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• W2111452470 abstract "Background & Aims: The extensive infiltration of CD8+ T cells in the intestinal mucosa of celiac disease (CD) patients is a hallmark of the disease. We identified a gliadin peptide (pA2) that is selectively recognized by CD8+ T cells infiltrating intestinal mucosa of HLA-A2+ CD patients. Herein, we investigated the phenotype, the tissue localization, and the effector mechanism of cells responsive to pA2 by using the organ culture of CD intestinal mucosa. The target of pA2-mediated cytotoxicity was also investigated by using the intestinal epithelial cell lines Caco2 and HT29, A2+ and A2−, respectively, as target cells. Methods: Jejunal biopsy specimens from CD patients were cultured in vitro with pA2, and cellular activation was evaluated by immunohistochemistry and cytofluorimetric analysis. Cytotoxicity of pA2-specific, intestinal CD8+ T cells was assayed by granzyme-B and interferon-γ release and by apoptosis of target cells. Results: pA2 challenge of A2+ CD mucosa increased the percentage of CD8+CD25+ and of CD80+ cells in the lamina propria, the former mainly localized beneath the epithelium, as well as the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-positive cells (TUNEL+) in the epithelium. Intraepithelial CD3+ cells and enterocyte expression of Fas were also increased. CD8+CD25+ and CD8+FASL+ T cells were significantly increased in cell preparations from biopsy specimens cultured with pA2. CD8+ T-cell lines released both granzyme-B and interferon-γ following recognition of pA2 when presented by Caco2 and not by HT29. Conclusions: These data indicate that gliadins contain peptides able to activate, through a TCR/HLA class I interaction, CD8-mediated response in intestinal CD mucosa and to induce the enterocyte apoptosis. Background & Aims: The extensive infiltration of CD8+ T cells in the intestinal mucosa of celiac disease (CD) patients is a hallmark of the disease. We identified a gliadin peptide (pA2) that is selectively recognized by CD8+ T cells infiltrating intestinal mucosa of HLA-A2+ CD patients. Herein, we investigated the phenotype, the tissue localization, and the effector mechanism of cells responsive to pA2 by using the organ culture of CD intestinal mucosa. The target of pA2-mediated cytotoxicity was also investigated by using the intestinal epithelial cell lines Caco2 and HT29, A2+ and A2−, respectively, as target cells. Methods: Jejunal biopsy specimens from CD patients were cultured in vitro with pA2, and cellular activation was evaluated by immunohistochemistry and cytofluorimetric analysis. Cytotoxicity of pA2-specific, intestinal CD8+ T cells was assayed by granzyme-B and interferon-γ release and by apoptosis of target cells. Results: pA2 challenge of A2+ CD mucosa increased the percentage of CD8+CD25+ and of CD80+ cells in the lamina propria, the former mainly localized beneath the epithelium, as well as the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-positive cells (TUNEL+) in the epithelium. Intraepithelial CD3+ cells and enterocyte expression of Fas were also increased. CD8+CD25+ and CD8+FASL+ T cells were significantly increased in cell preparations from biopsy specimens cultured with pA2. CD8+ T-cell lines released both granzyme-B and interferon-γ following recognition of pA2 when presented by Caco2 and not by HT29. Conclusions: These data indicate that gliadins contain peptides able to activate, through a TCR/HLA class I interaction, CD8-mediated response in intestinal CD mucosa and to induce the enterocyte apoptosis. Celiac disease (CD) is one of the most thoroughly investigated and characterized forms of food intolerance. Ingestion of wheat gliadins and related prolamines leads to enteropathy in genetically susceptible individuals.1Sollid L.M. Celiac disease: dissecting a complex inflammatory disorder.Nat Rev Immunol. 2002; 2: 647-655Crossref PubMed Scopus (787) Google Scholar The characteristic features of CD inflammation are villous atrophy, crypt hyperplasia, and increased number of both intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). Although CD4+ T-cell-mediated hypersensitivity to gluten plays a crucial role in tissue injury in CD, its pathogenesis still remains unclear.2Lundin K.E. Scott H. Hansen T. et al.Gliadin-specific HLA-DQ(a1*0501,b1*0201) restricted T-cells isolated from the small intestinal mucosa of celiac disease patients.J Exp Med. 1993; 178: 187-196Crossref PubMed Scopus (523) Google Scholar, 3van de Wal Y. Kooy Y. van Veelen P. et al.Small intestinal T cells of celiac disease patients recognize a natural peptide fragment of gliadin.Proc Natl Acad Sci U S A. 1998; 95: 10050-10054Crossref PubMed Scopus (218) Google Scholar, 4Troncone R. Gianfrani C. Mazzarella G. et al.Majority of gliadin-specific T-cell clones from celiac small intestinal mucosa produce interferon-γ and interleukin-4.Dig Dis Sci. 1998; 43: 156-161Crossref PubMed Scopus (54) Google Scholar Although the role of HLA class II-restricted CD4+ T cells in CD pathogenesis is well documented, recent evidence also supports the involvement of both HLA class I-restricted CD8+ T cells5Gianfrani C. Troncone R. Magione P. et al.Celiac disease association with CD8+ T-cell responses: identification of a novel gliadin-derived HLA-A2 restricted epitope.J Immunol. 2003; 170: 2719-2726Crossref PubMed Scopus (69) Google Scholar and of the innate immune system.6Oberhuber G. Vogelsang H. Stolte M. et al.Evidence that intestinal intraepithelial lymphocytes are activated cytotoxic T cells in celiac disease but not in giardiasis.Am J Pathol. 1996; 148: 1351-1357PubMed Google Scholar, 7Ciccocioppo R. Di Sabatino A. Parroni R. et al.Cytolytic mechanisms of intraepithelial lymphocytes in coeliac disease (CoD).Clin Exp Immunol. 2000; 120: 235-240Crossref PubMed Scopus (49) Google Scholar, 8Di Sabatino A. Ciccocioppo R. D’Alò S. et al.Intraepithelial and lamina propria lymphocytes show distinct patterns of apoptosis, whereas both populations are active in Fas based cytotoxicity in coeliac disease.Gut. 2001; 49: 380-386Crossref PubMed Scopus (74) Google Scholar, 9Meresse B. Chen Z. Ciszewski C. et al.Coordinated induction by IL15 of a TCR-independent NKG2D signaling pathway converts CTL into lymphokine-activated killer cells in coeliac disease.Immunity. 2004; 21: 357-366Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar, 10Hue S. Mention J.J. Monteiro R. et al.A direct role for NKG2D/MICA interaction in villous atrophy during coeliac disease.Immunity. 2004; 21: 367-377Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar, 11Jabri B. Patey-Mariaud De Serre N. Cellier C. et al.Selective expansion of intraepithelial lymphocytes expressing the HLA-E-specific natural killer receptor CD94 in coeliac disease.Gastroenterology. 2000; 118: 867-879Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 12Gianfrani C. Auricchio S. Troncone R. Adaptive and innate immune responses in coeliac disease.Immunol Lett. 2005; 99: 141-145Crossref PubMed Scopus (108) Google Scholar Several studies reported a marked increase of activated CD8+ T cells containing large granzyme-B (GrB)-positive granules and of cell surface expression of FASL in intestinal mucosa of untreated CD patients.6Oberhuber G. Vogelsang H. Stolte M. et al.Evidence that intestinal intraepithelial lymphocytes are activated cytotoxic T cells in celiac disease but not in giardiasis.Am J Pathol. 1996; 148: 1351-1357PubMed Google Scholar, 7Ciccocioppo R. Di Sabatino A. Parroni R. et al.Cytolytic mechanisms of intraepithelial lymphocytes in coeliac disease (CoD).Clin Exp Immunol. 2000; 120: 235-240Crossref PubMed Scopus (49) Google Scholar Additionally, FAS-FASL-mediated killing of enterocytes by CD8+ lymphocytes has been reported in mucosa from patients with active disease.8Di Sabatino A. Ciccocioppo R. D’Alò S. et al.Intraepithelial and lamina propria lymphocytes show distinct patterns of apoptosis, whereas both populations are active in Fas based cytotoxicity in coeliac disease.Gut. 2001; 49: 380-386Crossref PubMed Scopus (74) Google Scholar Finally, recent studies have demonstrated a marked expression of the stress molecule MICA on enterocytes in patients with active disease and the involvement of MICA-NKG2D interaction in the enterocyte lysis by CD8+ IELs.9Meresse B. Chen Z. Ciszewski C. et al.Coordinated induction by IL15 of a TCR-independent NKG2D signaling pathway converts CTL into lymphokine-activated killer cells in coeliac disease.Immunity. 2004; 21: 357-366Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar, 10Hue S. Mention J.J. Monteiro R. et al.A direct role for NKG2D/MICA interaction in villous atrophy during coeliac disease.Immunity. 2004; 21: 367-377Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar Interestingly, the activation of these killer IELs, although gliadin-triggered, is T-cell receptor (TCR) independent and most likely mediated by interleukin (IL)-15, an inflammatory cytokine highly up-regulated in patient-derived mucosa.11Jabri B. Patey-Mariaud De Serre N. Cellier C. et al.Selective expansion of intraepithelial lymphocytes expressing the HLA-E-specific natural killer receptor CD94 in coeliac disease.Gastroenterology. 2000; 118: 867-879Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 13Maiuri L. Ciacci C. Auricchio S. et al.Interleukin 15 mediates epithelial changes in coeliac disease.Gastroenterology. 2000; 119: 996-1006Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 14Mention J.J. Ahmed M.B. Begue B. et al.Interleukin 15: a key to disrupted intraepithelial lymphocyte homeostasis and lymphomagenesis in coeliac disease.Gastroenterology. 2003; 125: 730-745Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar We have previously reported a TCR-dependent activation of intestinal CD8+ T cells by a gliadin-derived peptide (pA2) in patients either on a gluten-free or gluten-containing diet.5Gianfrani C. Troncone R. Magione P. et al.Celiac disease association with CD8+ T-cell responses: identification of a novel gliadin-derived HLA-A2 restricted epitope.J Immunol. 2003; 170: 2719-2726Crossref PubMed Scopus (69) Google Scholar This peptide was selectively recognized in the context of the HLA-A*0201 molecule and induced interferon (IFN)-γ production and lysis of target cells by specific CD8+ T cells. Importantly, IFN-γ is a proinflammatory cytokine dominant in untreated and in gliadin-challenged treated CD mucosa,15Nilsen E.M. Jahnsen F.L. Lundin K.E.A. et al.Gluten induces an intestinal cytokine response strongly dominated by interferon-γ in patients with coeliac disease.Gastroenterology. 1998; 115: 551-563Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 16Salvati V. Mazzarella G. Gianfrani C. et al.Recombinant human IL-10 suppresses gliadin-dependent T-cell activation in ex vivo cultured coeliac intestinal mucosa.Gut. 2005; 54: 46-53Crossref PubMed Scopus (115) Google Scholar and it is produced by TCR α/β CD8+ T cells in both epithelium and lamina propria.17Forsberg G. Hernell O. Hammarström S. et al.Concomitant increase of IL-10 and pro-inflammatory cytokines in intraepithelial lymphocyte subsets in celiac disease.Int Immunol. 2007; 19: 993-1001Crossref PubMed Scopus (46) Google Scholar, 18Leon F. Sanchez L. Camarero C. et al.Cytokine production by intestinal intraepithelial lymphocytes subsets in celiac disease.Dig Dis Sci. 2005; 50: 593-600Crossref PubMed Scopus (29) Google Scholar The aim of the present study was to characterize further the gliadin-specific, HLA class I-restricted CD8+ T cells and their role in CD pathogenesis by using the organ culture system of treated CD mucosa and combined immunohistochemistry (IHC) and flow cytometry (FACS) ex vivo analysis. Both IHC and FACS were complementarily used to investigate immunologic activation following mucosal challenge with the cytotoxic gliadin peptide. Expression of activation (CD25), effector (FASL, FAS), and costimulatory (CD80) molecules, as well as mucosal localization of activated cells, were evaluated in intestinal explants cultured for 24 hours in presence of pA2, a peptic-tryptic digested gliadin (PT-gliadin) or an HLA-A2-restricted, control peptide. Finally, the role of pA2-activated intestinal CD8+ T cells in damage of the epithelial layer was also investigated. Twenty-two patients with CD (5 male and 17 female; mean age, 35 years; range 20–57 years) on a gluten-free diet for at least 2 years were recruited (see Supplemental Table 1 online at www.gastrojournal.org). Jejunum biopsy specimens were also obtained from 8 non-CD individuals (4 male and 4 female; mean age, 43 years; range 21–58 years): 3 had esophagitis, 3 gastritis without Helicobacter pylori infection, and 2 gastritis with Helicobacter pylori infection. All controls had a normal intestinal mucosa and a negative serology for antigliadin/antibodies and antiendomysium antibodies. All patients and controls were serologically typed for major histocompatibility complex (MHC) class I alleles using commercial kit (EsseMedical, Milan, Italy); 11 of 22 patients and 2 of 8 controls were HLA-A2+. DQA1, DQB1, and DRB1 genotypes were determined using a commercial kit (Dynal, Oslo, Norway) (see Supplemental Table 1 online at www.gastrojournal.org). All patients and controls were recruited from the Avellino area and gave their informed consent to the study approved by the local ethical committee of Hospital Moscati. Peptides were synthesized as previously reported.19Gulukota K. Sidney J. Sette A. et al.Two complementary methods for predicting peptides binding major histocompatibility complex molecules.J Mol Biol. 1997; 267: 1258-1267Crossref PubMed Scopus (232) Google Scholar pA2 corresponds to the residues 123–132 (QLIPCMDVVL) of A-gliadin. The A2-restricted, HIVgag17 peptide (SLYNTVATL) was used as a negative control (pctl); PT-gliadin was used as a positive control. Intestinal biopsy specimens were cultured as described elsewhere.20Mazzarella G. Maglio M. Paparo F. et al.An immunodominant DQ8 restricted gliadin peptide activates small intestinal immune response in in vitro cultured mucosa from HLA-DQ8 positive but not HLA-DQ8 negative coeliac patients.Gut. 2003; 52: 57-62Crossref PubMed Scopus (74) Google Scholar Briefly, biopsy specimens were placed on iron grids in an organ culture dish in the presence of medium alone, pA2, or pctl (100 μg/mL) or PT-gliadin (1 mg/mL). After 24 hours, biopsy specimens were embedded in OCT and snap frozen in liquid nitrogen. Acetone-fixed sections (5 μm) were stained with the monoclonal antibodies (mAb) (see Supplemental Table 2 online at www.gastrojournal.org), and peroxidase-antiperoxidase immunodetection was used for CD3 and Fas; alkaline-phosphatase/antialkaline-phosphatase was used for CD25 and CD80. Mouse immunoglobulins were used as isotype control. The sections were finally stained with Mayer’s hematoxylin. In experiments to detect CD8+CD25+ cells, immunofluorescence with confocal microscopy (Leica TCS-SP, Heidelberg, Germany) was used. Cryosections were fixed in acetone and incubated with mouse anti-CD25 and rat anti-CD8 antibodies, followed by a mixture of horse anti-mouse fluorescein isothiocyanate (FITC) (1:200; Vector Laboratories, Milan, Italy) and rabbit anti-rat tetramethylrhodamine isothiocyanate (TRITC) conjugated (1:300; Dako, Milan, Italy). Sections were counterstained with ToPro-3 (Molecular Probes, Leiden, The Netherlands) and mounted in phosphate-buffered saline (PBS)/glycerol (1:1). The density of intraepithelial CD3+ cells was calculated on 100 enterocytes; the number of CD25+ and CD80+ cells was evaluated within 1 mm2 of lamina propria. Percentage of CD25+CD8+ T cells was calculated on total of CD8+ LPLs. Staining of intestinal epithelial cells (IEC) expressing Fas was graded as weak, moderate, or strong. Cell apoptosis was detected on frozen tissue fixed with 4% paraformaldehyde using the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay (Roche, Mannheim, Germany). Reagents were incubated for 1 hour at 37°C. To highlight the epithelium, the same slides were next incubated with mouse anti-human epithelial antigen (DAKO) for 1 hour at room temperature (RT), followed by 1 hour of incubation with horse anti-mouse FITC. Finally, the samples were counterstained with ToPro-3 and imaged with confocal microscope. All slides were blindly analyzed by 2 observers. Jejunal biopsy specimens, either following 24 hours of peptides/gliadin challenge or freshly processed, were digested with collagenase-A, as previously described.4Troncone R. Gianfrani C. Mazzarella G. et al.Majority of gliadin-specific T-cell clones from celiac small intestinal mucosa produce interferon-γ and interleukin-4.Dig Dis Sci. 1998; 43: 156-161Crossref PubMed Scopus (54) Google Scholar Immediately after the isolation from mucosal explant, cells were analyzed for surface phenotype by fluorescence-activated cell sorter (FACS) using 2- or 3-color immunostainings: 2 × 104 cells were washed in FACS buffer (PBS containing 2% fetal calf serum [FCS] and 0.05% sodium azide) and stained for 30 minutes at RT with fluorochrome-conjugated antibodies (see Supplemental Table 2 online at www.gastrojournal.org). Isotype control immunoglobulins (Igs) were also used in each set of analysis. Cell fluorescence was analyzed using a FACSalibur (Becton Dickinson). Viable cells were delimited by the exclusion of propidium iodide (Sigma); lymphocytes were gated on the base of the forward/side scattered (FCS/SSC) profile or CD45 expression. Analysis was performed on gated cells using the Cell Quest software (Becton Dickinson). pA2-specific CD8+ cytotoxic T lymphocytes (CTLs) lines were generated from treated mucosa as previously described.12Gianfrani C. Auricchio S. Troncone R. Adaptive and innate immune responses in coeliac disease.Immunol Lett. 2005; 99: 141-145Crossref PubMed Scopus (108) Google Scholar Briefly, mucosal cells were suspended at 2–5 × 105 in 1 mL of complete medium (RPMI plus 10% human serum and antibiotics, purchased from Bio-Whittaker, Bergamo Italy) and plated in the presence of 1 × 106 irradiated (3000 rad) autologous peripheral blood mononuclear cells (PBMC) and pA2 (20 μg/mL). Twenty-four hours later and every 3 days, cultures were fed with 1 mL fresh medium and 10 ng/mL IL-15 (R&D Systems, Minneapolis, MN). Every 7 days, CTLs were restimulated with pA2/PBMC. CTLs were tested for pA2 recognition by both IFN-γ and GrB release. HLA-A2+ EBV-transformed B-cell line, JY, were used as antigen-presenting cells (APCs). T cells were plated at 3 × 104 in the presence of 1 × 105 APCs and pA2 at 20 μg/mL and incubated for 20–48 hours, as indicated. IFN-γ enzyme-linked immunospot (ELISPOT) was performed as previously described.5Gianfrani C. Troncone R. Magione P. et al.Celiac disease association with CD8+ T-cell responses: identification of a novel gliadin-derived HLA-A2 restricted epitope.J Immunol. 2003; 170: 2719-2726Crossref PubMed Scopus (69) Google Scholar Levels of IFN-γ in cell supernatants were determined by sandwich enzyme-linked immunosorbent assay (ELISA), as described.21Gianfrani C. Levings M.K. Sartirana C. et al.Gliadin-specific type 1 regulatory T cells from the intestinal mucosa of treated celiac patients inhibit pathogenic T cells.J Immunol. 2006; 177: 4178-4186Crossref PubMed Scopus (99) Google Scholar ELISPOT/ELISA commercial kits for GrB detection were purchased from Sanquin (Amsterdam, The Netherlands). Spot-forming cells (SFC) were counted by an immunospot analyzer (A.EL.VIS, Hannover, Germany). In the experiments with blocking mAbs, T-cells and JY were preincubated for 10 minutes with anti-human NKG2D (10 μg/mL; clone 1D11; Biolegend, San Diego, CA) and anti-HLA class I (10 μg/mL; clone W6-32; Biolegend), respectively, before addition of peptide. For pentamer experiments, 96-well round-bottom plates were coated with pA2- or pctl-pentamers (ProImmune, Oxford, UK) at concentration of 1.0, 0.5, and 0.1 μg/mL in 100 μL PBS overnight at 4°C. Following 2 washings with PBS, 0.3 × 105 cells were plated in 200 μL complete medium. After 48 hours of incubation, supernatants were collected and assayed for IFN-γ ELISA. All experiments were performed in duplicate. In some experiments, human colon adenocarcinoma Caco2 (HLA-A2+) and HT29 (HLA-A2−) cells were used as APCs. EC were grown in Dulbecco’s modified Eagle medium (DMEM)/10% FCS medium supplemented with glucose 4.5 g/mL and harvested at subconfluence by a 10-minute incubation with trypsin-EDTA (Bio-Whittaker). In all coculture experiments, except those for apoptosis evaluation, 1–2 × 105 EC were irradiated (10,000 rad) before being added to 0.5–1 × 105 T cells (T/EC ratio, 1:2). Apoptosis of Caco2 was determined using the FITC-Annexin-V/propidium staining kit (Sigma). Percentage of apoptotic cells was calculated in the region of epithelial (EpCAM+) cells. Student 2-tailed t test was used to compare responses of specimens cultured with peptides/PT-gliadin with those cultured with medium alone. A P value ≤.05 was considered statistically significant. Consistent with previous results,16Salvati V. Mazzarella G. Gianfrani C. et al.Recombinant human IL-10 suppresses gliadin-dependent T-cell activation in ex vivo cultured coeliac intestinal mucosa.Gut. 2005; 54: 46-53Crossref PubMed Scopus (115) Google Scholar, 20Mazzarella G. Maglio M. Paparo F. et al.An immunodominant DQ8 restricted gliadin peptide activates small intestinal immune response in in vitro cultured mucosa from HLA-DQ8 positive but not HLA-DQ8 negative coeliac patients.Gut. 2003; 52: 57-62Crossref PubMed Scopus (74) Google Scholar the number of cells/mm2 expressing CD25 was significantly higher in biopsy specimens cultured with PT-gliadin than in those cultured with medium alone, both in HLA-A2+ (n = 8) and HLA-A2− (n = 6) CD patients: (mean ± SD): 126 ± 51 and 114 ± 41 in PT-gliadin-challenged biopsy specimens and 30 ± 11 and 26 ± 3 in medium-challenged biopsy specimens in A2+ and A2− CD patients, respectively (P < .001) (Figure 1A). The number of CD25+ cells in A2+ CD patients was significantly higher in specimens cultured with pA2 (84 ± 25, P < .001) than in those cultured with medium. By contrast, no significant differences were noted in A2− patients in biopsy specimens cultured with pA2 (43 ± 31, compared with medium, P > .05; Figure 1A), although in 2 patients CD25+ cells appeared increased. Furthermore, CD25+ cells did not increase in biopsy specimens cultured with pctl both in A2+ (25 ± 10) and A2− (33 ± 7) CD patients (P > .2), as well as in healthy controls (n = 4) (Figure 1A). In accordance with previous studies,16Salvati V. Mazzarella G. Gianfrani C. et al.Recombinant human IL-10 suppresses gliadin-dependent T-cell activation in ex vivo cultured coeliac intestinal mucosa.Gut. 2005; 54: 46-53Crossref PubMed Scopus (115) Google Scholar, 20Mazzarella G. Maglio M. Paparo F. et al.An immunodominant DQ8 restricted gliadin peptide activates small intestinal immune response in in vitro cultured mucosa from HLA-DQ8 positive but not HLA-DQ8 negative coeliac patients.Gut. 2003; 52: 57-62Crossref PubMed Scopus (74) Google Scholar CD25+ activated cells were not found in the epithelium layer but were mainly localized beneath the epithelium, both in pA2− and/or PT-gliadin-challenged mucosa (Figure 2A and B).Figure 2Activated CD25+ cells localized in the subepithelial region. (A) Mononuclear cells expressing CD25 were evaluated by IHC on 5-mm cryostat sections of jejunal mucosa cultured as described in Figure 1 legend. Mucosal sections from a representative A2+ CD patient cultured in vitro with medium only or with pA2 are illustrated. Activated CD25+ cells were observed only in the lamina propria and in the subepithelial region. (B) Immunofluorescence staining for CD8 and CD25. Number of CD8+ intraepithelial lymphocytes (red) were increased following pA2 challenge compared with medium alone; also evident is the increase of CD25+ cells (green) and of CD8+CD25+ activated T cells (yellow), particularly in the subepithelial region (arrows). A similar pattern was observed when the mucosa were cultured with the peptic-tryptic digest of gliadin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A significant increase of CD8+CD25+ double positive cells was observed in the lamina propria of biopsy specimens challenged with PT-gliadin in both A2+ (4.2 ± 1.3 vs 1.2 ± 0.3, respectively, n = 8, P < .001) and A2− (3.5 ± 1.2 vs 1.2 ± 0.6, respectively, n = 6, P < .001) CD patients; only the A2+ CD mucosa showed a significant increase of CD8+CD25+ cells in response to pA2 (2.8 ± 1.2 vs 1.2 ± 0.3 with medium, respectively, P < .01) (Table 1, Figure 2B). Conversely, no significant increase of CD8+CD25+ cells was observed in biopsy specimens cultured with pctl both in A2+ (1.3 ± 0.2) and A2− (1.0 ± 0.3) patients.Table 1Percentage of Lamina Propria CD8+CD25+ CellsHLA-A2+ CD patients (n = 8)HLA-A2− CD patients (n = 6)Medium1.2 ± 0.31.2 ± 0.6PT-gliadin4.2 ± 1.3aP < .05.3.5 ± 1.2aP < .05.pA22.8 ± 1.2aP < .05.1.8 ± 0.9pctl1.3 ± 0.21.0 ± 0.3NOTE. Values represent mean ± SD.a P < .05. Open table in a new tab NOTE. Values represent mean ± SD. The number of CD80+ cells/mm2 (mean ± SD), mainly lamina propria mononuclear cells, was significantly increased in A2+ (86 ± 51, n = 7) and A2− (86 ± 52, n = 5) CD biopsy specimens cultured with PT-gliadin, compared with those cultured with medium (11 ± 4, P < .001, and 16 ± 8, P < .01, in A2+ and A2−, respectively) (Figure 1B). Importantly, only in A2+ biopsy specimens, CD80+ cells were significantly enhanced in the presence of pA2 (46 ± 30, P < .01). No differences were obtained in biopsy specimens from A2− patients cultured with pA2 (38 ± 41) compared with medium (16 ± 8, P > .3) (Figure 1B), although in 1 A2− patient the CD80+ cells were increased. Finally, in the case of the control mucosa (n = 4), no significant variation of CD80+ cells, compared with the medium control, was observed in any condition analyzed. The pA2-induced cell maturation was also investigated on monocyte-derived dendritic cells generated from 5 A2+ patients, as previously described.22Terrazzano G. Sica M. Gianfrani C. et al.Gliadin regulates the NK-dendritic cell cross-talk by HLA-E surface stabilization.J Immunol. 2007; 179: 372-381Crossref PubMed Scopus (41) Google Scholar With the exception of 1 patient, in which percentage of CD80+ dendritic cells markedly increased following an overnight pA2-incubation (42.3% vs 67.5%, respectively), no variation was observed in the remaining 4 patients (mean ± SD: 54.9% ± 3.8% vs 52.4% ± 4.2%, respectively, data not shown). Collectively, our data suggested that the cell maturation occurring in pA2-challenged biopsy specimens is a phenomenon not dependent from gliadin peptide itself but most likely secondary to the local activation of CD8+ T-cell response.23Palová-Jelínková L. Rozková D. Pecharová B. et al.Gliadin fragments induce phenotypic and functional maturation of human dendritic cells.J Immunol. 2005; 175: 7038-7045Crossref PubMed Scopus (83) Google Scholar, 24Acuto O. Michel F. CD28-mediated costimulation: a quantitative support for T-cell signaling.Nat Rev Immunol. 2003; 3: 939-951Crossref PubMed Scopus (520) Google Scholar To investigate further the immune response arising in CD intestinal mucosa upon pA2 stimulation, we used FACS analysis of T cells soon after their isolation from primary tissue. Specifically, we evaluated the percentage of different cell subsets infiltrating the intestinal mucosa. The frequency of CD3+CD8+ cells was significantly higher than CD3+CD4+ cells in CD patients (mean ± SD): CD3+CD8+: 23.7% ± 10.6; CD3+CD4+: 11.8% ± 5.6% (P < .005, n = 11) (Figure 3A). By contrast, both cell subsets did not differ in control mucosa: CD3+CD8+: 15.8% ± 3.2; CD3+CD4+: 12% ± 5.7% (P < .5, n = 4). Next, the percentage of CD8+ cells expressing CD25 and CD95(FAS)L was evaluated in organ cultured biopsy specimens. The percentage of CD8+CD25+ and CD8+CD95L+ cells was normalized to the total CD8+ cells (gated in R1; Figure 3B and C). CD8+CD25+ cells were significantly increased in A2+ CD biopsy specimens cultured with pA2: (mean ± SD) 11.6% ± 4.3% compared with a baseline value of 5.9% ± 3.0% (P < .02, n = 6). A significant increase of CD8+CD25+ cells was also observed in PT-gliadin-challenged biopsy specimens: 15.0% ± 3.12% (P < .001, n = 4). No increment of CD8+CD25+ cells was observed in A2+ mucosa challenged with pctl: 7.3% ± 1.3% (P = .4, n = 4) (Figure 4A). In A2− patients, a significant variation of CD8+CD25+ cells was observed only in biopsy specimens cultured with PT-gliadin: 18.8% ± 18.7% compared with a baseline value of 3.6% ± 3.1% (P < .05, n = 4). No significant activation of CD8+ cells was detected in biopsy specimens from controls in any experimental condition tested. Similarly to CD25, a marked increase of CD8+ cells expressing the CD95(FAS)L occurred in A2+ biopsy specimens cultured with pA2: 9.3% ± 5.3% vs baseline: 3.2% ± 2.8%; (P < .03, n = 6); in PT-gliadin-cultured biopsy specimens, CD8+CD95L+ cells were also increased, but this trend did not reach statistical significance: 9.1% ± 6.7% (P < .08, n = 4) (Figure 3B and Figure 4B). No significant variation in the number of CD8+CD95L+ cells was found in A2+ biopsy specimens in response to pctl (5.6% ± 4.3%, P < .3, n = 4) and in biopsy specimens from either A2− patients or controls in any condition analyzed. A significant increase in the number of CD3+ cells was observed in the epithelium of A2+" @default.
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• W2111452470 title "Gliadin Activates HLA Class I-Restricted CD8+ T Cells in Celiac Disease Intestinal Mucosa and Induces the Enterocyte Apoptosis" @default.
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