CD3 copotentiation is dependent on TCR-HLA and CD8 co-receptor engagement. (A-B) PBMCs were cultured with or without exogenous NLV peptide in the presence of mono-OKT3-Fab or control Ms-IgG-Fab for 9 days as in Figure 2A. Counts of A2/NLV-tetramer(−) CD8 T cells are shown for exog-NLV-bulk-responsive donors (A) and exog-NLV-bulk-nonresponsive donors (B). Each symbol represents the average of ≥3 independent experiments per donor (mean ± SD, 2-tailed paired Student t test). (C-D) PBMCs were cultured with or without exogenous NLV peptide in the presence of Ms-IgG-Fab (control), mono-OKT3-Fab, or mono-UCHT1-Fab for 9 days. Mono-UCHT1-Fab dampened the copotentiation effect as compared with mono-OKT3-Fab in both A2/NLV-tetramer(+) (C) and A2/NLV-tetramer(−) (D) CD8 T cells. (E-F) PBMCs were cultured with or without exogenous NLV peptide in the presence of Ms-IgG-Fab or mono-OKT3-Fab and with or without the CD8 blocking antibody, DK25, for 7 days. Blocking CD8 reduced the copotentiation effect of mono-OKT3-Fab for both A2/NLV-tetramer(+) (E) and A2/NLV-tetramer(−) (F) CD8 T cells. One representative experiment of donor 47M is shown for 3 replicates (C-F) (mean ± SD from triplicate samples, 2-tailed unpaired Student t test).