*Purpose: Pregnancy is a common immune sensitizing event, which creates an access barrier to organ transplantation and can impact transplant outcomes. While most research has focused on how fetal antigen primes conventional maternal T cells, pregnancy also primes the expansion of protective alloreactive regulatory T cells (Tregs). Pregnancy thus represents a unique model of Treg expansion, which may yield insight into the therapeutic use of Tregs in transplantation. Importantly, prior studies have characterized maternal Treg responses to transgenic fetal antigens, which necessarily provide a limited view of pregnancy-induced expansion. Here we use bulk TCR sequencing to assess the effect of pregnancy alloimmunization on the endogenous polyclonal Treg repertoire.

*Methods: Female Foxp3^GFP Treg reporter mice were mated with either syngeneic B6 or allogeneic Balb/c males or left as naive virgin controls. Maternal Tregs were sorted from peripheral blood collected prior to mating and late in pregnancy (embryonic day 18). TCR sequencing was performed on genomic DNA using the ImmunoSeq^TM assay (Adaptive Biotechnologies).

*Results: An average of 478 (SD=206) productive Treg clones/mouse were identified at each timepoint. Despite the limitations imposed by sampling depth, we detected expansion of a subset of Treg clones during allogeneic pregnancy, which produced an increase in the overall clonality of the Treg repertoire [Figure]. Notably, no change in clonality was observed in either syngeneic pregnancy or naive controls. We identified 22 Treg clones that expanded in response to allogeneic mating compared with 2 clones that expanded in syngeneic pregnancy. These 22 clones represent 0.46% of all Treg clones sampled at either timepoint (vs. 0.087% in the syngeneic condition). We did not detect any clones that expanded in the naive controls. Collectively, these results indicate alloantigen-driven expansion of select Treg clones as a result of pregnancy-induced immune sensitization.

*Conclusions: Pregnancy is one of few immunization events that promotes the expansion of allogeneic Tregs. Using TCR sequencing, we were able to demonstrate this expansion over the course of allogeneic pregnancy, and characterize the resulting shift in clonality that occured in the endogenous Treg repertoire. Further study of Treg biology during pregnancy may help to guide the development of Treg-based therapies that regulate immune responses to alloantigen in transplantation.

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