CD4+NKG2D+ T Cells in Crohn’s Disease Mediate Inflammatory and Cytotoxic Responses Through MICA Interactions
2007; Elsevier BV; Volume: 132; Issue: 7 Linguagem: Inglês
10.1053/j.gastro.2007.03.025
ISSN1528-0012
AutoresMatthieu Allez, Vannary Tieng, Atsushi Nakazawa, Xavier Tréton, Vincent Pacault, Nicolas Dulphy, Sophie Caillat‐Zucman, Pascale Paul, Jean‐Marc Gornet, Corinne Douay, Sophie Ravet, Ryad Tamouza, Dominique Charron, Marc Lémann, Lloyd Mayer, Antoine Toubert,
Tópico(s)T-cell and B-cell Immunology
ResumoBackground & Aims: Crohn's disease (CD) is an inflammatory bowel disease characterized by uncontrolled immune responses to bacterial flora, with excessive activation of T lymphocytes. MICA is a stress-induced major histocompatibility complex–related molecule expressed on normal intestinal epithelial cells (IECs) and recognized by the NKG2D-activating receptor on CD8+ T cells, γδ T cells, and natural killer cells. We examined the role of MICA-NKG2D interactions in the activation of T lymphocytes in CD. Methods: MICA expression was analyzed by flow cytometry on IECs isolated from patients with active inflammatory bowel disease and controls. NKG2D expression and function were analyzed on lamina propria and peripheral blood lymphocytes. Results: MICA expression was significantly increased on IECs in CD, with higher expression in macroscopically involved areas. A subset of CD4+ T cells expressing NKG2D was increased in the lamina propria from patients with CD compared with controls and patients with ulcerative colitis. CD4+NKG2D+ T cells with a Th1 cytokine profile and expressing perforin were increased in the periphery and in the mucosa in CD. CD4+NKG2D+ T-cell clones were functionally active through MICA-NKG2D interactions, producing interferon-γ and killing targets expressing MICA. IECs from patients with CD had the ability to expand this subset in vitro. CD4+NKG2D+ lamina propria lymphocytes from patients with CD highly expressed interleukin-15Rα, and interleukin-15 increased NKG2D and DAP10 expression in CD4+NKG2D+ T-cell clones. Conclusions: These findings highlight the role of MICA-NKG2D in the activation of a unique subset of CD4+ T cells with inflammatory and cytotoxic properties in CD. Background & Aims: Crohn's disease (CD) is an inflammatory bowel disease characterized by uncontrolled immune responses to bacterial flora, with excessive activation of T lymphocytes. MICA is a stress-induced major histocompatibility complex–related molecule expressed on normal intestinal epithelial cells (IECs) and recognized by the NKG2D-activating receptor on CD8+ T cells, γδ T cells, and natural killer cells. We examined the role of MICA-NKG2D interactions in the activation of T lymphocytes in CD. Methods: MICA expression was analyzed by flow cytometry on IECs isolated from patients with active inflammatory bowel disease and controls. NKG2D expression and function were analyzed on lamina propria and peripheral blood lymphocytes. Results: MICA expression was significantly increased on IECs in CD, with higher expression in macroscopically involved areas. A subset of CD4+ T cells expressing NKG2D was increased in the lamina propria from patients with CD compared with controls and patients with ulcerative colitis. CD4+NKG2D+ T cells with a Th1 cytokine profile and expressing perforin were increased in the periphery and in the mucosa in CD. CD4+NKG2D+ T-cell clones were functionally active through MICA-NKG2D interactions, producing interferon-γ and killing targets expressing MICA. IECs from patients with CD had the ability to expand this subset in vitro. CD4+NKG2D+ lamina propria lymphocytes from patients with CD highly expressed interleukin-15Rα, and interleukin-15 increased NKG2D and DAP10 expression in CD4+NKG2D+ T-cell clones. Conclusions: These findings highlight the role of MICA-NKG2D in the activation of a unique subset of CD4+ T cells with inflammatory and cytotoxic properties in CD. Crohn's disease (CD) is an inflammatory bowel disease (IBD) characterized by uncontrolled immune responses toward the intestinal flora.1Bouma G. Strober W. The immunological and genetic basis of inflammatory bowel disease.Nat Rev Immunol. 2003; 3: 521-533Crossref PubMed Scopus (1537) Google Scholar Breakdown of immune tolerance toward the intestinal contents results in dysregulated and/or constant activation of the immune system. The identification of susceptibility genes for CD, such as NOD2/CARD15, suggests an important role of innate immunity in the pathogenesis of uncontrolled inflammation, but it remains clear that adaptive immunity also plays a crucial role in chronic intestinal inflammation. Indeed, a number of experimental systems show that altered regulation of intestinal T-cell function can result in chronic intestinal inflammation.2Mizoguchi A. Mizoguchi E. Bhan A.K. Immune networks in animal models of inflammatory bowel disease.Inflamm Bowel Dis. 2003; 9: 246-259Crossref PubMed Scopus (65) Google Scholar, 3Elson C.O. Cong Y. McCracken V.J. Dimmitt R.A. Lorenz R.G. Weaver C.T. Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota.Immunol Rev. 2005; 206: 260-276Crossref PubMed Scopus (425) Google Scholar In these models, antigenic stimuli from the gut lumen drive the expansion of both effector cells and regulatory T cells, the latter keeping the immune response under control. Chronic intestinal inflammation may be due either to impaired regulatory T-cell activity or excessive effector T-cell function.3Elson C.O. Cong Y. McCracken V.J. Dimmitt R.A. Lorenz R.G. Weaver C.T. Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota.Immunol Rev. 2005; 206: 260-276Crossref PubMed Scopus (425) Google Scholar, 4Allez M. Mayer L. Regulatory T cells: peace keepers in the gut.Inflamm Bowel Dis. 2004; 10: 666-676Crossref PubMed Scopus (106) Google Scholar An essential role for CD4+ T cells has been shown in different animal models of experimental colitis.5Neurath M.F. Finotto S. Glimcher L.H. The role of Th1/Th2 polarization in mucosal immunity.Nat Med. 2002; 8: 567-573Crossref PubMed Scopus (559) Google Scholar While both Th1 and Th2 cells have been shown able to induce chronic intestinal inflammation in vivo, a Th1 cytokine profile is clearly demonstrated in CD. Intestinal epithelial cells (IECs) can play a role in the activation of mucosal T cells in IBD. IECs can take up and process antigen and function as antigen-presenting cells.6Hershberg R.M. Mayer L.F. Antigen processing and presentation by intestinal epithelial cells—polarity and complexity.Immunol Today. 2000; 21: 123-128Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar IECs express surface molecules and restriction elements (classic and nonclassic major histocompatibility complex [MHC] molecules) that allow them to interact with unique subsets of T cells.7Allez M. Brimnes J. Dotan I. Mayer L. Expansion of CD8+ T cells with regulatory function after interaction with intestinal epithelial cells.Gastroenterology. 2002; 123: 1516-1526Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 8Nakazawa A. Dotan I. Brimnes J. Allez M. Shao L. Tsushima F. Azuma M. Mayer L. The expression and function of co-stimulatory molecules B7h and B7-H1 on colonic epithelial cells.Gastroenterology. 2004; 126: 1347-1357Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar In the context of IBD, IECs may drive the expansion of effector T cells with a lack of activation of regulatory T cells.9Brimnes J. Allez M. Dotan I. Ling S. Nakazawa A. Mayer L. Defects in CD8+ regulatory T cells in the lamina propria of patients with inflammatory bowel disease.J Immunol. 2005; 174: 5814-5822PubMed Google Scholar, 10Mayer L. Eisenhardt D. Lack of induction of suppressor T cells by intestinal epithelial cells from patients with inflammatory bowel disease.J Clin Invest. 1990; 86: 1255-1260Crossref PubMed Scopus (174) Google Scholar MICA, an MHC-related class Ib molecule expressed on normal IECs, could be involved in the activation of mucosal lymphocytes. Under normal conditions, expression of MIC molecules (MICA and MICB) is restricted to intestinal and thymic epithelium. This expression can be induced by stress in different epithelial cells and is up-regulated in tumors and upon exposure to intracellular pathogens.11Groh V. Bahram S. Bauer S. Herman A. Beauchamp M. Spies T. Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.Proc Natl Acad Sci U SA. 1996; 93: 12445-12450Crossref PubMed Scopus (909) Google Scholar, 12Groh V. Rhinehart R. Randolph-Habecker J. Topp M.S. Riddell S.R. Spies T. Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells.Nat Immunol. 2001; 2: 255-260Crossref PubMed Scopus (840) Google Scholar, 13Groh V. Rhinehart R. Secrist H. Bauer S. Grabstein K.H. Spies T. Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB.Proc Natl Acad Sci U S A. 1999; 96: 6879-6884Crossref PubMed Scopus (930) Google Scholar MICA is a ligand of the NKG2D activating receptor preferentially expressed on CD8+ T cells, γδ T cells, and natural killer (NK) cells.14Raulet D.H. Roles of the NKG2D immunoreceptor and its ligands.Nat Immunol. 2003; 3: 781-789Crossref Scopus (1106) Google Scholar, 15Steinle A. Li P. Morris D.L. Groh V. Lanier L.L. Strong R.K. Spies T. Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family.Immunogenetics. 2001; 53: 279-287Crossref PubMed Scopus (399) Google Scholar, 16Li P. Morris D.L. Willcox B.E. Steinle A. Spies T. Strong R.K. Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA.Nat Immunol. 2001; 2: 443-451Crossref PubMed Scopus (321) Google Scholar MIC molecules function as signals of cellular stress and trigger a range of immune effector mechanisms, including cellular cytotoxicity and cytokine secretion.17Das H. Groh V. Kuijl C. Sugita M. Morita C.T. Spies T. Bukowski J.F. MICA engagement by human Vgamma2Vdelta2 T cells enhances their antigen-dependent effector function.Immunity. 2001; 15: 83-93Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 18Jamieson A.M. Diefenbach A. McMahon C.W. Xiong N. Carlyle J.R. Raulet D.H. The role of the NKG2D immunoreceptor in immune cell activation and natural killing.Immunity. 2002; 17: 19-29Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar In CD8+ T-cell receptor (TCR) αβ+ cells, MIC/NKG2D interaction delivers a costimulatory signal that complements TCR-mediated antigen recognition on target cells.12Groh V. Rhinehart R. Randolph-Habecker J. Topp M.S. Riddell S.R. Spies T. Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells.Nat Immunol. 2001; 2: 255-260Crossref PubMed Scopus (840) Google Scholar NKG2D associates in humans with the DAP10 adaptor protein, allowing transduction of activating signals.19Wu J. Song Y. Bakker A.B. Bauer S. Spies T. Lanier L.L. Phillips J.H. An activating immunoreceptor complex formed by NKG2D and DAP10.Science. 1999; 285: 730-732Crossref PubMed Scopus (894) Google Scholar, 20Diefenbach A. Tomasello E. Lucas M. Jamieson A.M. Hsia J.K. Vivier E. Raulet D.H. Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D.Nat Immunol. 2002; 3: 1142-1149Crossref PubMed Scopus (386) Google Scholar, 21Gilfillan S. Ho E.L. Cella M. Yokoyama W.M. Colonna M. NKG2D recruits two distinct adapters to trigger NK cell activation and costimulation.Nat Immunol. 2002; 3: 1150-1155Crossref PubMed Scopus (362) Google Scholar We previously showed that MICA expression can be markedly increased by exposure to different bacterial strains, including adherent-invasive Escherichia coli strains, which have been identified as colonizing the intestinal mucosa of patients with CD.22Tieng V. Le Bouguenec C. du Merle L. Bertheau P. Desreumaux P. Janin A. Charron D. Toubert A. Binding of Escherichia coli adhesin AfaE to CD55 triggers cell-surface expression of the MHC class I-related molecule MICA.Proc Natl Acad Sci U S A. 2002; 99: 2977-2982Crossref PubMed Scopus (207) Google Scholar, 23Darfeuille-Michaud A. Neut C. Barnich N. Lederman E. Di Martino P. Desreumaux P. Gambiez L. Joly B. Cortot A. Colombel J.F. Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease.Gastroenterology. 1998; 115: 1405-1413Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar The data obtained in the present study suggest that up-regulation of MICA on IECs in CD may play a role in the activation of a subset of CD4+ effector T cells. Indeed, we identified a subset of CD4+ T cells expressing NKG2D in both mucosa and peripheral blood (PB) of patients with CD. This CD4+ subset exhibits a Th1 cytokine profile and exerts cytotoxic activity against targets expressing MICA. In vitro data suggest a role for IECs and interleukin (IL)-15 in expansion and functional modulation of CD4+NKG2D+ T cells. Forty-one patients with moderate to severely active IBD (CD, n = 25; ulcerative colitis [UC], n = 16) were included in this prospective study. Characteristics of the patients are given in Table 1. Peripheral blood lymphocytes (PBLs) were isolated from 34 patients (CD, n = 21; UC, n = 13). IECs and mucosal lymphocytes (lamina propria lymphocytes [LPLs] and intraepithelial lymphocytes [IELs]) were isolated from surgical specimens (n = 15) or endoscopic biopsy specimens (n = 9) of 24 patients (CD, n = 16; UC, n = 8). With surgical specimens of patients with CD, tissues were taken from inflammatory sites and noninflammatory sites when available.Table 1Clinical Characteristics of Patients With IBD and ControlsPatients With CD (n = 25)Patients With UC (n = 16)Controls (n = 24)Age (y)aMean ± SD.34 ± 536 ± 1352 ± 14Sex (M/F)10/1511/511/13Location of lesions Ileum180— Colon1516— Anus60—Active disease2516— Harvey–BradshawaMean ± SD.10 ± 4.2—— Truelove–WittsbMedian (extremes).,cNumber of criteria.—3 (1–4)—C-reactive protein (mg/L)bMedian (extremes).32 (9–333)27 (1–105)Not determinedTreatmentsdAt study inclusion. 5-ASA16— Corticosteroids48— Antibiotics10— Azathioprine/6-mercaptopurine, methotrexate83—a Mean ± SD.b Median (extremes).c Number of criteria.d At study inclusion. Open table in a new tab The control group consisted of patients undergoing bowel resection for cancer (n = 4), healthy patients undergoing screening colonoscopy for colorectal cancer (n = 6), or healthy blood donors (n = 16). IECs and mucosal lymphocytes (LPLs and IELs) were isolated from 10 controls, and PBLs were isolated from 20 controls (including 16 healthy blood donors and 4 patients undergoing colonoscopy). This study was approved by the ethical committee of Hospital Saint-Louis, and all subjects gave written informed consent. Isolation from surgical specimens was performed as described previously.7Allez M. Brimnes J. Dotan I. Mayer L. Expansion of CD8+ T cells with regulatory function after interaction with intestinal epithelial cells.Gastroenterology. 2002; 123: 1516-1526Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar Briefly, surgical specimens were washed extensively with phosphate-buffered saline (PBS). The mucosa was stripped off from the submucosa, minced into small pieces, and placed in 1 mmol/L dithiothreitol for 10 minutes at room temperature. The pieces were washed in PBS and incubated in medium (RPMI 1640) containing 1.5 mmol/L MgCl2 and 1 mmol/L EDTA for 30 minutes at 37°C and vortexed every 5 minutes. The supernatant, containing IECs and IELs, was passed through a nylon filter (Falcon 2360; Becton Dickinson, BD Biosciences, Le Pont de Claix, France). Cells were washed twice in PBS and resuspended in RPMI 1640. For isolation from endoscopic biopsy specimens, the same technique was applied but biopsy specimens were incubated directly in medium (RPMI 1640) containing EDTA, without incubation in dithiothreitol. LPLs were isolated from the remaining tissue. The tissue was incubated for 1 hour at 37°C in medium containing 1 mg/mL collagenase (Clostridiopeptidase A). The cell suspension was collected, centrifuged, washed, and resuspended in PBS. Heparinized venous blood was collected from patients or controls, diluted 1:3 with PBS, layered on a Ficoll-Hypaque density gradient, and centrifuged for 30 minutes at 900g. The PB mononuclear cells were collected from the interface, washed 3 times with PBS, and resuspended in RPMI 1640. Deparaffinized sections of resected specimens or biopsy specimens were treated with 40% normal human serum for 20 minutes at room temperature and incubated with the monomorphic anti-MICA SR99 monoclonal antibody (mAb) for 1 hour or isotype-matched control immunoglobulin (Ig).24Hue S. Mention J.J. Monteiro R.C. Zhang S. Cellier C. Schmitz J. Verkarre V. Fodil N. Bahram S. Cerf-Bensussan N. Caillat-Zucman S. A direct role for NKG2D/MICA interaction in villous atrophy during celiac disease.Immunity. 2004; 21: 367-377Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar Antibody binding was visualized by using biotinylated goat antimouse Ig and the peroxidase EnVision System (Dako, Carpinteria, CA). IECs were incubated with mAbs AMO-1 (anti-MICA mAb; Immatics, Biotechnologies, Tubingen, Germany), L243 (anti-MHC II), and W6/32 (anti-MHC I) and IgG isotype controls at 4°C for 30 minutes and then washed and incubated with a phycoerythrin-conjugated goat anti-mouse Ig mAb and then with a fluorescein isothiocyanate (FITC)-conjugated anti-epithelial specific antigen (ESA, Biomeda). Two color analyses, gated on ESA-positive cells (IECs), were performed using CellQuest software on a Becton Dickinson FACScalibur. Results were expressed as percentage of IECs expressing MICA as well as mean fluorescence intensity (MFI). Lymphocyte preparations (PBLs, LPLs, and IELs) were resuspended in PBS and incubated for 30 minutes with antibodies. The following antibodies either conjugated with FITC, phycoerythrin, peridin-chlorophyll protein, or allophycocyanin and directed against CD3, CD8, CD4, CD25, CD28, CD56, CD57, CD94, CD158b, CD161 (BD Biosciences), NKG2D (Beckman Coulter, Miami, FL); NKG2A, NKG2C, NKp30, NKp44, NKp46; integrins α4, β1, and β7 (BD Biosciences); and IL15Rα, IL21R, CCR2, CCR5, CCR7, CCR9 (R&D Systems, Abingdon, UK), and relevant isotype controls were used. Anti-DAP10 was a rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Four-color analyses were performed using CellQuest software (BD Biosciences). Lymphocytes (PBLs, LPLs) were stimulated for 4 hours with phorbol myristate acetate (PMA) and ionomycin before intracellular staining. Cells were incubated with surface markers (CD3, CD4, NKG2D) followed by 10-minute fixation in 3.5% formaldehyde solution. The cells were then incubated with FACS permeabilizing solution (BD Biosciences) for 10 minutes and with FITC-conjugated antibodies directed against interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-2, IL-4, IL-10 (R&D Systems), and isotype controls. The same protocol was used for intracellular anti-DAP10 staining. Intracellular staining for perforin was performed using saponin 0.2%. Different T-cell populations (CD3+CD4+NKG2D+ and CD3+CD4+NKG2D− T cells) were sorted from the PB of 2 patients with active CD, on a FACS Vantage (BD FACS Vantage, Franklin Lakes, NJ) machine, to generate T-cell lines and clones. For cloning, CD4+NKG2D+ and NKG2D− T cells were plated at 1 cell/well. T cells were stimulated every 10 days using IL-2 (150 UI/mL), phytohemagglutinin (PHA) 1 μg/mL, and feeder cells (allogeneic PB mononuclear cells from 3 healthy donors and a B-EBV line, irradiated at 50 Gray) in RPMI 1640 with 10% human serum. Fifteen CD4+NKG2D+ T-cell clones (4 used for functional analysis), 2 CD4+NKG2D− T-cell clones, and 2 CD4+NKG2D+ and 2 CD4+NKG2D− T-cell lines were obtained from the 2 patients with CD. CD3+CD4+NKG2D+ and CD4+NKG2D− T-cell clones were cultured for 12 hours at 37°C in presence of a C1R line transfected with MICA complementary DNA or a vector control,22Tieng V. Le Bouguenec C. du Merle L. Bertheau P. Desreumaux P. Janin A. Charron D. Toubert A. Binding of Escherichia coli adhesin AfaE to CD55 triggers cell-surface expression of the MHC class I-related molecule MICA.Proc Natl Acad Sci U S A. 2002; 99: 2977-2982Crossref PubMed Scopus (207) Google Scholar and supernatants were collected for measurement of IFN-γ (Diaclone, Stamford, CT). In blocking experiments, C1R-MICA cells were preincubated with mAbs (10 μg/mL) directed against MICA (AMO1), MHC class II (L243), or isotype controls for 30 minutes before the culture with CD4+ T-cell clones. T-cell clones were also preincubated for 30 minutes with mAbs directed against NKG2D (1D11; Pharmingen, BD Biosciences Pharmingen, San Diego, CA) or isotype controls before culture with C1R-MICA targets. Clones were also incubated in the presence of coated soluble MICA. Soluble MICA (Prospect-Tany TechnoGene, Rehovot, Israel) was coated at different concentrations (2–10 μg/mL in PBS 1× overnight at 4°C on 96-well plates (Maxisorp Polylabo, Strasbourg, France). CD4+NKG2D+ T-cell clones (2 × 105 cells) were incubated for 24 hours, and IFN-γ was measured in the supernatant. Clones were also preincubated with anti-NKG2D blocking mAb (1D11) or isotype control. 51Chromium-release assays were performed using P815 cells (an FcγR+ mouse mastocytoma; ATCC) and C1R-MICA or control C1R transfectants as targets at a 50:1 effector/target ratio in triplicate wells. For Fc-dependent redirected cytotoxicity, effectors and targets were incubated with an anti-NKG2D mAb (1D11 at 5 μg/mL) and/or an anti-CD3 mAb (OKT3 at 0.05 μg/mL) and/or anti–killer Ig-like receptor 2DS2 mAb (anti-CD158b at 5 μg/mL; BD Biosciences) or control mAbs. Chromium release was measured after a 4-hour incubation using a scintillation counter (Packard, Meriden, CT). The percentage of specific cytotoxicity was calculated using the following formula: 100 × (cpm experimental − cpm spontaneous)/(cpm maximum − cpm spontaneous). Mixed cell culture was performed as previously described using irradiated IECs as stimulator cells and allogeneic PB T cells as responder cells.7Allez M. Brimnes J. Dotan I. Mayer L. Expansion of CD8+ T cells with regulatory function after interaction with intestinal epithelial cells.Gastroenterology. 2002; 123: 1516-1526Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar Carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells and IECs were cocultured at 1 × 106/mL and 0.5 × 106/mL, respectively, in culture medium at 37°C in a 5% co2 humidified incubator for 5–10 days. CFSE-labeled T cells from healthy donors were cultured in the presence of the following recombinant cytokines: IL-15 (15 ng/mL), IL-12 (5 μg/mL), IL-7 (10 ng/mL), or IL-2 (30 U/mL) (all from Pharmingen). T-cell proliferation was analyzed by flow cytometry at 5 days. T-cell clones and lines were lysed in TRIzol (Invitrogen Life Technologies, Cergy Pontoise, France). Total RNA was isolated according to the manufacturer's instructions. After reverse transcription, the complementary DNA sample was subjected to real-time polymerase chain reaction analysis using primer sets specific for the various genes (KIR2DS1-5, KIR2DL1-5, KIR3DL1-3, NKG2A/B/C/D/E, NKp30/p44/p46, DAP10, DAP12, CD94, CD161) or housekeeping control genes as previously described.25Lafarge X. Pitard V. Ravet S. Roumanes D. Halary F. Dromer C. et al.Expression of MHC class I receptors confers functional intraclonal heterogeneity to a reactive expansion of gammadelta T cells.Eur J Immunol. 2005; 35: 1896-1905Crossref PubMed Scopus (25) Google Scholar For all studies, data are expressed as mean ± SD. Differences between groups were analyzed by using the Student t test with significance defined as P < .05. There was a low level of expression of MICA on IECs in normal controls, with a trend toward a higher expression of MICA in the ileum and/or right colon (MFI, 6 ± 5.4; percentage of cells expressing MICA, 4.1% ± 2.3%) compared with the left colon (MFI, 1.7 ± 1.3; percentage of cells expressing MICA, 3.1% ± 2.3%) (Figure 1A). Immunohistochemistry in normal controls showed that MICA expression was noted on epithelium on the surface, while the staining was negative in the crypts. In contrast, MICA expression was significantly increased on IECs isolated from patients with IBD. In CD, MICA expression was significantly increased on IECs isolated from inflammatory lesions in the ileum and/or right colon (MFI, 22.4 ± 15; percentage of cells expressing MICA, 28% ± 17%) compared with IECs isolated from the ileum and/or right colon in controls (P = .001). MICA expression was higher in macroscopically involved areas compared with noninvolved areas. The ratio for MICA MFI on IECs from macroscopically involved and noninvolved areas was 3.4 ± 2.1 (n = 5). MHC class II molecule expression was also increased in involved areas compared with uninvolved areas, while there was no difference in the expression of MHC class I molecules (Figure 1B). By immunohistochemistry, MICA was strongly expressed on both crypt and villous enterocytes of the macroscopically inflamed epithelium of patients with CD both at apical and basal surfaces (Figure 1D and E), contrasting with lower staining on macroscopically normal areas from the same individuals (observed mainly on villous enterocytes but not in the crypts) (Figure 1C). In UC, MICA expression was significantly increased on IECs isolated from inflammatory lesions in the left colon (MFI, 9.7 ± 9; 27% ± 19% of cells expressing MICA) compared with IECs isolated from the same location in controls (MFI, 1.7 ± 1.3; P = .02; 3.1% ± 2.3% of cells expressing MICA). Immunohistochemistry also showed an increased expression of MICA in inflamed areas (Figure 1I) compared with noninflamed areas (Figure 1H). In controls, the NKG2D expression was observed on the majority of CD8+ PBLs (>95%) but not on CD4+ PBLs (1.6% ± 3.3%). In the mucosa of controls, CD8+ T cells frequently expressed NKG2D (>95%), as well as γδ (>95%) and double negative IELs (CD3+CD4−CD8−) (86% ± 15%). In contrast, few CD4+ mucosal T cells (LPLs and IELs) from controls expressed NKG2D, with the percentage of CD4+ LPLs expressing NKG2D being 2.1% ± 1.3%, 3.2% ± 1.3%, and 2.1% ± 0.9% in the left colon, right colon, and ileum, respectively. There was no difference between patients with CD, patients with UC, and controls regarding the percentage of CD8+ T cells expressing NKG2D as well as the level of NKG2D expression on PB and lamina propria CD8+ T cells (data not shown). In contrast, as illustrated in Figure 2A and B, the percentage of CD4+ T cells expressing NKG2D isolated from the mucosa of patients with CD was increased compared with CD4+ LPLs isolated from ileum or right colon in controls (9% ± 5.3% vs 2.9% ± 1.6%; P = .003). Also, the percentage of CD4+ T cells expressing NKG2D in the PB was significantly increased in patients with CD (6.7% ± 7%; n = 21) compared with normal controls (1.5% ± 2.7%; n = 18; P = .01) (Figure 2B). However, there was no correlation between percentages of CD4+ T cells expressing NKG2D in the periphery and the mucosa. In contrast to patients with CD, the expression of NKG2D on CD4+ LPLs isolated from the inflamed mucosa of patients with UC (2.1% ± 1%) was similar to the expression of NKG2D on CD4+ LPLs isolated from controls. The percentage of CD4+ T cells expressing NKG2D in the PB of patients with UC (1.6% ± 1.3%) was also similar to the percentage observed in controls and significantly lower than in patients with CD (P = .003). We then studied the phenotype of CD4+NKG2D+ T cells in the lamina propria of patients with CD (Table 2). All CD4+NKG2D+ T cells expressed TCRαβ. This subset was α 4+ (74% ± 15%), β 7+ (>80%) and β 1+ (>90%) and expressed CD103 (αEβ7) (75% ± 13%). They also expressed CCR9 (54% ± 27%) but did not express CCR2 (<5%), CCR5 (<5%), or CCR7 (<10%). CD4+NKG2D+ T cells expressed perforin (66% ± 28%), suggesting that this subset might have cytotoxic activity.Table 2The Phenotype of CD4+NKG2D+ and CD4+NKG2D− T Cells in the Lamina Propria and the PB of Patients With CDCD4+ LPLs (%)CD4+ PBLs (%)NKG2D−NKG2D+NKG2D−NKG2D+CD2870 ± 1956 ± 3289 ± 915 ± 9CD10321 ± 775 ± 13<1 90%). The phenotype of PB CD4+NKG2D+ T cells also differed from CD4+NKG2D+ LPLs, with a low or negative expression of CCR9, CD103 (αEβ7), and β7. Perforin staining was high in CD4+NKG2D+ PBLs as well as in CD4+NKG2D+ LPLs (Figure 3B). Intracellular cytokine expression was analyzed by flow cytometry on PBLs and LPLs freshly isolated from 8 patients with CD comparatively in the CD4+NKG2D+ and CD4+NKG2D− lymphocyte populations. CD4+NKG2D+ T cells were highly positive for IFN-γ and TNF-α intracellular staining after 4-hour stimulation with PMA and ionomycin both in the PB (Figure 3C) and in the mucosa (Figure 3D). Only a low percentage of CD4+NKG2D+ T cells expressed IL-2 (Figure 3C and D), while no expression of IL-4 and IL-10 was observed (data not shown). To further investigate the function of CD4+NKG2D+ T cells, T-cell clones and lines were derived from the PB of 2 patients with CD by limited dilution. The CD4+NKG2D+ T-cell clones had the same phenotype as ex vivo PB CD4+NKG2D+ T cells isolated from the s
Referência(s)