Pregnancy imprints regulatory memory that sustains anergy to fetal antigen

Abstract

Pregnancy is an intricately orchestrated process where immune effector cells with fetal specificity are selectively silenced. This requires the sustained expansion of immune-suppressive maternal FOXP3(+) regulatory T cells (T(reg) cells), because even transient partial ablation triggers fetal-specific effector T-cell activation and pregnancy loss. In turn, many idiopathic pregnancy complications proposed to originate from disrupted fetal tolerance are associated with blunted maternal T(reg) expansion. Importantly, however, the antigen specificity and cellular origin of maternal T(reg) cells that accumulate during gestation remain incompletely defined. Here we show that pregnancy selectively stimulates the accumulation of maternal FOXP3(+) CD4 cells with fetal specificity using tetramer-based enrichment that allows the identification of rare endogenous T cells. Interestingly, after delivery, fetal-specific T(reg) cells persist at elevated levels, maintain tolerance to pre-existing fetal antigen, and rapidly re-accumulate during subsequent pregnancy. The accelerated expansion of T(reg) cells during secondary pregnancy was driven almost exclusively by proliferation of fetal-specific FOXP3(+) cells retained from prior pregnancy, whereas induced FOXP3 expression and proliferation of pre-existing FOXP3(+) cells each contribute to T(reg) expansion during primary pregnancy. Furthermore, fetal resorption in secondary compared with primary pregnancy becomes more resilient to partial maternal FOXP3(+) cell ablation. Thus, pregnancy imprints FOXP3(+) CD4 cells that sustain protective regulatory memory to fetal antigen. We anticipate that these findings will spark further investigation on maternal regulatory T-cell specificity that unlocks new strategies for improving pregnancy outcomes and novel approaches for therapeutically exploiting T(reg) cell memory.

Figure 1. Accumulation of maternal CD4 and Foxp3⁺ Tregs with fetal-specificity during gestation

a, Total 2W1S⁺ or 2W1S⁺Foxp3⁺ CD4 cells in B6 females impregnated by Balb/c-2W1S males. b, Percent Foxp3⁺ among 2W1S⁺ or 2W1S⁻ CD4 cells. c, Percent Ki67⁺ among 2W1S⁺Foxp3⁺ or 2W1S⁺Foxp3⁻ CD4 cells. d, Percent Helios^hi among 2W1S⁺Foxp3⁺or 2W1S⁻Foxp3⁺ CD4 cells. e, Percent Foxp3⁺ among Foxp3^DTR/DTR donor (CD45.1⁺) or Foxp3^WT/WT recipient (CD45.2⁺) 2W1S⁺ CD4 cells midgestation (E11.5) by Balb/c-2W1S males, with DT treatment (top) or no DT controls (bottom). Bar, mean ± one standard error.

Figure 2. Accelerated expansion of maternal Tregs with fetal-specificity during secondary pregnancy

a, Percent Foxp3⁺ among virgin (primary pregnancy) or postpartum (secondary pregnancy) females before mating or midgestation (E11.5) by Balb/c-2W1S males. b, Percent Foxp3⁺ among postpartum donor (CD45.1⁺) or naïve recipient (CD45.2⁺) 2W1S⁺ CD4 cells midgestation by Balb/c-2W1S males. c, Percent Foxp3⁺ among Treg-ablated Foxp3^DTR/DTR postpartum donor (CD45.1⁺) or naïve recipient (CD45.2⁺) 2W1S⁺ CD4 cells midgestation. Bar, mean ± one standard error.

Figure 3. The postpartum environment maintains anergy for maternal CD4 cells with pre-existing fetal-specificity

a, IFN-γ producing 2W1S⁺ CD4 cells five days after Lm-2W1S inoculation in virgin, or pregnant females midgestation by Balb/c-2W1S or Balb/c males. b, IFN-γ producing 2W1S⁺ CD4 cells five days after Lm-2W1S inoculation in postpartum mice previously impregnated by Balb/c-2W1S or Balb/c males. c, IFN-γ producing postpartum donor (CD45.2⁺CD90.2⁺), naive donor (CD45.2⁺CD90.1⁺), or naive recipient (CD45.1⁺) CD4 cells five days after Lm-2W1S inoculation and stimulation with PMA/ionomycin. Bar, mean ± one standard error.

Figure 4. Maternal postpartum Tregs mitigate IFN-γ responsiveness and mediate resiliency to fetal resorption in secondary pregnancy

a, Representative plots illustrating the majority (>96%) of Foxp3⁺ cells are derived from adoptively transferred CD4 in DT treated Foxp3^DTR/DTR mice. b, IFN-γ producing 2W1S⁺ cells among CD45.1⁺CD45.2⁻ cells, accumulation of 2W1S⁺Foxp3⁺ cells, and Helios expression among 2W1S⁺Foxp3⁺ Tregs five days after Lm-2W1S inoculation. c, Percent fetal resorption during primary (open) or secondary (shaded) allogeneic pregnancy for Foxp3^WT/WT, Foxp3^DTR/WT, or Foxp3^DTR/DTR females five days after DT initiation beginning midgestation. Bar, mean ± one standard error.