Fig. 5: Modulation of CD4⁺ regulatory T (Treg) cell number and antigen-specificity during pregnancies.

a Age-matched DEREG mice (2^nd: n = 4, 2^nd + DT: n = 6) were allogenically mated to Balb/c males twice. Some second pregnancy mice were injected with diphtheria toxin (DT) after the first delivery to deplete CD4⁺ Treg cells. On gestational day (gd) 15.5, frequencies of CD4⁺ Treg cells in (b, c) lymph node (2^nd vs 2^nd + DT: p = 0.0008) and (d) uterus were assessed by flow cytometry. e 150,000 CD4⁺ Treg cells – either harvested from virgin mice (termed naïve (n)Tregs) or from parous mice (termed memory (m)Tregs) – were adoptively transferred (AT) into first pregnancy C57Bl6/J mice mated to OVA-Balb/c males on the day of plug (1^st: n = 6, 1^st+nTregs: n = 5, 1^st+mTregs: n = 4). f Histograms of fetal antigen (OVA)-specific CD4⁺ Treg cells in first (black line) and second (blue line) in lymph node (LN, top) and uterus (bottom). Numbers represent the frequency of OVA⁺ cells in the CD4⁺ Treg cell compartment. Flow cytometry analysis of CD4⁺ Treg frequencies was performed in (g) lymph node and (h) uterus (1^st+nTregs vs 1^st+mTregs: p = 0.0403). i Age-matched Fir/Tiger mice were allogenically mated to Balb/c or DBA male mice for their second pregnancy (DF = different father; 2^nd: n = 8, 2^nd + DF: n = 6), respectively. On gd 15.5, frequencies of CD4⁺ Treg cells in (j, k) lymph node (2^nd vs 2^nd + DF: p = 0.0279) and (l) uterus (2^nd vs 2^nd + DF: p = 0.0456) were assessed by flow cytometry. m Age-matched Fir/Tiger mice were mated to Balb/c males once or twice and prenatally sound-stressed mid-gestationally (1^st: n = 7, 1^st+stress: n = 6, 2^nd: n = 5, 2^nd+stress: n = 5). On gd 15.5, flow cytometry analysis assessed CD4⁺ Treg cell frequencies in (n, o) lymph node (1^st+stress vs 2^nd+stress: p = 0.0071) and (p) uterus. Data are presented as violin plots with individual point, median and quartiles, and the statistical significance between groups was calculated using Student’s t-test when comparing two groups or One-way ANOVA when comparing three or more groups (* p < 0.05, ** p < 0.01, *** p < 0.001). Additional data are provided in Supplementary Fig. 4 and 5. Source data are provided as a Source Data file.