Mef2 proteins, required for muscle differentiation, bind an essential site in the Ig lambda enhancer.

The Ig lambda light chain gene enhancer has two unique essential motifs, lambdaA and lambdaB. The transcription factors that bind the AB motif have been identified as Pu.1 and Pu.1-interacting partner (Pip). We report here that the lambdaA site includes a binding site for the myocyte-specific enhancer factor 2 (Mef2) family of transcription factors. Mef2 proteins were first described in muscle cells and, in vertebrates, include four known members designated A to D. Using a AA electrophoretic-mobility shift assay (EMSA), in conjunction with a high affinity Mef2 binding site and anti-Mef2 Abs, we show that members of the Mef2 family are present in nuclear extracts of lambda-producing B cells and bind the AA site. Functional assays using the chloramphenicol acetyltransferase (CAT) reporter construct containing three copies of the lambdaA motif demonstrate that the AA sequence can function as an enhancer in conjunction with the thymidine kinase (TK) promoter and is regulated by Mef2 proteins. Extrapolating from other systems where transcriptional regulation by Mef2 has been studied, other transcription factors may be involved along with Mef2 in transcriptional regulation at the lambdaA site.

Differential regulation of T cell activation for primary versus secondary proliferative responses.

T cell activation in vivo results in proliferation and generation of effector cytokine-secreting cells, as also in development of memory cells that mount enhanced responses upon restimulation. However, differences in the signals promoting generation of effector vs memory T cells are not yet characterized. In this study, using various strategies to modulate an allorecognition system for priming human T cells in vitro, we show that there are indeed differences between the signaling requirements for a first proliferative response and those for priming T cells for enhanced recall proliferative responses. Using APCs fixed with varying concentrations of paraformaldehyde, we show that the loss of ability of these APCs to generate a first response is not matched by a similar loss in their ability to prime responder T cells for recall responses. Prevention of DNA replication during T cell priming with aphidicolin, a DNA polymerase inhibitor, is not inimical to successful T cell priming. Thus, clonal expansion during priming is less crucial than the primed activation status of T cells for the enhanced recall response. We also show that pentoxifylline, a phosphodiesterase inhibitor, inhibits the primary proliferative response, but its presence during priming enhances the recall response capabilities of T cells. On the other hand, the presence of the calcineurin inhibitor cyclosporin A during priming reduces the efficiency of priming, but at low concentrations it induces, like pentoxifylline, enhancement in recall response capability. These findings have significant implications in designing immunosuppressive therapy and in the analysis of signals for T cell memory commitment.

Induction of tolerance in freshly isolated alloreactive T cells by activated T cell stimulators is not due to the absence of CD28-B7 interaction.

Human T cells express MHC class II molecules on activation, and this makes them potential APCs for responder CD4 T cells. We have shown earlier that MHC class II-expressing human T cell APCs induce specific tolerance in freshly isolated alloreactive responder CD4 T cells. In this study, we show that this induction of tolerance does not depend on the expression of a specific coreceptor on the stimulator T cell APCs, because both CD4 and CD8 T cell stimulators efficiently induce tolerance. Such a tolerant cell population responds significantly better to IL-2 than unprimed cells, indicating the physical presence of T cells expressing higher levels of IL-2 receptors. The addition of exogenous IL-2 during priming with activated T cell APCs effectively blocks the induction of tolerance and permits successful priming of alloreactive responder cells. We have investigated the possible role of the CD28/CTLA4-B7 family interaction in this model of induction of T cell anergy in T-T interactions. The freshly activated T cell APCs used in this study express CTLA4-binding ligand(s). Furthermore, cross-linking CD28 on responder T cells does not enable activated T cell APCs to elicit a primary proliferative alloresponse, nor does it prevent the induction of tolerance in responder T cells. The CD28/CTLA4-B7 family interaction is thus unlikely to be involved in the induction of T cell anergy by T cell APCs.

Induction of tolerance in freshly isolated alloreactive CD4+ T cells by activated T cell stimulators.

Activated human T cells express major histocompatibility complex class II proteins, and their potential to present antigens to T cell clones has been documented extensively. The effect of such TT presentation on responder T cell clones has been shown to be the induction of tolerance, sometimes accompanied by activation. To investigate whether freshly isolated responder T cells are also susceptible to such induction of tolerance by activated T cells functioning as antigen-presenting cells (APC), we have used the capability of unprimed ex vivo T cells to respond in a proliferation assay in vitro to alloligands on professional APC. We show that purified human T cells ex vivo, when exposed to alloligand on activated T cells for primary allorecognition in vitro, fail to mount a proliferative response. Priming of responder CD4+ T cells with alloligand expressed on activated T cells results in the induction of nonresponsiveness to a subsequent challenge by competent allo-APC. This ability of activated, HLA-DR+ T cells to induce nonresponsiveness to subsequent challenge in bulk CD4+ T cell populations ex vivo has interesting implications for infections involving T cells such as human immunodeficiency virus.