Tissue-specific improvements in CD4 ⁺ T cell responses after treatment for visceral leishmaniasis

Visceral leishmaniasis (VL) is a life-threatening parasitic disease that requires robust CD4 ⁺ T cell-mediated immunity for parasite control. However, the heterogeneity and transcriptional dynamics of CD4 ⁺ T cell responses in VL remain poorly defined. In this study, we use a model of experimental VL with tissue-specific immunity and single-cell RNA sequencing to provide a high-resolution assessment of CD4 ⁺ T cell responses. Our analysis reveals the complexity of CD4 ⁺ T cell differentiation in VL, identifying distinct Th1 subsets with transcriptional heterogeneity that may reflect functional specialisation. Despite minimal transcriptional differences between splenic and hepatic CD4 ⁺ T cells, we identified shifts in subset composition, including the emergence of a stem-like CD4 ⁺ T cell population in the spleen, which was suppressed by the transcription factor Bhlhe40. Bhlhe40 deficiency skewed CD4 ⁺ T cell differentiation, impairing Th1 responses while promoting Tr1 cells, resulting in defective parasite control in the liver. Additionally, AmBisome treatment induced a profound transcriptional shift in CD4 ⁺ T cells, leading to the maintenance of stem-like CD4 ⁺ T cells in the spleen and the expansion of tissue resident memory-like cells in the liver. These findings uncover key regulatory mechanisms that shape CD4 ⁺ T cell differentiation in VL and provide insights into how immune-modulatory strategies could enhance long-term immunity.