Fezf2 promotes differentiation of Aire-expressing and mimetic epithelial populations regulating thymic homeostasis

Fezf2 has been implicated in shaping tissue-restricted antigen (TRA) expression and as an “Aire-like” factor in the thymus. Here we demonstrate loss of Fezf2 results primarily in a developmental block in mTEC development with pronounced expansion of Ccl21a-expressing mTECs at the expense of thymic tuft cells and most Aire-high and recently described post-Aire “mimetic” populations. While loss of either Aire or Fezf2 leads to a loss of post-Aire mimetic subtypes, their transcriptional programs are distinct; whereas Aire positively regulates the expression of thousands of genome-wide targets, Fezf2 orchestrates a restricted transcriptional program balanced between induction and repression that largely impacts TEC development. Strikingly, most Fezf2-repressed genes map to Ccl21a-expressing mTECs, suggesting Fezf2 maintains a cellular state conducive to the development of Aire-expressing mTECs and downstream access to terminally differentiated mimetic programs. At least some mimetic cell programs appear to have functions beyond simply serving as antigen reservoirs; thymic microfold cells (M cells) organize the medullary B cell niche in a Ccr6-dependent manner and promote IgA class-switching, analogous to Peyer's Patches. In Aire and Fezf2 KO mice, where development of thymic M cells is impaired, B cell class-switching is similarly disrupted. Therefore, our data reveal Fezf2 as a gatekeeper of mTEC terminal development which cooperates with Aire to allow the full program of medullary epithelial diversity, supporting unexpected layers of mimetic cell function that impact thymic homeostasis beyond antigen diversity. Overall design: WT control (3 replicates), Fezf2 KO, and Aire KO CD45-, EpCAM+ TECs were sorted from C57BL/6 mice for scRNA-seq analysis of major TEC subpopulations