(A) Western blot analysis. Representative blots show Bcl-2, Bcl-xL, and Mcl-1 in 2 different RCDII IEL lines cultured for 72 hours with the indicated concentrations of IL-15. Below, comparison by densitometry of Bcl-2, Bcl-xL, and Mcl-1 levels in 4 RCDII IEL lines is shown normalized to β-actin. Only Bcl-xL expression significantly decreased upon IL-15 deprivation (paired t test). (B) FACS analysis. Percentages of positive RCDII IELs and MFI of Bcl-2 and Bcl-xL decreased with IL-15 concentrations, but their expression remained detectable at 0.5 ng/ml, the lowest concentration allowing cell survival. (C) mRNA levels for Bcl-2, Bcl-xL, and Mcl-1. Only Bcl-2 and Bcl-xL mRNA levels decreased after IL-15 starvation in RCDII IEL lines. Shown is 1 representative experiment of 3. Values were normalized to ribosomal Protein Large PO (RLPO). (D) Apoptosis induction by ABT-737. RCDII IEL lines were cultured with 5 ng/ml IL-15 in the presence of ABT-737 at the indicated concentrations for 48 hours. Cell survival was assessed by counting viable cells in trypan blue (top) and by flow cytometry after staining with Annexin V/PI (bottom). Shown is 1 representative of 4 experiments with 4 RCDII IEL lines.

(A and B) Analysis of RCDII IEL lines cultured with 5 ng/ml IL-15 in the presence of anti–IL-15Rα or anti–IL-15Rβ blocking mAbs or control isotype for 72 hours. (A) Flow cytometry analysis of apoptosis in 1 representative cell line, and mean fold increase in percent apoptotic (annexin V/PI⁺) cells in 4 RCDII IEL lines. Only anti–IL-15Rβ mAb significantly increased apoptosis. *P = 0.02 versus IgG, paired t test. (B) Analysis of Bcl-2, Bcl-xL, and Mcl-1 expression by Western blot and of Bcl-2 and Bcl-xL expression (black histograms and text) by flow cytometry. Blockade of IL-15Rβ, but not IL-15Rα, decreased Bcl-xL, and to a lesser degree Bcl-2, compared with cells cultured in the presence of control isotype (gray filled histograms and text); Mcl-1 expression was unchanged. Shown is 1 representative experiment of 4. (C and D) Analysis of IL-15Rβ–dependent signaling in RCDII IEL lines. (C) Comparison of cell lines cultured with or without IL-15 for 72 hours demonstrated IL-15–induced phosphorylation of Jak3, STAT5, STAT3, and AKT. (D) Flow cytometry showed that IL-15 starvation and IL-15Rβ blockade, but not IL-15Rα blockade, decreased phosphorylation of STAT5, STAT3, AKT, and ERK (black histograms and text) compared with control cells in IL-15 (gray filled histograms and text). Dashed lines in A, B, and D denote isotype controls.

(A) Quantification of apoptosis in 5 RCDII IEL lines cultured for 72 hours with IL-15 in the presence of kinase inhibitors. Percentages of apoptotic cells, determined by flow cytometry as in Figure A, increased significantly after treatment with the Jak3 inhibitor WHIP-131 (JAK3i) and IL-15 starvation (0), but not with AG490, the PI3K inhibitors LY294002 and wortmannin, or the ERK inhibitor U0126 (ERKi). *P < 0.001 versus IL-15 alone, 1-way ANOVA with Tukey’s multiple comparison test. (B) Flow cytometry indicated that WHIP-131, but not LY294002 (black histograms and text), reduced expression of Bcl-2 and Bcl-xL compared with control cells (gray filled histograms and text). (C) In contrast, proliferation of RCDII IEL lines to 10 ng/ml IL-15 assessed by ³H thymidine incorporation was inhibited by both LY294002 and WHIP-131. Shown is 1 representative experiment of 3. (D) WHIP-131 decreased expression of phospho-STAT5 and phospho-STAT3 in treated cells (black histograms and text) compared with untreated cells (gray filled histograms and text). (E and F) Specific inhibition of STAT5 and STAT3 expression by shRNA in 1 RCDII IEL line cultured in IL-15. shLuc, control shRNA directed against the luciferase protein. (E) Analysis of signaling molecules and caspase 3 activation by Western blot. (F) Cell survival, measured as the number of viable cells and the percentage of trypan blue–positive (i.e., dead) cells. Inhibition of STAT5, but not STAT3, reduced expression of Bcl-2 and Bcl-xL and induced caspase 3 activation and cell death. Dashed lines in B and D denote isotype controls.

(A) Freshly isolated IELs from RCDII or ACD patients labeled with annexin V/PI after a 72-hour culture in the presence or absence of IL-15. All cells were protected from apoptosis by IL-15. (B and C) Percent of apoptotic cells, determined as in Figure A, after a 48-hour culture of normal IEL lines (n = 4) in the presence or absence of 5–10 ng/ml IL-15 and (B) ABT-737 at the indicated concentrations or (C) blocking anti–IL-15Rα or –IL-15Rβ mAbs and control isotype or pharmacological inhibitors. *P < 0.05, **P < 0.01, ***P < 0.001 versus respective control, 1-way ANOVA with Tukey’s multiple comparison test.

(A) Flow cytometry analysis of Bcl-2, Bcl-xL, and phospho-STAT5 in 2 normal and 2 RCDII IEL lines cultured in the absence (black histograms) or presence of 5 ng/ml IL-15 (gray filled histograms). MFI was higher in RCDII than in normal IEL lines. Dashed lines denote isotype controls. (B) Flow cytometry analysis of IL-15Rβ (CD122) in freshly isolated IELs from 8 RCDII patients. MFI of CD122 significantly increased in abnormal IELs compared with residual normal IELs (paired t test). Below, representative FACS staining of freshly isolated IELs from 1 RCDII patient showed higher expression of CD122 on gated abnormal CD103⁺sCD3^– IELs compared with normal residual CD3⁺CD103⁺ IELs. (C) Flow cytometry analysis of apoptosis in normal control (CT) and RCDII IEL lines after a 72-hour culture with the indicated concentrations of IL-15. Rescue from apoptosis required lower IL-15 concentrations in RCDII than in control IEL lines.

(A) Western blot analysis of Bcl-xL and Bcl-2 in duodenal extracts from 5 controls, 4 CD patients on GFD, 6 ACD patients, and 5 RCDII patients. *P < 0.05. (B) Flow cytometry analysis of Bcl-xL and Bcl-2 in freshly isolated IELs (closed symbols) and LPLs (open symbols). Bcl-xL expression significantly increased in all CD patients, whereas Bcl-2 significantly decreased in ACD and RCDII patients, in IELs but not in LPLs. *P < 0.05 versus control, Mann-Whitney test.

(A and B) Representative immunohistochemical staining of duodenal biopsies from control (n = 4), ACD (n = 5), and RCDII (n = 4) individuals with anti–phospho-Jak3 (A) or anti–phospho-STAT5 (B) Abs. (C) Immunostaining of duodenal biopsies from 1 RCDII patient with anti–phospho-Jak3 or anti–phospho-STAT5 Abs after a 48-hour organ culture in the presence of 10 μg/ml AMG714 or control IgG. Shown is 1 representative of 4 experiments. (A–C) Panels with outlines show higher-magnification views of the boxed regions in the adjacent images. Original magnification, ×10; ×40 (higher magnification). (D) STAT5 phosphorylation and Bcl-xL expression were investigated using Western blot in biopsies from 1 RCDII and 1 ACD patient cultured for 48 hours with AMG714 or control IgG.

(A) Frozen duodenal sections of IL-15Tg_E mice and B6 littermates labeled with anti-CD3 (red) and anti-KI67 (green) Abs. In IL-15Tg_E mice, intestinal accumulation of CD3⁺KI67^– lymphocytes (middle) suggested impaired lymphocyte apoptosis. Numbers of CD3⁺ lymphocytes decreased markedly after 2 weekly i.p. injections of 50 μg AMG714, but not of control human IgG. (B) Flow cytometry determination, using Trucount beads, of CD3⁺ lymphocyte number in LPLs and IELs from B6 and IL-15Tg_E mice. Treatment with AMG714 (as in A), but not with control IgG, reduced IEL numbers to those observed in B6 littermates. *P < 0.5, **P < 0.01, Mann-Whitney. (C) Flow cytometry analysis demonstrated increased apoptosis of IELs and LPLs in IL-15Tg_E mice 2 days after 1 i.p. injection of AMG714. Shown is 1 representative of 3 experiments.

(A) 3 IL-15Tg_E and 2 B6 mice were treated with daily i.p. injections of 75 mg/kg ABT-737 or vehicle, and absolute numbers of CD8⁺ T, NK, and NKT cells were determined in the blood on days 7 and 14. Treatment of IL-15Tg_E mice markedly decreased the number of circulating CD8⁺ T, NK, and NKT cells, which returned to that observed in B6 mice. No changes in the numbers or percentages of cells were observed in mice that received vehicle only (not shown). (B) For comparison, IL-15Tg_E mice were treated with 2 i.p. injections of AMG714 or control human IgG (n = 3 per group). AMG714, like ABT-737, preferentially decreased the number of CD8⁺ T, NK, and NKT cells in the blood of IL-15Tg_E mice, whereas control human IgG had no effect (not shown). (C) Treatment of IL-15Tg_E mice with ABT-737 induced a marked decrease of CD3⁺CD8⁺ T cells in spleen and mesenteric lymph nodes (MLNs), but had little or no effect on LPLs and IELs, compared with mice treated with vehicle. (D) IELs isolated from IL-15Tg_E mice were cultured in vitro for 48 hours in medium alone and supplemented with 20 ng/ml IL-15 in the presence of different concentrations of ABT-737. Results are representative of 2 independent experiments.