Figure 1
Figure 1. T cells primed with either APC-HLA-G1s or soluble HLA-G5 are suppressors whose generation and function occur in an IL-10 microenvironment. (A) T cells primed with either APCs or APC-HLA-G1s for 6 days were used in a suppression assay as γ-irradiated third-party cells with HLA-mismatched PBMCs as γ-irradiated stimulatory cells at a responder–stimulator–third-party cell ratio of 1:1:1. Results are expressed as the mean of thymidine incorporation (cpm) in triplicate wells plus or minus SEM from 10 allogeneic combinations and corrected for background values (Δcpm). (Box) Both APCs and APC-HLA-G1s were analyzed for cell surface expression of HLA-G1 by flow cytometry using the MEM-G/9 antibody. Dashed lines correspond to APCs and solid lines to APC-HLA-G1s. Numbers on the right correspond to the mean fluorescence intensity (MFI) observed with APCs (top) and APC-HLA-G1s (bottom). Cells were stained with an isotype-matched antibody as negative control (filled histograms). (B) Concomitantly, IL-10 and IL-2 concentrations were measured by ELISA at day 6 in suppression assay supernatants. Results from 3 independent experiments are shown. (C) Similar experiment was performed with T cells primed with supernatant from either M8-HLA-G5 (HLA-G5+ SNs) or M8-pcDNA (HLA-G5− SNs). Results are expressed as the mean of thymidine incorporation (cpm) in triplicate wells plus or minus SEM from 3 allogeneic combinations and corrected for background values (Δcpm). (D) Concomitantly, IL-10 and IL-2 concentrations were measured by ELISA at day 6 in suppression assay supernatants. Results from 4 independent experiments are shown. (E,F) Suppression assays were performed by adding HLA-G5–primed T cells at the top chamber of a transwell culture system, while MLR was at the bottom chamber. (E) One representative combination is shown. Tritiated thymidine incorporation was measured after 6 days of MLR. Results are expressed as the mean of thymidine incorporation (cpm) in triplicate wells, corrected for background values (Δ cpm). (F) IL-10 and IL-2 concentrations were measured by ELISA at day 6 in suppression assay supernatants from 3 independent experiments. (G) Suppression assays were performed at various ratios of third-party T cells primed with APC-HLA-G1s or HLA-G5+ SNs. Results are expressed as percentage of alloproliferation inhibition (%) ± SEM from 3 independent experiments. Alloproliferation inhibition (%) observed with third-party T cells primed with APC-HLA-G1s or HLA-G5+ SNs was calculated, based on the alloproliferation observed with third-party T cells primed with APCs or HLA-G5− SNs, respectively. (H) T cells primed with either APCs or APC-HLA-G1s for 6 days were used in a suppression assay at a responder–stimulator–third-party cell ratio of 1:1:1. Neutralizing IL-10 mAb (B-S10) or isotype control Ab was added on days 1 and 3 during the suppression assay. Results are expressed as the mean of thymidine incorporation (cpm) in triplicate wells plus or minus SEM from 4 allogeneic combinations and corrected for background values (Δcpm). (I,J) IL-10 and IL-2 concentrations were determined by ELISA in supernatants of MLR after 6 days of allostimulation with either (I) APCs or APC-HLA-G1s, or (J) HLA-G5− or HLA-G5+ SNs. Concentrations are given as mean plus or minus SEM of 4 (I) or 3 (J) independent experiments.

T cells primed with either APC-HLA-G1s or soluble HLA-G5 are suppressors whose generation and function occur in an IL-10 microenvironment. (A) T cells primed with either APCs or APC-HLA-G1s for 6 days were used in a suppression assay as γ-irradiated third-party cells with HLA-mismatched PBMCs as γ-irradiated stimulatory cells at a responder–stimulator–third-party cell ratio of 1:1:1. Results are expressed as the mean of thymidine incorporation (cpm) in triplicate wells plus or minus SEM from 10 allogeneic combinations and corrected for background values (Δcpm). (Box) Both APCs and APC-HLA-G1s were analyzed for cell surface expression of HLA-G1 by flow cytometry using the MEM-G/9 antibody. Dashed lines correspond to APCs and solid lines to APC-HLA-G1s. Numbers on the right correspond to the mean fluorescence intensity (MFI) observed with APCs (top) and APC-HLA-G1s (bottom). Cells were stained with an isotype-matched antibody as negative control (filled histograms). (B) Concomitantly, IL-10 and IL-2 concentrations were measured by ELISA at day 6 in suppression assay supernatants. Results from 3 independent experiments are shown. (C) Similar experiment was performed with T cells primed with supernatant from either M8-HLA-G5 (HLA-G5+ SNs) or M8-pcDNA (HLA-G5− SNs). Results are expressed as the mean of thymidine incorporation (cpm) in triplicate wells plus or minus SEM from 3 allogeneic combinations and corrected for background values (Δcpm). (D) Concomitantly, IL-10 and IL-2 concentrations were measured by ELISA at day 6 in suppression assay supernatants. Results from 4 independent experiments are shown. (E,F) Suppression assays were performed by adding HLA-G5–primed T cells at the top chamber of a transwell culture system, while MLR was at the bottom chamber. (E) One representative combination is shown. Tritiated thymidine incorporation was measured after 6 days of MLR. Results are expressed as the mean of thymidine incorporation (cpm) in triplicate wells, corrected for background values (Δ cpm). (F) IL-10 and IL-2 concentrations were measured by ELISA at day 6 in suppression assay supernatants from 3 independent experiments. (G) Suppression assays were performed at various ratios of third-party T cells primed with APC-HLA-G1s or HLA-G5+ SNs. Results are expressed as percentage of alloproliferation inhibition (%) ± SEM from 3 independent experiments. Alloproliferation inhibition (%) observed with third-party T cells primed with APC-HLA-G1s or HLA-G5+ SNs was calculated, based on the alloproliferation observed with third-party T cells primed with APCs or HLA-G5− SNs, respectively. (H) T cells primed with either APCs or APC-HLA-G1s for 6 days were used in a suppression assay at a responder–stimulator–third-party cell ratio of 1:1:1. Neutralizing IL-10 mAb (B-S10) or isotype control Ab was added on days 1 and 3 during the suppression assay. Results are expressed as the mean of thymidine incorporation (cpm) in triplicate wells plus or minus SEM from 4 allogeneic combinations and corrected for background values (Δcpm). (I,J) IL-10 and IL-2 concentrations were determined by ELISA in supernatants of MLR after 6 days of allostimulation with either (I) APCs or APC-HLA-G1s, or (J) HLA-G5− or HLA-G5+ SNs. Concentrations are given as mean plus or minus SEM of 4 (I) or 3 (J) independent experiments.

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