1. CD8+ Cytotoxic T Cells Induce Relapsing Colitis in Normal Mice
Stéphane Nancey, Sébastien Holvöet, Ivan Graber, Grégoire Joubert, David Philippe, Stefan Martin, Jean–François Nicolas, Pierre Desreumaux, Bernard Flourié, Dominique Kaiserlian 
Gastroenterology 
Volume 131, Issue 2, Pages (August 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
Colitis results from a hapten-specific DTH response. (A and B) Percentage of body weight change. (A) Balb/C mice were sensitized intracolonically with DNBS in ethanol and challenged 5 days later with 1 mg of either DNBS (filled circle) or the irrelevant hapten TNBS (open square). Control (unsensitized) mice received ethanol vehicle alone on day 0 and 1 mg DNBS on day 5 (open circles). (B) DNBS-sensitized Balb/C mice challenged on day 5 with DNBS were rechallenged on day 30 with DNBS (filled circle) or ethanol alone (open circle). Results are expressed as the percentage of body weight change at various times post-challenge as compared with body weight before challenge. Data are representative of 1 of 6 experiments using 5 to 7 mice per group. (C) Histology of the colon was performed in either naive mice (panel 1) or at 72 hours post-challenge in control ethanol-injected/day 5–DNBS challenged mice (panel 2), DNBS-sensitized and day 5–DNBS-challenged mice (panel 3), DNBS-sensitized and day 5–DNBS-challenged mice injected on day 30 with ethanol alone (panel 4), or with DNBS (panel 5) (H&E staining, final magnification ×100). (D) Semiquantitative RT-PCR analysis of messenger RNA transcripts of TNF-α and IL-1β in colon samples from naive mice and day 5–DNBS-sensitized mice harvested at 24 or 48 hours after either ethanol (left) or DNBS (right) challenge.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
CD8+ CTL are primed by colonic DNBS sensitization. (A) T-cell proliferation assay. CD8+ and CD4+ T cells (2.105/well) purified from MLN and spleen 5 days after colonic DNBS sensitization were cultured in vitro with medium (white bar) or with mitomycin C–treated naive spleen cells (5.105/well) either unpulsed (grey bar) or pulsed with DNBS (black bar). Hapten-specific proliferation was determined on day 3 of culture by 3H-thymidine incorporation over the last 12 hours of culture. The results were expressed as mean cpm ± standard deviation of triplicate wells. (B) Frequency of hapten-specific IFN-γ–producing SFC in MLNs, CLNs, and the spleen. Total and CD8-depleted (>90% CD8 negative) cells were cultured for 24 hours at 37°C with either medium alone (white bars), TNBS (gray bars), or DNBS (black bars). Results are expressed as number of IFN-γ SFC/106 cells. (C) Hapten-specific in vivo CTL assay. Mice sensitized intracolonically on day 0 with DNBS and treated on days −1, 0, +1, and +4 with a depleting anti-CD8 mAb or control IgG mAb were injected intravenously with a 1/1 mixture of DNBS-pulsed and unpulsed spleen cells as target cells, respectively, and stained with .5 μmol/L or 5 μmol/L of CFSE. Twenty-four hours later, the percentage of stained cells in pooled MLN (black bars) and spleen (white bars) was analyzed by FACS as indicated in the materials and methods section. Results are expressed as mean ± standard deviation percentage of hapten-specific cytotoxicity.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
In vivo depletion of CD8+ T cells abrogates colitis. (A) Body weight change of mice was recorded at various times after colonic challenge of DNBS-sensitized mice injected with anti-CD8 mAb (open triangle), anti-CD4 mAb (open circle), or control IgG (filled circle). (B) Macroscopic (Wallace) score and (C) histologic (Ameho) score were evaluated at 72 hours after DNBS challenge in DNBS-sensitized mice treated with either control IgG (black bar) or with an anti-CD4 (white bar) or anti-CD8 mAb (shaded bar). Results are expressed as the mean score ± standard deviation in 5 mice per group and are representative of 2 experiments. (D) H&E staining of distal colon at 72 hours after DNBS challenge in DNBS-sensitized mice treated with either control IgG mAb (left panel), anti-CD4 mAb (middle panel), or anti-CD8 mAb (right panel) (final magnification ×100).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
Adoptive transfer of colitis by CD8+ T cells from DNBS-sensitized mice. Nude athymic mice received an enema of 1 mg of DNBS and were either untransferred (A: open triangle; B: left panel) or transferred intravenously with CD8+ T cells from colon-draining lymph nodes harvested from Balb/C mice 5 days after ethanol injection (A: open circle; B: middle panel) or DNBS sensitization (A: filled circle; B: right panel) without challenge. (A) Body weight change is expressed as mean ± standard deviation of 1 representative experiment using 5 mice per group. (B) H&E of colon section performed at 72 hour post-challenge (final magnification ×100).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
CD4+ T cells from sensitized mice do not transfer colitis. Nude athymic mice received an enema of 1 mg DNBS and were either untransferred (A: filled triangle; B: panel 1) or transferred intravenously with purified CD4+ T cells (A: open circle; B: panel 2) or CD8+ T cells (A: filled circle; B: panel 3) pooled from MLNs and the spleen from day 5 DNBS sensitized mice (no challenge). (A) Body weight change of recipient mice at various times after colonic challenge is expressed as mean of percent body weight change ± standard deviation [SD] in 5–7 mice per group. (B) H&E staining of paraffin-embedded colon sections was performed at 48 hours post-challenge (final magnification ×100). (C) Real-time RT-PCR analysis of CD4 (white) and CD8 (black) messenger RNA transcripts in distal colon of mice either untransferred or transferred with CD4+ or CD8+ T cells from DNBS-sensitized mice and harvested at 48 hours after DNBS challenge.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

7. Figure 6
Recruitment of IFN-γ–producing CD8+ T cells in challenged colon. Semiquantitative RT-PCR analysis of CD8 and IFN-γ transcripts (A) and immunohistochemical staining for CD8 (B) in mice DNBS-sensitized mice challenged with ethanol alone (A and B: left panels) or DNBS (A and B: right panels). Mice were sacrificed at 48 and 72 hours (A) or at 48 hours (B) after challenge (final magnification ×200).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

8. Figure 7
CD8+T cells recruited into the colon are specific cytotoxic effectors. Leukocytes extracted from the colon lamina propria of either ethanol- or DNBS-sensitized mice at 48 hours after DNBS challenge were cultured in vitro for 4 hours at 37°C with DNBS-pulsed syngeneic C26 colon epithelial cells. (A) FACS analysis of granzyme B–expressing CD8+ T cells on gated CD45+ cells. (B) Direct ex vivo cytotoxicity against DNBS-pulsed (black circle) or unpulsed (open circle) 51Cr-labeled C26 cells (E/T, effector to target cell ratio). Results are expressed as the percentage of specific cytotoxicity.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions