Fetal T_regs suppress fetal T cell responses to maternal alloantigens. (A) Fetal T cell proliferation after stimulation with allogeneic APCs from an unrelated donor for 5 days (3:1 ratio of fetal lymphocytes:allogeneic APCs). (B) Proliferative responses to autologous, maternal, or unrelated APCs after a 5-day MLR. Histograms depict proliferation in the presence (no depletion) or absence (CD25 depletion) of fetal T_regs. CFSE, carboxy-fluorescein diacetate succinimidyl ester. (C) Summary of all experiments addressing fetal T cell proliferative responses to autologous (n = 6), maternal (n = 9), or unrelated (n = 9) APCs in the presence or absence of fetal T_regs. Statistical significance was determined by paired Student’s t test. mLN, mesenteric lymph nodes. (D) Comparison of T_regs suppression against autologous, maternal, or unrelated APCs.

T_regs specific for non-inherited maternal alloantigens persist long after birth. (A) CD8+ T cell proliferation in mock-depleted (gray histograms) and CD25-depleted (unshaded histograms) T cells after an 8-day MLR with maternal (left) and paternal (right) APCs. Three children from a single family are represented. (B) Summary of all children tested, comparing the relative increase in proliferation after depletion of CD25+ cells from MLRs against maternal (left) or paternal (right) APCs (1:3 ratio of APCs: responders). Statistical analysis calculated by Mann-Whitney U rank sum test. (C) Summary of individual responses of all children tested, showing proliferation of mock-depleted (PBMC) or CD25-depleted (CD25−) T cells in response to maternal or paternal APCs. Two different dilutions of APCs:responders (1:3, top; 1:30, bottom) are depicted. Statistical significance was determined by paired Student’s t test.

Fetal T cells differentiate into T_regs upon stimulation with alloantigens. (A) Fetal T cells depleted of CD25+FoxP3+ cells were stimulated for 5 days with unrelated APCs, and FoxP3 expression was measured in proliferating T cells. (B) Kinetic analysis of CD25 and FoxP3 up-regulation by adult and fetal CD4+ T cells after stimulation with alloantigens. (C) Fold difference in cytokine mRNA expression in normal adult (n = 4) and fetal (n = 5) LNs. Genes found to be significantly different are labeled in red (fetal) or blue (adult) (P < 0.05, unpaired Student’s t test). Asterisks denote TNF family members involved in organogenesis. (D) Inhibition of TGFβ signaling by addition of the activin receptor–like kinase inhibitor, SB-431542 (1 uM), which blocks FoxP3 up-regulation by fetal T cells stimulated with unrelated APCs. (E) TGFβ-dependent acquisition of suppressive function after stimulation of fetal T cells with alloantigens. Error bars indicate SD observed in three separate experiments.