Volume 142, Issue 2, Pages 326-334.e2 (February 2012) Dysregulation of CD1d-Restricted Type II Natural Killer T Cells Leads to Spontaneous Development of Colitis in Mice  Chia–Min Liao, Michael I. Zimmer, Sharmila Shanmuganad, Hon–Tsen Yu, Susanna L. Cardell, Chyung–Ru Wang  Gastroenterology  Volume 142, Issue 2, Pages 326-334.e2 (February 2012) DOI: 10.1053/j.gastro.2011.10.030 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 CD1d overexpression affects 24αβ T-cell development in CD1dTg/24αβTg mice. (A) Thymocytes and splenocytes from indicated mice were stained with monoclonal antibody against Vα3.2 and Vβ9 and analyzed by flow cytometry. Data are representative of 6 experiments. (B and C) Bar graphs depict the mean ± SEM of the (B) percentages and (C) absolute numbers of Vα3.2+Vβ9+ cells from 24αβTg (n = 6) and CD1dTg/24αβTg (n = 6) mice. **P < .01; ***P < .001. Gastroenterology 2012 142, 326-334.e2DOI: (10.1053/j.gastro.2011.10.030) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Increased CD1d expression leads to enhanced negative selection of 24αβ T cells in CD1dTg/24αβTg mice. (A) Thymocytes from indicated mice were stained with monoclonal antibody against Vα3.2, Vβ9, CD4, and CD8 and analyzed by flow cytometry. Data shown are representative of 3 experiments. (B) Bar graphs depict the number of DN, DP, CD4SP, and CD8SP Vα3.2+Vβ9+ cells in the thymus of 24αβTg (n = 8) and CD1dTg/24αβTg (n = 8) mice. (C) Histograms depict the expression of Nur77 on thymocytes (solid line) compared with isotype control staining (filled histogram). Data shown are representative of 2 experiments. **P < .01, ***P < .001. Gastroenterology 2012 142, 326-334.e2DOI: (10.1053/j.gastro.2011.10.030) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Altered 24αβ T-cell surface phenotype and function observed in CD1dTg/24αβTg mice. (A) Flow cytometric analysis of Vα3.2 and Vβ9 expression on splenocytes from 24αβTg and CD1dTg/24αβTg mice. (B) Expression of NK1.1, CD4, and CD8 on Vα3.2+Vβ9+ cells in the spleen and mesenteric lymph nodes of indicated mice. Data shown are representative of 5 experiments. (C) Mesenteric lymph node lymphocytes from indicated mice were stimulated with anti-CD3 and intracellularly stained for the indicated cytokines. Data shown are representative of 3 experiments. Gastroenterology 2012 142, 326-334.e2DOI: (10.1053/j.gastro.2011.10.030) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 CD1dTg/24αβTg mice develop severe spontaneous inflammation of the large intestine. (A) 24αβTg and CD1dTg/24αβTg mice (n = 45 each) were monitored from 3 to 24 weeks of age for symptoms of IBD. (B) Gross observation of representative colon and cecum from 24αβTg and CD1dTg/24αβTg mice (n = 10; scale bar = 1 cm). (C) H&E-stained sections of representative colon samples from indicated mice (original magnification 200×; scale bars = 100 μm). (D) Histologic score of colon samples from 24αβTg (n = 15) and CD1dTg/24αβTg mice (n = 21). (E) Colon mononuclear cells from 24αβTg (n = 4) and CD1dTg/24αβTg (n = 6) mice were stained with various monoclonal antibodies to identify 24αβ T cells, non–type II NKT cells (T cells expressing the Tg TCR α or β chain alone), B cells, granulocytes (TCRβ−Gr1hiCD11b+), and dendritic cells (TCRβ−CD11b+CD11c+), respectively. Bar graphs depict the mean ± SEM for the proportion of cells within the indicated gate. (F) Colon mononuclear cells from indicated mice were stimulated and intracellularly stained for IFN-γ and IL-17 by Vα3.2+Vβ9+ cells. Data shown are representative of 3 experiments. *P < .05, **P < .01, and ***P < .001. Gastroenterology 2012 142, 326-334.e2DOI: (10.1053/j.gastro.2011.10.030) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 CD1dTg/24αβTg/RAG−/− mice spontaneously develop IBD. (A) Splenocytes and colon mononuclear cells from indicated mice were stained with monoclonal antibody to Vα3.2 and Vβ9 and analyzed using flow cytometry. Data are representative of 3 experiments. (B) 24αβTg/RAG−/− (n = 29) and CD1dTg/24αβTg/RAG−/− mice (n = 27) were monitored from 4 to 28 weeks of age for signs of IBD. (C) Dot plots and histograms depict the expression of indicated markers on Vα3.2+Vβ9+ cells in the spleen from both genotypes of mice. Data shown are representative of 5 experiments. (D) Hepatic leukocytes from 24αβTg/RAG−/− (n = 7) and CD1dTg/24αβTg/RAG−/− mice (n = 8) were stimulated with phorbol myristate acetate/ionomycin and intracellularly stained for IFN-γ production by Vα3.2+Vβ9+ cells. Data shown are representative of 4 experiments. *P < .05. Gastroenterology 2012 142, 326-334.e2DOI: (10.1053/j.gastro.2011.10.030) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Dysregulated type II NKT cell responses directly contribute to intestinal inflammation. (A) Sorted T cells from 24αβTg/RAG−/− or CD1dTg/24αβTg/RAG−/− mice were adoptively transferred into either CD1+RAG−/− or CD1dTg/RAG−/− recipient mice. H&E-stained sections are representative of colons from recipient mice (original magnification 100×; scale bars = 100 μm). (B) Histologic scores of colon samples from recipient mice in each experimental group. Gastroenterology 2012 142, 326-334.e2DOI: (10.1053/j.gastro.2011.10.030) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 CD4+ Foxp3+ T cells can be detected in 24αβTg and CD1dTg/24αβTg mice. Lymphocytes from 24αβTg and CD1dTg/24αβTg mice were isolated from various organs and stained with mAb to Vα3.2 and Vβ9, together with CD4. Cells were then intracellularly stained for Foxp3 and analyzed by flow cytometry. Data shown are representative of three individual experiments. Gastroenterology 2012 142, 326-334.e2DOI: (10.1053/j.gastro.2011.10.030) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 A diminished population of Vα3.2+ GFP+ cells is found in CD1dTg/24αβTg/4get mice. Lymphocytes were isolated from the thymus and spleen of 24αβTg/4get and CD1dTg/24αβTg/4get mice, stained with mAb to Vα3.2 and analyzed by flow cytometry. Numbers indicate the proportion of 4get+ cells in the Vα3.2+ gate in the indicated organs of 24αβTg/4get (n=4) and CD1dTg/24αβTg/4get (n=6) mice. Results are representative of three independent experiments. Gastroenterology 2012 142, 326-334.e2DOI: (10.1053/j.gastro.2011.10.030) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 A potential pathogenic role of 24αβ T cells in DSS-induced colitis. (A) Wild type and 24αβTg mice were treated with 3% DSS in the drinking water for six days and then switched to distilled, deionized water. Body weight was measured daily. Relative body weight (%) is compared to that from day 0 with standard errors. Untreated WT (n=3), 24αβTg (n=4), DSS-WT (n=11), DSS-24αβTg (n=9). (B) IFN-γ was determined by supernatant of whole colon organ culture for 24 hr from wild type or 24αβTg mice treated with DSS compared to untreated groups. WT (n=5), 24αβTg (n=4), DSS-WT (n=6), DSS-24αβTg (n=6). *, P < .05. Gastroenterology 2012 142, 326-334.e2DOI: (10.1053/j.gastro.2011.10.030) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 Antibiotic treatment reduced the severity of IBD in CD1dTg/24αβTg mice. CD1dTg/24αβTg mice were treated and untreated with broadspectrum antibiotic water for six weeks. Mice were monitored for the symptoms of IBD. (A) H&E stained sections of representative colon samples from treated and untreated CD1dTg/24αβTg mice (100×; Scale bar =100 μm). (B) Histological score of colon samples (based on the severity and extent of inflammation and crypt damage) taken from treated (n=6) and untreated (n=4) CD1dTg/24αβTg mice. **, P < .01. Gastroenterology 2012 142, 326-334.e2DOI: (10.1053/j.gastro.2011.10.030) Copyright © 2012 AGA Institute Terms and Conditions