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

Strong tolerogenic signalling recalibrates CD4+ T cell activation in part via the PD-1 pathway. However, the contribution of other checkpoints is unknown. Here, we reveal that Lag3 and PD-L1 pathways are the major regulators of TCR signalling in adaptively tolerised CD4⁺ T cells. Lag3 and PD-L1 co-blockade (CB) drove additive and synergistic changes in T cell activation, resulting in a transcriptional profile dominated by a NFAT-biased/ T follicular helper cell signature. Mechanistically, CB led to an enhanced duration of TCR signalling, which drove a transcriptional motif of TCR signal duration-sensitive genes. In addition, CB synergistically upregulated CCR6, leading to enhanced CCL20-dependent migration. Analysis of datasets from humans on anti-PD-1 and anti-Lag3 immunotherapy revealed upregulation of CCR6 and ICOS in blood effector memory CD4⁺ T cells, underscoring the translational relevance of our findings. The PD-L1 and Lag3 pathways, therefore, are major regulators of TCR signal duration in adaptively tolerised CD4⁺ T cells in vivo.