pMHC tetramer⁺ CD8⁺ T cells exhibit different degrees of CD79b peptide sensitivity and specificity. T cells were clonally expanded by single-cell sorting of CD8⁺ T cells binding to pMHC tetramers containing CD79b peptides. (A) Shown are pMHC-tetramer staining of 5 representative clones (K110, K64, K68, K116, and K308) isolated using pMHC tetramer CD79b₁₇₃:A2. Control includes clone HSS12 specific for USP11-derived peptide FTWEGLYNV bound to HLA-A2. Shown are CD8⁺ cells. (B-C) The same T-cell clones as in (A) were coincubated with K562-A2 cells loaded with decreasing concentrations of CD79b₁₇₃ peptide (B) or were cocultured with 3 HLA-A2/B7–positive B-LCLs endogenously expressing CD79b (C). Controls included CD79b-negative K562-A2 cells. (D-E) K562-A2 cells peptide-pulsed with decreasing concentration of peptide CD79b₁₇₃ or CD79b₂₀ as indicated were coincubated with clone K308 isolated using pMHC tetramer CD79b₁₇₃:A2 (D) or clone S100 isolated using pMHC tetramer CD79b₂₀:A2 (E). (F) K562 cells retrovirally transduced with HLA-B7 (K562-B7) were peptide-pulsed with decreasing concentration of peptide CD79b₉ and coincubated with clone A23 isolated with pMHC tetramer CD79b₉:B7. (G-I) Clones K308 (G), S100 (H), and A23 (I) were cocultured with HLA-A2 and HLA-B7–positive B-LCLs. CD79b-negative K562-A2 or K562-B7 cells were included as controls. IFN-γ secretion was measured using standard ELISA. Axes were adjusted to maximum cytokine release measured. (J-L) K308 was stained with pMHC tetramer CD79b₁₇₃:A2 (J), S100 with CD79b₂₀:A2 (K), and A23 with CD79b₉:B7 (L). Staining with an irrelevant pMHC tetramer USP11:A2 was used as control. Dot plots are shown with biexponential axis.