1. Volume 130, Issue 2, Pages 424-434 (February 2006)
CD48 Controls T-Cell and Antigen-Presenting Cell Functions in Experimental Colitis 
Ana C. Abadía–Molina, Honbing Ji, William A. Faubion, Aimée Julien, Yvette Latchman, Hideo Yagita, Arlene Sharpe, Atul K. Bhan, Cox Terhorst 
Gastroenterology 
Volume 130, Issue 2, Pages (February 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Terms and Conditions

2. Figure 1
Colitis does not develop after adoptive transfer of CD48−/−CD45RBhi cells into CD48−/− × Rag-2−/− mice. (A) Disease Activity Index of mice with chronic colitis. wt or CD48−/−CD45RBhi T cells were transferred into CD48−/− × Rag-2−/− mice or Rag-2−/− recipients. Median and independent values of each group are indicated. *P < .05, calculated comparing DAI scores of CD48−/−CD45RBhi→CD48−/− × Rag-2−/− mice with the mice in each other group. (B) Histology score of mice with chronic colitis. *P < 0.05, calculated comparing histology scores of CD48−/−CD45RBhi→CD48−/− × Rag-2−/− mice with the mice in each other group. (C) Representative histological analysis of colon tissue from wt or CD48−/− CD45RBhi→Rag-2−/− recipients or CD48−/− × Rag-2−/− recipients (original magnification 10×, except for CD48−/−→CD48−/− × Rag−/− [20×]). (D) IFN-γ detected by ELISA in the supernatant of CD4+ T cells from MLN of wt CD45RBhi→Rag-2−/− and wt or CD48−/−CD45RBhi→CD48−/− × Rag-2−/− mice. CD4+ T cells were activated with plate-bound anti-CD3 for 36 hours (see Materials and Methods). Representative data are shown of 1 mouse out of 3 mice of each group analyzed. *P < .05. (E) IFN-γ measured by ELISA in the supernatant of the colonic tissue incubated in vitro for 36 hours of the indicated groups of mice: wt CD45RBhi→Rag-2−/− (n = 3) or CD48−/− CD45RBhi→CD48−/− × Rag-2−/− (n = 4). *P < .05. (F) DAI score of wt CD45RBhi→Rag-2−/− and wt CD45RBhi + CD48−/−CD45RBlow→Rag-2−/− mice. CD48−/− Treg were capable to prevent colitis. Independent values and medians for each group of mice are shown. *P < .05. (G) Histology score of wt CD45RBhi→Rag-2−/− and wt CD45RBhi + CD48−/−CD45RBlow→Rag-2−/− mice. CD48−/− Treg were able to prevent colitis. Independent values and medians for each group of mice are shown. *P < .05. (H) CD4+CD25+ Treg cells from CD48−/− mice suppress CD4+CD25− cells in vitro. CD4+CD25+ T cells isolated from the spleen of CD48−/− mice were incubated at different ratios with wt CD4+CD25− splenocytes, soluble anti-CD3, and 1 × 105 syngeneic APCs from wt mice. CPM, counts per minute.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

3. Figure 2
The CD48 surface receptor modulates CD4+ T-cell stimulation and cytokine production in vitro. (A) Proliferation of CD48−/−CD4+ T cells in response to macrophages pulsed with cecal antigen. Proliferation of CD4+ T cells isolated from the spleen of CD48−/− mice, by using negative selection columns as described in Materials and Methods, was compared with that of wt mice. CD4+ T cells were stimulated in vitro with pMφ pulsed with cecal antigen for 3 days. The cultures were pulsed with H3 thymidine for the last 12 hours. Representative data out of 3 independent experiments are shown. *P < .05. (B) Proliferation of CD48−/− CD4+ T cells upon in vitro stimulation with T-cell–depleted splenocytes or stimulated with soluble anti-CD3 and anti-CD28. CD4+ T cells isolated from the spleen by using negative selection columns as described in Materials and Methods were stimulated for 3 days and were pulsed with H3 thymidine for the last 12 hours. Data are representative of 3 independent experiments. *P < .05. (C) Production of IL-2. wt or CD48−/− purified DO11.10-TCRtg CD4+CD25− T cells were incubated with either wt or CD48−/− APCs pulsed with ovalbumin peptide for 24 hours. IL-2 was detected in the supernatant of cell cultures by ELISA. Data represent mean ± SD of triplicates. Data are representative of 2 independent experiments. *P < .05. (D) Production of IL-10 by CD48−/− CD4+ T cells. CD4+ wt or CD48−/− T cells were stimulated in vitro with plate-bound anti-CD3 and soluble anti-CD28. Irrelevant IgG or anti-CD48 was also added. Supernatants were collected after 72 hours of culture. Data represent mean ± SD of triplicate wells. Data are representative of 2 independent experiments. *P < .05. (E) Production of IL-4 by CD48−/− CD4+ T cells stimulated as described previously. Data represent mean ± SD of triplicate wells. Data are representative of 2 independent experiments. *P < .05. (F) Production of IFN-γ CD48−/−CD4+ T cells stimulated as described previously. Ag, antigen; CPM, counts per minute.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

4. Figure 3
CD48−/− pMφ are defective in antigen processing and cytokine production. (A) pMφ were incubated with ovalbumin protein overnight, and CD4+ T cells were added to the culture the next day and incubated for 3 days. Proliferation of the T cells was measured as described in Figure 2. Data represent mean ± SD of triplicate wells. Data are representative of 2 independent experiments. *P < .05. (B) pMφ were incubated with ovalbumin peptide and CD4+ T cells and incubated for 3 days. Proliferation of the T cells was measured as described in Figure 2. Data represent mean ± SD of triplicate wells. Data are representative of 2 independent experiments. *P < .05. (C, D, and E) pMφ were stimulated in vitro with LPS at 100 ng/mL or the amount indicated. Supernatants were collected and analyzed for relevant cytokines at the time point indicated. CD48−/− pMφ produced less TNF-α, and IL-12 than wt pMφ, whereas production of IL-6 was similar in both wt and CD48−/− pMφ. Data represent mean ± SD. Data are representative of 3 independent experiments. *P < .05. (F) CD48−/− pMφ are less able to clear bacteria than wt pMφ. In a gentamicin-protection assay, pMφ were incubated with E coli F18 as described in Materials and Methods. Data represent mean ± SEM of recovered bacteria from 106 wt or CD48−/− pMφ seeded with 107 bacteria at the indicated time points. Data are representative of 3 independent experiments. CPM, counts per minute.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

5. Figure 4
Treatment with anti-CD48 prevents colitis in CD45RBhi→Rag-2−/− mice. (A) DAI and (B) histology score of CD4+ CD45RBhi→Rag-2−/− mice treated with anti-CD48 (HM48-1) or control hamster IgG. Independent values and medians are indicated in each group. *P < .05. (C) Representative histology slides of CD4+ CD45RBhi→Rag-2−/− mice treated with hamster IgG (original magnification, 10×) or anti-CD48 (HM48-1; original magnification, 20×). (D) Production of IFN-γ is reduced in the serum of CD45RBhi→Rag-2−/− mice treated with anti-CD48. IFN-γ was measured by ELISA in the serum of CD45RBhi→Rag-2−/− mice treated with hamster IgG (n = 6) or anti-CD48 (HM48-1) (n = 5). Data represent mean ± SD of the mice analyzed from each group. (E) IFN-γ production detected by ELISA in the supernatant of stimulated CD4+ T cells from MLN of CD45RBhi→Rag-2−/− treated with hamster IgG (n = 2) or anti-CD48 (n = 4) as described in Figure 1C. Data represent mean ± SD of the mice analyzed from each group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

6. Figure 5
Anti-CD48 (HM48-1) therapy of colitis. (A) DAI and (B) histology score of CD45RBhi→Rag-2−/− mice treated with hamster IgG or anti-CD48 (HM48-1). The treatment started when sign of colitis appeared after 3 weeks of transfer. *P < .05. (C) Histology slides representative of CD45RBhi→Rag-2−/− mice treated with hamster IgG or anti-CD48 (HM48-1) (original magnification, 10×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions