The mission of the Clinical and Molecular Immunology Group within the Clinical Immunology Section is to perform cellular, molecular and clinical studies aimed at understanding the basis of immune tolerance. A long-term objective is to develop new therapies for allograft transplantation and the treatment of autoimmune and inflammatory diseases. I. 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. We?ve demonstrated that sequences within the 3' untranslated region of the mouse IL-2 mRNA are responsible for mRNA instability but cannot confer CD28 responsiveness upon a heterologous reporter mRNA. Additional mRNA stability elements located in exon 2 and the coding region of exon 4 are required for CD28 mediated IL-2 mRNA stabilization. Reporter constructs designed to test the role of these sequences in CD28- mediated stabilization unexpectedly revealed that IL-2 mRNA stability appears to be coupled to splicing of the pre-mRNA in the nucleus. While this finding has complicated our studies on the role of the exonic sequences, we plan to pursue this novel finding to determine how and why these two processes are coupled. Biochemical studies carried out to identify proteins that bind the IL-2 mRNA have focused on the 3'UTR of the mRNA. We identified one such protein to be 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 its? CD28-mediated stabilization. This work has been published in the Journal of Biological Chemistry. The controversial nature of this observation prompted us to generate IL-2 deletion mutants expected not to bind HuR. Characterization of this mutant is incomplete, but it exhibits a partial defect in CD28-mediated stabilization indicating that HuR, if not sufficient, may be necessary for IL-2 mRNA stabilization. Bioinformatic analysis of the HuR binding site in the IL-2 mRNA reveals several potential stem-loop structures in the region and we?ve identified a second, unknown 50kD protein that binds the same sequence. We are currently assessing the potential role of these stem-loop structures in HuR binding and working to identify and determine the function of the 50kD IL-2 mRNA binding protein. II. Molecular Consequences of IL-2 Receptor Blockade Laboratory investigations to understand the mechanism(s) by which blockade of CD25, the high affinity IL-2 receptor (a therapeutic modality in transplantation, allergic, and autoimmune disease) inhibits immune activation have completed their initial phase of study. Our results revealed 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 of IL-2R signaling. These findings 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. These observations are being extended to understand at the molecular level the activation pathways involved in CD40L expression. Others have shown that blocking CD40L alone can induce long-term tolerance in a primate transplant model. As this has never been accomplished by blocking any other molecule, it will be of critical importance to understand the pathways controlling the expression of this important determinant of immune tolerance. We expected early expression to occur on memory CD4 cells and late expression to be on naive cells. Though CD40L expression on resting cells is restricted to the CD4 memory population, early expression on activated cells occurred proportionately on naive and memory T cells while late expression was predominately on naive cells. Early expression, like late expression, is dependent on cell-cell contact with monocytes, but through different cell surface receptors. In contrast, early expression is independent of cytokines while late expression is wholly dependent on IL-2 and partially dependent on INF-gamma. Furthermore, the late phase can be completely restored in cells that have not been CD28 costimulated by exogenous IL-2. These results have been submitted for publication. Our efforts are currently focused on identifying the unknown surface ligand on monocytes that is necessary for the induction of early CD40L expression. III. Induction of Clinical Immunotolerance in Uveitis We?ve initiated an IRB approved Phase I/II Clinical Study to Evaluate the Induction of Immune Tolerance in Patients with Sight Threatening Autoimmune Uveitis (04-EI-0115). To date, autoimmune disease and transplant graft rejection has 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. The observation that immunosuppressive monotherapy with daclizumab, a monoclonal antibody against the interluekin-2 receptor, can control autoimmune uveitis but does not appear to block the induction of tolerance, presents us with a unique opportunity to clinically test the hypothesis that sirolimus therapy can result in immune tolerance. Sirolimus is a macrocyclic lactone produced by Streptomyces hygroscopicus that inhibits T lymphocyte activation and proliferation in response to both antigenic and cytokine stimulation by a mechanism that is distinct from that of other immuno-suppressants. In cells, sirolimus binds to the immunophilin, FKBP-12, to generate an immunosuppressive complex. This complex binds to and inhibits the activation of mTOR, a key regulatory kinase. This inhibition suppresses cytokine-driven T-cell proliferation, inhibiting the progression from the G 1 to the S phase of the cell cycle, which is believed to responsible for tolerance induction. The study is currently 60% enrolled.
