Human uterine lymphocytes acquire a more experienced and tolerogenic phenotype during pregnancy

Abstract

Pregnancy requires a delicate immune balance that nurtures the allogeneic fetus, while maintaining reactivity against pathogens. Despite increasing knowledge, data is lacking on the transition of pre-pregnancy endometrial lymphocytes to a pregnancy state. Here, we immunophenotyped lymphocytes from endometrium (MMC), term decidua parietalis (DPMC), and PBMC for direct comparison. We found that the immune cell composition of MMC and DPMC clearly differ from each other, with less NK-cells, and more NKT-cells and T-cells in DPMC. An increased percentage of central memory and effector memory T-cells, and less naive T-cells in DPMC indicates that decidual T-cells are more experienced than endometrial T-cells. The increased percentage of CD4⁺CD25^highCD127^- Treg in DPMC, including differentiated Treg, is indicative of a more experienced and tolerogenic environment during pregnancy. The Th cell composition of both MMC and DPMC was different from PBMC, with a preference for Th1 over Th2 in the uterine environment. Between MMC and DPMC, percentages of Th cell subsets did not differ significantly. Our results suggest that already before pregnancy a tightly controlled Th1/Th2/Th17 balance is present. These findings create opportunities to further investigate the underlying immune mechanism of pregnancy complications using menstrual blood as a source for endometrial lymphocytes.

Figure 1

Lymphocyte composition of peripheral blood, menstrual blood, and term decidua. Mononuclear cells were isolated from peripheral blood (PBMC, n = 17), menstrual blood (MMC, n = 17), and decidua parietalis (DPMC, n = 12). Percentages of lymphocytes (CD45⁺) based on side scatter (SS) and forward scatter (FS), T cells (CD56⁻CD3⁺; CD4⁺ and CD8⁺), NKT cells (CD3⁺CD56⁺), NK cells (CD3⁻CD56⁺; CD56⁺CD16⁺ and CD56⁺CD16⁻), and B cells (CD19⁺) were obtained by flow cytometric analysis. *P < 0.05, **P < 0.01, and ***P < 0.001 (lines indicate mean, non-parametric Kruskal-Wallis with Dunns post-hoc test).

Figure 2

CD4⁺ and CD8⁺ T cells subsets in peripheral blood (PBMC), menstrual blood (MMC) and term decidua parietalis (DPMC). (a) Representative staining for CD45RA and CD45RO on CD3⁺ T cells and CD45RA and CCR7 on CD4⁺ and CD8⁺ T cells from PBMC, MMC, and DPMC. (b) Expression of CD45RA and CD45RO on CD3⁺ T cells (PBMC n = 17, MMC n = 16, and DPMC n = 12. (c) CD4⁺ and CD8⁺ T cells are separated into four subsets based on the expression of CD45RA and CCR7. Naive T cell (CD45RA⁺CCR7⁺); effector T cell, Teff (CD45RA⁺CCR7⁻); effector memory T cell, EM (CD45RA⁻CCR7⁻); central memory T cell, CM (CD45RA⁻CCR7⁺) (PBMC n = 17, MMC n = 11, and DPMC n = 8). Lines indicate mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 (non-parametric Kruskal-Wallis with Dunns post-hoc test).

Figure 3

Distribution of regulatory T cells. (a) Representative staining of CD25, CD127, and CD45RA on CD4⁺ T cells from peripheral blood (PBMC, n = 17), menstrual blood (MMC, n = 16), and term decidua parietalis (DPMC, n = 12). (b) Percentage of CD25^highCD127⁻ Treg within CD4⁺ T cells in PBMC, MMC, and DPMC. (c) Distribution of naive Treg (CD45RA⁺CD25⁺) and differentiated Treg (CD45RA⁻CD25⁺⁺) in CD4⁺ T cells. **P < 0.01, and ***P < 0.001 (lines indicate mean, non-parametric Kruskal-Wallis with Dunns post-hoc test).

Figure 4

Distribution of CD4⁺ Th cell subsets in peripheral blood (PBMC), menstrual blood (MMC), and term decidua parietalis (DPMC) and production of IFN-γ and IL-17 by CD4⁺ T cells. (a,c) Representative gating for CCR6, CXCR3, and CCR4 on CD4⁺ T cells and for IFN-γ and IL-17 by CD4⁺ T cells from PBMC, MMC, and DPMC. (b) Th1, Th2, Th17, and nonconventional Th1 (Th1-like) CD4⁺ T cells can be classified based on the expression of CCR6, CXCR3, and CCR4. CCR6⁻CXCR3⁺CCR4⁻, Th1; CCR6⁺CXCR3⁺CCR4⁻, Th1-like; CCR6⁻CXCR3⁻CCR4⁺, Th2; CCR6⁺CXCR3⁻CCR4⁺, Th17 (PBMC n = 17, MMC n = 11, DPMC n = 7). (d) Production of IFN-γ and IL-17 by CD4⁺ T cells from PBMC (n = 12), MMC (n = 15), and DPMC (n = 7). *P < 0.05, **P < 0.01, and ***P < 0.001 (lines indicate mean, non-parametric Kruskal-Wallis with Dunns post-hoc test).