Project 1: During this period we initiated a study of a rare but immunologically informative primary immunodeficiency disease known as X-linked hyper-IgM syndrome (XHIM). This disease has recently been shown to be due to a defect in the gene encoding CD40L, a key T cell signaling molecule that is essential for interactions between T cells and B cells or T cells and antigen-presenting cells. It is the role of CD40L in the T cell-B cell interaction that is responsible for the obvious immunoglobulin abnormality characterizing the disease. Activated T cells expressing CD40L and signaling B cells via CD40 is a necessary antecedent to B cell isotype switching. Hence, in the absence of CD40L, IgM+ B cells fail to switch to IgG+ and IgA+ B cells and one observes increased production of IgM and greatly reduced or absent production of IgG and IgA. For many years, the consequences of failure to signal via CD40L or T cell function has been largely overlooked in XHIM. This, despite the fact that CD40L has been shown to be critical for Th1 T cell differentiation and CTL induction. That a defect in this area of the immune system may in fact be present in XHIM was suggested by the observation that patients are prone to certain types of opportunistic infection and the development of neoplasia. We have filled in this gap in our knowledge of XHIM by conducting an extensive series of studies of T cell function in XHIM patients. In initial studies we determined if patient antigen-presenting cells (APC?s) could produce IL-12 is stimulated by activated patient T cells, recognizing that in normals, the latter express CD40L and signal APC?s to produce this cytokine. We found that, indeed, patient cells stimulated with either anti-CD3epsilon or PHA plus IFN-gamma induced greatly reduced IL-12 production as compared to normals. This defect in IL-12 production, however, was not due to an intrinsic inability to produce IL-12 since direct stimulation of APC?s with LPS plus IFN-gamma or CD40L-trimer plus IFN-gamma led to normal or even increased IL-12 production. In further studies we probed the consequences of this reduced IL-12 production and showed that XHIM patient T cells stimulated by anti-CD3epsilon or PHA produced markedly decreased amounts of TNF-alpha. In contrast, understimulation in which patients APC?s were stimulated directly to produce IL-12 by CD40L-trimer and patient T cell independently stimulated by anti-CD3epsilon, IFN-gamma production was almost normal. Thus, again, there was no intrinsic defect of cytokine production. In further studies, we demonstrated that the above cytokine production defect was accompanied by markedly reduced T cell maturation in ?memory? CD45RO+ T cells. This defect was present in both the CD4+ and CD8+ T cell subsets. In studies to explore the basis of this defect, we showed that normal ?na?ve? CD4+/CD45RA+ T cells undergo maturation into CD4+/CD45RO+ T cells when stimulated by SAC, but that this maturation is blocked by the presence of CTLA-Fc, a substance that interferes with CD28/B7 interactions. It thus becomes apparent that such maturation depends on signaling of T cells via B7 (CD80 and CD86). Relating this finding in normals to patients with XHIM, we then showed that patient B cells in PBMC failure to manifest surface B7 expression when stimulated with PHA, presumably due to lack of CD40-CD40L interactions. One can therefore conclude that the CD40L defect in patients leads to T cell immaturity because APC?s fail to express B7 upon stimulation. Project 2: Studies of the rare syndrome intestinal lymphangectasia (IL) has been a longstanding interest of scientists in the Mucosal Immunity Sections as these patients represent a unique experiment of nature with which to probe immunologic processes in humans. IL is a primary/congenital or a secondary/acquired disorder due and generalized but unevenly distributed peripheral abnormalities of lymphocyte development and a characteric asymmetric edema or a variety of abnormalities which lead to blockage of lymph flow in the gastrointestinal tract. Previous studies have revealed that the loss process in IL is characterized not only by lymphocytopenia, but also by depletion of lymphocytes in lymphoid tissues and diminished delayed skin test reactivity. In addition, these studies have shown that patient T cells manifest poor in vitro proliferative responses to antigens and mitogens even when T cell numbers are normalized in in vitro cultures. This implies that a qualitative as well as a quantitative disorder of T cells is present in IL, on perhaps due to preferential loss of responsive lymphocyte subpopulations. Evidence in favor of this possibility has come from studies showing that in IL, CD4+ T cells are more reduced than CD8+ T cells. In the present study, we re-examined the question of selective lymphocyte loss in IL utilizing recently available monoclonal antibodies to identify and isolate lymphocyte subsets. In the present studies we initially found that patients with IL have greatly reduced total T cell counts largely due to reduced CD4+ T cell numbers rather than CD8+ T cell numbers. On this basis, patients with IL had reversed CD4/CD8 T cell ratios. Of great interest, the reduced CD4+ T cell levels were due to a selective decrease in CD45RA+ T cells (na?ve T cells) rather than a decrease in CD45RO+ T cells which were more or less normal. A similar but lesser imbalance in the CD8+ T cell CD45RA/RO ratio was also observed. In addition to these T cell subset abnormalities, patients also had reduced numbers of B cells (CD20+/CD19+ cells); however, they had normal numbers of NK cells (CD16+/CD56+ cells). In further studies, both CD45RA+ and CD45RO+ T cells bearing the CD27 and CD31 markers was assessed in patients with IL. These markers are associated with more na?ve T cells that have not undergone extensive prior stimulation. We found that cells bearing these markers in either the CD45RA+ or CD45RO+ subsets. This again indicates that IL is associated with a selective decrease in more na?ve T cells. In a final series of studies, we evaluated the functional capabilities of circulatory cells in IL. Here we found that patient T cells manifested reduced proliferation when stimulated by monoclonal antibodies providing polyclonal stimulation and such reduction persisted even when patient cells were compared with normal cells of the same subsets. In addition, we found that patient cells produced less IFN-gamma and IL-2 and more IL-4 and IL-5 than similarly stimulated control cells.