A STAT3-dominant signalling signature associated with immune suppression in human visceral leishmaniasis is reversed by checkpoint blockade and STAT3 inhibition.

Visceral leishmaniasis (VL) is associated with impaired Th1 responses, yet the intracellular signalling events linking immunosuppressive pathways to effector dysfunction remain poorly defined. We studied 150 adults in endemic northern Nigeria (50 active VL, 50 healed VL, 50 endemic controls) using flow cytometry, cytokine profiling, targeted RT-qPCR of 13 JAK-STAT pathway genes with dual reference gene normalisation, ex vivo checkpoint blockade, and phospho-flow cytometry for STAT1/STAT3 in both CD4+ T cells and CD14+ macrophages. Active VL was associated with reduced antigen-specific IFN-γ (220 versus 940 pg/mL in healed VL; P < 0.001), elevated IL-10, expanded regulatory T cells, transcriptional upregulation of STAT3 (2.66-fold, adjusted P < 0.001), and downregulation of its negative regulator SOCS3 (0.56-fold, adjusted P = 0.001). A STAT3-dominant phosphorylation profile was present in both CD4+ T cells (pSTAT1/pSTAT3 ratio 1.33) and, more prominently, CD14+ macrophages (ratio 0.51; Kruskal-Wallis P < 0.001 across disease groups). CD14+ pSTAT3 correlated with macrophage infection index (Spearman ρ = 0.52, P < 0.001), linking signalling state to effector function. Combined IL-10 and PD-1 blockade reversed the signalling imbalance in both compartments, restored IFN-γ production up to 4.8-fold, and reduced macrophage infection index by 42%. Pharmacological STAT3 inhibition with Stattic confirmed pSTAT3 reduction by phospho-flow cytometry (-33%, P < 0.001), reproduced transcriptional reprogramming comparable to checkpoint blockade for 9 of 13 genes, and enhanced macrophage parasite clearance (infection index -36%, P < 0.001). These findings across phosphoprotein, transcript, and functional levels identify a STAT3-dominant signalling signature spanning T-cell and macrophage compartments as a consistent feature of immune suppression in human VL and a candidate therapeutic target warranting investigation.