NR4A family members regulate T cell tolerance to preserve immune homeostasis and suppress autoimmunity
Ryosuke Hiwa, Hailyn V. Nielsen, James L. Mueller, Ravi Mandla, Julie Zikherman
Ryosuke Hiwa, Hailyn V. Nielsen, James L. Mueller, Ravi Mandla, Julie Zikherman
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Research Article Immunology

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–/– (double-knockout, DKO) bone marrow developed autoantibodies and a systemic inflammatory disease despite a replete Treg compartment of largely WT origin. This disease differs qualitatively from that seen with Treg deficiency and is B cell extrinsic. 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 accumulated in chimeric mice. Nevertheless, we observed upregulation of genes encoding inflammatory mediators in anergic DKO T cells, and DKO T cells exhibited 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.

Authors

Ryosuke Hiwa, Hailyn V. Nielsen, James L. Mueller, Ravi Mandla, Julie Zikherman

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Figure 6

Accumulation of anergic DKO CD4+ T cells in competitive chimeras.

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Accumulation of anergic DKO CD4+ T cells in competitive chimeras. (A) Sp...
(A) Splenocytes from WT, Nr4a3–/–, Nr4a1–/–, and gDKO mice were stained to detect CD4+ T cell subsets on the basis of CD44 and CD62L expression. Plots are representative of ≥5 mice/genotype. (B) Quantification of splenic CD4+CD44hiCD62Llo T cells as gated in A (n ≥ 5, 3- to 4-week-old gDKO and 5- to 6-week-old mice of other genotypes). (C) Quantification of splenic FOXP3–CD4+CD44hiCD62Llo T cells as gated in Supplemental Figure 6A from competitive chimeras at indicated time points posttransplant (n ≥ 3, pooled from 2 sets of independently generated chimeras). (D) Splenocytes from 12 weeks posttransplant DKO:WT = 1:5 chimera were stained to detect anergic T cell subsets. Flow plots depict CD73hiFR4hi (anergic) T cells within CD44loCD62Lhi (naive), CD44hiCD62Lhi, and CD44hiCD62Llo (memory) compartments of CD4+FOXP3– cells of each donor genotype. Representative of 7 chimeras, generated in 1 set. (E–G) Ratio of CD45.2 to CD45.1/2 within CD73hiFR4hi gate among naive (E), CD44hiCD62Lhi (F), or memory (G) CD4+ T cell compartments, as gated in D. Shown are WT:WT = 1:1 and DKO:WT = 1:5 chimeras at indicated time points posttransplant (n ≥ 3 pooled from 2 sets of independently generated chimeras). Ratios were normalized to naive CD4+ T cells. Graphs depict mean ± SEM. Statistical significance was assessed by 1-way (B) ANOVA with Tukey’s test, 2-way ANOVA with Dunnett’s test (C), or 2-tailed unpaired Student’s t test with the Holm-Šídák method (E–G). *P < 0.05; **P < 0.01; ****P < 0.0001. NS, not significant.