Umbilical cord blood (UCB) stem cells have emerged as novel treatment for patients requiring allogeneic grafting. The incidence and severity of graft-versus-host disease (GVHD) after UCB transplantation in these early clinical trials compares favorably to that observed in recipients of matched unrelated donors (MUD) or partially mismatched family member allogeneic grafts. It has been shown that stimulated lymphocytes from UCB have reduced production of cytokines including IFN-gamma and TNF-alpha, known to play an important role in GVHD pathophysiology. This laboratory has investigated the molecular mechanisms underlying this reduced cytokine production by analyzing expression of nuclear factor of activated T cells-1 (NFAT1) in UCB T cells. The applicants detected no constitutive expression of NFAT1 protein in non-stimulated UCB T cells, and although NFAT1 expression in UCB T cells was upregulated after prolonged (40 hr) T cell stimulation, it was only partially upregulated when compared to adult controls. These observations of minimal NFAT1 expression after stimulation correlated with reduced cytoplasmic IFN-gamma and TNF-alpha production in UCB T cells studied simultaneously. The first hypothesis underlying the work outlined in this application is that gene regulation of NFAT1, a central transcription factor involved in cytokine gene expression, differs between neonatal and adult T cells. Secondly, they hypothesize that that these fundamental differences in NFAT1 regulation between neonatal and adult T cells will elucidate underlying regulatory pathways mediating hyporesponsiveness of UCB T cells. To test these hypotheses, they will [Aim 1] compare in detail expression kinetics and gene regulation of NFAT1 in UCB and adult T cells. Then [Aim 2] in vitro studies will be performed to compare NFAT1 protein levels with surface expression of immunomodulatory molecules including CD40L, CTLA-4, and FasL on UCB T cells, and cytoplasmic expression of IFN-gamma and TNF-alpha, in the presence or absence of cyclosporine or FK506 (tacrolimus). Finally, [Aim 3] NFAT1 protein levels and surface expression T cells of immunomodulatory molecules will be compared in donor UCB and HLA-matched sibling T cells emerging during immune recovery after transplant. These studies may provide further insight into the regulation and expression of above described genes during neonatal and adult life. Moreover, since NFAT1 regulates the gene expression of cytokines and immunomodulatory molecules known to mediate GVHD, their studies may reveal new therapeutic approaches to the treatment and/or prophylaxis of GVHD.
