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.

Keywords: CD4; NKT cells; immune checkpoint pathways; pregnancy; trimester.

Figure 1

Frequency of CD4⁺ and CD4⁻ NKT cells in peripheral blood across pregnancy trimesters and non-pregnant controls. The frequency of the CD4⁺ and CD4⁻ NKT cells in the lymphogate (A) and in the total NKT population (B) in the three trimesters of healthy pregnancy and non-pregnant women. The solid bars represent medians of 35, 34, 31 and 36 determinations, respectively; the boxes indicate the interquartile ranges; and the whiskers represent the lower and upper 25% of the data between the minimum and maximum values. At the same time, the outliers are values that are at least one and a half times smaller or larger than the interquartile range from the lower or upper quartile. Statistically significant differences with p-values < 0.01 *** and <0.03 ** are indicated. NP: non-pregnant, 1st: first trimester, 2nd: second trimester, 3rd: third trimester.

Figure 2

PD-1 and PD-L1 expression by CD4⁺ and CD4⁻ NKT subpopulations during healthy pregnancy and non-pregnant controls. PD-1 receptor expression (A) and PD-L1 ligand expression (B) by the CD4⁺ and CD4⁻ NKT subpopulations in the three trimesters of healthy pregnancy and in non-pregnant women. The solid bars represent medians of 14, 13, 11, and 18, determinations respectively; the boxes indicate the interquartile ranges; and the whiskers represent the lower and upper 25% of the data between the minimum and maximum values, while the outliers are values that are at least one and a half times smaller or larger than the interquartile range from the lower or upper quartile. Statistically significant differences with p-values <0.01 ***, <0.03 ** and <0.05 * are indicated. Representative FACS plots show the PD-1 surface marker (C) and PD-L1 surface molecule (D) expression by cells in the lymphocyte gate. To determine the positivity of PD-1 and PD-L1, a fluorescent minus one (FMO) control was used. NP: non-pregnant, 1st: first trimester, 2nd: second trimester, 3rd: third trimester.

Figure 3

LAG-3 and Gal-3 expression by CD4⁺ and CD4⁻ NKT subpopulations during healthy pregnancy and non-pregnant controls. LAG-3 receptor expression (A) and Gal-3 ligand expression (B) by the CD4⁺ and CD4⁻ NKT subpopulations in the three trimesters of healthy pregnancy and in non-pregnant women. The solid bars represent medians of 19, 20, 19 and 18 determinations, respectively; the boxes indicate the interquartile ranges; and the whiskers represent the lower and upper 25% of the data between the minimum and maximum values, while the outliers are values that are at least one and a half times smaller or larger than the interquartile range from the lower or upper quartile. Representative FACS plots show the LAG-3 surface marker (C) and Gal-3 molecule (D) expression by cells in the lymphocyte gate. To determine the positivity of LAG-3 and Gal-3, FMO control was used. NP: non-pregnant, 1st: first trimester, 2nd: second trimester, 3rd: third trimester.

Figure 4

TIGIT and CD226 expression by CD4⁺ and CD4⁻ NKT subpopulations during healthy pregnancy and non-pregnant controls. TIGIT receptor expression (A) and CD226 receptor expression (B) by the CD4⁺ and CD4⁻ NKT subpopulations in the three trimesters of healthy pregnancy and in non-pregnant women. The solid bars represent medians of 19, 20, 20, and 17 determinations, respectively; the boxes indicate the interquartile ranges; and the whiskers represent the lower and upper 25% of the data between the minimum and maximum values, while the outliers are values that are at least one and a half times smaller or larger than the interquartile range from the lower or upper quartile. Statistically significant differences with p-values <0.01 ***, <0.03 ** and <0.05 * are indicated. Representative FACS plots show the TIGIT surface marker (C) and CD226 surface molecule (D) expression by cells in the lymphocyte gate. To determine the positivity of TIGIT and CD226, FMO control was used. NP: non-pregnant, 1st: first trimester, 2nd: second trimester, 3rd: third trimester.

Figure 5

Ratio of TIGIT/CD226 double-positive cells and the ratio of CD226 MFI/TIGIT MFI by different NKT cells during healthy pregnancy and non-pregnant controls. Frequency of the TIGIT/CD226 double-positive NKT cell subpopulations (A) and the ratio of the CD226 MFI/TIGIT MFI by the TIGIT/CD226 double-positive NKT cell subpopulation (B) receptors in the three trimesters of healthy pregnancy and non-pregnant women. The solid bars represent medians of 17, 20, 18, and 17 determinations, respectively; the boxes indicate the interquartile ranges; and the whiskers show the most extreme observations. The middle square within the box represents the mean value. Statistically significant differences with p-values <0.01 *** and <0.03 ** are indicated. NP: non-pregnant, 1st: first trimester, 2nd: second trimester, 3rd: third trimester.

Figure 6

CD112 and CD155 expression by CD4⁺ and CD4⁻ NKT subpopulations during healthy pregnancy and non-pregnant controls. CD112 receptor expression (A) and CD155 receptor expression (B) by the CD4⁺ and CD4⁻ NKT subpopulations in the three trimesters of healthy pregnancy and non-pregnant women. The solid bars represent medians of 18, 19, 19, and 16 determinations, respectively; the boxes indicate the interquartile ranges; and the whiskers represent the lower and upper 25% of the data between the minimum and maximum values, while the outliers are values that are at least one and a half times smaller or larger than the interquartile range from the lower or upper quartile. Statistically significant differences with p-values <0.01 ***, and <0.05 * are indicated. Representative FACS plots show the CD112 surface marker (C) and CD155 surface molecule (D) expression by cells in the lymphocyte gate. To determine the positivity of CD112 and CD155, FMO control was used. NP: non-pregnant, 1st: first trimester, 2nd: second trimester, 3rd: third trimester.

Figure 7

NKG2D expression by CD4⁺ and CD4⁻ NKT subpopulations during healthy pregnancy and in non-pregnant controls. NKG2D receptor expression (A) by the CD4⁺ and CD4⁻ NKT subpopulations in the three trimesters of healthy pregnancy and in non-pregnant women. The solid bars represent medians of 31, 34, 31, and 30 determinations, respectively; the boxes indicate the interquartile ranges; and the whiskers represent the lower and upper 25% of the data between the minimum and maximum values, while the outliers are values that are at least one and a half times smaller or larger than the interquartile range from the lower or upper quartile. Statistically significant differences with p-values <0.01 *** are indicated. Representative FACS plots show the NKG2D surface marker (B) expression by cells in the lymphocyte gate. To determine the positivity of NKG2D, FMO control was used. NP: non-pregnant, 1st: first trimester, 2nd: second trimester, 3rd: third trimester.

Figure 8

Flow cytometric identification of CD4⁺ and CD4⁻ of NKT subpopulations. The flow cytometric gating strategy to separate the examined CD4⁺ and CD4⁻ NKT subpopulations. After a doublet exclusion step (A,B) using FSC-A/SSC-A parameters, the lymphocyte gate was created (C). From the lymphocyte gate, the NKT cell population was gated based on the CD3⁺/CD56⁺ combination (D). From the NKT subpopulations, the CD8⁻ NKT subset was gated (E). From the CD8⁻ NKT cell subsets, CD4⁻ NKT and CD4⁺ NKT subpopulations were separated (F).