Normal human pregnancy is associated with an elevation in the immune suppressive CD25+ CD4+ regulatory T-cell subset

Abstract

Summary CD4+ CD25+ T regulatory cells (TReg), suppress antigen-specific immune responses and are important for allograft tolerance. During pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus) requiring substantial changes in immune regulation over a programmed period of time. We analysed whether immune-suppressive TReg cells were altered during pregnancy and therefore might play a part in this tolerant state. The presence of TReg cells was assessed in the blood of 25 non-pregnant, 63 pregnant and seven postnatal healthy women by flow cytometry. We observed an increase in circulating TReg cells during early pregnancy, peaking during the second trimester and then a decline postpartum. Isolated CD25+ CD4+ cells expressed FoxP3 messenger RNA, a marker of TReg cells, and suppressed proliferative responses of autologous CD4+ CD25- T cells to allogeneic dendritic cells. These data support the concept that normal pregnancy is associated with an elevation in the number of TReg cells which may be important in maintaining materno-fetal tolerance.

Figure 1

Changes in the blood T_Reg cell population size during pregnancy. Box and whisker plot showing CD25⁺ CD4⁺ T_Reg population as a percentage of total CD4⁺ cells, giving medians, quartiles and ranges for each trimester (T1–T3). T1 = < 13 weeks, T2 = 14–26 weeks, T3 = > 27 weeks gestation, Postnatal = 6–8 weeks. Scatter plot with regression line showing percentageT_Reg for individual pregnancies at given weeks of gestation. *P < 0·05; **P < 0·01; n.s., not significant (Mann–Whitney test).

Figure 2

Three-colour flow cytometry analysis of CD4⁺ CD25⁺ cells during normal pregnancy. (a) Dot plot for fluorescence intensity of CD4 versus side light scatter showing gating for identification of CD4⁺ lymphocytes. (b) Dot plot for forward versus side light scatter of gated CD4⁺ lymphocytes demonstrating small size of these cells during pregnancy (black) and large size following mitogen stimulation (grey). (c) Dot plot for CD3 PE versus CD25 FITC of gated CD4⁺ lymphocytes demonstrating that all of these cells are T cells (CD3⁺) and that 22% during pregnancy (black) have weak CD25 expression (upper right quadrant) whilst all (grey) have strong CD25 expression after activation by mitogen. The quadrants are set according to isotypematched, negative control antibodies. (d–g, i) Dot plots for CD25 PE versus CD69 FITC, DR FITC, CD45RA FITC, CD45RO FITC, CD11a FITC, respectively, for gated CD4⁺ lymphocytes. (h) Dot plot for CD25 FITC versus CXCR3 PE for gated CD4⁺ lymphocytes.

Figure 3

FoxP3 is up-regulated in CD4⁺ CD25⁺ T cells. Quantitative PCR analysis was carried out for FoxP3 and β-actin. Relative expression was calculated from the cycle threshold for FoxP3 relative to an internal β-actin standard, for purified CD25⁺ and CD25⁻ T cells.

Figure 4

T_Reg from pregnant women suppress alloresponses. T_Reg cells from a pregnant (a) and a non-pregnant (b) woman were tested for their ability to inhibit proliferation of autologous CD25⁻ T cells stimulated by allogeneic DCs for 5 days. The ratio of DCs to CD4 CD25⁻T cells is shown on the x axis and the ratio of T_Reg to CD25⁻ T cells is given for each bar. Responses (counts per minure; c.p.m.) represent the mean [³H]thymidine incorporation plus the standard error of triplicate cultures. Data shown are representative of five separate paired experiments.