NR4A family members regulate T cell tolerance to preserve immune homeostasis and suppress autoimmunity

Abstract

The NR4A family of orphan nuclear receptors (Nr4a1-3) plays redundant roles to establish and maintain Treg identity; deletion of multiple family members in the thymus results in Treg deficiency and a severe inflammatory disease. Consequently, it has been challenging to unmask redundant functions of the NR4A family in other immune cells.Here we use a competitive bone marrow chimera strategy, coupled with conditional genetic tools, to rescue Treg homeostasis and unmask such functions. Unexpectedly, chimeras harboring Nr4a1 -/-Nr4a3 -/-(DKO) bone marrow develop autoantibodies and a systemic inflammatory disease despite a replete Treg compartment of largely wild-type origin. This disease differs qualitatively from that seen with Treg-deficiency and is B cellextrinsic. Negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO T cells with functional, phenotypic, and transcriptional features of anergy accumulate in chimeric mice. Nevertheless, we observe upregulation of genes encoding inflammatory mediators in anergic DKO T cells, and DKO T cells exhibit enhanced capacity for IL-2 production. These studies reveal cell-intrinsic roles for the NR4A family in both central and peripheral T cell tolerance, and demonstrate that each is essential to preserve immune homeostasis.(memory) compartment and upregulate well-established markers of anergy (CD73 and FR4) in a cell-intrinsic manner (Figures 6D-G) (39). Similarly, expansion of anergic CD4 + T cells was exaggerated in 1:1 DKO:WT chimeras relative to both control chimeras and SKO mice (Supplementary Figures 6A-H). Moreover, even phenotypically 'naive' DKO CD44 lo CD62L hi CD4 + T cells in mixed chimeras upregulated CD73 and FR4, suggestive of antigen encounter (Figure 6D, E, Supplementary

NR4A nuclear receptors in T and B lymphocytes: Gatekeepers of immune tolerance*

“…3,17,37,65,68,79 It remains to be fully defined whether these processes exhibit differential dependence on the NR4A family. However, given the magnitude of the phenotype we observed in DKO:WT chimeras, 16 it is likely that the collective function of the NR4A family is required for deletion in both settings, consistent with prior work.…”

“…We postulated that DKO cells escaping negative selection should be enriched for self‐reactivity in the periphery. Indeed, in DKO:WT chimeras, we observed accumulation of DKO (but not WT) peripheral T cells that expressed markers associated with chronic antigen stimulation and anergy, including CD44 and PD‐1 in the CD8 compartment, and CD44, FR4, PD‐1, LAG3, and CD73 in the CD4 compartment 16,77 . These DKO T cells also exhibited impaired TCR signal transduction, consistent with functional anergy.…”

“…By contrast to autoantibodies that develop even in DKO:WT chimeras, we observed that the myeloproliferative disorder described in both our germline DKO mice and a previously reported, independent germline DKO line 69 was almost entirely suppressed in DKO:WT competitive chimeras even after 12 weeks. 16 Furthermore, DKO myeloid cells exhibited no competitive advantage over WT cells in these chimeras. This suggested to us that myeloid expansion in germline DKO mice is cell-extrinsic and most likely attributable to loss of Treg.…”

“…T cell homeostasis was rescued. 16 This implied that accumulation of antigen-experienced peripheral T cells in DKO:WT chimeras was due to escape from negative selection. Importantly, in contrast to prior studies that have relied upon TCR Tg mice and model self-an tigens, 1,3,6,17,26,65,67,68,79 this phenotype is apparent with a physiologic TCR repertoire in the face of bona fide endogenous antigen encounter.…”

“…To that end, we recently generated an independent line of Nr4a1 –/– Nr4a3 –/– mice (germline DKO) that exhibit near‐complete loss of Treg, severe runting, and death before 4 weeks of age, 16 consistent with genetic models described earlier 5,69 . We used a distinct line of Nr4a1 ‐deficient mice derived from a conditional allele which, in contrast to the previously generated Nr4a1 –/– , does not express exon 2 16,66 . We next generated competitive radiation chimeras using both DKO and WT donor bone marrow (input ratios of 1:1 or 1:5 DKO:WT) in order to allow WT donor bone marrow (BM) to reconstitute a functional Treg compartment when DKO cells could not.…”