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. 2013 Aug 13;21(9):1727–1737. doi: 10.1038/mt.2013.166

Figure 5.

Figure 5

Antigen-specificity of Tregitopes-induced suppression of CTL responses is mediated by MHC I. (a) CTL assay in which target cells were loaded with the MHC I epitope VPQYGYLTL from AAV and incubated with HLA-matched AAV-specific Teff alone (AAV, dashed line), or Teff mixed at a 1:1 ratio with negatively-selected CD4+ T cells from AAV+hTreg167 restimulated PBMC (AAV+[AAV-hTreg167 CD4+], black line), or Teff mixed at a 1:1 ratio with negatively-selected CD4+ T cells from EBV+hTreg167 restimulated PBMC (AAV+[EBV-hTreg167 CD4+], gray line). (b) CTL assay as in (a) in which target cells were loaded with the MHC I epitope RPPIFIRRL from EBV and incubated with HLA-matched EBV-specific Teff alone (EBV, dashed line), or mixed 1:1 with negatively-isolated CD4+ T cells from EBV+hTreg167 cultures (EBV+[EBV-hTreg167 CD4+], gray line) or with negatively-isolated CD4+ T cells from AAV+hTreg167 cultures (EBV+[AAV-hTreg167 CD4+], black line). Teff:target ratio 10:1. Results are expressed as % cytotoxicity compared to a max cytotoxicity (cells treated with 10% SDS) after background subtraction. (c) MHC I blockade experiment. Black bars: PBMC were restimulated in vitro with AAV+hTreg167 and CD4+ T cells were negatively selected and incubated with increasing amounts of soluble AAV-specific TCR. After washing, CD4+ T cells were mixed 1:1 with AAV-specific Teff and added to targets. Grey bar: PBMC restimulated with AAV MHC I epitope only, used as positive control (AAV only). White bar: PBMC restimulated with AAV MHC I only mixed 1:1 with CD4+ T cells derived from cell restimulated with hTreg167 only, without AAV (hTreg167 only). AAV+AAV-TCR control, CTL in which AAV-only Teff were used the CTL assay in the presence of 20 µg/ml of AAV-TCR. Shown is the % cytotoxicity. Error bars represent the standard error of the mean of quadruplicate testing. Comparison of results with the AAV-only condition gave the following p values (unpaired, two-tailed t test): AAV-TCR 0 µg/ml P = 0.00262, 1 µg/ml P = 0.00016, 5 µg/ml P = 0.00112, 20 µg/ml P = 0.03335; hTreg167 only P = 0.86133; AAV+AAV-TCR P < 0.00001. Experiments shown were repeated at least twice. Error bars represent SEM.