Bulk RNAseq of Aire-Expressing Cells from Pregnant vs Virgin Mice

We had previously found that ablating autoimmune regulator (Aire)-expressing cells during pregnancy lead to fetal loss, intruterine growth restriction, and the specific expansion of Tfh populations. To investigate transcriptional changes in Aire+ cells, specifically in medullary thymic epithelial cells (mTECs) and extrathymic Aire-expressing cells (eTACs), we used the Aire-Driven Igrp-GFP (Adig) reporter mouse. We sorted mTECs and eTACs from virgin Adig females and E9.5 pregnant Adig dams for bulk RNA sequencing. While we found no differences in the frequency or absolute number of mTECs or eTACs, or of Aire expression in mTECs or eTACs during pregnancy, we did find a significant number of differentially expressed (DE) transcripts during pregnancy in splenic eTACs, and almost no changes in mTECs. Pathway analysis of splenic DE genes revealed enrichment for erythrocyte development and included a number of putative PAAs such as embryonic hemoglobin theta. While these transcriptional changes may represent eTAC-intrinsic expression of PAAs, pregnancy also causes increased maternal hematopoiesis as well as passage of fetal nucleated RBCs into the maternal circulation and the immunologic relevance of this phenomenon requires further study. Seven Adig females were bred to BALB/c males and sacrificed on day 9 of pregnancy, and six age-matched virgin females were sacrified on the same day. Stromal cells were prepared for cell sorting by mincing thymus, lymph nodes, and spleen with razor blades and sujected to a Percoll gradient. Isolated cells from these tissues were stained for CD3, CD19, NK1.1, MHCII, and live/dead marker. CD3- CD19- NK1.1- MHCII+ GFP+ cells were sorted via FACS, resuspended in lysis bffer, and snap frozen for RNA isolation and sequencing.  We employed standard mRNA-seq protocols used by the UCSF Functional Genomics sequencing core. 52 samples were spread across 8 conditions, each with 6-7 replicates. 49 samples are represented in this dataset.