1. Volume 142, Issue 3, Pages 582-591.e8 (March 2012)
Glucocorticoid-Induced Tumor Necrosis Factor Receptor Family-Related Protein Regulates CD4+T Cell–Mediated Colitis in Mice 
Gongxian Liao, Cynthia Detre, Scott B. Berger, Pablo Engel, Rene de Waal Malefyt, Roland W. Herzog, Atul K. Bhan, Cox Terhorst 
Gastroenterology 
Volume 142, Issue 3, Pages e8 (March 2012)
DOI: /j.gastro
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2. Figure 1
CD4+ T cells induce colitis in GITR−/− × Rag−/− but not in Rag−/− recipients. CD4+ T cells obtained from wt spleens by FACS were intraperitoneally injected into Rag−/− (n = 17) or GITR−/− × Rag−/− (n = 14) hosts (1 × 106 cells/mouse). Mice were euthanized when signs of diarrhea, hunching, and wasting disease manifested. Statistical significance was determined by 2-tailed Student t test. (A) Weight loss as a percentage of the initial weight. Values represent the mean weight ± SD. (B and C) DAI and histology score. Mean and individual values of each group are indicated. (D) Representative histology. Original magnification 10×. (E) Cytokine production by short-term colon cultures. Colon tissue was isolated from GITR−/− × Rag−/− (n = 7) and Rag−/− (n = 6) mice with wt CD4+ T-cell transfer and cultured for 24 hours. Supernatants were collected after centrifugation and analyzed for inflammatory cytokines with the multiplex cytometric bead array assay. (F) MLN CD4+ T cells differentiate into Th1 cells upon transfer into GITR−/− × Rag−/− mice. MLN cells isolated from GITR−/− × Rag−/− (n = 5) and Rag−/− (n = 5) mice into which wt CD4+ T cells had been transferred were stimulated with 10 μg/mL of plate-bound αCD3ϵ for 24 hours. Cells were stained with monoclonal antibodies against CD4, IFN-γ, and IL-17A. Representative staining of each group is shown.
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3. Figure 2
GITR−/− CD4+ T cells induce colitis upon transfer into GITR−/− × Rag−/− recipients. CD4+ T cells obtained from GITR−/− spleens by FACS were intraperitoneally injected into GITR−/− × Rag−/− or Rag−/− hosts (1 × 106 cells/mouse). Disease parameters were determined as described in Materials and Methods and Figure 1. (A) DAI. (B) Histology score. (C) Cytokine production by short-term colon cultures. Ex vivo colon cultures from GITR−/− × Rag−/− (n = 8) and Rag−/− (n = 8) mice into which GITR−/− CD4+ T cells had been transferred were prepared for determining the inflammatory cytokines as in Figure 1.
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4. Figure 3
Treg cells do not suppress colitis after their cotransfer with CD45RBhi cells into GITR−/− × Rag−/− recipients. GITR+/+ or GITR−/− CD4+CD45RBhi cells alone (5 × 105 cells/mouse) or together with CD4+CD25+ cells (5 × 104 cells/mouse) were transferred to Rag−/− or GITR−/− × Rag−/− recipients. Mice were euthanized at week 4 after cell transfer. DAI and histology score were assigned as described in Figure 1. Each filled or open circle represents one mouse. Statistical significance was determined by the 2-tailed Student t test. Mean and individual values of each group are indicated. (A and B) DAI and histology score upon cell transfer into GITR−/− × Rag−/− recipients. (C and D) DAI and histology score upon cell transfer into Rag−/− recipients. (E) Weight loss as a percentage of the initial weight after transfer of CD4+CD45RBhi cells into Rag−/− and GITR−/− × Rag−/− recipients. Values represent the mean weight ± SD. (F) DAI on transfer of CD4+CD45RBhi cells into Rag−/− and GITR−/− × Rag−/− recipients.
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5. Figure 4
GITR deficiency in Rag−/− recipients caused an overexpansion of naïve T cells as well as a loss of FoxP3 expression on Treg cells. CD4+CD45RBhi naïve T cells sorted from Thy1.1+ B6 congenic mice were mixed with CD4+FoxP3+ Treg cells from Thy1.2+ FoxP3-IRES-GFP reporter mice at a 9:1 ratio and adoptively transferred to GITR−/− × Rag−/− or Rag−/− hosts (5 mice for each group). Mice were weighed weekly and euthanized for colitis activity as described in Figure 1. Spleen, MLN, and lamina propria cells from GITR−/− × Rag−/− or Rag−/− recipients were isolated and evaluated for FoxP3+, Thy1.1+, and Thy1.2+ cells. (A) Weight loss as a percentage of the initial weight. Values represent the mean weight ± SD. (B) DAI. Mean and individual values of each group are indicated. (C) Ratio of Thy1.1+ and Thy1.2+ cells after cell transfer. Thy1.1+ cells represent CD4+CD45RBhi naïve T cells. Thy1.2+ cells represent FoxP3+ (GFP+) cells. (D) Percentage of FoxP3(GFP)+/Thy1.2+ cells after cell transfer. (E) Percentage of FoxP3+ cells expressing Thy1.1+ and Thy1.2+. Splenocytes from GITR−/− × Rag−/− or Rag−/− recipients were stained with CD4, Thy1.1, and FoxP3. Thy1.1- and FoxP3-expressing cells were compared among gated CD4+ T cells.
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6. Figure 5
GITR expression by non-T hematopoietic cells, but not GITR-L by CD4+ T cells, abrogated the Treg-mediated suppression. (A) DAI. (B) Histology score. GITR-L−/− CD4+CD45RBhi (5 × 105 cells/mouse) and CD4+CD45RBlo (5 × 105 cells/mouse) T cells were coinjected into GITR−/− × Rag−/− or Rag−/− hosts. DAI and histology scores were determined as in Figure 1. Mean and individual values of each group are indicated.
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7. Figure 6
GITR expression on the surface of lamina propria DCs and macrophages of colitic mice and on splenic monocyte-derived cells upon activation. (A) DCs and macrophages isolated from the lamina propria of colitic Rag−/− mice express GITR. CD4+CD45RBhi wt T cells were transferred to Rag−/− recipients (5 × 105 cells/mouse). Once diarrhea started, monocyte-derived cells were isolated from the colonic lamina propria and stained with αCD11c, αCD11b, αLy6C, and αGITR. Different cell subsets were gated to compare the GITR expression levels. The bold black line represents αGITR, and the gray shaded area represents immunoglobulin G isotype control. (B and C) Lipopolysaccharide (LPS) induces GITR expression on splenic DCs and macrophages. Freshly isolated wt or GITR−/− splenocytes (upper panel) or those stimulated with LPS (lower panel) for 24 hours were stained with monoclonal antibodies directed against CD11c, Ly6C, and GITR. The expression levels of GITR on different cell subpopulations (gated as in Figure 6B) were compared. The bold black line represents GITR+/+ cells, and the gray shaded area represents GITR−/− cells. (D and E) IDO protein levels and enzymatic activity in the colon of GITR−/− × Rag−/− mice are identical to that of Rag−/− mice. IDO protein levels and enzymatic activity in the colon extracts of GITR−/− × Rag−/− and Rag−/− mouse were measured by immunoblot with an αIDO antibody and by the production of L-kynurenine, respectively.
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8. Figure 7
DCs from MLN of colitic GITR−/− mice preferentially drive Th1 responses instead of Treg expansion. (A) GITR−/− DCs from MLN preferentially stimulate Th1 cells. CD11c+ DCs from MLN of wt and GITR−/− mice were primed with 2 mg/mL of chicken OVA for 18 hours. OVA-loaded DCs were irradiated (3000 rad) and cocultured with OTII CD4+ T cells at a 1:5 ratio for 3 days. Supernatants were used for assessing the cytokines by cytometric bead array. Data represent mean ± SD of triplicate. Results are representative of 3 individual experiments. (B) Cytokines secreted by CD4+ T cells in the presence of splenic DCs. CD11c+ DCs from spleen of wt and GITR−/− mice were loaded with OVA protein and used to stimulate OTII CD4+ T cells as described in A. Supernatants were used for assessing the cytokines by cytometric bead array. Data represent mean ± SD of triplicate. (C and D) Percentage of MLN CD103+ DCs and splenic PDCA1+CCR9+ pDCs of GITR−/− mice was reduced under steady state. (C) MLN and (D) spleen cells from wt and GITR−/− were stained for comparing the percentage of different DC subsets. Each filled or open circle represents one mouse. Mean and individual values of each group are indicated. (E) Reduced number of pDCs in spleen of GITR−/− × Rag−/− mice without transfer of CD4+ T cells. Splenocytes from Rag−/− and GITR−/− × Rag−/− mice (5 for each group) were stained for MHCII, CD11c, and PDCA1. (F) Reduced number of pDCs in GITR−/− × Rag−/− mice during inflammation induced by CD4+ T cells. CD4+ T cells from wt mice were adoptively transferred into GITR−/− × Rag−/− and Rag−/− recipients (5 for each group). Mice were euthanized at week 4. Spleen, MLN, or colon lamina propria cells were pooled for CD11c, PDCA1 staining. The result represents 2 separate experiments.
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9. Supplementary Figure 1
Generation of GITR-L−/− C57BL/6 mouse. (A) Schematic strategy to generate GITR-L−/− C57BL/6 mouse. Briefly, GITR-L knockout vector was electroporated into ∼107 B6-3 ES cells and selected with 200 μg/mL G418. One G418-resistant positive clone P9 was electroporated with 35 μg of pCAGGS-FLPe supercoil plasmid to remove the neomycin-resistant gene. G418-sensitive clones were selected for further genotyping. (B) Protocol to genotype GITR-L−/− deficiency. Tail DNA was isolated to analyze the genotype of the GITR-L−/− deficiency by polymerase chain reaction. WT band is 101 base pairs, and knockout band is 217 base pairs.
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10. Supplementary Figure 2
Histology. Different CD4+ T-cell subsets were adoptively transferred into GITR−/− × Rag−/− and/or Rag−/− recipients. Representative photomicrographs show H&E-stained sections of colons. Original magnification 10×. (A) GITR−/− CD4+ T cells induce colitis upon transfer into GITR−/− × Rag−/− recipients. GITR−/− CD4+ T cells were adoptively transferred into Rag−/− and GITR−/− × Rag−/− recipients as described in Figure 2. (B) Treg cells do not prevent colitis after cotransfer with CD45RBhi cells into GITR−/− × Rag−/− recipients. WT and GITR−/− CD4+CD25+ T cells were adoptively cotransferred with GITR−/− CD45RBhi cells to GITR−/− × Rag−/− recipients as described in Figure 3A. (C) Treg cells suppress colitis after their cotransfer with CD45RBhi cells into Rag−/− recipients. WT and GITR−/− CD4+CD25+ Treg cells were adoptively cotransferred with wt CD45RBhi cells into Rag−/− recipients as described in Figure 3C. (D) GITR-L−/− CD4+CD45RBlo T cells prevent colitis after their cotransfer with CD45RBhi cells into Rag−/− but not into GITR−/− × Rag−/− recipients. GITR-L−/− CD45RBhi and/or CD45RBlo cells were adoptively transferred into Rag−/− and/or GITR−/− × Rag−/− recipients as described in Figure 4A.
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11. Supplementary Figure 3
The percentage of CD4+FoxP3+ cells was reduced after adoptive transfer of CD4+ T cells from FoxP3-IRES-GFP mice into GITR−/− × Rag−/− mice. CD4+ T cells isolated from FoxP3-IRES-GFP mice were adoptively transferred into Rag−/− (n = 6) and GITR−/− × Rag−/− (n = 5) recipients. DAI is assessed as in Figure 1. Four weeks after cell transfer, spleens, MLN, and lamina propria cells were isolated and stained with monoclonal antibody against CD4. FoxP3(GFP)+/CD4+ ratios were evaluated. (A) Schematic strategy of transfer of CD4+ T cells from FoxP3-IRES-GFP reporter mice to GITR−/− × Rag−/− and Rag−/− mice. (B) Unfractionated CD4+ T cells derived from FoxP3 reporter mice induce colitis in GITR−/− × Rag−/− but not in Rag−/− recipients. DAI, mean, and individual values of each group are shown. (C) Representative staining of FoxP3+CD4+ Treg cells in GITR−/− × Rag−/− and Rag−/− recipients upon transfer of CD4+ T cells. (D) Statistics of FoxP3+CD4+ Treg cells. Data represent mean ± SD of triplicate.
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12. Supplementary Figure 4
GITR−/− mice do not spontaneously develop colitis. Representative photomicrographs show H&E-stained sections of colons from 20-week-old GITR−/− mice. Original magnification 10×.
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13. Supplementary Figure 5
GITR−/− × Rag−/− mice do not spontaneously develop colitis. Representative photomicrographs show H&E-stained sections of colons from 20-week-old GITR−/− × Rag−/− mice. Original magnification 10×.
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14. Supplementary Figure 6
GITR−/− CD4+CD45RBhi T cells induce colitis in Rag−/− mice. WT (n = 5) and GITR−/− (n = 7) CD4+CD45RBhi T cells were adoptively transferred into Rag−/− mice. Mice were euthanized when signs of diarrhea, hunching, and wasting disease manifested. DAI and histology score were assigned as described in Materials and Methods. Each filled or open circle represents one mouse. Statistical significance was determined by the 2-tailed Student t test. (A) DAI. Mean (denoted by horizontal bar) and individual values. (B) Histology score. Mean (denoted by horizontal bar) and individual values. (C) Weight as a percentage of the initial weight. Values represent the mean weight ± SD. (D and E) IFN-γ and IL-6 level of colon explant cultures. Colon explants were cultured for 24 hours. Supernatants were collected for analyzing the cytokine IFN-γ by cytometric bead array.
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15. Supplementary Figure 7
CD4+FoxP3+ Treg cells do not induce colitis in GITR−/− × Rag−/− recipients. CD4+FoxP3+ Treg cells were sorted from FoxP3-IRES-GFP reporter mice and adoptively transferred to GITR−/− × Rag−/− and Rag−/− recipients as described in Figure 1. Mice were weighed weekly and euthanized to analyze the DAI at week 9 after cell transfer. (A) Weight as a percentage of the initial weight. Values represent the mean weight ± SD. (B) DAI. Mean (denoted by horizontal bar) and individual values. (C) The percentage of CD4+FoxP3+ Treg cells in the spleen. Splenocytes from GITR−/− × Rag−/− and Rag−/− recipients after transfer of CD4+FoxP3+ Treg cells were stained for CD4. The percentage of CD4+FoxP3+ Treg cells was evaluated. Mean and individual values are indicated. (D) Representative staining result in C.
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16. Supplementary Figure 8
CD4+CD25+ Treg cells efficiently inhibit the proliferation of CD4+CD25− Teff cells in vitro in the presence of GITR−/− × Rag−/− splenocytes as APCs. CD4+CD25− Teff cells from C57BL/6 spleen were labeled with carboxyfluorescein diacetate succinimidyl ester and cocultured with different doses of CD4+CD25+ Treg cells in the presence of 0.25 μg/mL of soluble αCD3ϵ and irradiated (3000 rad) Rag−/− or GITR−/− × Rag−/− splenocytes as APC. Cells were stained with CD4 after activation for 72 hours. (A) The numbers of proliferated CD4+ cells were normalized to those with CD4+CD25− Teff cells alone. Representative staining result is shown in B.
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17. Supplementary Figure 9
CD4+ T cells do not express GITR-L. (A) Neither Th1 nor Th-17 T cells in the lamina propria of colitic mice express GITR-L. WT CD4+CD45RBhi T cells were adoptively transferred into Rag−/− (n = 5) mice. Mice were euthanized when signs of diarrhea, hunching, and wasting disease became apparent. Lamina propria cells were isolated from the colons. After restimulation with phorbol myristate acetate/ionomycin for 4 hours, cells were stained with monoclonal antibodies against CD4, IFNγ, IL-17A, and 3 different anti–GITR-L monoclonal antibodies. CD4+IFNγ+ and CD4+IL-17A+ cells were gated to compare expression of GITR-L. The gray shaded area represents the immunoglobulin G isotype control, and the bold black line represents αGITR-L. Four different monoclonal anti–GITR-L antibodies, including clone #12.27 from Merck, clone #YGL386 from eBioscience and Biolegend, and clone #386.2 generated in our own laboratory, were used for the staining. (B) CD4+ T cells do not express GITR-L as judged by FACS. WT CD4+ T cells were isolated and activated with 5 μg/mL of plate-bound αCD3ϵ for 24 hours. Fresh or αCD3ϵ-activated CD4+ T cells were gated to compare expression of GITR-L. The gray shaded area represents the immunoglobulin G isotype control, and the bold black line represents αGITR-L. (C) 293T cells expressing mGITR-L are positive for staining using αGITR-L monoclonal antibody. 293T cells expressing mouse GITR-L were stained with immunoglobulin G isotype control (black line) and monoclonal antibody against GITR-L (cyan line, 0.1 μg/mL; orange line, 1 μg/mL). (D) CD4+ T cells do not express GITR-L as judged by polymerase chain reaction. RNA from fresh wt CD4+CD25+ and CD4+CD25− T cells or αCD3ϵ-activated CD4+CD25− T cells was isolated and used for reverse-transcription polymerase chain reaction to analyze the expression of GITR-L. GAPDH and CD25 were used as a housekeeping gene and activation-inducible gene controls, respectively. A GITR-L expression vector pST-GITR-L was used for positive control.
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18. Supplementary Figure 10
CD11c+ DCs isolated from the lamina propria of Rag−/− colon do not express GITR. Lamina propria cells were isolated from the colon of GITR−/− × Rag−/− and Rag−/− mice without inflammation. CD11c+ cells were gated to compare the expression of GITR. The gray shaded area represents GITR−/− × Rag−/− cells, and the bold black line represents Rag−/− cells.
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19. Supplementary Figure 11
Anti-CD40 induces colitis in GITR−/− × Rag−/− mice. Anti-CD40 (FGK45) was intraperitoneally injected into Rag−/− (n = 5) and GITR−/− × Rag−/− (n = 4) cells at 200 μg/mouse. DAI and weight were analyzed as described in Figure 1. (A) DAI. Mean and individual values of each group are indicated. (B) Weight as a percentage of the initial weight. Values represent the mean weight ± SD.
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20. Supplementary Figure 12
GITR deficiency affects the function and composition of tolerogenic DC subsets. (A) Increased number of IFN-γ–producing CD4+ T cells in the presence of GITR−/− DCs from MLN. Cells activated as in Figure 7A were stained with TCRvβ5-PerCP and IFN-γ-PE. TCRvβ5+ cells were gated to compare the cell proliferation and expression of IFN-γ. Representative of 3 separate experiments. (B) Combined results. The percentage of IFN-γ+/TCRvβ5+ was compared. Data represent mean ± SD of triplicate. (C and D) Gating strategy for staining tolerogenic DC subsets shown in Figure 7C and D. Spleen and MLN cells from wt and GITR−/− were stained for comparing the percentage of different DC subsets. (C) Gating strategy for staining MLN DCs shown in Figure 7C. (D) Gating strategy for staining splenic DCs shown in Figure 7D.
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