Although bulk cultures from A2/NLV-tetramer(−) donors appear nonresponsive to exogenous NLV, single-clone analysis reveals weak NLV-responsive clones amplified by CD3 copotentiation in a unique expansion signature. (A) Gold/silver-bronze analysis was applied to exog-NLV-bulk-nonresponsive donors for the top clones showing greater abundance in NLV + Ms-IgG-Fab vs no peptide + Ms-IgG-Fab conditions. It was observed that for each of these clones, the NLV + mono-OKT3-Fab combination condition yielded greatest abundance. (B) Among top NLV-specific clones, those from exog-NLV-bulk-responsive donors respond more than those from exog-NLV-bulk-nonresponsive donors to exogenous NLV. NLV-specific fold increase in TCR abundance was determined for gold-response clones from exog-NLV-bulk-responsive donors vs those from nonresponsive donors (supplemental Table 5). (C) NLV-specific fold-increase in TCR abundance was also assessed when gold-response clones from both types of donors were cultured in the presence of mono-OKT3-Fab. (D) Exog-NLV-bulk-nonresponsive donors respond more than exog-NLV-bulk-responsive donors to CD3 copotentiation when it is driven by exogenous NLV. Mono-OKT3-Fab-specific fold increase in TCR abundance was determined for gold-response clones from exog-NLV-bulk-responsive donors vs those from nonresponsive donors (supplemental Table 5). Data are included for gold-response clones in exog-NLV-bulk-responsive and nonresponsive donors. Each dot represents the fold-increase of a TRBV-CDR3-bearing clone (B-D) (mean ± SEM, 1-tailed unpaired Student t test).