Project Summary The cytokine IL-27 is a critical mechanism for restraining immune hyperactivity during infection. During Toxoplasma gondii infection, the loss of IL-27 results in a lethal, CD4+ T cell mediated immune response as well as elevated inflammatory cytokine responses and systemic thrombosis. How these mechanisms mediate pathology, and possibly intersect with one another, is unclear. GM-CSF production by CD4+ T cells is enhanced in IL-27 deficient mice, and we have recently observed that blockade of this cytokine leads to survival of infected IL-27-/- mice. GM-CSF can enhance monocyte and macrophage responses as well as contribute to immunothrombosis, suggesting that this may be a potential central mechanism by which IL-27 mediated protection is achieved. Additionally, we have observed that the loss of IL-27 results in enhanced monopoiesis and monocyte responses to infection. Monocytes do not express the IL-27 receptor, but long-term hematopoietic stem cells (LT-HSCs) do and can be skewed towards several differentiation pathways by IL-27. Thus, we hypothesize that the loss of IL-27 leads to enhanced monocyte development during infection which may in turn be impacted by GM-CSF during later stages of infection to mediate pathology. To test this, we will analyze if HSC development and monocyte phenotypes are impacted by IL-27 during the early stages of toxoplasmosis and if enhanced monocytes responses are pathological in this setting. This will be achieved through a combination of cell transfers, high-dimensional flow cytometry, and scRNA-seq as well as selective depletion experiments. We will then determine the mechanisms by which GM-CSF mediates pathology in the absence of IL-27. This will be done by blocking GM-CSF during infection of IL-27-/- mice and analyzing the immune parameters connected to pathology. Following this, the GM-CSF receptor will be selectively removed from potential cell types to determine if this rescues IL-27 deficient mice during infection. Together, these studies will enhance our understanding of cytokine driven pathologies and mechanisms of immune protection.