Figure 5. Frequency of CD3⁺CD8⁺TCRγδ⁺ IELs expressing intracellular TGF-β1 increases in patients on GFD.

(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.