Single-cell RNA sequencing of CTLA-4 and PD-1 blockade in pulmonary paracoccidioidomycosis highlights a protective transcriptional program mediated by activated Th17 cells, neutrophils and macrophages

Abstract

Pulmonary paracoccidioidomycosis (PCM) relies on a finely balanced lung-immune network in which Th17, Treg, neutrophils, and macrophages orchestrate fungal control and tissue integrity. Furthermore, within the context of single-cell sequencing, little is known about how immune checkpoint inhibition modulates this balance during systemic mycosis. In a previous study we verified that the blockade of checkpoint molecules (CTLA-4 and PD-1) restores protective immunity that reduces fungal loads, tissue pathology and mortality of infected mice. Here, we have further studied this model by single-cell RNA sequencing on lung leukocytes from mice infected with Paracoccidioides brasiliensis and treated with anti-CTLA-4 or anti-PD-1 antibodies to define the cellular and molecular consequences of checkpoint blockade in vivo. We generated a high-resolution atlas covering T cells, neutrophils, macrophages/monocytes, B cells, NK cells, and epithelial subsets. Checkpoint inhibition consistently remodeled the CD4 T cell compartment toward a Th17-enriched program, with reduced interleukin-10 expressing Treg frequencies and a prominent interleukin-17A (IL-17), C-C chemokine receptor type 2, CXC chemokine (CXC) receptor type 6, CD44 (Il17aCcr2Cxcr6Cd44) signature. This shift was accompanied by the expansion and activation of neutrophils and macrophages expressing tumor necrosis factor and chemokines (including CXC motif chemokine ligand (Cxcl1, Cxcl2 and Ccl4), microbicidal-associated genes (S100 calcium-binding protein A8 and A9), and regulatory mediators such as secretory leukocyte protease inhibitor and interleukin-15. Ligand-receptor and trajectory analyses revealed a reinforced Th17-myeloid communication axis, particularly under CTLA-4 blockade, converging on IL17-centered inflammatory circuits while attenuating canonical Treg checkpoints (cytotoxic T-lymphocyte-associated protein 4, programmed cell death protein 1, interleukin-10). Together, these data demonstrate that anti-CTLA-4 and anti-PD-1 immunotherapies profoundly rewire the lung-immune microenvironment during PCM, amplifying effector Th17-neutrophil-macrophage networks that benefits protective antifungal activity Our work provides a mechanistic framework for evaluating immune checkpoint inhibitors in chronic fungal infections.