I. Induction of Clinical Immunotolerance. To date, autoimmune disease and transplant graft rejection have been managed using a barrage of immunosuppressive drugs. These medications often require life-long administration and have a plethora of serious side effects. With few exceptions, these same drugs block the induction of immune tolerance, a likely prerequisite for long-term graft acceptance in the absence of continued immunosuppression or for an autoimmune disease cure. We are currently completing a Phase I/II Clinical Trial to induce immune tolerance. Study 04-EI-0115 has now been underway for 2 years. In this study we utilize two pharmacological agents; daclizumab, a monoclonal antibody against the interluekin-2 receptor that can control autoimmune uveitis but does not appear to block the induction of tolerance, and sirolimus (rapamycin), a drug that can induce experimental immune tolerance. To date three subjects have completed the study. Two reached the primary study endpoint and the third had a partial response. Two additional subjects are approaching the primary study endpoint.? ? One important challenge in these clinical studies is to develop tools that will allow us to detect and monitor the fate of autoreactive T cells that have become tolerized. Studies of host protein incorporation into HIV might lend insight into the development of such tools.? ? II. Molecular Consequences of IL-2 Receptor Blockade. Laboratory investigations to understand the mechanism(s) by which blockade of the high affinity IL-2 receptor (a therapeutic modality in transplantation, allergic, and autoimmune disease) inhibits immune activation have demonstrated for the first time that both IFN-gamma production and CD40L expression are biphasic and that the latter, but not the initial phase of expression, is highly dependent on IL-2R signaling. Weve found that nave and memory CD4 T cells exhibit biphasic CD40L expression and in both, the late phase is CD28-dependent and inhibited by daclizumab independently of cell division. In contrast to mouse, human late phase CD40L is a consequence of CD28 signaling and IL-2, not the principal Th1/Th2 polarizing cytokines. This fundamental difference between man and mouse in the regulation of CD40L has profound implications for mouse models of B cell maturation, transplant tolerance, allergy and autoimmune disease. Our findings also have important implications for the choice of immunosuppressive regimen (e.g. anti- IL-2R vs. anti- IL-12) employed in the setting of transplantation or autoimmune disease. Others have shown that blocking CD40L alone can induce long-term tolerance in a primate transplant model, which has never been accomplished by any other means. Unfortunately, CD40L blocking antibodies had serious adverse effects when used in human trials. Our observations suggest IL-2R blockade could represent one component of an alternative strategy to anti-CD40L immunotherapy for the induction of immune tolerance. Collectively, our results also indicate that IL-2 has a broader immunologic role than the expansion and maintenance of CD25+Tregs. In contrast to late CD40L expression, early expression is completely independent of cytokines. However, weve discovered that early CD40L expression, like late expression, is dependent on the presence of antigen presenting cells (APC), but through different and unknown cell surface receptors. The CD40L costimulatory activity of APC is dependent upon cell-cell contact with monocytes and does not require monocyte activation. Our current efforts are focused on identifying the unknown surface ligand on APC that augments the induction of early CD40L expression and the molecular mechanisms underlying its biphasic expression.? ? III. CD28 Signaling. Signaling through the CD28 receptor during T cell activation exerts a profound influence on the outcome of T cell receptor (TCR) engagement. Failure to receive a costimulatory signal through CD28 results in an unresponsive state termed anergy or in T cell death; both of which contribute to the induction and maintenance of immune tolerance. The CD28 signaling pathway is poorly understood. To decipher this pathway, we've utilized a mouse model system in which CD28 signaling is responsible for greater than 99% of T cell IL-2 production. Earlier work has shown that this CD28 dependent upregulation of IL-2 is a consequence of increased IL-2 mRNA stability. Biochemical studies carried out to identify proteins that bind the IL-2 mRNA have focused on the 3'UTR of the mRNA, which contains a cis-dominant instability element. We have identified a half-dozen RNA binding proteins that specifically interact with distinct sequence elements within the 3UTR. One such protein is HuR, the mammalian homolog of the Drosophila ELAV (embryonic lethal abnormal vision) gene. It is widely held that HuR binding stabilizes labile mRNAs such as c-myc and IL-3, however, we concluded that HuR binding to the IL-2 mRNA is not sufficient for CD28-mediated stabilization. The controversial nature of this observation prompted us to generate an IL-2 deletion mutant expected not to bind HuR. Characterization of this mutant reveals a partial defect in CD28-mediated stabilization, indicating that HuR, if not sufficient, may be necessary for IL-2 mRNA stabilization. However, we subsequently identified additional HuR binding sites downstream of the one we initially characterized and deleted. This discovery both complicates our interpretation of the mutant phenotype and raises the possibility that HuR binding to the IL-2 mRNA is in part regulated by differential polyadenylation as these newly identified HuR binding sites that are located downstream of the 5 most polyadenylation signal (the IL-2 transcript has 3 potential polyadenylation signals). We are currently assessing the role of these additional HuR binding sites and working to characterize the binding and function of the other IL-2 mRNA binding proteins weve identified.
