggesting a potential role for PKC in the regulation of signaling via CD43. To address this issue, we have analyzed the effect of PKC activation by the tumor promoter PMA on L10-triggered rise in intracellular free Ca2+ concentrations ([Ca2+]i). Treatment of mononuclear cells with PMA profoundly inhibited the increase in [Ca2+]i induced by L10. The inhibition of CD43-mediated signaling by PMA was due, in part, to uncoupling of CD43 from the signal-transducing G protein. This was evidenced by the comparatively modest inhibition by PMA of the increase in [Ca2+]i induced by the direct G protein activator AlF4-. PMA treatment did not affect the surface expression of CD43. However, it induced the hyperphosphorylation of CD43, the extent of which correlated with the inhibition of CD43-mediated increase in [Ca2+]i. Staurosporine, a potent inhibitor of PKC, abrogated the hyperphosphorylation of CD43 and normalized CD43-mediated signaling in PMA-treated cells. Significantly, in the absence of PMA, staurosporine enhanced the rise in [Ca2+]i triggered by L10, suggesting that engagement of CD43 by activating ligands results in feedback inhibition by PKC. It is concluded that activation of PKC inhibits signaling via CD43 by mechanisms involving phosphorylation and uncoupling of CD43 from the signal-transducing apparatus and by distal, post-receptor events.     

Developmental regulation of transmembrane signaling via the T cell antigen receptor/CD3 complex in human T lymphocytes.

We have examined transmembrane signaling events via the TCR/CD3 complex (TCR/CD3) at various stages of T cell development for evidence of developmental regulation. Engagement of TCR/CD3 induced defective activation of phospholipase C (PLC) in thymocytes relative to peripheral blood T lymphocytes. The defect in PLC activation via TCR/CD3 was restricted to immature thymocytes (CD3low, CD4+CD8+). Mature thymocytes (CD3high, CD4+CD8-/CD8+CD4-) were similar to PBL in signaling via TCR/CD3. Both immature and mature thymocytes expressed a similar profile of PLC isoenzyme mRNA species, indicating that the defect in signaling in immature thymocytes was not due to altered expression of PLC isoenzymes. Activation of tyrosine phosphorylation pathways implicated in the coupling of TCR/CD3 to PLC was impaired in immature thymocytes, as evidenced by depressed phosphorylation of CD3 zeta subunit after stimulation with anti TCR/CD3 mAb. This was associated with lower levels of p59fyn tyrosine kinase and minimal or undetectable stimulus-induced kinase activation in immature thymocytes relative to mature thymocytes. We conclude that the capacity to signal via TCR/CD3 is regulated during T cell development by mechanisms acting at the level of TCR/CD3-associated tyrosine phosphorylation pathways.     

Signaling via the inositol phospholipid pathway by T cell antigen receptor is limited by receptor number.

Engagement of the TCR initiates at least two transmembrane signaling pathways, the phosphatidylinositol pathway and a tyrosine kinase pathway. The T cell leukemic line Jurkat was used to study the relationship between the number of occupied TCR on the cell surface and the TCR-mediated activation of phosphatidylinositol-specific phospholipase C. We characterized a series of Ti beta-chain transfectants of the Jurkat mutant J.RT3-T3.5, in which surface expression of the TCR is limited by expression of the TCR beta-chain. Calibrated flow cytometry was used to determine the number of binding sites for anti-CD3 mAb on the surface of these cells, which was less than 1.2 x 10(3) to 1.2 x 10(4) sites/cell. In the presence of lithium chloride, the accumulation of inositol phosphates (InsP) in these cell lines in response to saturating concentrations of anti-CD3 mAb was proportional to the calculated surface TCR number. This result was consistent with dose-response studies using anti-CD3 mAb in Jurkat cells, in which ligand concentration, rather than number of binding sites, was limiting. Increase in intracellular free calcium concentration was a sensitive indicator of TCR engagement and correlated with the level of TCR expression, but less closely than did InsP levels. Induction of the early lymphocyte activation marker CD69 by anti-CD3 mAb also correlated with surface expression of TCR. In order to test whether limitation of this signaling pathway by TCR number may be relevant to signal transduction in the wild-type cell, we compared PLC activity in Jurkat cells during soluble anti-CD3 mAb-induced internalization of the TCR and also in response to immobilized mAb. The net accumulation of InsP per min decreased linearly with TCR number during the rapid phase of TCR internalization, confirming the limiting role of TCR number in this system. When internalization was prevented by immobilization of the stimulus, there was no decrease in the net accumulation of InsP per minute over time. In a Jurkat cell line transfected with the heterologous human muscarinic receptor, subtype 1, the InsP response to a muscarinic agonist was unaffected by TCR internalization, indicating that the distal phosphatidylinositol pathway was not affected by prolonged stimulation of the TCR. We conclude that transmembrane signaling through the TCR may be regulated by the number of surface TCR-ligand complexes. This observation has implications for transmembrane signaling in both mature T cells and thymocytes.     

Introduction of T cell receptor (TCR)-alpha cDNA has differential effects on TCR-gamma delta/CD3 expression by PEER and Lyon-1 cells

A TCR heterodimer composed of a TCR gamma-chain and a TCR delta-chain was found to be expressed in association with CD3 by a small population of human peripheral blood T cells, thymocytes, and certain leukemic T cell lines. The leukemic T cell lines PEER and Lyon-1 express such a TCR-gamma delta/CD3 complex at the cell surface. In addition, PEER and Lyon-1 cells transcribe a productively rearranged TCR-beta gene. Introduction of TCR alpha-chain cDNA of human or murine origin resulted in cell surface expression of a TCR-alpha beta/CD3 complex on PEER and Lyon-1 cells. The expression of the TCR-gamma delta/CD3 complex on PEER cells was not affected by introduction of TCR-alpha cDNA. In contrast, introduction of a TCR-alpha cDNA and expression of the TCR-alpha beta/CD3 complex in Lyon-1 cells resulted in the disappearance of the TCR-gamma delta/CD3 complex. These data were confirmed by indirect immunofluorescence, at the protein level and by gene expression analysis. Triggering of the TCR-alpha beta/CD3 complexes by anti-CD3 mAb or anti-TCR mAb resulted in increased internal Ca2+ levels, indicating that these receptors were functional in signal transduction. These results indicate that, besides TCR gene rearrangements, membrane expression of TCR-alpha beta heterodimers may be important in regulating TCR-gamma delta cell surface expression.     

PKCtheta is required for the activation of human T lymphocytes induced by CD43 engagement

The turnover of phosphoinositides leading to PKC activation constitutes one of the principal axes of intracellular signaling. In T lymphocytes, the enhanced and prolonged PKC activation resulting from the engagement of the TcR and co-receptor molecules ensures a productive T cell response. The CD43 co-receptor promotes activation and proliferation, by inducing IL-2 secretion and CD69 expression. CD43 engagement has been shown to promote phosphoinositide turnover and DAG production. Moreover, PKC activation was found to be required for the activation of the MAP kinase pathway in response to CD43 ligation. Here we show that CD43 engagement led to the membrane translocation and enzymatic activity of specific PKC isoenzymes: cPKC (alpha/beta), nPKC (epsilon and theta;), aPKC (zeta) and PKCmu. We also show that activation of PKCtheta; resulting from CD43 ligation induced CD69 expression through an ERK-dependent pathway leading to AP-1, NF-kappaB activation and an ERK independent pathway promoting NFAT activation. Together, these data suggest that PKCtheta; plays a critical role in the co-stimulatory functions of CD43 in human T cells.     

Mechanisms of T cell activation by the calcium ionophore ionomycin

We have investigated signaling mechanisms that may underlie the T cell mitogenic properties of the Ca2+ ionophore ionomycin. Ionomycin induces highly purified resting human T cells to proliferate in the presence of monocytes with accompanying IL-2R expression and IL-2 synthesis. Treatment of T cells with ionomycin triggers the hydrolysis of phosphoinositides, as evidenced by the accumulation of the hydrolytic by-products phosphatidic acid and inositol phosphates. Ionomycin also induces the activation of protein kinase C (PKC), as demonstrated by the auto-phosphorylation of PKC and the phosphorylation of the PKC target proteins CD4 and CD8. Ionomycin synergizes with PMA in enhancing the activation of PKC. It is concluded that, in addition to its putative activation of Ca2+/calmodulin-dependent signaling pathways, ionomycin induces the hydrolysis of phosphoinositides and the activation of PKC in human T cells. The synergy of ionomycin with phorbol esters in triggering T cell activation may relate, at least in part, to enhanced activation of PKC.     

Activation of T lymphocytes through the Ly-6 pathway is defective in A strain mice

The Ly-6 alloantigens have been shown to participate in the process of T cell activation based on the ability of anti-Ly-6 mAb to induce IL-2 production and proliferation of T lymphocytes. In the present investigation we have demonstrated that peripheral T lymphocytes from A strain mice exhibited abnormally low proliferative responses after stimulation through Ly-6A/E and Ly-6C molecules when compared to responses of T cells from numerous other mouse strains. The abnormal activation of the Ly-6 pathway of A strain T cells was not due to ineffective FcR cross-linking of the anti-Ly-6 mAb, to inappropriate cellular expression of the Ly-6A/E alloantigen in A strain T cells, or to an active suppressive phenomenon. T lymphocytes from A strain mice proliferated normally when the cells were activated by mAb to Thy-1 or the CD3/TCR complex suggesting that A strain mice did not exhibit a generalized T cell activation defect. Cell separation studies of T cells and accessory cells demonstrated that this defect was quantitative, rather than qualitative, and that it was complex, residing at both the T cell and accessory cell levels. These results suggest that activation of T lymphocytes via the Ly-6 molecule may involve a signaling pathway and/or cell-cell interactions distinct from those required for optimal activation via CD3/TCR.     

CD38 is associated with lipid rafts and upon receptor stimulation leads to Akt/protein kinase B and Erk activation in the absence of the CD3-zeta immune receptor tyrosine-based activation motifs.

T lymphocytes can be activated via the T cell receptor (TCR) or by triggering through a number of other cell surface structures, including the CD38 co-receptor molecule. Here, we show that in TCR+ T cells that express a CD3-zeta lacking the cytoplasmic domain, cross-linking with CD38- or CD3-specific monoclonal antibodies induces tyrosine phosphorylation of CD3-epsilon, zeta-associated protein-70, linker for activation of T cells, and Shc. Moreover, in these cells, anti-CD38 or anti-CD3 stimulation leads to protein kinase B/Akt and Erk activation, suggesting that the CD3-zeta-immunoreceptor tyrosine-based activation motifs are not required for CD38 signaling in T cells. Interestingly, in unstimulated T cells, lipid rafts are highly enriched in CD38, including the T cells lacking the cytoplasmic tail of CD3-zeta. Moreover, CD38 clustering by extensive cross-linking with an anti-CD38 monoclonal antibody and a secondary antibody leads to an increased resistance of CD38 to detergent solubilization, suggesting that CD38 is constitutively associated with membrane rafts. Consistent with this, cholesterol depletion with methyl-beta-cyclodextrin substantially reduces CD38-mediated Akt activation while enhancing CD38-mediated Erk activation. CD38/raft association may improve the signaling capabilities of CD38 via formation of protein/lipid domains to which signaling-competent molecules, such as immunoreceptor tyrosine-based activation motif-bearing CD3 molecules and protein-tyrosine kinases, are recruited.     

TCR dynamics in human mature T lymphocytes lacking CD3 gamma

The contribution of CD3gamma to the surface expression, internalization, and intracellular trafficking of the TCR/CD3 complex (TCR) has not been completely defined. However, CD3gamma is believed to be crucial for constitutive as well as for phorbol ester-induced internalization. We have explored TCR dynamics in resting and stimulated mature T lymphocytes derived from two unrelated human congenital CD3gamma-deficient (gamma(-)) individuals. In contrast to gamma(-) mutants of the human T cell line Jurkat, which were selected for their lack of membrane TCR and are therefore constitutively surface TCR negative, these natural gamma(-) T cells constitutively expressed surface TCR, mainly through biosynthesis of new chains other than CD3gamma. However, surface (but not intracellular) TCR expression in these cells was less than wild-type cells, and normal surface expression was clearly CD3gamma-dependent, as it was restored by retroviral transduction of CD3gamma. The reduced surface TCR expression was likely caused by an impaired assembly or membrane transport step during recycling, whereas constitutive internalization and degradation were apparently normal. Ab binding to the mutant TCR, but not phorbol ester treatment, caused its down-modulation from the cell surface, albeit at a slower rate than in normal controls. Kinetic confocal analysis indicated that early ligand-induced endocytosis was impaired. After its complete down-modulation, TCR re-expression was also delayed. The results suggest that CD3gamma contributes to, but is not absolutely required for, the regulation of TCR trafficking in resting and Ag-stimulated mature T lymphocytes. The results also indicate that TCR internalization is regulated differently in each case.     

Assembly, intracellular processing, and expression at the cell surface of the human alpha beta T cell receptor/CD3 complex. Function of the CD3-zeta chain

The TCR/CD3 complex is a multimeric protein complex composed of a minimum of seven transmembrane chains (TCR alpha beta-CD3 gamma delta epsilon zeta 2). Whereas earlier studies have demonstrated that both the TCR-alpha and -beta chains are required for the cell surface expression of the TCR/CD3 complex, the role of the CD3 chains for the TCR/CD3 expression have not been experimentally addressed in human T cells. In this study the function of the CD3-zeta chain for the assembly, intracellular processing, and expression of the TCR/CD3 complex in the human leukemic T cell line Jurkat was investigated. The results indicate that: 1) CD3-zeta is required for the cell surface expression of the TCR/CD3 complex; 2) the pentameric form (TCR alpha beta-CD3 gamma delta epsilon) of the TCR/CD3 complex and single TCR chains associated with CD3 (TCR alpha-CD3 gamma delta epsilon and TCR beta-CD3 gamma delta epsilon) are produced in the endoplasmic reticulum in the absence of CD3-zeta; 3) the CD3-zeta does not associate with TCR alpha-CD3 gamma delta epsilon or TCR beta-CD3 gamma delta epsilon complexes; 4) CD3-zeta associate with the pentameric form of the TCR/CD3 complex in the endoplasmic reticulum to form the heptameric complex (TCR alpha beta-CD3 gamma delta epsilon----TCR alpha beta-CD3 gamma delta epsilon 2); and 5) CD3-zeta is required for the export of the TCR/CD3 complex from the endoplasmic reticulum to the Golgi apparatus for subsequent processing.     

Activation of c-jun N-terminal kinase by 4-1BB (CD137), a T cell co-stimulatory molecule

It has been widely accepted that T cell activation requires two signals; one from the binding of the antigen/major histocompatibility complex to the T-cell receptor (TCR)/CD3 complex and the other from the interaction between a surface molecule on antigen presenting cells and its receptor on T cells. The second signal is considered as co-stimulatory and the B7/CD28 pair has been well studied as a prototype. Recently 4-1BB (CD137) has been characterized as another co-stimulatory molecule for T cell activation. However, unlike the CD28/B7 pair, 4-1BB and its ligand 4-1BBL constitute a member of the tumor necrosis factor (TNF) receptor/TNF pair superfamily. The signaling mechanism of 4-1BB has not been revealed in detail. To investigate whether 4-1BB takes the signaling pathways analogous to those for TNF receptors, we generated polyclonal antibodies against human 4-1BB and 4-1BBL and established stable transfectants of the receptor and the ligand with a high level of cell surface expression. Over-expression of h4-1BB was found to result in the activation of c-Jun N-terminal kinase (JNK) in the human embryonic kidney cell line 293. In T cells, it has been previously demonstrated that JNK activation requires dual signals such as the ligation of TCR/CD3 complex plus CD28 co-stimulation or PMA plus ionomycin. The JNK activation by 4-1BB in Jurkat T cells was also found to require stimulation of the TCR/CD3 complex, consistent with the notion that 4-1BB functions as a co-stimulatory molecule for T cell activation.     

Restoration of T cell receptor-mediated signal transduction by transfection of CD45 cDNA into a CD45-deficient variant of the Jurkat T cell line.

The TCR is a multimeric structure comprised of distinct Ag recognition and signal transduction components. Although none of the molecules that make up the TCR possess intrinsic protein tyrosine kinase (PTK) activity, stimulation of T cells via the TCR results in the rapid appearance of newly tyrosine phosphorylated proteins in cell lysates. Evidence suggests ligation of the TCR induces activation of a PTK that may be a member of the src family. One early consequence of this TCR-mediated PTK activation is the phosphorylation of the gamma 1 isoform of phospholipase C. This phosphorylation event is associated with increased enzymatic activity resulting in the hydrolysis of phosphatidylinositol 4,5 bisphosphate into two second messengers, inositol 1,4,5 trisphosphate and diacylglycerol. Recently, our laboratory and others have isolated mutant T cells that lack surface expression of CD45, the major surface tyrosine phosphatase expressed on lymphoid cells. Stimulation of the TCR on these cells fails to result in the expected activation events. We demonstrate that reconstitution of surface expression of the 180-kDa isoform of CD45 by gene transfer into a CD45-deficient mutant of the Jurkat T cell leukemic line restores the ability of the TCR to couple fully to its signal transduction machinery. These results support the role of CD45 tyrosine phosphatase activity in regulating the TCR-activated PTK.     

The CD3 zeta subunit contains a phosphoinositide-binding motif that is required for the stable accumulation of TCR-CD3 complex at the immunological synapse

T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 ε subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3) with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell-APC interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation.     

Heterodimeric complex formation with CD8 and TCR by bispecific antibody sustains paracrine IL-2-dependent growth of CD3+ CD8+ T cells.

During physiologic activation of mature CD8+ T cells, TCR and CD8 bind to the same Ag-complexed MHC class I molecule. Thereby, close proximity is induced between CD8 and the TCR/CD3 complex. During this engagement, CD8 may deliver TCR-independent signals via its associated protein tyrosine kinase, p56lck. We studied the potential biologic effects of close association between CD8 and TCR/CD3 complexes by using a bispecific antibody (bsAb) directed against both TCR and CD8 molecules. This hybrid hybridoma (quadroma)-produced bsAb binds as a monomeric molecule to CD3+ CD8+ but not CD3+ CD4+ T cells. The bsAb proved capable of inducing the cytotoxic effector function of cloned CD3+ CD8+ T cells but not of CD3+ CD4+ T cells. When the bsAb was presented to resting T cells by monocytes, proliferation of the CD3+ CD4+ but not the CD3+ CD8+ subset of T lymphocytes was induced. Parental anti-TCR antibody induced vigorous growth of cells of both subsets. Essentially identical results were obtained when bsAb was presented in an immobilized fashion. The unresponsiveness of the CD3+ CD8+ T cells with respect to mitogenesis could be restored by exogenous rIL-2. The data suggest that bsAb-induced activation differs from activation by monospecific anti-TCR antibody. The former appears to more closely mimic physiologic Ag-induced signaling, because it leads to a similar paracrine IL-2-dependent growth pattern. The bsAb may, therefore, be instrumental in studying T cell signaling pathways, in particular the role of CD8-associated p56lck therein.