CD8+ Cytotoxic T Cells Induce Relapsing Colitis in Normal Mice

Background & Aims: Most mouse models of IBD have emphasized an effector role of type-1 CD4+ T cells in colitis. The aim of this study was to develop a model of antigen-specific relapsing colitis to investigate the relative contribution of CD4+ and CD8+ effectors.Methods: Balb/C mice were sensitized and challenged with a suboptimal dose of 2.4 dinitrobenzene sulfonic acid to generate a colonic delayed-type hypersensitivity response. The respective role of CD4+ and CD8+ T cells in the initiation of colitis was analyzed by in vivo monoclonal antibody depletion and cell-transfer experiments. Dynamic and function of the colitogenic effectors were studied by immunohistochemistry, fluorescence-activated cell sorter analysis, enzyme-linked immunospot assay, quantitative polymerase chain reaction, and in vivo CTL assays.Results: Relapsing colitis rapidly occurred only after challenge of previously sensitized mice. Interferon-γ–producing cytotoxic CD8+ T cells (Tc1) specific for hapten-modified self-proteins were generated in colon-draining lymph nodes on day 5 after sensitization, before the onset of disease. These CD8+ T cells were rapidly recruited upon challenge into colon lamina propria as granzyme B–expressing effectors exerting ex vivo cytotoxicity against syngeneic hapten-modified colonic epithelial cells. Colitis was prevented by in vivo antibody depletion of CD8+, but not of CD4+, T cells and could be induced in naive recipients within 48 hours after transfer of CD8+, but not CD4+, T cells purified from sensitized mice.Conclusions: Our data show that antigen-specific CD8+ T cells can induce relapsing colitis in normal mice and suggest that the cytolytic function of CD8 Tc1 against epithelial cells may initiate the intestinal inflammatory process. Background & Aims: Most mouse models of IBD have emphasized an effector role of type-1 CD4+ T cells in colitis. The aim of this study was to develop a model of antigen-specific relapsing colitis to investigate the relative contribution of CD4+ and CD8+ effectors. Methods: Balb/C mice were sensitized and challenged with a suboptimal dose of 2.4 dinitrobenzene sulfonic acid to generate a colonic delayed-type hypersensitivity response. The respective role of CD4+ and CD8+ T cells in the initiation of colitis was analyzed by in vivo monoclonal antibody depletion and cell-transfer experiments. Dynamic and function of the colitogenic effectors were studied by immunohistochemistry, fluorescence-activated cell sorter analysis, enzyme-linked immunospot assay, quantitative polymerase chain reaction, and in vivo CTL assays. Results: Relapsing colitis rapidly occurred only after challenge of previously sensitized mice. Interferon-γ–producing cytotoxic CD8+ T cells (Tc1) specific for hapten-modified self-proteins were generated in colon-draining lymph nodes on day 5 after sensitization, before the onset of disease. These CD8+ T cells were rapidly recruited upon challenge into colon lamina propria as granzyme B–expressing effectors exerting ex vivo cytotoxicity against syngeneic hapten-modified colonic epithelial cells. Colitis was prevented by in vivo antibody depletion of CD8+, but not of CD4+, T cells and could be induced in naive recipients within 48 hours after transfer of CD8+, but not CD4+, T cells purified from sensitized mice. Conclusions: Our data show that antigen-specific CD8+ T cells can induce relapsing colitis in normal mice and suggest that the cytolytic function of CD8 Tc1 against epithelial cells may initiate the intestinal inflammatory process. See editorial on page 667 See editorial on page 667 Idiopathic inflammatory bowel diseases (IBDs), encompassing Crohn’s disease (CD) and ulcerative colitis, are complex chronic relapsing inflammatory diseases of the intestine whose etiology and pathophysiology remain unclear. It is widely believed that disease pathogenesis is multifactorial, involving genetic predisposition, environmental factors such as the bacterial intestinal flora, and dysregulation of the intestinal immune system. Although the primary mechanisms leading to initiation and perpetuation of the inflammatory process are not completely understood, there is increasing evidence that CD is driven by overproduction of the proinflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-12 resulting in high numbers of interferon gamma (IFN-γ)-producing Th1-type cells in the lamina propria1Fuss I.J. Neurath M. Boirivant M. Klein J.S. de la Motte C. Strong S.A. Fiocchi C. Strober W. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn’s disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5.J Immunol. 1996; 157: 1261-1270PubMed Google Scholar, 2Podolsky D.K. Inflammatory bowel disease.N Engl J Med. 2002; 347: 417-429Crossref PubMed Scopus (3217) Google Scholar, 3Bouma G. Strober W. The immunological and genetic basis of inflammatory bowel disease.Nat Rev Immunol. 2003; 3: 521-533Crossref PubMed Scopus (1524) Google Scholar and abnormal CD4+ T-cell reactivity to the endogenous microflora.4Duchmann R. Schmitt E. Knolle P. Meyer zum Buschenfelde K.H. Neurath M. Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12.Eur J Immunol. 1996; 26: 934-938Crossref PubMed Scopus (338) Google Scholar Insight into the pathophysiology of CD has benefited from a large number of experimental mouse models of colitis, in which sustained intestinal inflammation resulted from excessive effector function of Th1-biased CD4+ T cells or deficient regulatory T-cell function and required the presence of endogenous gut flora. The most extensively used system is the well-established model of IBD induced by T-cell reconstitution of immunocompromised SCID- or RAG-deficient mice. In this model, adoptive transfer of naive (CD45RBhi)5Powrie F. Leach M.W. Mauze S. Caddle L.B. Coffman R.L. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice.Int Immunol. 1993; 5: 1461-1471Crossref PubMed Scopus (938) Google Scholar or memory (CD25−CD45RBlow)6Asseman C. Read S. Powrie F. Colitogenic Th1 cells are present in the antigen-experienced T cell pool in normal mice control by CD4+ regulatory T cells and IL-10.J Immunol. 2003; 171: 971-978PubMed Google Scholar CD4+ T cells from normal mice generates chronic colitis that is prevented by the cotransfer of regulatory CD4+CD25+CD45RBlow T cells.7Powrie F. Leach M.W. Mauze S. Menon S. Caddle L.B. Coffman R.L. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells.Immunity. 1994; 1: 553-562Abstract Full Text PDF PubMed Scopus (1016) Google Scholar In Th1-type colitis that develops spontaneously at the age of 3 to 5 months in the SAMP1/Yit mutant8Kosiewicz M.M. Nast C.C. Krishnan A. Rivera-Nieves J. Moskaluk C.A. Matsumoto S. Kozaiwa K. Cominelli F. Th1-type responses mediate spontaneous ileitis in a novel murine model of Crohn’s disease.J Clin Invest. 2001; 107: 695-702Crossref PubMed Scopus (217) Google Scholar and C3H/HeJBir strain,9Cong Y. Brandwein S.L. McCabe R.P. Lazenby A. Birkenmeier E.H. Sundberg J.P. Elson C.O. CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice increased T helper cell type 1 response and ability to transfer disease.J Exp Med. 1998; 187: 855-864Crossref PubMed Scopus (335) Google Scholar CD4+ T cells harvested during active disease can transfer colitis into SCID-recipient mice. Likewise, colitis associated with increased mucosal infiltration with IFNγ-producing CD4+ T cells was also shown to develop with time in mice with genetically disrupted IL-210McDonald S.A. Palmen M.J. Van Rees E.P. MacDonald T.T. Characterization of the mucosal cell-mediated immune response in IL-2 knockout mice before and after the onset of colitis.Immunology. 1997; 91: 73-80Crossref PubMed Scopus (76) Google Scholar or IL-1011Singh U.P. Singh S. Taub D.D. Lillard Jr, J.W. Inhibition of IFN-gamma-inducible protein-10 abrogates colitis in IL-10-/- mice.J Immunol. 2003; 171: 1401-1406PubMed Google Scholar gene housed in a conventional environment. The well-validated SCID transfer model of colitis has clearly shown the colitogenic potential of CD4+ T cells. However, intestinal inflammation routinely develops around 2 months after T-cell reconstitution and homeostatic expansion of transferred cells and thus hampers analysis of the contribution of antigen specific T cells in the onset of disease. Alternatively, the hapten-induced colitis model appears suitable to dissect the early events of the IBD process because colitis develops in normal mice within days after intrarectal administration of contact sensitizing haptens including trinitrobenzene sulfonate (TNBS), 2,4 dinitrobenzene sulfonate (DNBS), and oxazolone. Indeed, TNBS or DNBS covalently bind to lysine residues of proteins and generate modified self-antigens,12Martin S. Ortmann B. Pflugfelder U. Birsner U. Weltzien H.U. Role of hapten-anchoring peptides in defining hapten-epitopes for MHC-restricted cytotoxic T cells. Cross-reactive TNP-determinants on different peptides.J Immunol. 1992; 149: 2569-2575PubMed Google Scholar including tissue proteins and components of the microbial flora.4Duchmann R. Schmitt E. Knolle P. Meyer zum Buschenfelde K.H. Neurath M. Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12.Eur J Immunol. 1996; 26: 934-938Crossref PubMed Scopus (338) Google Scholar These modified self-antigens can be presented by both major histocompatibility complex class I and class II molecules and induce efficient in vivo priming of CD8+ and CD4+ T cells, respectively.13Krasteva M. Kehren J. Horand F. Akiba H. Choquet G. Ducluzeau M.T. Tedone R. Garrigue J.L. Kaiserlian D. Nicolas J.F. Dual role of dendritic cells in the induction and down-regulation of antigen-specific cutaneous inflammation.J Immunol. 1998; 160: 1181-1190PubMed Google Scholar, 14Kehren J. Desvignes C. Krasteva M. Ducluzeau M.T. Assossou O. Horand F. Hahne M. Kagi D. Kaiserlian D. Nicolas J.F. Cytotoxicity is mandatory for CD8+ T cell-mediated contact hypersensitivity.J Exp Med. 1999; 189: 779-786Crossref PubMed Scopus (244) Google Scholar Studies using different haptens, experimental protocols, and mice strains have shown that several immune pathways can lead to intestinal inflammation. Initial studies using TNBS showed that local exposure of the colonic mucosa to TNBS resulted in a Th1-type colitis with similarities with CD in susceptible SJL and Balb/C mice.15Neurath M.F. Fuss I. Kelsall B.L. Stuber E. Strober W. Antibodies to interleukin 12 abrogate established experimental colitis in mice.J Exp Med. 1995; 182: 1281-1290Crossref PubMed Scopus (1210) Google Scholar, 16Fuss I.J. Marth T. Neurath M.F. Pearlstein G.R. Jain A. Strober W. Anti-interleukin 12 treatment regulates apoptosis of Th1 T cells in experimental colitis in mice.Gastroenterology. 1999; 117: 1078-1088Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 17Neurath M. Fuss I. Strober W. TNBS-colitis.Int Rev Immunol. 2000; 19: 51-62Crossref PubMed Scopus (182) Google Scholar Disease could develop after a single administration of TNBS in 50% ethanol and induced transmural lesions with severe diarrhea, weight loss, and wasting disease associated with a Th1 pattern of cytokine production in colon.15Neurath M.F. Fuss I. Kelsall B.L. Stuber E. Strober W. Antibodies to interleukin 12 abrogate established experimental colitis in mice.J Exp Med. 1995; 182: 1281-1290Crossref PubMed Scopus (1210) Google Scholar Chronic colitis driven by IL-12 and requiring CD40-CD40L interaction18Stuber E. Strober W. Neurath M. Blocking the CD40L-CD40 interaction in vivo specifically prevents the priming of T helper 1 cells through the inhibition of interleukin 12 secretion.J Exp Med. 1996; 183: 693-698Crossref PubMed Scopus (327) Google Scholar could be induced with TNBS at either a low15Neurath M.F. Fuss I. Kelsall B.L. Stuber E. Strober W. 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Hapten-induced model of murine inflammatory bowel disease mucosa immune responses and protection by tolerance.J Immunol. 1996; 157: 2174-2185PubMed Google Scholar These studies provided indirect evidence that CD4+ T cells play a major role in TNBS colitis. Interestingly, more recent studies revealed that intrarectal administration of oxazolone in C57Bl/6 mice induces a type 2 colitis mediated by natural killer (NK-T) cells via IL-13 and reminiscent of ulcerative colitis (UC).21Heller F. Fuss I.J. Nieuwenhuis E.E. Blumberg R.S. Strober W. Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells.Immunity. 2002; 17: 629-638Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar Thus, besides CD4+ T cells, other cell types can contribute to experimental colitis. In this study, we developed a model of DNBS-induced colitis based on induction of a colonic delayed-type hypersensitivity (DTH) response that allowed identification of the T-cell subset responsible for initiation of disease soon after challenge. Our data show that relapsing colitis can be induced in normal immunocompetent mice and that hapten-specific cytolytic CD8+ Tc1-type effector cells are responsible for initiation of colonic lesions. Balb/C male mice and syngeneic athymic nude male mice (6–10 weeks old) were purchased from Charles River laboratories (L’Arbresle, France). All experiments were previously approved by the Animal Care and Use Committee according to governmental guidelines and were performed in the accredited establishment, PBES of Ecole Normale Superieure de Lyon. All mice were fed with standard mice chow pellets ad libidum. The model of hapten-specific colonic DTH was set up by using DNBS, a hapten comparable but not cross-reactive to the more widely used hapten TNBS.22Wallace J.L. MacNaughton W.K. Morris G.P. Beck P.L. Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease.Gastroenterology. 1989; 96: 29-36Abstract PubMed Scopus (0) Google Scholar The optimal doses of DNBS for colonic sensitization and elicitation were selected from preliminary dose-response experiments as the minimal dose of hapten unable to elicit colitis in naive mice but able to induce colitis in sensitized mice without mortality or toxicity. Stock solutions, prepared immediately before use, were made by dissolving 20 mg (for sensitization) or 10 mg (for elicitation) of DNBS in 50% ethanol (1/1, vol/vol) in water. Ethanol was used as a mucosal barrier-breaking agent that enhances intestinal permeability.23Morris G.P. Beck P.L. Herridge M.S. Depew W.T. Szewczuk M.R. Wallace J.L. Hapten-induced model of chronic inflammation and ulceration in the rat colon.Gastroenterology. 1989; 96: 795-803Abstract Full Text PDF PubMed Scopus (1597) Google Scholar Sensitization (ie, the afferent phase of the colonic DTH response) was performed as follows. Mice anesthetized by an intraperitoneal injection 2/1 (vol/vol) solution of 50 mg/mL of ketamine and 1.5% of xylazine were sensitized on day 0 by infusion into the distal 4 cm of the colon of 100 μL of the 20-mg/mL stock solution, containing 2 mg of DNBS, by using a soft plastic catheter. The control unsensitized mice received 100 μL of 50% ethanol alone. Mice were left in an upside down position for 30 seconds. For elicitation of colitis (ie, efferent phase of the colonic DTH), sensitized mice were challenged on day 5 with 1 mg of either DNBS or TNBS as an irrelevant control hapten diluted in 50% ethanol or with 50% ethanol alone. Mice were examined daily with respect to their general condition, body weight, and consistency of stools. Seventy-two hours after elicitation of DTH, macroscopic colonic lesions were analyzed in a double-blinded fashion by using the Wallace score,22Wallace J.L. MacNaughton W.K. Morris G.P. Beck P.L. Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease.Gastroenterology. 1989; 96: 29-36Abstract PubMed Scopus (0) Google Scholar taking into account hyperemia, thickening of the bowel wall, extent of colonic inflammation, and presence of mucosal ulcerations. Microscopic damage was assessed on a segment of 1 cm of colon located 3 cm above the anal canal, after fixation in 4% paraformaldehyde and H&E staining of paraffin-embedded sections (4 μm). Histological grading of colonic lesions, including thickness of the bowel wall, extent and severity of the inflammatory cell infiltration, and gland distortion, was performed by using the Ameho score.24Ameho C.K. Adjei A.A. Harrison E.K. Takeshita K. Morioka T. Arakaki Y. Ito E. Suzuki I. Kulkarni A.D. Kawajiri A. Yamamoto S. Prophylactic effect of dietary glutamine supplementation on interleukin 8 and tumour necrosis factor alpha production in trinitrobenzene sulphonic acid induced colitis.Gut. 1997; 41: 487-493Crossref PubMed Scopus (218) Google Scholar To deplete CD4+ or CD8+ T cells, mice were injected intraperitoneally on days −1, 0, +1, and +3, (with respect to day 0 of DNBS sensitization) with the anti-CD4 mAb (clone GK1.5) or the anti-CD8 mAb (clone H 35.17.2), kindly provided by G. Milon (Institut Pasteur, Paris, France). Control mice were injected with an irrelevant β-Gal–specific rat immunoglobulin IgG (clone GL113) monoclonal antibody (mAb). Fluorescence-activated cell sorter (FACS) analysis of peripheral blood mononuclear cell and secondary lymphoid organs confirmed depletion of >95% of CD4+ or CD8+ T cells, respectively. In some experiments, depletion of CD8+ T cell during the sensitization phase was performed by injecting the anti-CD8 mAb daily from day −12 to −9. Mice were then sensitized on day 0 and challenged on day 10. FACS analysis confirmed depletion of >75% of CD8+ T cells up to 5 days after the last injection and recovery of 70% CD8+ T cells on day 10. Cryostat sections (5 μm) of the distal colon were incubated for 1 hour with the anti-CD8 rat mAb (clone Ly2, BD Biosciences, Pharmingen, San Diego, CA) or an irrelevant IgG rat mAb as control; followed by a biotinylated mouse-adsorbed goat antirat IgG Ab. Specific binding was revealed with a streptavidin peroxidase kit (Dako, Trappes, France), according to the manufacturer’s instruction. The reaction was developed by using 3-amino-9-ethylcarbazole in the presence of H2O2. Sections were counterstained with hematoxylin. No staining was obtained with the control rat mAb. Total RNA from the distal colon was isolated with trizol reagent (Invitrogen, Cergy Pontoise, France). DNAse treatment of total RNA and reverse transcription into complementary DNA were performed by using Superscript II reverse transcriptase (Invitrogen, Cergy Pontoise, France). Polymerase chain reaction (PCR) was performed by using 1 μg of total RNA in a Perkin Elmer apparatus. Sense and antisense primers sequences were as follows: TNF-α Sense, 5′-TCT-CAT-CAG-TTC-TAT-GGC-CC-3′ and TNF-α Antisense, 5′-GGG-AGT-AGA-CAA-GGT-ACA-AC-3′; IL-1-β Sense, TTG-ACG-GAC-CCC-AAA-AGA-TG-3′ and IL-1-β Antisense, 5′-AGA-AGG-TGC-TCA- TGT- CCT-CAT-3′; IFN-γ Sense, 5′-AAA-GAG-ATA-ATC-TGG-CTC-TGC- 3′ and IFN-γ antisense 5′-GCT-CTG-AGA-CAA-TGA-ACG-CT-3′; CD8 Sense, 5′-TCA-CAG-GCG-AAG-TCC-AAT-CC-3′ and CD8 Antisense 5′-AGG-ATG-CTC-TTG-GCT-CTT-CC-3′; β-actin Sense, 5′-GGG-TCA-GAA-GGA-TTC-CTA-TG-3′; and β-actin Antisense, 5′-GGT-CTC-AAA-CAT-GAT-CTG-GG-3′. Annealing temperature and cycle number were as follows: TNF-α, IL-1β, and β-actin, 55°C and 40 cycles, and IFN-γ and CD8, 61°C and 32 cycles. The amplifications were conducted with 40 cycles for the β-actin, TNF-α, IL-1-β, and with 32 cycles for CD8- and IFN-γ–amplified genes, and the PCR products were finally loaded onto 1% agarose gel and analyzed with a Kodak Digital Science apparatus (Kodak, Rochester, NY). Results were normalized by using the housekeeping gene β-actin in the same sample. Quantitative polymerase chain reaction (Q-PCR) analysis was conducted as follows. Reverse-transcription reaction mixture was amplified by real-time PCR (platinium Sybr Green qPCR Supermix, UDG; Invitrogen). Mouse specific sense and antisense primers used were as follows: CD8α Sense, 5′-CTT- GTG-CCT-CAA-ACT-GCA-AG-3′, CD8α Antisense, 5′-CCG-CTA-AAG-GCA-GTT-CTC-C-3′; CD4 Sense, 5′-TGA-ACC-TGG-TGG-TGA-TGA-AA-3′; CD4 Antisense, 5′-TCT-CCT-GCT-TCA- GGG-TCA-GT-3′, 3 housekeeping genes: β-actine Sense, 5′-AAG-ATC-TGG-CAC-CAC-ACC-TTC-T-3′; β-actine Antisense 5′-TTT-TCA-CGG-TTG-GCC-TTA-GG-3′; HPRT Sense, 5′-TCA-TTA-TGC-CGA-GGA-TTT-GGA-3′; and HPRT Antisense 5′-CAG-AGG-GCC-ACA-ATG-TGA-TG-3′; G3PDH Sense, 5′-GCA-TGG-CCT-TCC-GTG-TTC-3′; G3PDH Antisense 5′-TGT-CAT-CAT-ACT-TGG-CAG-GTT-TCT-3′. Results were expressed as arbitrary units with respect to expression of transcripts of the 3 housekeeping genes (QR). Spleen, mesenteric lymph nodes (MLNs) and caudal lymph nodes (CLNs) were harvested on day 5 after sensitization. CD8+ T cells were purified by MACS-positive selection by using anti-CD8 mAb-coated microbeads (Miltenyi Biotec, Paris, France). CD4+ T cells were negatively sorted by magnetic adsorption cell sorting selection by using a cocktail of mAbs including anticlass II (clone M5/114), anti-CD8 (clone H35), anti-CD19, and anti-CD11b (M1/70). FACS analysis revealed that viable CD8+ and CD4+ T-cell enrichment was >90% and 70% for CLNs/MLNs and spleen, respectively. Pooled CD8+ or CD4+ T cells from MLNs, CLNs, and the spleen were used for adoptive transfer experiments. CD8+ or CD4+ T cells (2.105 per well) were cocultured for 3 days at 37°C in a 96-well round bottomed microculture plate, with 5.105 DNBS- or TNBS-derivatized or unpulsed naive syngeneic spleen cells as antigen-presenting cells in a final volume of 200 μL, as described previously.25Dubois B. Chapat L. Goubier A. Papiernik M. Nicolas J.F. Kaiserlian D. Innate CD4+CD25+ regulatory T cells are required for oral tolerance and inhibition of CD8+ T cells mediating skin inflammation.Blood. 2003; 102: 3295-3301Crossref PubMed Scopus (159) Google Scholar Antigen-presenting cells were hapten derivatized by incubation for 20 minutes at 37°C with either 1.6 mmol/L of DNBS (pH 8) or 2 mmol/L of the irrelevant hapten TNBS, diluted in serum-free RPMI-1640, and then treated for 20 minutes at 37°C with 25 μg/mL of mitomycin C. Proliferation was assessed on day 3 of culture by 3H-thymidine incorporation (1 μCi /well) during the last 12 hours of culture. Radioactivity was counted by using a β-plate liquid scintillation counter Topcount NXT (Packard instrument, Rungis, France). The frequency of DNBS-specific IFN-γ–producing cells was determined by an enzyme-linked immunospot (ELISPOT) assay, as previously described26Desvignes C. Etchart N. Kehren J. Akiba I. Nicolas J.F. Kaiserlian D. Oral administration of hapten inhibits in vivo induction of specific cytotoxic CD8+ T cells mediating tissue inflammation a role for regulatory CD4+ T cells.J Immunol. 2000; 164: 2515-2522Crossref PubMed Scopus (55) Google Scholar with minor modifications. Briefly, 96-well nitrocellulose microplate (MAHA, Millipore, Molsheim, France) wells were coated with an IFN-γ–specific mAb (clone R46-A2, BD Pharmingen). Lymphoid cell suspensions from the spleen, MLNs, and CLNs were added to triplicate wells in the presence of .4 mmol/L of DNBS or .2 mmol/L of TNBS (as control) for analysis of DNBS-specific spot forming cells (SFCs). After overnight incubation at 37°C, plates were washed 3 times with PBS-containing .05 % Tween 20 and incubated for 2 hours at RT with a biotinylated anti–IFN-γ antibody (XMG 1.2, BD Pharmingen). IFN-γ SFCs were revealed with a streptavidin peroxidase kit (Dako) and AEC + H2O2. The number of IFN-γ SFCs in each well was counted by using a microscope and the results expressed as number of IFN-γ SFCs/106 cells. In vivo analysis of DNBS-specific cytotoxic T cells (CTLs) was assessed on day 5 after immunization by using the previously described in vivo CTL assay27Beloeil L. Tomkowiak M. Angelov G. Walzer T. Dubois P. Marvel J. In vivo impact of CpG1826 oligodeoxynucleotide on CD8 T cell primary responses and survival.J Immunol. 2003; 171: 2995-3002PubMed Google Scholar with modifications. Immunized and naive mice were injected intraperitoneally with a mixture of DNBS-pulsed and unpulsed naive syngeneic spleen cells as targets, previously labeled with a low and high concentration of carboxy-fluorescein succinimidyl ester (CFSE), respectively. DNBS-pulsed target cells were prepared by 10 minutes of incubation at 37°C with 1.6 mmol/L with DNBS in RPMI 1640 medium and labeled by 8 minutes of incubation at 37°C in PBS 1% FCS with .5 μmol/L CFSE (Molecular Probes, Leiden, the Netherlands). Unpulsed targets were incubated with medium alone and labeled with 5 μmol/L of CFSE (control targets). After extensive washing, 10.106 Ag-pulsed and 10.106 unpulsed target cells were mixed and transferred intravenously into immunized or naive recipient mice. MLNs and CLNs were harvested 24 hours after target cell transfer and 10,000 CFSE positive cells were acquired by using a FACSCalibur (BD Biosciences, San José, CA). In vivo cytotoxicity was assessed by calculating the ratio of unpulsed/pulsed targets in immunized versus naive mice. Leukocytes from the lamina propria were isolated from the 4-cm distal portion of colons of ethanol-injected or DNBS-sensitized BALB/c mice 48 hours after colonic challenge with DNBS. Briefly, colon fragments were cut in small pieces and incubated for 45 minutes at 37°C in Ca++/Mg++-free phosphate-buffered saline (PBS) containing 5 mmol/L EDTA, 25 mmol/L HEPES, and 10% fetal calf serum to remove epithelial cells. Lamina propria cells were extracted by 45 minutes at 37°C in 35 μg/mL liberase (Roche Diagnostics, Meylan, France) and 10 U/mL DNAse (Roche Diagnostics), as described. The resulting cell suspension contained >90% viable cells. Serial dilutions of cells from colon lamina propria were incubated for 4 hours at 37°C with 51Cr-labeled and hapten-derivatized or unpulsed C26 Balb/C adenocarcinoma (from American Type Culture Collection) (2.104 cells/well) as targets. Targets were simultaneously pulsed with 1.6 mmol/L DNBS or medium alone and labeled with 100 μCi of Na2CrO4 (Amersham, Orsay, France), by 1 hour incubation at 37°C. Spontaneous and maximal 51Cr release by unpulsed or DNBS-pulsed C26 targets was assessed from wells incubated with either medium alone or .05% Triton ×100, respectively. The radioactivity in supernatant was counted with a Topcount NXT (Packard Instrument, Rungis, France). The percentage of specific lysis was calculated as follows: (experimental counts per minute (cpm) − spontaneous cpm/maximal cpm − spontaneous cpm) × 100. The ratio of spontaneous/maximal cpm was routinely <15%. FACS analysis was performed on lamina propria cells cultured for 4 hours at 37°C with hapten-pulsed or unpulsed C26 adenocarcinoma cells at a 100/1 ratio in the presence of 1 μg/mL of brefeldin A for the last 2 hours of culture. Cells were surface stained by using anti-CD45 fluorescein isothiocyanate and anti-CD8 Cy5 (Pharmingen), then fixed and permabilized with cytofix-cytoperm (Becton Dickinson, Pont de Claix, France), and stained with the mouse anti-human granzyme B-PE mAbs (Caltag) or a mouse isotype control. Staining was analyzed by flow cytometry on a FACScan Becton Dickinson analyzer and Cellquest software (BD Biosciences, San José, CA). To induce DNBS-specific colonic DTH, Balb/C mice were immunized by intracolonic administration (enema) of a nontoxic dose of 2 mg of DNBS in 50% ethanol. This dose of hapten was determined in preliminary dose-response experiments as the minimal nontoxic dose of hapten unable to induce colitis or mortality in naive recipient (not shown). Five days later, colonic DTH was elicited by intracolonic challenge with 1 mg of DNBS. No sign of colitis or body weight loss was detected in day 5–DNBS-immunized mice either before hapten challenge (not shown) or at any time point after challenge with the irrelevant hapten TNBS (Figure 1A); likewise, no colitis developed in day 5–ethanol-injected (unsensitized) control mice either before or after DNBS challenge (Figure 1A), although a mild edema of colonic wall in the absence of inflammatory infiltrate was seen in both groups (Figure 1C, panel 2). In contrast, DNBS challenge of sensitized mice resulted in wasting disease with progressive body weight loss (Figure 1A). At 72 hours post-DNBS challenge, mice exhibited macroscopic signs of colitis including mild edema and hyperemia of the colon and sometimes adhesions to the small bowel. Histologic evidence of colitis included colon wall thickening; superficial erosion