(A) Representative flow cytometry histograms showing percentage of CD3⁺TCRαβ⁺ IELs sorted from the jejunal biopsies of an ACD patient expressing intracellular (IC) IFN-γ and granzyme B and cell-surface NKG2D in the absence and presence of 25 ng/ml of rhIL-15 (top and middle rows, respectively); and in the presence of 25 ng/ml of rhIL-15 without (middle row) and after addition of sorted CD3⁺CD8⁺TCRγδ⁺ IELs from the same patient at a 1:1 ratio (bottom row). CD3⁺TCRαβ⁺ IELs were cultured for 36 hours prior to flow cytometric analysis. Similar results were seen with samples from GFD patients. (B) Representative flow cytometry dot plots showing percentage of CD3⁺TCRαβ⁺ IELs that express CD107a/b after 4 hours of stimulation with plate-bound anti-CD3 in the presence (lower panels) or absence (upper panels) of CD3⁺TCRγδ⁺ IELs. (C) Percentage (mean ± SEM) of CD3⁺TCRαβ⁺ IELs expressing intracellular IFN-γ and granzyme B in the presence of rhIL-15 to which CD3⁺CD8⁺TCRγδ⁺ IELs had been added at different TCRγδ/TCRαβ IEL ratios. (D) Percentage (mean ± SEM) of CD3⁺TCRαβ IELs expressing intracellular IFN-γ and granzyme B in cocultures of CD3⁺TCRαβ IELs and ESA⁺ ECs, in the presence of rhIL-15. In parallel wells, CD3⁺CD8⁺TCRγδ⁺ IELs or CD3⁺CD8^–TCRγδ⁺ IELs were added at a 1:1 TCRγδ/TCRαβ IEL ratio (from 4 independent experiments). A statistically significant decrease (*) in the percentage of CD3⁺TCRαβ⁺ IELs expressing intracellular IFN-γ (P = 0.004) and granzyme-B (P = 0.003) was seen in wells to which CD3⁺CD8⁺TCRγδ⁺ IELs were added compared with wells with only CD3⁺TCRαβ⁺ IELs. No significant decrease (^‡) in the percentage of CD3⁺TCRαβ⁺ IELs expressing intracellular IFN-γ (P = 0.06) and granzyme B (P = 0.06) was observed in wells to which CD3⁺CD8^–TCRγδ⁺ IELs were added compared with wells with CD3⁺TCRαβ⁺ IELs only (P values determined by Wilcoxon’s signed-rank test).