Aire+ Cell Depletioin Results in Expansion of Activated T Cells in Pregnant Mice

We explored the role of the autoimmune regulator (Aire) gene in supporting fetal tolerance during pregnancy with transgenic diphtheria toxin receptor (Aire-DTR) mice, in which Aire-expressing populations can be deleted by injection of diptheria toxins (DT). Aire-expressing cell depletion was found to cause intruterine growth restriction (IUGR) in both allogenic and syngeneic pregnancies. This phenotype is immune-mediated, as IUGR is rescued in Rag1-deficient mice, and involves a memory response, as demonstrated by severe IUGR in second pregnancies. We further explored this phenotype through single-cell RNA sequencing of leukocytes obtained from the secondary lymphoid organs (uterine and non-uterine-draining lymph nodes) of wild-type (WT) and Aire-DTR pregnant dams during allogeneic pregnancy. Transciptomic data reveals a shift in population distributions from naïve to effector CD4+ and CD8+ T cells in Aire-DTR mice. Within the effector CD4+ compartment, there was a significant shift away from Tregs and toward mature T effectors, most notably T follicular helper (Tfh) and Th17 cells. These findings support the idea that Aire is essential in maintaining immune homeostasis. Overall design: Seven Aire-DTR or WT dams were bred to BALB/c males and given DT for the first 9 days of pregnancy. Uteruses, uterine-draining lymph nodes (udLN) and brachial lymph nodes (brLN) from each mouse were harvested at E9.5 and subjected to Percoll gradient. Isolated cells from these tissues were stained for CD45, and CD3, and sorted to keep live, single CD45+ cells. Cells from multiple mice and tissues were multiplexed into single sequencing runs by hash-tagging cells with totalseq-C anti-mouse hashtags. Three cell pools were created: (i) Pool 1 (ii) Pool 2, and (iii) Pool LN. Pool 1 and Pool 2 consisted of cells isolated from uterus, udLN and brLN tissues from one Aire-DTR and one WT mouse each. In each pool, 160,000 cells were split into two wells for sequencing.. Pool LN consisted of cells isolated from udLN and brLN tissues from two Aire-DTR mice and one WT mouse. 13,200 cells from Pool LN were split into two wells for sequencing. Gene expression profiles from all batches were analyzed together after demultiplexing.