The host defense related cytokine IFN-gamma induces the expression of a broad array of genes in both hematopoietic and nonhematopoietic cells. A dominant set of these genes belong to the p47kDa GTPase family. Mice deficient in one gene in this family, Irgm1 (LRG47), are highly susceptible to a wide variety of different intracellular bacterial and protozoan pathogens and we have been investigating the immunologic mechanisms underlying this profound and far reaching defect in host resistance and its relationship to IFN function. Although Irgm1 has been shown to regulate the killing of pathogens inside IFN-gamma-activated macrophages in vitro, this is not its only function in host resistance. Thus, we have shown previously that Mycobacterium avium and Trypanosoma cruzi infected Irgm1-/- mice undergo a profound loss in lymphocytes, platelets and other bone marrow derived cells, a phenomenon referred to as 'bone marrow failure". In work completed this year we analyzed the mechanism responsible for this defect in blood cell generation/survival in Irgm-1 deficient animals focussing on both the bone marrow itself and the periphery where immune responses to pathogens occur. With a collaborator Margaret Goodell at Baylor, we first examined the role of hematopoietic stem cells (HSCs) which are self-renewing bone marrow cells that give rise to all blood lineages and retain the capacity to proliferate in response to different forms of exogenous stress. We found that Irgm1 inhibits baseline HSC proliferation and is required for a normal HSC response to chemical and infectious stimuli. HSCs from IRGm-1 deficient mice were found to be severely impaired in functional repopulation assays, and when subjected in vivo to hematopoietic ablation with the drug 5-fluorouracil or infection with Mycobacterium avium, the mice failed to undergo the expected expansion in stem and progenitor cell populations. This impaired expansion of HSC and progenitor cells likely contributes to the defective host resistance Irgm1-/- to infection by limiting the generation of new effector cells needed to replace those attacking the pathogen. In the second part of this project, we addressed the possible role of Irgm1 in the IFN-gamma dependent regulation of cell survival in the periphery. Focusing on CD4+ T lymphocytes we showed that IFN-gamma induced Irgm1 is critical for the survival of activated mature lymphocytes since in the absence of Irgm1, IFN-gamma triggers death of CD4+ T cells in vitro by a novel mechanism involving autophagy. Importantly, the lymphopenia and mortality previously observed in M. avium-infected Irgm1-/- animals was completely abrogated in Irgm1-/- IFN-gamma -/-double deficient mice, demonstrating that the presence or absence of Irgm1 determines the host protective vs detrimental outcome of IFN-gamma induction during pathogen encounter. These findings reveal an unexpected regulatory function for Irgm1 in T lymphocyte survival and suggest that the GTPase, in addition to its role in regulating stem cell/progenitor cell renewal, is essential for the peripheral expansion of immune effector cells during Th1 responses to intracellular pathogens Additional projects undertaken during the year addressed the innate pathogen signals responsible for triggering Th2 and Th17 effector cell development. We and other groups had previously shown that Schistosoma mansoni eggs contain factors that trigger potent Th2 responses in vivo and condition murine dendritic cells (DC) to promote Th2 lymphocyte differentiation in vitro. Using the latter assay which involves bystander polarization by DC of OVA specific TCR transgenic T cells, we purified the major Th2-inducing component from egg extracts and identified it by its peptide sequence as the secreted T2 ribonuclease, omega-1 an egg glycoprotein previously characterized by another group. Importantly, omega-1-exposed DC displayed pronounced cytoskeletal changes and exhibited decreased antigen dependent conjugate formation with CD4 T cells an in vitro assay. Based on this evidence we propose that S. mansoni omega-1 acts on DC and limits their interaction with CD4 T lymphocytes. By so doing the molecule lowers the strength of the activation signal that the DC deliver to the cells thereby creating a setting that favors Th2 differentiation. In a related study, we investigated the mechanism by which mycobacteria promote Th17 differentiation. Th17 cells represent a newly discovered CD4+ T cell subset that appears to play an important role in host inflammatory responses and immunity to certain pathogens. Th17 responses have been shown to function in vaccine induced protection against Mycobacterium tuberculosis. In addition heat killed M. tuberculosis is a critical component in Complete Freunds' Adjuvant (CFA) where it promotes the development of Th17 as well as Th1 responses to protein antigens co-administered with it. Indeed, CFA immunization forms the basis of many autoimmune disease models that require Th17 induction. In our work this year we investigated the specific role played by MyD88 dependent signaling pathways in the induction of Ag specific Th17 versus Th1 responses following immunization with ovalbumin in CFA. MyD88 is in adaptor molecule required for signaling by both toll-like receptors (TLR) and the Interleukin-1 (IL-1) receptor and mice deficient in this protein are typically used to assess the role of TLR in immune reponses. As predicted, we observed a major requirement for MyD88 signaling in the induction of Ag specific CD4+ T cell IFN-gamma and IL-17 responses following CFA/Ova immunization. Mice doubly deficient in TLR2 and TLR9, two TLR previously shown to be stimulated by live M. tuberculosis, displayed only partial defects in these CFA induced Th1 and Th17 responses. In contrast, mice deficient in IL-1R, showed a near complete loss in Th17 and a major reduction in Th1 responses. Taken together these findings suggest that TLR signaling may play a limited role in the effects of CFA on Th17 polarization and only partially explain its effects on Th1 induction. Instead they argue that CFA triggers Th17 polarization through an IL-1R dependent signaling cascade. Preliminary data implicate an upstream requirement for inflammasome mediated IL-1 processing in this function. In addition to providing information concerning the innate recognition pathways stimulated by mycobacteria that contribute to adaptive CD4+T responses, these findings establish a convenient and easily manipulated in vivo model for dissecting the unique signals involved in Th17 polarization.