A single 3' alpha hs1,2 enhancer in the rabbit IgH locus

Multiple cis-acting elements including the intronic enhancer and the 3'alpha enhancer (3'alphaE) regulate expression of the Ig heavy chain genes during B cell development. A 3'alphaE is composed of DNase I-hypersensitive sites, hs1,2, hs3a,b, and hs4, found 3' of the murine Calpha gene as well as 3' of both human Calpha genes, Calpha1 and Calpha2. Rabbits have 13 Calpha genes, and we tested whether a 3'alphaE is associated with each of these genes. To identify 3'alphaE regions we developed a rabbit hs1,2 probe and used this to search for enhancer homologues of human hs1,2 in a genomic fosmid library. We identified a single hs1,2 fragment 8-kb downstream of Calpha13, the presumed 3'-most Calpha gene. We also identified and partially sequenced a new Calpha gene, Calpha14, located 6 kb upstream of Calpha13. Genomic Southern blot analysis confirmed that the rabbit genome contains only one hs1,2 enhancer region. We tested the enhancer activity of the hs1,2 with the SV40, V(H), and Ialpha promoters using the luciferase reporter gene in transient transfection assays and found that it significantly enhanced the activity of SV40 and V(H) promoters and slightly enhanced an Ialpha promoter. We conclude that the rabbit has a single hs1,2 enhancer that resides at the 3' end of the IgH gene cluster and may constitute one of the cis-elements regulating the expression of IgH genes.     

Interactions between CD3 and Thy1 T cell activation pathways: blockade of CD3-mediated T lymphocyte activation induced by immobilized anti-Thy1 antibodies.

Resting murine T cell activation induced by either CD3 complexes or Thy1 molecules was investigated in vitro, using surface-bound anti-CD3 mAb as the stimulus. One mitogenic anti-Thy 1 mAb (G7) lost mitogenicity when presented to T cells immobilized on a plastic surface, even in the presence of phorbol ester. Moreover, T cell activation induced by immobilized anti-CD3 was potently blocked by coimmobilized anti-Thy 1 mAb. Nonmitogenic anti-Thy 1 mAb also blocked CD3-induced activation when coimmobilized with anti-CD3. Control experiments showed that anti-Thy 1 specifically blocked T cell activation, even in the presence of measurable and functional concentrations of plastic-bound anti-CD3. Coimmobilized anti-Thy 1 potently blocked IL2 secretion stimulated by anti-CD3. Addition of exogenous rIL2 completely prevented anti-Thy 1-mediated blockade. On the other hand, while completely blocking T cell proliferation, immobilized anti-Thy 1 only partially blocked secretion of IL3-like activity by the T cells. One IgM anti-Thy 1 mAb (2A3) induced secretion of IL3-like activity by T cells when immobilized in the absence of bound anti-CD3. These results indicate that extensive aggregation of Thy 1 molecules delivers a potent negative signal which antagonizes CD3-mediated T cell activation and growth.     

Herpesvirus saimiri replaces ZAP-70 for CD3- and CD2-mediated T cell activation.

The protein tyrosine kinase ZAP-70 plays a pivotal role involved in signal transduction through the T cell receptor and CD2. Defects in ZAP-70 result in severe combined immunodeficiency. We report that Herpesvirus saimiri, which does not code for a ZAP-70 homologue, can replace this tyrosine kinase. H. saimiri is an oncogenic virus that transforms human T cells to stable growth based on mutual CD2-mediated activation. Although CD2-mediated proliferation of ZAP-70-deficient uninfected T cells was absent, we could establish H. saimiri-transformed T cell lines from two unrelated patients presenting with ZAP-70 deficiencies. In these cell lines, CD2 and CD3 activation were restored in terms of [Ca(2+)](i), MAPK activation, cytokine production, and proliferation. Activation-induced tyrosine phosphorylation of zeta remained defective. The transformed cells expressed very high levels of the ZAP-70-related kinase Syk. This increased expression was not observed in the primary T cells from the patients and was not due to the transformation by the virus because transformed cell lines established from control T cells did not present this particularity. In conclusion, wild type H. saimiri can restore CD2- and CD3-mediated activation in signaling-deficient human T cells. It extends our understanding of interactions between the oncogenic H. saimiri and the infected host cells.     

CD40-mediated activation of vascular smooth muscle cell chemokine production through a Src-initiated, MAPK-dependent pathway

The interaction between CD40 ligand (CD154) expressed on activated T cells and its receptor, CD40, has been shown to play a role in the onset and maintenance of autoimmune inflammation. Recent studies suggest that CD154+T cells also contribute to the regulation of atherogenesis due to their capacity to activate CD40+cells of the vasculature, including vascular smooth muscle cells (VSMC). The present study evaluated the signalling events initiated through CD40 ligation which culminate in VSMC chemokine production. CD40 ligation resulted in the phosphorylation/activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38, but not c-jun N-terminal kinase. Inhibition of both ERK1/2 and p38 activity abrogated CD40 stimulation of IL-8 and MCP-1 production. CD40-mediated induction of chemokines also showed dependence on the Src family kinase activity. The Src kinase inhibitor, PP2, was found to inhibit CD40-induced phosphorylation of ERK1/2 as well as activation of IkappaB kinase. An evaluation of Src kinases that may be important in CD40 signalling identified Lyn as a potential candidate. These data indicate that CD40 signalling in VSMC activates a Src family kinase-initiated pathway that results in the induction of MAPK activities required for successful induction of chemokine synthesis.     

IL-6 is an accessory signal in the alternative CD2-mediated pathway of T cell activation

Recent studies have demonstrated that IL-1 and IL-6 are synergistic accessory signals for activation of T cells. In this study, highly purified human T cells were cultured with either a stimulating pair of anti-CD2 mAb or with immobilized anti-CD3 mAb. Monocytes, a cellfree monocyte culture supernatant or IL-1 were required for anti-CD2-stimulated T cell proliferation, and they each strongly enhanced anti-CD3-induced T cell growth. IL-6 was synergistic with IL-1 as a helper factor for T cell growth after activation via CD2, but we could not demonstrate any effect of IL-6 in the CD3 pathway. The mechanism of the synergistic helper activity of IL-1 and IL-6 on T cell activation in the CD2 pathway was further examined. IL-1 (but not IL-6) was required for induction of IL-2 production. Both IL-1 and IL-6 enhanced IL-2R (p55) expression and the proliferative response to IL-2. T cell proliferation after stimulation with anti-CD2 and IL-1 or IL-1/IL-6 proceeded through an autocrine IL-2-dependent pathway. Moreover we found that, in the absence of IL-1, IL-6 still supported a transient and limited proliferation of anti-CD2- (but not of anti-CD3-) stimulated T cells, which apparently was independent of the autocrine growth factors IL-2 or IL-4. Our data suggest that IL-6 is important as an accessory signal for T cell growth in the CD2 pathway of T cell activation.     

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

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

Regulation of the CD2 alternate pathway of T cell activation by CD3. Evidence for heterologous desensitization

Normal resting T cells were stimulated through the alternate CD2 pathway. A CD3 mAb VIT3 completely blocked their proliferative response. The time interval for 50% inhibition lasted for 24 h after the onset of CD2 stimulation. Mitogen-activated cloned long term cultured T cells could also be stimulated via CD2. This proliferative response was again inhibitable by VIT3, indicating that CD3 regulates the CD2 pathway not only in resting cells, but also in lymphocytes actively involved in an Ir. T cells were further loaded with Quin2 and their free cytoplasmic Ca2+ levels were monitored in response to CD3 and CD2 stimulation. Antibodies directed against both surface R triggered a rapid elevation of Ca2+ levels. Both responses were abrogated when the cells had been treated overnight with VIT3. The free cytoplasmic Ca2+ levels of VIT3-pretreated cells, however, were not higher than those of control cells. These results point to a functional interaction between CD3 and CD2 possibly at the level of signal transducing proteins. Finally, cholera toxin was found to inhibit the Ca2+ response in Jurkat T cells. Both the CD3 and CD2 stimulation were sensitive to cholera toxin, indicating that a GTP-binding protein may be involved in signal transduction for both surface structures.     

Relevance of CD6-mediated interactions in T cell activation and proliferation

CD6 is a cell surface receptor expressed on immature thymocytes and mature T and B1a lymphocytes. The ultimate function of CD6 has not been deciphered yet, but much evidence supports a role for CD6 in T cell activation and differentiation. In this study, we show that a fraction of CD6 molecules physically associates with the TCR/CD3 complex by coimmunoprecipitation, cocapping, and fluorescence resonance energy transfer experiments. Image analysis of Ag-specific T-APC conjugates demonstrated that CD6 and its ligand, activated leukocyte cell adhesion molecule (CD166), colocalize with TCR/CD3 at the center of the immunological synapse, the so-called central supramolecular activation cluster. The addition of a soluble rCD6 form significantly reduced the number of mature Ag-specific T-APC conjugates, indicating that CD6 mediates early cell-cell interactions needed for immunological synapse maturation to proceed. This was in agreement with the dose-dependent inhibition of CD3-mediated T cell proliferation induced by soluble rCD6. Taken together, our data illustrate the important role played by the intra- and intercellular molecular interactions mediated by CD6 during T cell activation and proliferation processes.     

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.     

Enhancement of CD2-mediated T cell activation by the interaction of VLA-4 with fibronectin.

Human fibroblasts were shown to enhance the proliferation of peripheral T cells in the presence of suboptimal concentrations of anti-CD2 antibodies (anti-T112 and anti-T113) and agonistic anti-VLA-4 antibody. Evidence is provided that the interaction of VLA-4 with immobilized fibronectin can enhance the proliferation of T cells subjected to suboptimal stimulation via CD2. Our results suggest that the fibroblast-stimulated T cell proliferative response to low doses of anti-T112 and T113 antibodies is due to the interaction of VLA-4 with fibroblast fibronectin. These findings also suggest a role for the fibronectin/VLA-4 interaction in the inflammatory process.     

Kinetics of anti-CD4-induced T helper cell depletion and inhibition of function. Activation of T cells by the CD3 pathway inhibits anti-CD4-mediated T cell elimination and down-regulation of cell surface CD4.

In vivo treatment with anti-CD4 antibody profoundly suppresses a number of T cell-dependent responses and is clinically useful in the treatment of certain mouse models of autoimmune disease. Treatment with anti-CD4 antibody will inactivate and can deplete CD4 T cells, but the mechanisms responsible for these effects are incompletely understood. When mouse spleen cells were exposed in vitro to both SRBC and monoclonal anti-CD4, there was 55% reduction of the anti-SRBC response. If cultures were preincubated with anti-CD4 for 48 h before in vitro challenge, the reduction was greater than 80%. When unfractionated spleen cells were cultured with anti-CD4 for 96 h, there was actual elimination of CD4 cells in these cultures since virtually all CD3+ cells were CD8+. Activation of T cells by exposure to anti-CD3 rendered them resistant to antibody-mediated CD4 depletion. This resistance to CD4 depletion was seen even in cultures that were pretreated with anti-CD4 for as long as 24 h before anti-CD3 exposure. In cultures of purified T cells, anti-CD4 did not eliminate CD4 T cells. However, culture of T cells with macrophage-rich adherent cells and anti-CD4 resulted in elimination of CD4 T cells. Thus, it appears that macrophages play a role in anti-CD4-induced T cell elimination. While anti-CD4 did not eliminate CD4 cells from a population of purified T cells, there was profound down-regulation of cell surface CD4. Activating T cells with immobilized anti-CD3 before addition of anti-CD4 prevented down-regulation of CD4. These experiments demonstrate that T cell activation by anti-CD3 renders the activated cells resistant to antibody-induced CD4 down-regulation and to antibody-induced CD4 T cell depletion. These findings may have relevance to the application of anti-CD4 therapy in human diseases that are mediated by activated Th cells.     

Effect of T3 modulation on pokeweed mitogen-induced T cell activation: evidence for an alternative pathway of T cell activation

Modulation of the T3 molecule on human T cells with monoclonal anti-T3 antibodies has been shown to result in the disappearance of the T3-Ti complex from the membrane and to preclude subsequent T cell activation by various mitogenic and antigenic stimuli. We have examined the effect of T3 modulation on pokeweed mitogen (PWM)-induced T cell activation. T3 modulation was accomplished by incubating peripheral blood mononuclear cells (PBMC) or mixtures of T cells and non-T cells at 37 degrees C for 18 hr in the presence of UCHT-1, a mouse IgG1 anti-T3 monoclonal antibody. Only donors whose PBMC were unresponsive to the mitogenic activity of this antibody were selected. Although T3 modulation resulted in complete to substantial inhibition of T cell proliferation induced by low PWM concentrations of 5 or 50 ng/ml, it had no effect on T cell proliferation when PWM was added at a concentration of 0.5 and 5 micrograms/ml. The results demonstrate that the higher doses of PWM can induce T cell proliferation via an alternative pathway that does not involve participation of the T3-Ti complex. In contrast, irrespective of the PWM dose added, T3 modulation almost totally inhibited PWM-induced interleukin 2 (IL 2) production. The differential effect of T3 modulation on IL 2 production and on T cell proliferation induced by high doses of PWM suggests that this alternative pathway of T cell proliferation is IL 2 independent. This suggestion was additionally substantiated by the lack of effect of anti-Tac, and anti-IL 2 receptor antibody, on PWM-induced proliferation of T3-modulated T cells. In conclusion our data demonstrate that high doses of PWM can induce T cells to proliferate via an alternative pathway that does not involve perturbation of the T3-Ti complex.     

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.     

The roles of CD28 and CD40 ligand in T cell activation and tolerance

Costimulation of T cell activation involves both the B7:CD28 as well as the CD40 ligand (CD40L):CD40 pathway. To determine the importance of these pathways to in vitro and in vivo T cell activation, a direct comparison was made of the responses of TCR transgenic T cells lacking either CD28 or CD40L. In vitro, CD28-/- T cells showed a greater reduction in proliferative responses to Ag than did CD40L-/- T cells. The absence of CD28 resulted in defective Th2 responses, whereas CD40L-/- T cells were defective in Th1 development. In vivo, CD28-/- T cells failed to expand upon immunization, whereas CD40L-/- T cells could not sustain a response. These results suggest that CD28 is critical for initiating T cell responses, whereas CD40L is required for sustained Th1 responses. The different functional roles of these costimulatory pathways may explain why blocking B7:CD28 and CD40L:CD40 interactions has an additive effect in inhibiting T cell responses.