Lag3 and PD-L1 govern T cell receptor signal duration in adaptively tolerised CD4+T cells

Abstract Anti-Lag3 and anti-PD-1 combination immunotherapy for melanoma has received recent regulatory approval, yet its mechanism of action is unclear, particularly for CD4 + T cells. Here, we determined the roles of the Lag3 and PD-1/PD-L1 pathways during CD4 + T cell activation in vivo . During primary immune responses, Lag3 played a redundant role with the PD1/PD-L1 axis dominant in regulating early CD4 + T cell activation. By exploiting an adaptive tolerance model, we reveal that Lag3 and PD-L1 co-blockade (CB) drove major changes in CD4 + T cells, resulting in a transcriptional profile dominated by a hybrid T follicular helper (Tfh)/Th17 cell phenotype. Mechanistically, CB enhanced T cell receptor (TCR) signal duration, thereby inducing an NFAT-biased transcriptional motif, previously linked to positive clinical outcomes for melanoma patients. Additionally, CB synergistically upregulated CCR6, leading to enhanced CCL20-mediated CD4 + T cell migration. Analysis of melanoma patients on anti-PD-1 pathway immunotherapies revealed that tumour CCR6 expression stratifies clinical outcomes and effector memory CD4 + T cells upregulate CCR6 in patients on anti-PD-1 and anti-Lag3 combination immunotherapy. Our data reveal that Lag3 and PD-1 pathways have context dependent roles in regulating the activation and migration of CD4 + T cells and highlights CCR6 as a biomarker for immunotherapy responses.