Changes in T Cell and Dendritic Cell Phenotype from Mid to Late Pregnancy Are Indicative of a Shift from Immune Tolerance to Immune Activation

Abstract

During pregnancy, the mother allows the immunologically distinct fetoplacental unit to develop and grow. Opinions are divided as to whether this represents a state of fetal-specific tolerance or of a generalized suppression of the maternal immune system. We hypothesized that antigen-specific T cell responses are modulated by an inhibitory T cell phenotype and modified dendritic cell (DC) phenotype in a gestation-dependent manner. We analyzed changes in surface markers of peripheral blood T cells, ex vivo antigen-specific T cell responses, indoleamine 2,3-dioxygenase (IDO) activity (kynurenine/tryptophan ratio, KTR), plasma neopterin concentration, and the in vitro expression of progesterone-induced blocking factor (PIBF) in response to peripheral blood mononuclear cell culture with progesterone. We found that mid gestation is characterized by reduced antigen-specific T cell responses associated with (1) predominance of effector memory over other T cell subsets; (2) upregulation of inhibitory markers (programmed death ligand 1); (3) heightened response to progesterone (PIBF); and (4) reduced proportions of myeloid DC and concurrent IDO activity (KTR). Conversely, antigen-specific T cell responses normalized in late pregnancy and were associated with increased markers of T cell activation (CD38, neopterin). However, these changes occur with a simultaneous upregulation of immune suppressive mechanisms including apoptosis (CD95), coinhibition (TIM-3), and immune regulation (IL-10) through the course of pregnancy. Together, our data suggest that immune tolerance dominates in the second trimester and that it is gradually reversed in the third trimester in association with immune activation as the end of pregnancy approaches.

Keywords: activation; immune modulation; immune response; pregnancy; tolerance.

Figure 1

Longitudinal functional responses to recall antigens and peptides show dampened IFN-γ and IL-10 in the second trimester that revert in the third. (A) Shows numbers of IFN-γ producing cells in pregnancy in response to EBV, measles, and tetanus toxoid (TTOX), compared to healthy controls. (B) Likewise, panel B shows pregnancy trimester changes of IL-10 cellular responses to CMV, EBV, HSV, purified protein derivative (PPD), and TTOX, compared to non-pregnant controls. (C,D) Granzyme and IL-4 responses to TTOX and measles, respectively. ^♯p < 0.05, ^♯♯p < 0.01 (Wilcoxon matched-pairs signed ranks test); *p < 0.05, **p < 0.01, ***p = 0.001 (Mann–Whitney-U). Abbreviation: PBMC, peripheral blood mononuclear cell.

Figure 2

Changes in T-cell maturation and exhaustion with normal pregnancy. (A) Shows the frequencies of naïve, EM, CM, and TEMRA CD4 and CD8 T cells as determined by flow cytometry in pregnant (Pr) and health control (HC). Additionally, we show proportions of CD4 recent thymic emigrants determined by CD31 and PTK7 coexpression. (B) Programmed death-1 (PD-1) and PD ligand 1 (PD-L1) expression measured by mean florescence intensity (MFI) on naïve, EM, CM, and TEMRA CD4 and CD8 T cells. Columns indicate median and error bars interquartile range. ^♯p < 0.05 and ^♯♯p < 0.01 (Kruskal–Wallis one way analysis of variance and Dunn’s test for multiple comparisons).

Figure 3

Gradual increase in T cell activation and pro-apoptotic marker expression during normal pregnancy. (A) Changes in activation markers CD38, HLA-DR, and CD69 expression in CD4 and CD8 T cells with trimesters and compared to controls. (B) Plasma neopterin concentration in pregnancy correlated with CD38 mean florescence intensity (MFI) on CD4⁺ T cells. (C) FAS and CD57 marker expression on CD4 and CD8 T cells during pregnancy and compared to health controls (HCs). Columns indicate median and error bars interquartile range. ^♯♯p < 0.01 (Kruskal–Wallis one way analysis of variance and Dunn’s test for multiple comparisons). Abbreviation: Pr, pregnant.

Figure 4

Tolerant T-cell phenotypes are favored in pregnancy and reflect changes in T-cell activation with increasing gestation. (A) Percentage expression of CTLA-4 on TIM-3 negative and positive, CD4 and CD8 T cells in pregnancy compared with controls. (B,C) Proportions of CD4 regulatory T cells (Tregs) in pregnancy. These cells were identified using the phenotype CD4⁺CD45RO⁺CD25^hi and CD4⁺CD45RO⁺CD25⁺CD127^lo. *p < 0.05 and **p < 0.01. Columns indicate median and error bars interquartile range. *p < 0.05 and **p < 0.01 (Mann–Whitney U). Abbreviation: HC, health control; Pr, pregnant.

Figure 5

Proportions of myeloid dendritic cell (mDC) and costimulatory CD86 expression on plasmacytoid DC (pDC) were reduced in pregnancy but positively correlated with indoleamine 2,3-dioxygenase (IDO) activity. (A) Proportions of mDC and pDC and their respective expression of CD86 in pregnancy and compared to controls. (B) Correlation between CD86 expression on DCs and plasma neopterin concentration. (C) Correlation between mDC proportions, CD86 expression, and plasma kynurenine/tryptophan ratio (KTR). (D) Plasma tryptophan, kyurenine concentration, and KTR (μmol/mmol) during pregnancy. Columns indicate median and error bars interquartile range. ^♯♯p < 0.01 (Kruskal–Wallis one way analysis of variance and Dunn’s test for multiple comparisons), *p < 0.05 (Mann–Whitney U). Abbreviations: HC, healthy control; MFI, mean florescence intensity; Pr, pregnant.

Figure 6

T cell receptor (TCR)-γδ⁺ T-cell proportions are reduced in pregnancy but they express greater progesterone-induced blocking factor (PIBF). These changes are primarily seen in the second trimester. (A) Shows changes in proportions of TCR-γδ CD4 and CD8 T cells in response to progesterone culture. (B) Alterations in PIBF expression on the aforementioned TCR-γδ T cells. (C) Peripheral blood mononuclear cell (PBMCs) were surface stained with anti-CD3, anti-CD8, anti-TCR-γδ, and anti-PIBF. Gating strategy used to phenotype CD4⁺/CD8⁺ TCR-γδ⁺ PIBF⁺ T-cells. Box and whiskers plots indicate median, interquartile range and 10th–90th centiles. ^♯p < 0.05 and ^♯♯p < 0.01 (Kruskal–Wallis one way analysis of variance and Dunn’s test for multiple comparisons). Abbreviations: HC, healthy control; Pr, pregnant.