(A) Flow cytom­etry dot plots showing gating strategy for CD3⁺TCRαβ⁺ IELs and CD3⁺TCRγδ⁺ IELs. (B and C) Box-and-whisker plots representing frequencies, as assessed by flow cytometry, of: CD3⁺TCRαβ⁺ IELs and CD3⁺TCRγδ⁺ IELs in jejunal biopsy samples from ACD patients (n = 15), GFD patients (n = 23), and controls (n = 10) (B); CD3⁺CD8⁺TCRγδ⁺ IELs and CD3⁺CD8^–TCRγδ IELs in jejunal biopsies from ACD patients, GFD pat­ients, and controls (C) (P values calculated using Mann-Whitney U test).

(A) Representative flow cytometry histograms showing percentage of CD3-gated CD8⁺TCRγδ^–, CD8⁺TCRγδ⁺, and CD8^–TCRγδ⁺ IELs (sorted from the jejunal biopsies of a GFD patient) expressing surface NKG2A. (B) Box-and-whisker plots comparing percentage of CD3⁺TCRαβ⁺NKG2A⁺ IELs with percentage of CD3⁺TCRγδ⁺NKG2A⁺ IELs. (C) Box and Whisker plots comparing frequencies of CD3⁺CD8⁺TCRγδ⁺ IELs and CD3⁺CD8^–TCRγδ⁺ IELs expressing NKG2A, from ACD patients (n = 15), GFD patients (n = 23), and controls (n = 10) (P values determined by Mann-Whitney U test).

(A) Representative flow cytometry histograms showing CD3⁺TCRαβ⁺ IELs and CD3⁺TCRγδ⁺ IELs expressing intracellular IL-10 and TGF-β1 and cell-surface LAP TGF-β1 in jejunal biopsies from a GFD patient. Cells were stimulated for 4 hours with PMA and ionomycin in the presence of brefeldin A. (B) Box-and-whisker plots comparing percentages of CD3⁺TCRαβ⁺ IELs, CD3⁺CD8⁺TCRγδ⁺ IELs, and CD3⁺CD8^–TCRγδ⁺ IELs expressing intracellular TGF-β1, from ACD patients (n = 15), GFD patients (n = 23), and controls (n = 10). (C) Box-and-whisker plots comparing percentages of CD3⁺TCRαβ⁺ IELs, CD3⁺CD8⁺TCRγδ⁺ IELs, and CD3⁺CD8^–TCRγδ⁺ IELs expressing cell-surface LAP TGF-β1, from ACD patients (n = 8), GFD patients (n = 11), and controls (n = 7). (D) Representative flow cytometry histograms showing percentage of CD3⁺TCRγδ⁺ NKG2A⁺ IELs and CD3⁺TCRγδ⁺ NKG2A^– IELs expressing intracellular TGF-β1 and cell-surface LAP TGF-β1 in jejunal biopsies from a GFD patient. (E) Representative flow cytometry histograms showing percentage of CD3⁺TCRγδ⁺ IELs (from an individual on GFD) expressing intracellular TGF-β1 (top row) and cell-surface LAP TGF-β1 (bottom row) after 24 hours culture in wells coated with 10 μg/ml mouse IgG1, 50 μg/ml anti-human NKG2A (clone 131411), or 50 μg/ml anti-human NKG2A plus 10 μg/ml anti-human CD3 (UCHT1; data representative of 3 independent experiments). (F) Levels (mean ± SEM pg/ml) of TGF-β1 in supernatants taken from cultures of CD3⁺TCRγδ⁺ IELs that were incubated for 24 hours in wells coated with 10 μg/ml mouse IgG1, 50 μg/ml anti-human NKG2A (clone 131411), or 50 μg/ml of anti-human NKG2C (clone 134522). (G) Levels (mean ± SEM pg/ml) of TGF-β1 in supernatants taken after 48 hours of cocultures of CD3⁺TCRγδ⁺ NKG2A⁺ IELs with HLA-E⁺CD3⁺TCRαβ IELs, CD3⁺TCRγδ⁺NKG2A^– IELs with HLA-E⁺CD3⁺TCRαβ IELs, or CD3⁺TCRγδ⁺NKG2A⁺ IELs with HLA-E^– CD3⁺TCRαβ IELs.

(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).

(A) Percentage (mean ± SEM) of CD3⁺TCRαβ IELs expressing intracellular IFN-γ and granzyme B in cocultures of CD3⁺TCRαβ IELs with ESA⁺ ECs, in the presence of rhIL-15. Addition of CD3⁺TCRγδ⁺ NKG2A⁺ IELs to these cultures resulted in a statistically significant decrease (*) in the percentage of CD3⁺TCRαβ⁺ IELs expressing intracellular IFN-γ (P = 0.002) and granzyme B (P = 0.001). No significant decrease (^‡) in the percentage of CD3⁺TCRαβ⁺ IELs expressing intracellular IFN-γ (P = 0.1) and granzyme B (P = 0.3) was seen in cultures to which CD3⁺TCRγδ⁺ NKG2A^– IELs were added. When either 10 μg/ml anti-human NKG2A, 10 μg/ml anti-human HLA-E (MEM-E/06), or a cocktail of anti-human NKG2A and anti-human HLA-E were added to cultures containing CD3⁺TCRγδ⁺NKG2A⁺ IELs, the percentage of CD3⁺TCRαβ IELs expressing intracellular IFN-γ increased by 38.5% ± 10.2%, 43.0% ± 9.8% and 46.2% ± 9.5%, respectively, as did intracellular granzyme B–expressing CD3⁺TCRαβ IELs (27.0% ± 7.4%, 38.2% ± 9.1%, and 44.2% ± 10.2%). (B) Representative flow cytometry dot plots showing percentages of CD3⁺TCRαβ⁺ IELs that express HLA-E, from healthy controls, patients with ACD, and patients on GFD. (C) Box-and-whisker plots representing frequencies of CD3⁺TCRαβ⁺ IELs expressing HLA-E in jejunal biopsy samples from ACD patients (n = 8), GFD patients (n = 12), and controls (n = 7). (D) Representative flow cytometry histogram showing an increase in the percent of HLA-E–expressing CD3⁺TCRαβ⁺ IELs sorted from the jejunal biopsy of an ACD patient, after 24 hours treatment with 25 ng/ml of rhIFN-γ. (E) Percentage (mean ± SEM) of CD3⁺TCRαβ IELs expressing intracellular IFN-γ and granzyme B in cocultures of CD3⁺TCRαβ IELs treated with 25 ng/ml of rhIL-15 in the bottom chamber of a diffusion chamber system. When CD3⁺TCRγδ⁺NKG2A⁺ IELs were added to the bottom chamber or CD3⁺TCRγδ⁺NKG2A⁺ IELs with sorted HLA-E⁺CD3⁺TCRαβ IELs (from an ACD patient) were added to the top chamber, a significant decrease (*) in the percentage of CD3⁺TCRαβ IELs expressing intracellular IFN-γ (P = 0.004 and P = 0.006, respectively) and granzyme B (P = 0.003 for both) was seen. No significant decrease (^‡) in the percentage of CD3⁺TCRαβ IELs expressing intracellular IFN-γ (P = 0.09) and granzyme B (P = 0.08) was seen when CD3⁺TCRγδ⁺NKG2A⁺ IELs and HLA-E^–CD3⁺TCRαβ IELs were added to the top chamber (P values determined by Wilcoxon’s signed-rank test).

(A) Representative flow cytometry histograms showing percentage of CD3⁺TCRαβ IELs expressing intracellular IFN-γ (left) and granzyme B (right) in cocultures of CD3⁺TCRαβ IELs with ESA⁺ ECs were stimulated with rhIL-15 in the presence (bottom row) or absence (top row) of rhTGF-β1. (B) Percentage (mean ± SEM) of CD3⁺TCRαβ IELs expressing intracellular IFN-γ and granzyme B in cocultures of CD3⁺TCRαβ IELs with ESA⁺ ECs, in the presence of rhIL-15 to which different concentrations of rhTGF-β1 were added. Data shown are from 3 independent experiments. (C) Percentage (mean ± SEM) of CD3⁺TCRαβ⁺ IELs expressing intracellular IFN-γ and granzyme B in cocultures of CD3⁺TCRαβ⁺ IELs with ESA⁺ ECs, in the bottom chamber of a diffusion chamber system. When CD3⁺TCRγδ⁺NKG2A⁺ IELs with HLA-E⁺ ECs (from an ACD patient) were added to the top chamber, a significant decrease (*) in the percentage of CD3⁺TCRαβ IELs expressing intracellular IFN-γ (P = 0.006) and granzyme B (P = 0.003) was seen. However, inhibition of intracellular IFN-γ and granzyme B expression by CD3⁺TCRαβ IELs in the bottom chamber was blocked when 1 μg/ml of anti-human TGF-β1 antibody was added to the bottom chamber. This led to an increase in the percentage of CD3⁺TCRαβ⁺ IELs expressing intracellular IFN-γ by 28.0% ± 4.1% and granzyme B by 21.2% ± 9.3%, compared with cultures with mouse IgG1 (control). Addition of anti-human IL-10 did not block the suppressive effect of CD3⁺TCRγδ⁺NKG2A⁺ IELs; data shown from 4 independent experiments (P values determined by Wilcoxon’s signed-rank test).