Innate cells and STAT1-dependent signals orchestrate vaccine-induced protection against invasive Cryptococcus infection

Fungal pathogens are underappreciated causes of significant morbidity and mortality worldwide. In previous studies we determined that a heat-killed, Cryptococcus neoformans fbp1-deficient strain (HK-fbp1) is a potent vaccine candidate. We determined that vaccination with HK-fbp1 confers protective immunity against lethal Cryptococcosis in an interferon γ (IFNγ)-dependent manner. In this study, we set out to uncover cellular sources and relevant targets of the protective effects of IFNγ in response to the HK-fbp1 vaccine. We found that early IFNγ production peaks at day 3 and that monocytes and neutrophils are important sources of this cytokine after vaccination. Neutralization of IFNγ at day 3 results in impaired CCR2+ monocyte recruitment and reduced differentiation into monocyte-derived dendritic cells (Mo-DC). In turn, depletion of CCR2+ cells prior to immunization results in impaired activation of IFNγ-producing CD4 and CD8 T cells. Thus, monocytes are important targets of innate IFNγ and help promote further IFNγ production by lymphocytes. We employed monocyte-fate mapper and conditional STAT1 knockout mice to uncover that STAT1 activation in CD11c+ cells, including alveolar macrophages, Mo-DCs, and monocyte-derived macrophages (Mo-Mac) is essential for HK-fbp1 vaccine-induced protection. Altogether, our aggregate findings suggest critical roles for innate cells as orchestrators of vaccine-induced protection against Cryptococcus infection. We hypothesize that the important roles observed for both CCR2+ and CD11c+ cells might be connected to the interrelationship of these populations where CCR2+ monocytes give rise to important CD11c+ effectors that no longer express CCR2. Thus, we set out to examine whether monocytes give rise to alveolar macrophages after HK-fbp1 immunization. To this end, we employed CX3CR1CreER tamoxifen- inducible cre mice crossed to Rosa26floxedTdTomato mice. This model allows us examine murine macrophage and monocyte fate mapping analysis by administration of tamoxifen. We injected mice with 2.5mg tamoxifen on day -1 on CX3CR1CreER x RosaR26TdTomato mice (monocyte fate mapper), and immunized mice with HK-fbp1 on day 0. On day 30 post vaccination, we sacrificed mice, and sorted tissue-derived alveolar macrophage (TD-AMs, CD45+DAPI-CD11c+SiglecFhi TdTomato-) and monocyte-derived AMs (Mo-AMs, CD45+DAPI-CD11c+SiglecFhi TdTomato+) the lung. Naive control is from Dr. Dane Park's group previous sorted naive AMs. We then performed gene expression profiling analysis on these sorted cell population.