Lag3 and PD-1 pathways preferentially regulate NFAT-dependent TCR signalling programmes during early CD4+ T cell activation.

Introduction: Lag3 and PD-1 are immune checkpoints that regulate T cell responses and are current immunotherapy targets. Yet how they function to control early stages of CD4⁺ T cell activation remains unclear.

Methods: Here, we show that the PD-1 and Lag3 pathways exhibit layered control of the early CD4⁺ T cell activation process, with the effects of Lag3 more pronounced in the presence of PD-1 pathway co-blockade (CB). RNA sequencing revealed that CB drove an early NFAT-dependent transcriptional profile, including promotion of ICOS^hi T follicular helper cell differentiation.

Results: NFAT pathway inhibition abolished CB-induced upregulation of NFAT-dependent co-receptors ICOS and OX40, whilst unaffecting the NFAT-independent gene Nr4a1. Mechanistically, Lag3 and PD-1 pathways functioned additively to regulate the duration of T cell receptor signals during CD4⁺ T cell re-activation. Phenotypic changes in peripheral blood CD4⁺ T cells in humans on anti-Lag3 and anti-PD-1 combination therapy revealed upregulation of genes encoding ICOS and OX40 on distinct CD4⁺ T cell subsets, highlighting the potential translational relevance of our findings.

Conclusion: Our data therefore reveal that PD-1 and Lag3 pathways converge to additively regulate TCR signal duration and may preferentially control NFAT-dependent transcriptional activity during early CD4⁺ T cell re-activation.