1. Blockade of Glucocorticoid-Induced Tumor Necrosis Factor–Receptor-Related Protein Signaling Ameliorates Murine Collagen-Induced Arthritis by Modulating Follicular Helper T Cells 
Jie Ma, Dingqi Feng, Yancai Wei, Jie Tian, Xinyi Tang, Ke Rui, Liwei Lu, Huaxi Xu, Shengjun Wang 
The American Journal of Pathology 
Volume 186, Issue 6, Pages (June 2016)
DOI: /j.ajpath
Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

2. Figure 1
Up-regulated glucocorticoid-induced tumor necrosis factor–receptor-related protein (GITR) expression in CD4+ follicular helper T (Tfh) cells compared with non-Tfh cells in type II collagen (CII)-immunized mice. Flow cytometry analysis of GITR expression in Tfh cells and non-Tfh cells from the spleens of collagen-induced arthritis (CIA) mice. DBA/1J mice were immunized with complete Freund's adjuvant plus CII. Purified CD4+ T cells from the spleens were stained for CXC receptor type 5 (CXCR5), inducible costimulator (ICOS), and GITR and analyzed by flow cytometry. n >3 independent experiments.
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3. Figure 2
Increased frequency and number of CD4+ follicular helper T (Tfh) cells that received glucocorticoid-induced tumor necrosis factor–receptor-related protein ligand (GITRL) treatment in vitro. Splenic CD4+ T cells from normal DBA/1J mice were purified by MACS beads and cultured with different concentrations of GITRL (0.1, 1.0, and 10.0 μg/mL) or control protein treatment. A: The frequencies of Tfh cells in cultures with different treatments are indicated in the representative flow cytometry profiles. B: The total number of Tfh cells in the culture. The expression levels of IL-21 (C) and Bcl-6 (D) mRNA in cultured CD4+ T cells were determined by quantitative RT-PCR analysis. The relative mRNA expression levels of IL-21 or Bcl-6/β-actin from three independent experiments are presented. E: The concentration of IL-21 in the supernatant of T-cell cultures was determined by enzyme-linked immunosorbent assay. The results are representative of three independent experiments. Data are expressed as means ± SD. n = 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
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Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

4. Figure 3
Glucocorticoid-induced tumor necrosis factor–receptor-related protein (GITR)-Fc protein decreases the severity of the disease in collagen-induced arthritis (CIA) mice. A total of 100 g of bovine type II collagen dissolved in 0.1 mol/L acetic acid was emulsified with an equal volume of complete Freund's adjuvant and administered intradermally at the base of the tail into DBA/1J mice. On day 21, a booster emulsion was administered intradermally. Following the same protocol, adjuvant-treated littermates that were given PBS in place of CII served as controls. Starting from day 22, GITRL protein, GITR-Fc protein, and control protein were injected into the tail vein of the mice (100 μg/mice) every 2 days. A: The development of arthritis among the immunized mice treated with PBS (PBS-CIA), control protein (CTL-CIA), GITRL (GITRL-CIA), and GITR- Fc (GITR-Fc-CIA) was monitored every 2 days. B: The clinical scores of arthritis severity were assessed in the PBS-CIA, CTL-CIA, GITRL-CIA, and GITR-Fc-CIA groups. C: The histopathologic scores of the joint tissue from the PBS-CIA, CTL-CIA, GITRL-CIA, and GITR-Fc-CIA groups. The values are expressed as the means ± SD. D: The joints were sectioned for H&E staining. n = 20 per group (A and B); n = 6 per group (C). ∗P < 0.05, ∗∗P < 0.01. Original magnification, ×100.
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5. Figure 4
Decreased CD4+ follicular helper T (Tfh) cell number in glucocorticoid-induced tumor necrosis factor–receptor-related protein (GITR)-Fc protein-treated collagen-induced arthritis (CIA) mice. A: The frequency of splenic CD4+ CXC receptor type 5 (CXCR5)+ inducible costimulator (ICOS)high cells was analyzed by flow cytometry. The percentages of CD4+CXCR5+ICOShigh cells are indicated in the representative flow cytometry profiles (boxed areas). B: The percentage of CD4+CXCR5+ICOShigh cells in the spleens from the four treatment groups. C: The percentage of CD4+CXCR5+ICOShigh cells in the lymph node (LN) from the four treatment groups. D: An immunofluorescence analysis of CD4+CXCR5+ cells in the spleens from the four treatment groups. The spleens of the mice were dissected 42 days after the first immunization. Staining was performed with anti-CXCR5 (green) and anti-CD4 (red) monoclonal antibody, and the combined staining is shown in yellow. The spleens from the GITR ligand (GITRL)-CIA group showed more CD4+CXCR5+ cells compared with the spleens from the CTL-CIA and PBS-CIA groups. The relative expression levels of IL-21 mRNA from cells in the spleen (E) and Bcl-6 mRNA from the CD4+ T cells in the spleen (F) were assessed by real-time quantitative PCR. The relative expression of IL-21 or Bcl-6/β-actin is presented as the means ± SD. G: The concentrations of IL-21 in the serum from immunized mice were determined by enzyme-linked immunosorbent assay. n = 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P <  Original magnification, ×200. CTL, control; CTL-CIA, mice treated with control protein; GITRL-CIA, mice treated with GITRL; PBS, phosphate-buffered saline; PBS-CIA, mice treated with PBS; GITR-Fc-CIA, mice treated with GITR-Fc.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

6. Figure 5
Decreased antibody production in the glucocorticoid-induced tumor necrosis factor–receptor-related protein (GITR)-Fc protein-treated collagen-induced arthritis (CIA) mice. The percentage (A) and number (B) of CD19+ cells in the spleens from the immunized mice. The percentage of CD138+ cells in the spleens (C) and bone marrow (BM; D) of the immunized mice. E: An immunohistochemistry analysis of the GC in the spleen of the immunized mice. The spleen was dissected out of the mice 42 days after the first immunization. F: Anti-CII–specific antibodies (IgG) were measured in the sera from the four treatment groups by enzyme-linked immunosorbent assay. G: Anti-CII–specific antibodies (IgG1) were measured in the sera from the four treatment groups by enzyme-linked immunosorbent assay. H: Anti-CII–specific antibodies (IgG2a) were measured in the sera from the four treatment groups by enzyme-linked immunosorbent assay. I: Anti-CII–specific antibodies (IgG2b) were measured in the sera from the four treatment groups by enzyme-linked immunosorbent assay. J: The number of IgG-secreting cells in the bone marrow was analyzed by ELISpot. K: The number of CII-specific IgG-secreting cells in the bone marrow. L: The number of IgG-secreting cells in the spleen. M: The number of CII-specific IgG-secreting cells in the spleen. n = 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P <  Original magnification, ×200. CII, type II collagen; CTL, control; CTL-CIA, mice treated with control protein; GITR-Fc-CIA, mice treated with GITR-Fc; GITRL-CIA, mice treated with GITRL; OD, optical density; PBS, phosphate-buffered saline; PBS-CIA, mice treated with PBS; SP, spleen.
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7. Supplemental Figure S1
Effects of glucocorticoid-induced tumor necrosis factor–receptor-related protein ligand (GITRL) on proliferation of CD4+ CXC receptor type 5 (CXCR5)+ T cells. Splenic CD4+CXCR5+ T cells were purified from mice spleen cell suspensions by MACS. After staining with carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE) the cells were treated with either 0.1 to 10.0 μg/mL GITRL protein or control protein for 72 hours. Representative flow plots of cells with CFSE are shown on the x axis, and count is shown on the y axis. n = 3 independent experiments.
The American Journal of Pathology  , DOI: ( /j.ajpath )
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8. Supplemental Figure S2
Effects of glucocorticoid-induced tumor necrosis factor–receptor-related protein ligand (GITRL) on apoptosis of CD4+ CXC receptor type 5 (CXCR5)+ T cells. Splenic CD4+ T cells were purified from mice spleen cell suspensions by MACS and then treated with 0.2 μg/mL dexamethasone (DEX), in the presence of either 1.0 to 10 μg/mL GITRL protein or control protein for 6 hours. A: Representative flow plots of cells with Annexin V on the x axis and propidium iodide on the y axis. Lower left shows normal cells, lower right, early apoptotic cells, and upper right, late apoptotic and necrotic cells. B: Percentage of early apoptotic cells in different groups. n = 3 independent experiments. ∗∗P < 0.01, ∗∗∗P < 0.001.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2016 American Society for Investigative Pathology Terms and Conditions