Transcriptional control of central T cell tolerance by NR4A family transcription factors

The diverse T cell receptor (TCR) repertoire – generated randomly through VDJ recombination early in development – is inherently self-reactive. Clonal deletion and diversion to regulatory T cell (Treg) fate are key central tolerance mechanisms engaged during thymic selection. The NR4A family of transcription factors has been implicated in both, but the extent of its contribution to clonal deletion and the transcriptional mechanisms at play remain unknown. Here, we investigated both NR4A-dependent and NR4A-independent transcriptional changes induced in developing thymocytes in response to model self-antigen presented by medullary thymic epithelial cells (mTECs). To do so, we took advantage of the MHCII-restricted TCR transgene OTII and model self-antigen (membrane-bound ovalbumin expressed under the control of the rat insulin promoter encoded by the RIP-mOVA transgene). Our results suggest that a surprisingly broad transcriptional program is enacted upon high affinity self-antigen encounter in the thymus. Furthermore, by investigating the expression profile of OTII Nr4a1-/- Nr4a3-/- double knock-out (OTII-DKO) thymocytes that receive high affinity antigen-dependent signal but escape both clonal deletion and Treg diversion, we reveal evidence for deletional and non-deletional central tolerance mechanisms. Overall design: We performed comparative gene expression profiling using bulk RNA sequencing from OTII Nr4a1-/-Nr4a3-/- double knock-out (OTII-DKO) and control OTII Nr4a1+/+ Nr4a3+/+ (OTII-ctl) transgenic thymocytes under conditions of positive and negative thymic selection (i.e. clonal deletion). Two or three biological replicates were analyzed for each selection type.