*negative cells to exclude non-viable cells subsets and on CD45+ to exclude GFP+ MODE-K cells, after 96?h. expansion of IELs in the intestinal mucosa. with resolution of 70,000 (200). Up to 12 most intense peaks (charge state 2) were fragmented and tandem mass spectrum was acquired with a resolution of 35,000 and dynamic exclusion 30?s. The tandem mass spectral data produced were searched against the NCBI database downloaded 29-May-2015 using the Mascot search program (Matrix Science) with search parameters set to: MS accuracy 5?ppm, MS/MS accuracy 0.5?Da, trypsin digestion with one missed cleavage allowed, EFNB2 and variable modifications were set for carbamidomethyl (C), propionamide (C), oxidation (M), and acetylation (protein N-terminal). T Cell Proliferation Assay Prior to coculture with IELs, MODE-K cells transfected with N-FLAG-Btnl6-pMX-IRES-GFP?+?N-HA-Btnl1-pMX-IRES-GFP, N-FLAG-Btnl1-pMX-IRES-GFP, or pMX-IRES-GFP were plated on 48- or 24-well flat-bottom tissue culture plates uncoated or precoated with 1?g/ml anti-CD3? (clone 145-2C11, BD Pharmingen). The following day, when the MODE-K monolayers were ~70% confluent, the medium was replaced with supplemented RPMI 1640 with or without IL-2 (10?U/ml) or IL-15 (50?ng/ml), to which CFSE (Molecular Probes?, Life Technologies) labeled IELs were added at 1??105 cells/well. IELs were left to proliferate for 72 or 96?h and were thereafter stained with anti-CD45 to exclude GFP+ MODE-K cells. Cells were gated on LIVE/DEAD? Fixable Red (Molecular Probes?, Life Technologies) negative cells to exclude non-viable cells. Splenocytes from C57BL/6 mice were depleted of B-cells by negative selection with anti-CD19 microbeads (Miltenyi Biotec) using an auto-MACS separator. The purity of cells was analyzed by flow cytometry and was 95% in all experiments performed. Splenocytes were labeled with CFSE and were stimulated with anti-CD3? (clone 145-2C11, BD Pharmingen) and anti-CD28 (clone 37.51, BD Pharmingen) in the presence of Btnl1-, Btnl1?+?6, or pMX transfected MODE-K cells. Proliferative response was assessed by flow cytometry after staining with anti-CD45 to exclude GFP+ MODE-K cells, and after gating on LIVE/DEAD? Fixable Red (Molecular Probes?, Life Technologies) negative cells to exclude non-viable cells. Cytokine Measurement in Cell Culture Supernatant Culture supernatants were analyzed by flow cytometry using Mouse Th1/Th2/Th17/Th22 13plex Kit FlowCytomix (eBioscience) according to the manufacturers instructions. The samples were acquired in LSR II flow cytometer. Analysis of data and quantification of cytokines was performed using the FlowCytomix Pro Software (eBioscience) on the basis of corresponding standards curves. Statistical Analysis All data were generated using GraphPad Prism version 6.04. Significance between conditions was determined by unpaired two-tailed T cell proliferation assay making use of a long-term culture system for intestinal IELs, which permits IELs to be rested as viable cells and then rapidly re-activated when stimulated via the EMT inhibitor-2 TCR (18, 21), and the fluorescent dye CFSE, which penetrates cell membranes and couples to proteins resulting in stable, long-term intracellular retention. Using costimulation with anti-CD3 mAb, and conditions without stimulation, the effect of Btnl proteins expressed by transfected MODE-K epithelial cells was assessed on IEL responses. Although IEL proliferation was not reproducibly affected by coculture with MODE-K-Btnl in the presence of anti-CD3 activation (Figure ?(Figure5A),5A), significant increase in proliferation was observed in the absence of TCR stimulation at both 72 and 96?hours of coculture (Figures ?(Figures5B,C).5B,C). The proliferative effect was dependent EMT inhibitor-2 on the presence of exogenous IL-2 or IL-15 as in the absence of these cytokines no proliferation was observed (Figure ?(Figure5B).5B). Although both Btnl1 and the Btnl1CBtnl6 heteromer were able to induce IEL proliferation, the expansion in IL-15-treated cells was considerably higher in the presence of Btnl1 (Figure ?(Figure5C).5C). The capacity to proliferate in the presence of Btnl proteins was specific for IELs as no proliferation was induced when unstimulated splenocytes EMT inhibitor-2 were cocultured in the presence of Btnl-transfected.