CTLA-4 blockade improves antifungal treatment outcomes in a murine model of pulmonary paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a systemic fungal infection endemic in the Americas, with the highest incidence in Brazil. Current treatment relies on antifungal agents such as amphotericin B (AmB), itraconazole, and fluconazole. However, long-term therapy frequently leads to treatment discontinuation and/or sequelae due to drug toxicity, chronic inflammation, and fibrosis, which can severely impair organ function and quality of life. Accordingly, immunomodulatory therapies have emerged, including the use of monoclonal antibodies against the checkpoint molecule CTLA-4. Previously, we demonstrated that CTLA-4 blockade reduced fungal burden, decreased lung lesion severity, and increased survival by enhancing protective immune responses. Therefore, this study aimed to evaluate a combined therapy of anti-CTLA-4 and amphotericin B. C57BL/6 mice were inoculated intratracheally with 1 × 10⁶ Paracoccidioides brasiliensis yeast cells and, after 6 weeks of infection, treated with anti-CTLA-4 and/or AmB. Two weeks after treatment, disease progression was evaluated by CFU counts, histopathology, and survival analysis. Immune responses were assessed by ELISA and flow cytometry. The combined anti-CTLA-4 + AmB therapy resulted in improved disease control, evidenced by reduced fungal burden in the lungs, liver, and spleen, decreased pulmonary tissue damage, and increased survival compared to monotherapies. Additionally, reduced infiltration of dendritic cells and neutrophils, lower expression of activation markers CD80 and CD86, and fewer CD4⁺ T cells in the lungs suggest controlled disease due to diminished antigenic stimulation. These findings demonstrate that CTLA-4-mediated immune modulation and the antifungal activity of amphotericin B act complementarity to control murine PCM, supporting combined therapy as superior to monotherapy.