Stage-dependent CD40 signalling defines a shift between protective and suppressive immunity in visceral leishmaniasis.

Visceral leishmaniasis (VL), caused by Leishmania donovani, is characterized by progressive immune dysregulation that supports parasite persistence. The CD40-CD40L axis is essential for antigen-presenting cell (APC) licensing and Th1-mediated nitric oxide-dependent parasite control; however, how its function evolves during infection remains incompletely understood. Here, we define the temporal regulation and functional polarity of CD40 signalling during experimental VL. Using BALB/c mice and primary macrophage assays, we investigated the temporal regulation of CD40 signalling during experimental VL. CD40 expression, cytokine production, nitric oxide levels, antibody responses, T-cell phenotypes, immune checkpoint expression, and macrophage activation were evaluated during progressive infection. Functional studies included CD40 agonism, CD40 knockdown, and TRAF-selective CD40 reconstitution to assess stage-specific effects on parasite control and immune regulation. Increasing parasite burden drove a gradual shift in CD40 signalling from protective to suppressive outcomes. In vitro infection reduced CD40 expression and IL-12 production while increasing IL-10, indicating early impairment of APC licensing. In vivo, progressive splenic infection was associated with diminished nitric oxide production, a declining IgG2a/IgG1 ratio, expansion of GATA3+ Th2 and FOXP3+ regulatory T-cell populations, and increased PD-L1 and TIM-3 expression, consistent with a regulatory and exhausted immune environment. Despite partial recovery of CD40 expression on myeloid APCs at later stages, declining CD40L+ T-cell availability and sustained IL-10/Arg1 signalling limited functional responsiveness. Early CD40 agonism failed to restore parasite control, whereas late activation exacerbated disease. CD40 knockdown increased parasite burden and reduced iNOS+ macrophages, confirming the requirement for CD40-dependent licensing. Finally, TRAF2-biased CD40 signalling promoted macrophage activation and parasite restriction, whereas TRAF6-biased signalling reinforced immunosuppression. These findings identify a stage-dependent functional threshold beyond which CD40 signalling no longer sustains protective immunity during VL, highlighting the importance of restoring APC competence and selectively directing CD40 signalling through TRAF2-linked pathways.