Cytotoxic CD4⁺ T Cells in Visceral Leishmaniasis Patients

Visceral leishmaniasis (VL), a parasitic disease caused by Leishmania donovani, requires a robust CD4⁺ T-cells response to control parasite replication by interferon-gamma (IFN-γ) production and activation of macrophages. However, in VL patients, the anti-parasitic CD4⁺ T-cell responses are ineffective for reasons that are still unclear. Our recent study reporting a transcriptional signature of CD4⁺ T-cell isolated from the peripheral blood of active VL patients showed enhanced expression of genes related to cytotoxicity. This study investigates the cytotoxic potential of CD4⁺ T-cells in VL patients, focusing on the expression of the key cytotoxic molecules granzyme B (GZMB), granulysin (GNLY), perforin (PRF), natural killer cell granule protein 7 (NKG7) and the degranulation capacity of CD4⁺ T-cells during infection. We observed significant upregulation of these cytotoxic markers in CD4⁺ T-cells from VL patients, particularly prior to anti-parasitic drug treatment (D0), suggesting activation of these cells. However, the degranulation capacity, as indicated by CD107a expression, was comparable between VL patients and endemic controls (ECs), suggesting potential functional impairment in the cytotoxic response. Yet, antigen-specific GZMB secretion in whole blood cultures and intracellular GZMB production in CD4⁺ T-cell subsets (notably Th1, Th9 and Th17/22 cells) were enhanced in VL patients, indicating a robust antigen-specific response, though this did not translate into effective parasite control. These findings highlight a paradox whereby CD4⁺ T-cells from VL patients have heightened production of cytotoxic molecules, but lack translocation of CD107a to the cell surface, which is associated with an inability to eliminate parasites. This study provides new insights into the immune dysfunction in VL, highlighting a potential role for the cytotoxic phenotype that develops in parasite-specific CD4⁺ T-cells. Targeting these pathways may offer novel therapeutic strategies to enhance immune responses and improve clinical outcomes in VL.