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.     

Regulatory role of CD4/L3T4 molecules in IL-2 production by affecting intracellular Ca2+ concentration of T cell clone stimulated with soluble anti-CD3

To elucidate the role of CD4 molecule in T cell activation, the effect of anti-CD4 on T cell IL-2 production was examined by using an alloreactive Th clone. The alloreactive T cell used in the present experiments produced IL-2 in response to soluble anti-CD3 epsilon-chain (anti-CD3) without accessory cell or insoluble antibody carrier. The IL-2 production was suppressed by the addition of anti-CD4 in cultures. An intracellular free Ca2+ concentration ([Ca2+]i) of the T cell clone was elevated by anti-CD3 stimulation, but the elevation was suppressed in the presence of anti-CD4. When the clone was stimulated in Ca2(+)-free medium, the elevation of [Ca2+]i was not observed. When Ca2+ influx was induced by calcium ionophore A23187 or ionomycin, the clone produced IL-2 in response to anti-CD3 in the presence of anti-CD4. When polyclonal T cell line or several other alloreactive T cell clones were examined for their anti-CD3 response, essentially the same results as mentioned above were obtained. Taken together, these results suggest that the slow and sustained elevation of [Ca2+]i is an essential signal for IL-2 production of T cells, and that anti-CD4 suppresses the IL-2 production by interfering the [Ca2+]i elevation. The significance of CD4 molecules in murine T cell activation was discussed.     

Cross-linking Fc receptors on monocytes triggers IL-6 production. Role in anti-CD3-induced T cell activation

IL-6 is a multifunctional cytokine which is produced by a variety of cells. Therefore it was examined whether anti-CD3-induced T cell activation was associated with the induction of functionally relevant IL-6 in human monocyte accessory cells. Significantly increased amounts of IL-6 were detected in supernatants of anti-CD3-treated PBMC. Stimulation of FACS-sorted greater than 98% pure monocyte accessory cells, but not of highly purified T cells with anti-CD3, resulted in an increased IL-6 production. Furthermore, anti-CD3 significantly enhanced IL-6 mRNA expression in monocyte accessory cells. IL-6 production was not limited to anti-CD3, inasmuch as equivalent IL-6 stimulation could be achieved with a mouse IgG2a isotype control antibody. In contrast to solid phase-bound mouse IgG2a, the soluble form of this antibody failed to induce IL-6 secretion indicating a requirement for Fc gamma RI receptor cross-linking. Moreover, this property may be specific for the Fc gamma RI receptor inasmuch as mouse IgG1 antibodies binding to the Fc gamma RII receptor did not significantly enhance IL-6 production. The role of IL-6 being an additional signal in T cell activation was confirmed by the finding that an anti-IL-6 antiserum was able to suppress anti-CD3-induced T cell activation. These data indicate that binding of anti-CD3 to Fc gamma RI may generate an activation signal towards the monocyte accessory cell leading to the production and secretion of monocyte IL-6, which in turn augments T cell activation, and also may be relevant to a variety of antibody-mediated immune responses against viral and bacterial infections.     

TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex

We show in this study that human T cells purified from peripheral blood, T cell clones, and Jurkat T cells release microvesicles in the culture medium. These microvesicles have a diameter of 50-100 nm, are delimited by a lipidic bilayer membrane, and bear TCR beta, CD3epsilon, and zeta. This microvesicle production is regulated because it is highly increased upon TCR activation, whereas another mitogenic signal, such as PMA and ionomycin, does not induce any release. T cell-derived microvesicles also contain the tetraspan protein CD63, suggesting that they originate from endocytic compartments. They contain adhesion molecules such as CD2 and LFA-1, MHC class I and class II, and the chemokine receptor CXCR4. These transmembrane proteins are selectively sorted in microvesicles because CD28 and CD45, which are highly expressed at the plasma membrane, are not found. The presence of phosphorylated zeta in these microvesicles suggests that the CD3/TCR found in the microvesicles come from the pool of complexes that have been activated. Proteins of the transduction machinery, tyrosine kinases of the Src family, and c-Cbl are also observed in the T cell-derived microvesicles. Our data demonstrate that T lymphocytes produce, upon TCR triggering, vesicles whose morphology and phenotype are reminiscent of vesicles of endocytic origin produced by many cell types and called exosomes. Although the exact content of T cell-derived exosomes remains to be determined, we suggest that the presence of TCR/CD3 at their surface makes them powerful vehicles to specifically deliver signals to cells bearing the right combination of peptide/MHC complexes.     

Cytokine-independent Jak3 activation upon T cell receptor (TCR) stimulation through direct association of Jak3 and the TCR complex

Jak3 is responsible for growth signals by various cytokines such as interleukin (IL)-2, IL-4, and IL-7 through association with the common gamma chain (gammac) in lymphocytes. We found that T cells from Jak3-deficient mice exhibit impairment of not only cytokine signaling but also early activation signals and that Jak3 is phosphorylated upon T cell receptor (TCR) stimulation. TCR-mediated phosphorylation of Jak3 is independent of IL-2 receptor/gammac but is dependent on Lck and ZAP-70. Jak3 was found to be assembled with the TCR complex, particularly through direct association with CD3zeta via its JH4 region, which is a different region from that for gammac association. These results suggest that Jak3 plays a role not only in cell growth but also in T cell activation and represents cross-talk of a signaling molecule between TCR and growth signals.     

Suppression of antibody synthesis by CD4+ T cell clones and normal T cells stimulated with monoclonal anti-CD3 antibody

CD4+ve Th1 clones, as well as normal splenic T cells, were found to suppress LPS-driven antibody secretion in a non-Ag-specific and non-MHC-restricted manner when the T cells were activated with the anti-CD3 mAb, 145-2C11. Suppression was observed with both primed and naive B cells, as well as with purified hapten-specific B cells, a result that suggests a direct effect of anti-CD3-activated T cells on B cell differentiation. Th1 clones activated by cognate Ag also suppressed LPS-driven antibody secretion. Furthermore, suppression of LPS-driven antibody secretion could be achieved across a cell-impermeable porous membrane when T cells were activated with anti-CD3. Suppression by Th1 clones and by normal T cells could not be attributed to a concomitant decrease in B cell proliferation or to a shift in the kinetics or isotype of the antibody response. These data demonstrate that CD4+ve Th1 clones, as well as normal T cells, can effect suppression of polyclonal antibody formation.     

Inhibitory effects of anti-CD2 monoclonal antibodies on interleukin 2 production and interleukin 2 receptor expression in anti-CD3-induced T cell activation

The effects of anti-CD2 monoclonal antibodies (mAb) on anti-CD3-driven interleukin 2 (IL2) production and IL2 receptor (IL2R) expression were investigated. Two anti-CD2 mAb, which had previously been shown to inhibit in vitro anti-CD3-induced T cell proliferation, also inhibited anti-CD3-induced IL2 production. However, it seemed unlikely that this was the crucial mechanism in the inhibition of anti-CD3-driven proliferation, since anti-CD2 mAb also partially inhibited T cell proliferation induced by the anti-CD3 mAb 446 which does not induce detectable IL2 levels. Anti-CD2 mAb also inhibited anti-CD3-induced surface IL2R expression as measured by immunofluorescence staining with an anti-IL2R mAb against the p55 chain. Inhibition of IL2R expression paralleled inhibition of proliferation. This anti-CD2-mediated inhibition involved a block in the generation of normal numbers of IL2R+ cells rather than a direct inhibitory effect on the IL2R+ cells themselves, since IL2R+ cells isolated from anti-CD2-containing cultures responded normally to IL2. Exogenous IL2 and IL4, singly or in combination, could reverse neither the anti-CD2-mediated inhibition of anti-CD3-induced proliferation nor the anti-CD2-mediated inhibition of anti-CD3-induced IL2R expression. Taken together, these observations suggest that anti-CD2 mAb inhibit anti-CD3-driven proliferation by inhibiting the generation of IL2R+ cells at a maturational stage proximal to their expression of surface IL2R. This inhibition cannot be overcome by exogenous IL2 or IL4, suggesting that the underlying biochemical mechanism involves an IL2- and IL4-independent pathway.     

Inhibition by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell-dependent B cell activation

Anti-CD3 mAb can activate T cells to help in B cell activation as detected by late events, such as maturation of B cells into Ig-secreting cells (IgSC), or by early events, such as B cell surface expression of the activation marker CD23. Two different anti-CD2 mAb each inhibited anti-CD3-induced T cell-dependent B cell activation in a dose-dependent fashion. Neither irradiation of the T cells prior to culture nor depletion of CD8+ cells abrogated the inhibitory effects of anti-CD2 mAb. Despite the ability of these anti-CD2 mAb to inhibit anti-CD3-induced IL2 production, addition of exogenous IL2 to anti-CD2 mAb-containing cultures could not fully reverse the inhibitory effects on IgSC generation. Furthermore, addition of various combinations of IL1, IL2, IL4, and IL6 or crude PBMC or monocyte culture supernatants also could not reverse anti-CD2-driven inhibition. In T cell-depleted cultures, anti-CD2 mAb had no effect on the ability of IL4 to induce B cell CD23 expression, confirming that anti-CD2 mAb had no direct effect on B cells. However, in cultures containing T+ non-T cells, anti-CD2 mAb did partially inhibit IL4-induced B cell CD23 expression. Taken together, these observations demonstrate that certain CD2 ligands can modulate T cell-dependent B cell activation by a mechanism which, at least in part, involves a direct effect by the CD2 ligand on the T cell itself.     

Differential in vitro activation of CD8-CD4+ and CD4-CD8+ T lymphocytes by combinations of anti-CD2 and anti-CD3 antibodies

Purified peripheral blood T lymphocytes and the CD8-CD4+ and CD4-CD8+ T cell subsets, exhaustively depleted of APC have been studied for their capacity to respond to mAb directed against the CD3-Ti Ag-specific TCR complex and against the CD2 SRBCR. It is demonstrated that high affinity IL-2R can be readily induced by either anti-CD3 and/or anti-CD2 stimulation. However, IL-2 production can be observed only in the CD4+CD8- T cell subset. These results clearly contrast data obtained with purified CD4-CD8+ T cells, which are able to proliferate when the CD3-Ti complex is activated in the presence of APC. The data presented in the present study demonstrate that a simplified model for T cell activation and clonal expansion of the two major T cell subsets involve only the CD3-Ti complex and the CD2 Ag. Under conditions where the activation signals for the T cells are restricted only to the activation of CD3-Ti and CD2, the CD4+CD8- T cells respond with IL-2 production and expression of high affinity IL-2R, whereas the CD4-CD8+ T cell subset depends on exogenous IL-2 provided by the CD4+CD8- cells. These data do not, however, exclude an involvement of other cell-surface signals for regulation and control of T cell activation and T cell effector functions.     

Essential role of NF-kappa B-inducing kinase in T cell activation through the TCR/CD3 pathway

NF-kappa B-inducing kinase (NIK) is involved in lymphoid organogenesis in mice through lymphotoxin-beta receptor signaling. To clarify the roles of NIK in T cell activation through TCR/CD3 and costimulation pathways, we have studied the function of T cells from aly mice, a strain with mutant NIK. NIK mutant T cells showed impaired proliferation and IL-2 production in response to anti-CD3 stimulation, and these effects were caused by impaired NF-kappa B activity in both mature and immature T cells; the impaired NF-kappa B activity in mature T cells was also associated with the failure of maintenance of activated NF-kappa B. In contrast, responses to costimulatory signals were largely retained in aly mice, suggesting that NIK is not uniquely coupled to the costimulatory pathways. When NIK mutant T cells were stimulated in the presence of a protein kinase C (PKC) inhibitor, proliferative responses were abrogated more severely than in control mice, suggesting that both NIK and PKC control T cell activation in a cooperative manner. We also demonstrated that NIK and PKC are involved in distinct NF-kappa B activation pathways downstream of TCR/CD3. These results suggest critical roles for NIK in setting the threshold for T cell activation, and partly account for the immunodeficiency in aly mice.     

B-Raf contributes to sustained extracellular signal-regulated kinase activation associated with interleukin-2 production stimulated through the T cell receptor

A T cell receptor (TCR) recognizes and responds to an antigenic peptide in the context of major histocompatibility complex-encoded molecules. This provokes T cells to produce interleukin-2 (IL-2) through extracellular signal-regulated kinase (ERK) activation. We investigated the roles of B-Raf in TCR-mediated IL-2 production coupled with ERK activation in the Jurkat human T cell line. We found that TCR cross-linking could induce up-regulation of both B-Raf and Raf-1 activities, but Raf-1 activity was decreased rapidly. On the other hand, TCR-stimulated kinase activity of B-Raf was sustained. Expression of a dominant-negative mutant of B-Raf abrogated sustained but not transient TCR-mediated MEK/ERK activation. The inhibition of sustained ERK activation by either expression of a dominant-negative B-Raf or treatment with a MEK inhibitor resulted in a decrease of the TCR-stimulated nuclear factor of activated T cells (NFAT) activity and IL-2 production. Collectively, our data provide the first direct evidence that B-Raf is a positive regulator of TCR-mediated sustained ERK activation, which is required for NFAT activation and the full production of IL-2.     

IL-7 maintains the T cell precursor potential of CD3-CD4-CD8- thymocytes.

We and other investigators have reported that IL-4 (in the presence of PMA) or IL-7 (used alone) induce proliferation of both adult and fetal (gestation day 15) CD4-CD8- thymocytes. These results suggested that these cytokines may be growth factors for pre-T cells. However, we recently observed that among adult CD4-CD8- thymocytes, only the CD3+ subset proliferates in response to IL-7, whereas IL-4 + PMA induces proliferative responses in both CD3- and CD3+ subsets. Thus, we concluded that IL-7 used alone is not a potent growth stimulus for adult thymic CD3-CD4-CD8- triple negative (TN) T cell precursors. Interestingly, the viability of adult TN thymocytes in culture was improved by IL-7 for up to 1 wk, in spite of the inability of IL-7 to induce significant [3H]TdR incorporation in these cells. After culture in IL-7 for 4 days, the viable cells remained CD4-CD8-, but 25 to 35% expressed CD3 whereas the rest remained CD3-. In contrast, most of the cells cultured with IL-4 + PMA for 4 days remained TN. To investigate whether adult TN thymocytes that survive in vitro in the presence of IL-4 + PMA or IL-7 retain T cell progenitor potential, we tested whether they could reconstitute lymphoid cell-depleted (2-deoxyguanosine-treated) fetal thymus organ cultures. Our results demonstrate that TN cells cultured in IL-7 retain T cell progenitor potential.     

sgp-60, a signal-transducing glycoprotein concerned with T cell activation through the T cell receptor/CD3 complex

T cell activation depends not only on the expression of a TCR, but also on that of accessory molecules that function in cell-cell adhesion and/or signal transduction. The subject of this report is the biochemical and functional characterization of what appears to be a novel murine lymphocyte cell surface antigen, provisionally termed sgp-60. Extensive, higher-order cross-linking of this glycoprotein with an anti-sgp-60 mAb and a second-step antibody reagent results in the activation of resting CD4+ T cells in the presence of a second signal. Monovalent or bivalent engagement of sgp-60 by the anti-sgp-60 antibody results in profound and direct inhibition of anti-CD3- or Con A-driven T cell activation, whereas alternative T cell activation via the phosphatidylinositol-linked proteins Thy-1 and TAP/Ly-6A is not affected. These findings raise the possibility that the sgp-60 molecule may be specifically involved in signal transduction through the TCR/CD3 complex and thus point to an important physiologic role for this protein in CD4+ T cells.     

Down-regulation of IL-2 production by activation of T cells through Ly-6A/E

Ly-6A/E molecules are expressed on the surface of T cells and have been shown to function in activation by the capacity of anti-Ly-6A/E mAb to induce T cell hybridomas or normal T cells to produce IL-2. Recent evidence suggests that activation through Ly-6A/E may be linked to the TCR signaling pathway. To further investigate the relationship between Ly-6- and TCR-induced T cell activation, we have examined whether an anti-Ly-6A/E mAb (D7) modulates TCR signaling in vitro. We now report that mAb D7 specifically inhibited IL-2 production by T cells also activated through TCR. Such inhibition was noted for normal T cells stimulated by soluble anti-CD3 or alloantigen and for T hybridomas stimulated by soluble anti-CD3. The ability of D7 to inhibit IL-2 production by T hybridomas was dependent on the nature of the TCR activating signal because IL-2 production was not inhibited when T hybridomas were stimulated with Ag or immobilized anti-CD3. Inhibition of IL-2 production by D7 apparently required cross-linking of the mAb because D7 F(ab')2 fragments were not effective for inhibition of IL-2 production. Similar to its ability to enhance anti-Ly-6A/E-induced activation of T and B cells, IFN-gamma enhanced the D7-induced inhibition of IL-2 production by alloantigen-activated normal T cells. These data further support the notion that Ly-6 and TCR signaling pathways are interrelated.     

Stimulation through the TCR/CD3 complex up-regulates the CD2 surface expression on human T lymphocytes.

Despite the well known interrelationship between the CD2- and CD3-mediated signal transduction pathways, it is not well established whether the CD2 surface expression can be regulated by triggering of TCR/CD3 complex. In this study we show that the stimulation of human PBMC with the Cris-7 (CD3) mAb, both in soluble and particulate form, results in hyperexpression of the CD2 surface Ag, as assessed by immunofluorescence and semi-quantitative immunoprecipitation assays. Similar effects on CD2 surface expression were obtained when different CD3 mAb (OKT3, RW2-8C8 and Leu-4) were tested. The CD3-mediated CD2 up-regulation was suppressed by cycloheximide and actinomycin D, indicating that it requires de novo protein and RNA synthesis. In agreement with this, increased CD2 RNA levels were observed after 3 h of stimulation, reaching a plateau at 24 h that was maintained for 72 h. The CD2 up-regulation was concomitant to other CD3-induced activation-related events such as induction of surface CD25 and CD71 and high RNA levels for c-myc, IL-2R alpha- and beta-chains, CD71, and IFN-gamma. CD2 up-regulation appeared to be elicited by a protein kinase C-dependent mechanism because it was abrogated by staurosporine, a potent protein kinase C inhibitor. Moreover, IL-2-dependent events may also help in enhancing CD2 hyper-expression because it was only partially inhibitable by cyclosporine, dexamethasone, or Mar-108 (CD25) mAb. In conclusion, our data suggest that CD2 up-regulation can be a relevant event in T cell activation triggered by the physiologic engagement of the TCR/CD3 complex.     

Interleukin-1-independent activation of human T lymphocytes stimulated by anti-CD3 and a Hodgkin's disease cell line with accessory cell activity

Antibodies directed against the human T cell receptor or the closely associated CD3 molecule stimulate polyclonal T cell proliferation via mechanisms that mimic a primary immune response. We have investigated the requirement for IL-1 production in anti-CD3 (OKT3)-mediated mitogenesis using a Hodgkin's disease cell line (L428) as the accessory cell. L428 cells did not produce detectable IL-1 following stimulation with lipopolysaccharide or phorbol ester (PMA), nor did they transcribe detectable levels of mRNA for IL-1 alpha or beta after such treatment. Despite their inability to produce IL-1, as few as 1 X 10(4) L428 cells reconstituted the proliferative response of accessory cell-depleted T cells to anti-CD3. Although larger numbers of non-rosette-forming (E-) cells were required for maximal responsiveness to anti-CD3, the maximal degree of proliferation was higher with E- cells than with L428 cells. L428-mediated T cell proliferation did not result from residual accessory cells in the responding population or an allogeneic effect since L428 cells were also capable of providing accessory cell activity for the anti-CD3-dependent generation of IL-2 by the Jurkat T cell line. Although the mechanism by which L428 cells provide accessory functions remains incompletely characterized, the ability of anti-HLA-DR F(ab')2 fragments to completely abrogate L428 and monocyte-mediated anti-CD3 mitogenesis, despite the addition of exogenous IL-1, provides evidence for the participation HLA-DR molecules in this response. These data indicate that anti-CD3-induced proliferation of unprimed human T lymphocytes can occur independently of IL-1 production by accessory cells and may involve the participation of HLA-DR molecules.     

Differential immunomodulation by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell activation: dependence upon the individual anti-CD3 monoclonal antibody used for activation

Two monoclonal antibodies (mAb) recognizing different CD2 epitopes each inhibited anti-CD3-induced proliferation and anti-CD3-induced increase in surface CD2 expression. The magnitude of inhibition by either anti-CD2 mAb was dependent upon which anti-CD3 mAb was used as the stimulus, being more pronounced when the anti-CD3 mAb 454 was used as the stimulus than when either anti-CD3 mAb 147 or 446 was the stimulus. The effects of neuraminidase-treated sheep erythrocytes (which bind to CD2) were also more pronounced on mAb 454-induced proliferation than on mAb 147- or 446-induced proliferation. Furthermore, the effects of preincubation with anti-CD2 mAb depended upon the responder status of the donor to IgG1 anti-CD3 mAb. Preincubation of high-responder cells with anti-CD2 mAb had little effect on subsequent IgG1 anti-CD3-induced proliferation. In contrast, preincubation of low-responder cells with anti-CD2 mAb usually augmented the otherwise small proliferative response to IgG1 anti-CD3 mAb. Taken together, these observations suggest that interaction of surface CD2 with ligand alters the response of T cells to anti-CD3 mAb, but these effects depend upon the individual anti-CD3 mAb used for stimulation. These studies raise the possibility that perturbation of different parts of the CD3-T cell antigen receptor complex may lead to different sequelae, and, as a result, the T cell may respond to a given immunomodulator in different ways.     

Human TIM-1 associates with the TCR complex and up-regulates T cell activation signals

The T cell, Ig domain, and mucin domain-1 (TIM-1) gene is associated with Th2 T cell responses and human atopic diseases. The mechanism by which TIM-1 influences T cell responses remains unknown. We demonstrate that TIM-1 is recruited to the TCR-signaling complex via association with CD3. TIM-1 up-regulates TCR-associated signaling events, including phosphorylation of Zap70 and IL-2-inducible T cell kinase. This activity requires TIM-1 tyrosine phosphorylation. TIM-1 expression induces formation of a novel complex that includes PI3K and ITK. Finally, the consequences of TIM-1 activation include increased expression of effector cytokines. These results demonstrate that TIM-1 is a critical component of the human T cell response and provide a mechanistic hypothesis for the role of TIM-1 in disease.     

IL-10 inhibits human T cell proliferation and IL-2 production.

Human IL-10 has been reported previously to inhibit the secretion of IFN-gamma in PBMC. In this study, we have found that human IL-10 inhibits T cell proliferation to either mitogen or anti-CD3 mAb in the presence of accessory cells. Inhibited T cell growth by IL-10 was associated with reduced production of IFN-gamma and IL-2. Studies of T cell subset inhibition by human IL-10 showed that CD4+, CD8+, CD45RA high, and CD45RA low cells are all growth inhibited to a similar degree. Dose response experiments demonstrated that IL-10 inhibits secretion of IFN-gamma more readily than T cell proliferation to mitogen. In addition, IL-2 and IL-4 added exogenously to IL-10 suppressed T cell cultures reversed completely the inhibition of T cell proliferation, but had little or no effect on inhibition of IFN-gamma production. Thus, in addition to its previously reported biologic properties, IL-10 inhibits human T cell proliferation and IL-2 production in response to mitogen. Inhibition of IFN-gamma production by IL-10 appears to be independent of the cytokine effect of IL-2 production.