Nuclear receptor Nr4a1 modulates both regulatory T-cell (Treg) differentiation and clonal deletion

Abstract

Immature thymocytes expressing autoreactive T-cell receptors (TCR) can adopt differing cell fates: clonal deletion by apoptosis or deviation into alternative lineages such as FoxP3(+) regulatory T cells (Treg). We revisited the role of the transcription factor Nr4a1 (Nur77), an immediate-early response gene induced by TCR engagement. Nr4a1KO mice show clear quantitative defects in antigen-induced clonal deletion. The impact of the Nr4a1 deletion is not enhanced by deletion of the proapoptotic factor Bim. In addition, Nr4a1 curtails initial differentiation into the Treg lineage in TCR transgenic mice and in nontransgenic mice. Transcriptional profiling of Nr4a1KO thymocytes under selection conditions reveals that Nr4a1 activates the transcription of several targets, consistent with these diverse actions: (i) Nr4a1 partakes in the induction of Bim after TCR triggering; (ii) perhaps paradoxically, Nr4a1 positively controls several transcripts of the Treg signature, in particular Ikzf2 and Tnfrsf9; (iii) consistent with its prosurvival and metabolic role in the liver, Nr4a1 is also required for the induction by TCR of a coordinated set of enzymes of the glycolytic and Krebs cycle pathways, which we propose may antagonize Treg selection as does activation of mTOR/Akt. Thus, Nr4a1 appears to act as a balancing molecule in fate determination at a critical juncture of T-cell differentiation.

Fig. 1.

Impaired clonal deletion of BDC2.5 TCR transgenic thymocytes in Nr4a1KO. E15.5 BDC2.5-tg FTOCs from B6g7 or B6g7.Nr4a1KO mice were cultured for 3 d to promote CD4+CD8+ differentiation and then incubated for 16 h with BDC mimotope peptide (BDCmi). (A) Representative cytometry plots after 16 h with 10 ng/mL BDCmi. Percentage of surviving DPs is indicated in upper right of each panel. (B) Differences in percentage (Upper) and absolute cell count (Lower) of DPs surviving after 16 h with titrated BDCmi; each point represents a single FTOC culture (representative experiment). (C) Compilation of five experiments comparing percentage of surviving DPs after 16 h FTOC with no or 3 ng/mL BDCmi; each point represents a single FTOC.

Fig. 2.

Nr4a1KO impairs clonal deletion of OTII TCR transgenic thymocytes. (A) Representative cytometry profiles of OTII TCR transgenic thymi on WT or Nr4a1KO backgrounds, without (Upper panels) or with (Lower panels) the RIP-mOVA transgene. (Left panels) CD4 vs. CD8. (Right panels) Anti-clonotypic antibodies. The gates drawn outline the OTII^hi and OTII^low populations, and numbers refer to OTII^hi cells. (B) Compilation of seven experiments comparing percentage of CD4SP (Left panels), Vα2Vβ5^hi of gated CD4SP (Center panels), and absolute cell count of CD4SP (Right panels).

Fig. 3.

No additional clonal deletion defect in Bim/Nr4a1 double KO. (A) Representative CD4xCD8 plots of OTII/B6 thymi from 6-wk-old male mice of WT (Left panels), Nr4a1KO (Center panels), and Nr4a1/Bim double KO (Right panels) genotypes, transgenic for OTII (Upper panels) or OTII/RIP-mOVA (Lower panels). (B) Compilation of three experiments comparing the percentage of CD4SP.

Fig. 4.

Fop3+ thymic Tregs populations increased in Nr4a1KO. (A) Representative plots showing increased FoxP3+ population in gated CD4SPs from 6-wk-old WT and Nr4a1KO mice. (B) Compilation of three experiments comparing percentage (Left) and number (Right) FoxP3+ cells within gated CD4SPs. (C) Representative FoxP3xCD25 plot of gated CD4SPs from OTII or OTII.RIP-mOVA on WT and Nr4a1KO background. (D) Compilation of eight experiments comparing FoxP3+ frequency within CD4SPs and absolute counts in WT and Nr4a1KO. (E) Representative CD25xFoxP3 staining of CD4SPs, with FoxP3-CD25+ “Treg precursor” population frequencies indicated (Left). (Right) Compilation from individual mice. (F) FC/FC plot depicting the ratio of expression in Treg vs. Tconv CD4SPs from WT (x axis) and Nr4a1KO (y axis) thymi. Treg up (red) and down (blue) signatures are highlighted. (G) Representative suppression assay at varying Treg:Teffector ratios, comparing Tregs from WT or Nr4a1KO.

Fig. 5.

Nr4a1-dependent transcripts identified by microarray profiling. (A) FoldChange vs. expression plots for WT vs. Nr4a1KO in resting DP thymocytes (Left) or DP thymocytes stimulated in vitro for 3 h with plate-bound anti-CD2/CD28/TCRβ. Transcripts highlighted indicate ratio of expression >2× (orange) or <0.5× (aqua) in WT/Nr4a1KO. (B) Volcano plots comparing WT and KO thymocytes (FoldChange: x axis; t test P value: y axis) for DPs stimulated 3 h with plate-bound anti-CD2/CD28/TCRβ (Left), DPs from OTII.OVA (Center), or CD4SPs from OTII.OVA (Right). (C) FC/FC plot showing the ratio of expression in CD4 SPs from OTII vs. OTII.OVA thymi from WT or Nr4a1KO mice. Treg up (red) and down (blue) signatures are highlighted. (D) FC/FC plot depicting the WT vs. KO ratio of expression in OTII.OVA CD4SPs (x axis) or in 3-h anti-TCR–stimulated DPs (y axis). Highlights as in C.