CD4+ CD25+ regulatory T cells in human pregnancy: development of a Treg-MLC-ELISPOT suppression assay and indications of paternal specific Tregs

Abstract

The current study was aimed at developing a one-way mixed leucocyte culture-enzyme-linked immunospot (MLC-ELISPOT) assay for the study of CD4(+) CD25(+) regulatory T (T(reg)) cells and applying this method in the study of antifetal immune reactions during human pregnancy. Twenty-one pregnant women and the corresponding fathers-to-be, and 10 non-pregnant control women and men, participated in the study. CD4(+) CD25(+) cells were isolated from peripheral blood mononuclear cells (PBMC) by immunomagnetic selection. Maternal/control PBMC were stimulated with paternal or unrelated PBMC in MLC. Secretion of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) from responder cells, with or without the presence of autologous T(reg) cells, was analysed by ELISPOT. PBMC from pregnant women showed increased secretion of IL-4 compared to controls. In pregnant and non-pregnant controls, T(reg) cells suppressed IFN-gamma reactivity against paternal and unrelated alloantigens. Interestingly, T(reg) cells suppressed IL-4 secretion against paternal but not unrelated alloantigens during pregnancy. We have successfully developed a model for studying T(reg) cells in antifetal cytokine reactions during pregnancy. Results indicate that T(reg) cells contribute to strict regulation of both T helper type 1-like and type 2-like antifetal immune reactions. Interestingly, T helper type 2-like cells specific to unrelated alloantigens are able to escape the suppression of T(reg) cells, which would allow for IL-4, alongside CD4(+) CD25(+) T(reg) cells, to control potentially detrimental IFN-gamma reactions during pregnancy.

Figure 1

Flow cytometric analysis of (a) total PBMC, unselected (CD25^bright gate), (b) CD4⁺ CD25⁺ depleted cells (CD25^bright gate), (c) immunomagnetically selected CD4⁺ CD25⁺ cells (CD25^bright gate), and (d) immunomagnetically selected CD4⁺ CD25⁺ cells (CD25⁺ gate). The CD25⁺ gate was set according to isotype controls and the CD25^bright gate was adjusted to contain CD4⁺ cells that expressed CD25 more brightly than CD4^– CD25⁺ cells. The x-axis shows expression of CD25 and the y-axis represents expression of CD4. (c, d) show that the immunomagnetically selected CD4⁺ CD25⁺ cells contained both CD25^bright and CD25^dim cells.

Figure 2

FOXP3 mRNA expression in CD4⁺ CD25⁺ depleted PBMC, PBMC and CD4⁺ CD25⁺ cells determined by real-time RT-PCR (n = 2). Results are presented as median FOXP3/18S rRNA relative expression and error bars represent the highest value.

Figure 3

Number of IL-4-secreting and IFN-γ-secreting cells in PBMC from pregnant women and non-pregnant controls. Results are displayed as plots with medians marked with a line. (a) Secretion of IL-4 from pregnant women (n = 10) and non-pregnant control women (n = 6) determined by ELISPOT. Differences between controls and pregnant women were always statistically significant (Bonferroni-corrected Mann–Whitney U-test; **P < 0·01, ***P < 0·001). In pregnant women, the difference between spontaneous and pooled alloantigen stimulated secretion was almost statistically significant (Friedman's test (P < 0·05) followed by Bonferroni-corrected Wilcoxon test; †P = 0·06). (b) Secretion of IFN-γ from pregnant women (n = 11) and non-pregnant control women (n = 10) determined by ELISPOT. In pregnant women, the difference between spontaneous and pooled alloantigen-stimulated secretion reached statistical significance while the difference between spontaneous and paternal alloantigen-stimulated secretion was almost statistically significant (Friedman's test; P < 0·05, followed by Bonferroni-corrected Wilcoxon test; *P < 0·05, ‡P = 0·06).

Figure 4

Effect of T_reg cells on the number of IFN-γ-secreting cells from non-pregnant controls (n = 10). Secretion was significantly inhibited by re-addition of T_reg cells in the presence of pooled alloantigens (Friedman's test; P < 0·05, followed by Bonferroni-corrected Wilcoxon test; *P < 0·05).

Figure 5

Effect of T_reg cells on the number of IFN-γ-secreting cells from pregnant women (n = 11 for all groups except pooled alloantigen stimulated T_reg-cell-depleted + T_reg cells n = 9). Secretion was significantly inhibited by the re-addition of T_reg cells both in the presence of paternal and pooled alloantigens (Friedman's test; P < 0·05, followed by Bonferroni-corrected Wilcoxon test; *P < 0·05).

Figure 6

Effect of T_reg cells on the number of IL-4-secreting cells from pregnant women (n = 10). Secretion was significantly, or almost significantly, inhibited by the re-addition of T_reg cells in the presence of paternal alloantigens in comparison to T_reg-cell-depleted cultures and total PBMC, respectively (Friedman's test; P < 0·01, followed by Bonferroni-corrected Wilcoxon test; *P < 0·05, †P = 0·059).