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. Thus CD28 signaling is a critical determinant of T cell activation and is an important factor in the development of both auto- and allo- immunity. While signal transduction through the T cell receptor has been extensively characterized, 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 had shown that this CD28 dependent regulation of IL-2 expression is not at the transcriptional or translational levels but rather a consequence of increased IL-2 mRNA stability. Our earlier work using sequence tagged genomic IL-2 reporter constructs 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. In addition, we discovered the presence of an additional mRNA instability element located within exon 3 and that sequences within exon 2 and the coding region of exon 4 are required for CD28 mediated IL-2 mRNA stabilization. Within these same regions of the IL-2 mRNA we have identified a sequence motif that may be critical for CD28 responsiveness. To test the role of this putative CD28 response element (RE) nearly a dozen mutants have been generated and stable cell lines carrying these mutations have been established. Unexpectedly, analyses of these constructs indicate that CD28 mediated stabilization of the IL-2 mRNA in the cytoplasm 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 CD28REs, we are actively pursuing this novel finding to determine how and why these two processes are coupled. The results of these studies will assist us in the identification of RNA binding proteins that interact with cytokine mRNAs in a CD28 specific fashion, thus providing us with a protein probe to help elucidate the CD28 signal transduction pathway. Biochemical studies carried out to identify proteins that bind the IL-2 mRNA initially 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 found no association between HuR binding to the IL-2 mRNA and its CD28-mediated stabilization. The controversial nature of our observation has prompted us to determine the biological significance of this finding. One hypothesis that is being pursued is that such binding is related to transport of the mRNA from the nucleus to the cytoplasm. This proposal is supported by our observation that HuR appears to translocate from the nucleus to the cytoplasm upon T cell activation. Along these lines we have generated several IL-2 deletions that would be postulated to abolish HuR binding and the phenotype of these mutants is being characterized. In addition to furthering our elucidation of the CD28 costimulatory pathway, these observations may have important consequences for our understanding of how IL-2 gene expression is regulated at both the transcriptional and post-transcriptional levels. The initial results of these studies have been submitted for publication. 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. We found that receptor blockade inhibits the expression of multiple cytokines (both Th1 and Th2) implicated in the pathogenesis of autoimmune disease and graft rejection. We have gone on to show that inhibition of IFN-gamma production occurs through both IL-12 dependent and IL-12 independent pathways. Furthermore, inhibition via the IL-12 dependent pathway is a consequence of blocking CD40L expression, which itself is critical for the induction of IL-12 from monocytes. Furthermore, our results reveal 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. 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. Since others have shown that blocking CD40L alone can induce long-term tolerance in a primate transplant model, it will be of critical importance to understand the pathways controlling the expression of this important determinant of immune tolerance. Recently, a great deal of scientific interest has focused on a subpopulation of CD4 T cells that are characterized in part by expressing CD25 on their surface. These resting cells, in contradistinction to activated CD25+ CD4 T cells, appear in the mouse to play an important role in the homeostasis of the immune system and in establishing tolerance. An analogous population has been defined in humans but their in vivo significance is unknown. We have a patient population that has been treated with a monoclonal antibody against CD25 for over 4 years, but contrary to what might have been predicted from the mouse experiments, these patients are healthy; showing no evidence of immune dysregulation. This patient population presents a unique opportunity to study the functional significance of these CD4/CD25+ regulatory cells in humans. We have begun to do so by further characterizing these cells in normal blood donors in an attempt to better define this population phenotypically. By using 4-color multiparameter flow cytometry, we?ve been able to enrich this population 20-fold. We are currently assessing whether this phenotypic population is also enriched for regulatory cell function.
