Research ArticleCELL BIOLOGY

The orphan nuclear receptor LRH-1/NR5a2 critically regulates T cell functions

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Science Advances  17 Jul 2019:
Vol. 5, no. 7, eaav9732
DOI: 10.1126/sciadv.aav9732
  • Fig. 1 Deletion of Nr5a2 leads to loss of mature T lymphocytes.

    (A) Representative picture of Nr5a2L2/L2 (L2/L2, ctrl) and Nr5a2L2/L2 CD4-Cre (cKO) spleens. ctrl, control. (B) Spleen organ weight relative to body weight (L2/L2, n = 17; cKO, n = 18 mice). (C) Spleen cellularity (n = 15). (D) Representative density plots of splenic CD4+ and CD8+ cells. The numbers indicate the percentage of cells. PE, phycoerythrin; FITC, fluorescein isothiocyanate. (E to G) Relative distribution of splenic CD3+ (E), B220+ (F), or NK1.1+ (G) CD3+ cells analyzed by flow cytometry (L2/L2, n = 11 mice; cKO, n = 12 mice per group). (H and I) Absolute numbers of splenic CD3+CD4+ and CD3+CD8+ T lymphocytes (L2/L2, n = 23; cKO, n = 17). (J) Representative hematoxylin and eosin (H&E) staining of L2/L2 or cKO spleens. (K) Representative immunohistology for CD3 (yellow) and B220 (blue) expression. (L) Representative staining for CD3ε (yellow), F4/80 (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 0.5 cm (A) and 300 μm (J to L). Mean values ± SD and individual values are shown in each graph. *P < 0.05 and ***P < 0.001. ns, not significant. Photo credit: Carina Seitz, University of Konstanz.

  • Fig. 2 LRH-1–deficient T cells are not anergic.

    (A and B) Up-regulation of the early activation marker CD69 (A) and CD25 (B) in unstimulated (unstim) or anti-CD3 (3 μg/ml) and anti-CD28 (1 μg/ml) (stim) CD4+ or CD8+ splenic T cells (n = 3 mice). (C and D) Production of interferon-γ (IFN-γ) (C) or interleukin-2 (IL-2) (D) in purified CD4+ or CD8+ splenic T cells. Cells were stimulated as indicated for 48 hours, and cytokine secretion was determined by enzyme-linked immunosorbent assay (ELISA) (n = 4 mice). Mean values ± SD of triplicates or quadriplicates of representative experiments are shown. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 Nr5a2 expression is up-regulated upon stimulation.

    (A) Nr5a2 mRNA expression in wild-type spleenocytes after 3 hours of phorbol myristate acetate (PMA) and ionomycin (iono) or ConA stimulation, normalized to unstimulated control (n = 3 mice per group). (B) NR5A2 mRNA expression in human PBMCs (hPBMCs) after stimulation with phytohemagglutinin (PHA-P) for 3 hours (n = 3 biological replicates). (C to E) Kinetics of (C) Myc, (D) Nr5a2, and (E) Cdne1 expression in splenocytes after anti-CD3/anti-CD28 stimulation normalized to unstimulated control (n = 3 mice). (F) Induction of NR5A2 promoter activity in Jurkat lT cells after stimulation with PMA and ionomycin. One representative experiment of five is shown (n = 3 technical replicates). (G) Effect of serum deprivation on NR5A2 promoter activity. One of three representative experiments is shown (n = 3 technical replicates). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 4 Impaired cell proliferation in LRH-1–deficient T cells.

    (A and B) Cell proliferation in response to anti-CD3 ± anti-CD28 stimulation of splenic CD4+ and CD8+ T cells was analyzed by CFSE dilution after 72 hours. Mean values ± SD of three individual experiments are shown. (C and D) Spleen cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of the LRH-1 antagonist 3d2 or control substance cd7 at indicated concentrations, and proliferation was analyzed by CFSE dilution at 72 hours. Values were normalized to stimulated cells without inhibitors (n = 3 mice). (E) Splenocytes were treated with or without the LRH-1 inhibitor SR1848 and stimulated with anti-CD3/anti-CD28; cell proliferation was analyzed by CFSE dilution at 72 hours (n = 3 mice). (F) Activation-induced 3H-thymidine incorporation in purified CD4+ and CD8+ splenic T cells after 72 hours, normalized to unstimulated cells. Mean values ± SD of a representative experiment are shown (n = 5 experiments). (G and H) Activation-induced Myc (G) and Cdne1 (H) mRNA expression in purified CD4+ and CD8+ splenic T cells after 24 hours. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 Impaired colitis induction by LRH-1–deficient CD4+T cells.

    (A) Scheme of transfer colitis induction in Rag1−/− recipient mice. (B) Colitis-induced loss of body weight (L2/L2 donor, n = 14; cKO donor, n = 9). (C to E) Representative histologies of colon samples from untreated Rag1−/− recipient mice (C) or after transfer of naïve L2/L2 (D) or cKO T cells (E). Top panels: Lower magnification. Bottom panels: Higher magnification. Scale bars, 150 μm. (F and G) Repopulation of spleen (F) or mesenteric lymph node (mLN) (G) by naïve L2/L2 or cKO T cells. (H to J) mRNA expression levels of the proinflammatory cytokines tumor necrosis factor–α (H), IL-1β (I), and IFN-γ (J) in colon samples or control recipients or recipients transferred with L2/L2 or cKO T cells. Mean ± SD and individual data points of n = 9 to 14 mice are shown. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 Impaired virus clearance by LRH-1–deficient cytotoxic T cells.

    (A) Virus-induced expansion of CD8αβ+ TCRαβ+ T cells [0 days post infection (dpi), n = 11; 6 dpi, n = 9; 8 dpi, n = 3 mice]. (B and C) Up-regulation of activation marker CD69 (B) and CD25 (C) in CD8+ T cells after LCMV infection (0 dpi, n = 11; 6 dpi, n = 9; 8 dpi, n = 6 mice). (D) Quantification of IFN-γ–producing virus-specific CD8+ T cells after ex vivo restimulation with buffer control, Adn5 control peptide or GP33 peptide (0 dpi: n = 8; 6 dpi: n = 9; 8 dpi: n = 6 mice). (E to H) Virus titers after LCMV infection of L2/L2 or cKO mice in the spleen (E), liver (F), small intestine (G), and serum (H) (6 dpi, n = 9; 8 dpi, n = 6; 10 dpi, n = 7 mice). (I) Immunohistological detection of LCMV-infected cells in liver sections in L2/L2 or cKO mice at days 6 and day 10 after infection. VL4 LCMV nucleoprotein, red; nuclei, blue (DAPI). Representative pictures are shown. Scale bars, 150 μm. (J) mRNA expression of the cytotoxic T cell effector molecule perforin (Prf1) (0 dpi, n = 5; 6 dpi, n = 5; 8 dpi, n = 6; 10 dpi, n = 2 mice). (K) DNA fragmentation of GP33 or Adn5-loaded EL4 cells by virus-specific T cells, 8 days after LCMV infection, measured by loss of 3H-thymidine (n = 3 mice; representative experiment of two is shown). *P < 0.05, **P < 0.01, ***P < 0.001. E:T, effector:target.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaav9732/DC1

    Fig. S1. Nr5a2 expression in immature and mature T lymphocytes.

    Fig. S2. Minor impact of Nr5a2 deletion on immature T lymphocytes.

    Fig. S3. Reduced mature T cells in axial and mesenteric lymph nodes of LRH-1–deficient mice.

    Fig. S4. Efficiency of LRH-1 deletion in immature and mature T cells.

    Fig. S5. Apoptosis induction in LRH-1–deficient thymocytes.

    Fig. S6. Apoptosis induction in LRH-1–deficient splenocytes.

    Fig. S7. Homeostatic expansion is impaired in LRH-1–deficient T cells.

    Fig. S8. Impaired ovalbumin-induced T cell expansion and antibody production in LRH-1–deficient mice.

    Fig. S9. Reduced Tregs function of LRH-1–deficient T cells.

    Fig. S10. Activation and cytotoxicity of LRH-1–deficient CD8+ T cells.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Nr5a2 expression in immature and mature T lymphocytes.
    • Fig. S2. Minor impact of Nr5a2 deletion on immature T lymphocytes.
    • Fig. S3. Reduced mature T cells in axial and mesenteric lymph nodes of LRH-1–deficient mice.
    • Fig. S4. Efficiency of LRH-1 deletion in immature and mature T cells.
    • Fig. S5. Apoptosis induction in LRH-1–deficient thymocytes.
    • Fig. S6. Apoptosis induction in LRH-1–deficient splenocytes.
    • Fig. S7. Homeostatic expansion is impaired in LRH-1–deficient T cells.
    • Fig. S8. Impaired ovalbumin-induced T cell expansion and antibody production in LRH-1–deficient mice.
    • Fig. S9. Reduced Tregs function of LRH-1–deficient T cells.
    • Fig. S10. Activation and cytotoxicity of LRH-1–deficient CD8+ T cells.

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