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 2

DKO thymocytes have a cell-intrinsic defect in negative selection.

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DKO thymocytes have a cell-intrinsic defect in negative selection. (A) F...
(A) Flow plots show thymic subsets in WT, Nr4a3–/–, Nr4a1–/–, and gDKO mice. Representative of n ≥ 4 mice/genotype. (B) Quantification of thymic subset cell number as gated in A; (n ≥ 4, 3 to 4-week-old gDKO and 5- to 6-week-old mice of other genotypes). (C) Ratio of CD45.2 to CD45.1/2 thymocytes among thymic DN3a and DN3b subsets (as gated in Supplemental Figure 2B), normalized to DN2 subset (n = 3–4 chimeras). (D) Flow plots show thymic subsets in competitive chimeras. Representative of ≥3 mice/genotype. (E) Ratio of CD45.2 to CD45.1/2 thymic subsets as gated in D normalized to DP subset (n ≥ 3). Data in C–E were from 6 to 7 weeks posttransplant chimeras pooled from 3 sets of independently generated chimeras. (F–K) Thymocytes from 1:1 DKO:WT chimeras were cultured with varying doses of plate-bound anti-CD3 and 2 μg/mL of anti-CD28 for 24 hours. Cells were stained to detect CD4/CD8 surface markers, followed by permeabilization and detection of active Caspase3 (aCasp3). Representative plots show aCasp3 expression in WT CD45.1/2 and DKO CD45.2 DP (F) and CD4SP (I) thymocytes from 1:1 DKO chimeras cultured with 10 μg/mL anti-CD3. Quantification percentage aCasp3+ cells among DP (G and H) or CD4SP (J and K) in 1:1 DKO:WT (G and J) or 1:1 WT:WT (H and K) chimeras (n = 3 from 1 chimera setup). Graphs depict mean ± SEM. Statistical significance was assessed by 1-way (B) or 2-way (C and E) ANOVA with Tukey’s test or 2-tailed unpaired Student’s t test with the Holm-Šídák method (G, H, J, and K). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS, not significant.