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 5

Reconstitution of WT Treg compartment does not restore CD8+ T cell homeostasis in competitive chimeras.

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Reconstitution of WT Treg compartment does not restore CD8+ T cell homeo...
(A) Splenocytes from WT, Nr4a3–/–, Nr4a1–/–, and gDKO mice were stained to detect CD8+ T cell subsets on the basis of CD44 and CD62L expression. Plots are representative of ≥5 mice/genotype. (B) Quantification of splenic CD44hiCD8+ 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) Flow plots showing the peripheral CD8+ T cell subsets in competitive chimeras, as described for A above. Representative of ≥7 chimeras of each type. (D) Quantification of splenic CD44hiCD8+ T cells from chimeras as gated in C at varied time points posttransplant (n ≥ 3). (E) Ratio of CD45.2 to CD45.1/2 for CD8+CD44hi population as gated in C, normalized to naive CD8+CD44loCD62Lhi gate (n ≥ 3). Data in C–E pooled from 2 sets of independently generated chimeras. (F and G) Thymocytes and splenocytes from CD8-cre and CD8-cre Nr4a1fl/fl Nr4a3–/– (cDKO) mice were stimulated with PMA and ionomycin (PMA/Io) for 2 hours. Flow plots show intracellular NUR77 expression following fixation and permeabilization within thymic and splenic T cell subsets (F). Quantification of NUR77 MFI in T cell subsets (G) (n = 3 mice/genotype). (H) Quantification of splenic CD8+CD44hi T cells from CD8-cre, Nr4a3–/–, CD8-cre Nr4a1fl/fl, and CD8-cre cDKO mice (n = 3 mice/genotype). Graphs depict mean ± SEM. Statistical significance was assessed by 1-way ANOVA with Tukey’s test (B and H) or 2-way ANOVA with Dunnett’s test (D and E). ***P < 0.001; ****P < 0.0001. NS, not significant.