Single-cell multiomics reveals primarily CD4+ TH1 cell clonal expansion leading to Pneumocystis murina clearance in immunocompetent hosts 3192

Description Objective To characterize the roles of pulmonary immune cells and identify immune mechanisms involved in clearance of Pneumocystis in immunocompetent hosts. Methods We used a co-housing exposure model of mouse Pneumocystis (P. murina) infection to determine gene expression levels in lungs of immunocompetent C57BL/6 mice. We applied single-cell multiomics combined with flow cytometry, qPCR, ELISA and pathway analyses, to characterize host immune responses during infection. Results P. murina organism load peaked at day 35 post-infection, and all infected C57BL/6 mice developed anti-P. murina antibodies. CD4+ TH1 cells showed the largest increase and clonal expansion, with smaller increases in TH17 and TfH cells. TCR diversity decreased post-infection and TRBV1-TRBJ2-3 was the most frequently used TCRβ gene segment in P. murina infected mice. Infected mice showed differential expression of pro-inflammatory cytokines (IL-21, Cxcl9), co-stimulatory molecule Ox-40, and regulatory genes (Ctla4, Ly6a), indicating a regulated immune response at peak of infection. Pathway analyses predicted IFN-γ-driven cytokine interactions, enhancing macrophage-mediated killing of Pneumocystis. Conclusion This study shows a predominant CD4+ TH1 response in immunocompetent hosts with Pneumocystis, suggesting that a regulated IFN-γ-rich milieu is necessary to enhance phagocytosis of Pneumocystis organisms by macrophages, leading to efficient clearance of Pneumocystis in immunocompetent individuals. Funding Sources This project was funded by the Intramural Research Program of the NIH Clinical Center, National Institutes of Health. Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)