Differential Immune Checkpoint Expression in CD4⁺ and CD4^- NKT Cell Populations During Healthy Pregnancy

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.