Volume 125, Issue 3, Pages 730-745 (September 2003) Interleukin 15: a key to disrupted intraepithelial lymphocyte homeostasis and lymphomagenesis in celiac disease  Jean-Jacques Mention, Mélika Ben Ahmed, Bernadette Bègue, Ullah Barbe, Virginie Verkarre, Vahid Asnafi, Jean-frédéric Colombel, Paul-henri Cugnenc, Frank M. Ruemmele, Elisabeth Mcintyre, Nicole Brousse, Chistophe Cellier, Nadine Cerf-Bensussan  Gastroenterology  Volume 125, Issue 3, Pages 730-745 (September 2003) DOI: 10.1016/S0016-5085(03)01047-3

Figure 1 IL-15 protein is increased in small intestinal biopsy specimens of patients with celiac disease. Intestinal sections from 14 patients with active celiac disease (ACD), 11 patients with celiac disease on a GFD, 10 patients with RCS, 11 histologically normal controls, and 6 control patients with active ileal Crohn’s disease were stained with anti-human IL-15 MAB647. (A) Typical staining in histologically normal controls, active Crohn’s disease, active celiac disease, and RCS is shown at 10× (left panel) and 40× magnification in the top epithelium (middle panel) and in the crypts (right panel). (B) Intensity of IL-15 expression was estimated by semiquantitative analysis using arbitrary units (AU) in lamina propria (LP) and at different levels of epithelium: apex, mid-level of villi and crypts in patients with normal villi or partial villous atrophy, and surface epithelium and crypts in patients with subtotal or total villous atrophy. For each patient group and localization, median was calculated and compared by the nonparametric Mann-Whitney test. Gastroenterology 2003 125, 730-745DOI: (10.1016/S0016-5085(03)01047-3)

Figure 2 IL-15 is expressed at the surface of enterocytes. (A) IL-15 was measured by enzyme-linked immunosorbent assay in supernatants of 24-hour organ cultures of biopsy specimens from 3 controls, 4 patients with active celiac disease (ACD), 6 patients with celiac disease on a GFD, and 3 patients with RCS stimulated or not with increasing concentrations of gliadin Frazer’s fraction. (B) IL-15 expression was studied by immunohistochemistry after 24 hours in the presence or not of 1 mg/mL gliadin Frazer’s fraction in 3 controls and 3 patients on a GFD. Expression of IL-15 was above controls in patients on a GFD and increased after 24 hours in the presence of gliadin. (C) Flow cytometry analysis of T84 cells: intracellular staining of permeabilized T84 cells (left panel) and surface staining of T84 cells (right panel) with control isotype (dotted line) and anti-IL-15 antibody in the absence (full line) or presence (hatched line) of 10 ng soluble recombinant IL-15. All T84 cells contain intracellular IL-15, and approximately 60% express it on their cell surface. (D and E) Flow cytometry analysis of enterocytes freshly isolated from small intestinal biopsy specimens of controls, patients on a GFD, and patients with active celiac disease or RCS shows expression of IL-15 on gated CD7(−) epithelial surface antigen(+) epithelial cells. Epithelial surface antigen(−) CD7(+) IELs were negative for IL-15 (data not shown). Histograms in 4 representative patients; dotted lines are isotype controls and thick lines are anti-IL-15 antibody. (E) Statistical representation of data analyzed by (D) Mann-Whitney test. Gastroenterology 2003 125, 730-745DOI: (10.1016/S0016-5085(03)01047-3)

Figure 3 Transcription of IFN-γ and granzyme B is selectively increased in patients with active celiac disease and RCS. Cytokine mRNA expression for IFN-γ, granzyme B, TNF-α, IL-2, and IL-12 was quantified by real-time PCR in duodenal samples of 9 patients with active celiac disease (ACD), 9 patients with celiac disease on a GFD, 6 patients with RCS, and 8 controls. Results were referred to expression of glyceraldehyde-3-phosphate dehydrogenase by calculating 2−ΔCT and expressed in arbitrary units. ∗P < 0.01 when compared with controls, ∗∗P < 0.01 when compared with patients on a GFD. Gastroenterology 2003 125, 730-745DOI: (10.1016/S0016-5085(03)01047-3)

Figure 4 IL-15 transcription is not modified in the small intestine of patients with celiac disease. (A) Total RNA was obtained from duodenal samples of patients with active celiac disease (lines 1 and 5), patients with celiac disease on a GFD (lines 2 and 6), patients with RCS (lines 3 and 7), and controls (lines 4 and 8). Reverse-transcription PCR using specific primers showed transcripts for both secreted and nonsecreted IL-15 isoforms in patients and controls. (B) Cytokine mRNA expression of IL-15 was quantified by real-time PCR in the same biopsy specimens as in Figure 3. Results were referred to expression of glyceralde-3-phosphate dehydrogenase by calculating 2−ΔCT and expressed in arbitrary units. Gastroenterology 2003 125, 730-745DOI: (10.1016/S0016-5085(03)01047-3)

Figure 5 IL-15 selectively promotes the growth of clonal CD103+sCD3− IELs in RCS. (A and B) Cell lines grown from duodenal biopsy specimens of patients with RCS cultured in IL-15 derived from abnormal IELs. (A) Surface staining shows membrane expression of CD103 but not of surface CD3ϵ (upper panel), whereas intracellular CD3ϵ (iCD3ϵ) is detected in permeabilized cells (lower panel; dotted line, control isotype). This phenotype is identical to that of freshly isolated IELs in RCS.11 (B) Sequencing of TcRγ chains amplified by PCR from DNA extracted from duodenal biopsy specimens (D2) and IL-15-dependent CD103+sCD3− IEL lines (IEL line) of patients with RCS showed the same rearrangements in each of the 5 patients, confirming the origin of the cell lines from the original abnormal IEL clones. (C) Biopsy specimens of 3 patients with RCS were cultured for several weeks in the presence of IL-15. Flow cytometry performed at the onset of cell outgrowth (T1) and 8–10 days later (T2) shows preferential expansion of lymphocytes lacking membrane CD3. (D) CD103+sCD3− cells and CD103−sCD3+ lymphocytes were cell sorted from peripheral blood in 1 patient with RCS and cultured for 5 days in the presence of the indicated concentrations of IL-15. (E) CD103+sCD3− lymphocytes cell sorted from peripheral blood in a second patient with RCS were cultured for 5 days with indicated cytokines. [3H]thymidine uptake in D and E is expressed as total cpm × 10−3. (F) Expression of IL-15Rα, CD122, and CD132 was analyzed on CD103+sCD3− lines before (thick lines) or after (bold lines) deprivation of IL-15 for 48 hours. Dotted lines are isotype controls (data representative of 4 independent experiments). (G) Expression of IL-15Rα was studied on total RNA extracted from cell-sorted CD103+sCD3− IELs freshly isolated from 4 RCS biopsy specimens. Reverse-transcription PCR using a couple of specific primers showed the presence of mRNA isoforms of IL-15Rα with exon 2 encoding the Sushi domain necessary for IL-15 binding (lower band). We also observed a spliced form (upper band) with an additional sequence of 153 base pairs between exon 1 and exon 2 derived from intron 1. This new isoform is not specific of RCS IELs because it was observed in control IELs as well as in other cell types (HT29 and T84 cell lines) (data not shown). Gastroenterology 2003 125, 730-745DOI: (10.1016/S0016-5085(03)01047-3)

Figure 7 IL-15 bound to T84 epithelial cells induces survival and IFN-γ secretion in RCS CD103+CD3− cells. CD103+CD3− cell lines deprived of IL-15 for 48 hours were cultured in medium supplemented or not with 1 ng/mL IL-15 or were cocultured with T84 cells for (A) 84 hours or (B) 48 hours. (A) Survival of CD103+CD3− cells was assessed using propidium iodure (PI) and fluorescein isothiocyanate/annexin V staining and by gating on cells labeled with APC-conjugated CD45. (B) IFN-γ secretion was analyzed by flow cytometry in CD7+ cells. (A and B) IL-15 effect was blocked by 10 μg/mL anti-IL-15 MAB647 or IgG1 isotype control. Cell-to-cell contact was inhibited either by anti-CD103 HML-1 antibody (or IgG2a control isotype) or by culture in Transwells (+T84/Transwell). Results are representative of 3–4 independent experiments. Gastroenterology 2003 125, 730-745DOI: (10.1016/S0016-5085(03)01047-3)

Figure 6 IL-15 triggers effector functions of clonal CD103+sCD3− lymphocytes in RCS. (A) Production of IFN-γ and TNF-α was analyzed by flow cytometry in freshly isolated lymphocytes cultured in medium supplemented or not with phorbol myristate acetate and ionomycin for 20 hours and IL-7, IL-12 + IL-18, IL-2, or IL-15 for 48 hours. Cytokine staining was coupled with membrane labeling using fluorescein isothiocyanate anti-CD103 and PE anti-CD3 mAbs. Percentages of CD103+sCD3− IEL-producing IFN-γ and TNF-α are shown. Results summarize experiments in 2 different patients. (B) CD103+sCD3− lines derived from patients with RCS were deprived or not of IL-15 for 72 hours. Cytotoxic activity was tested against K562 and HT29 by a 51Cr release assay. Results summarize experiments on 4 and 5 different cell lines, respectively. (C) Effector cells and HT29 lines (ratio 30:1) were incubated with 10 μg/mL of anti-CD103 antibody (HML1) or isotype IgG2a control (left panel). To inhibit perforin-mediated cytotoxicity against HT29, effector cells were preincubated with concanamycin A or chloroquine at different concentrations. Results summarize experiments on 3 different cell lines. (D) Freshly isolated CD3− IELs from duodenal biopsy specimens of 3 patients with RCS (90%–95% sCD3−) were cultured overnight with medium supplemented or not with IL-15 (20 ng/mL). Cytotoxic activity was tested against K562 and HT-29 by 51Cr release assay. Gastroenterology 2003 125, 730-745DOI: (10.1016/S0016-5085(03)01047-3)