Stage-dependent CD40 signalling defines a shift between protective and suppressive immunity in visceral leishmaniasis
Hima Mahor, Bhaskar SahaAbstract
Introduction
Visceral leishmaniasis (VL), caused by Leishmania donovani, is characterised 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.
Methods
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.
Results
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.
Conclusions
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.