Differential immune checkpoint expression in CD4+ and CD4− NKT cell populations during healthy pregnancy

Abstract

This study investigated the expression of immune checkpoint molecules on CD4+ and CD4− NKT cell subpopulations throughout healthy pregnancy trimesters and in non-pregnant condition to understand their role in maternal–fetal immunotolerance. Using flow cytometry, we found that CD4− NKT cells significantly outnumbered CD4+ NKT cells in all investigated groups. In the case of the immune checkpoint molecules, PD-1 receptor expression was significantly lower in CD4− NKT cells compared to CD4+ counterpart cells only in non-pregnant women, while the PD-L1 ligand expression on CD4+ NKT cells significantly decreased in the third trimester. In contrast, LAG-3 and Galectin-3 expressions remained stable across all subsets and trimesters. For the TIGIT/CD226 axis, CD226 expression was significantly higher in CD4+ NKT cells in the third trimester and in non-pregnant women. The two ligands CD112 and CD155 were consistently lower on CD4− NKT cells across all groups. The activating receptor NKG2D was significantly higher on CD4− NKT cells in all examined cohorts. These findings suggest that CD4+ NKT cells tend towards a more tolerogenic phenotype, while CD4− NKT cells maintain a balanced cytotoxic potential with reduced immunoregulation function. The dynamic regulation of immune checkpoints on NKT cell subsets, particularly the downregulation of PD-L1 and CD226 in late pregnancy, highlights their fine-tuned role in balancing maternal–fetal immune tolerance with readiness for parturition.