Functional analysis of CD4 and CD8 clones generated from MM and MGUS patients. (A,B) IFN-γ production and cytotoxicity properties of generated CD4⁺ T-cell clones (TCC) from MGUS and MM patients. (i) Peptide titration analysis of CD4⁺ T-cell clones from MM (Bi) and MGUS (Ai) patients. (ii) MAGE-A3–specific CD4⁺ T cells were stimulated with peptide-loaded or DMSO-loaded HLA-matched or -mismatched LCL target cells or target cells transfected with a recombinant MAGE-A3 protein (KS114d transfected with MAGE-A3 [DR52b⁺] or DESA mismatched DR52b⁻ LCL trans MAGE-A3). (Biii) T-cell clones from MM patient were screened against a panel of different LCLs to identify class II restriction of T-cell clones specific to MAGE-A3_145-160. Cytolytic activity of the CD4⁺ T-cell clones by standard 12-hour chromium release assay at an effector:target ratio of 10:1 (Biv) and (Aiii) against peptide-loaded HLA-matched target cells or target cells transfected with MAGE-A3. (Ci,ii) IFN-γ production and cytotoxicity properties of generated CD8 TCC from MM patients 4 and 7: (a) Peptide titration analysis of CD8⁺ T-cell clones from MM patients 4 and 7 represented as a percentage of IFN-γ release. The line represents 50% recognition of peptide concentration. (b) MAGE-A1_289-298–specific CD8⁺ T cells were stimulated with peptide-loaded or DMSO-loaded HLA-matched or -mismatched LCL target cells or MM cell lines U266, JJN3, and H929. (c) Cytolytic activity of the CD8⁺ T-cell clones by standard 6-hour chromium release assay at an effector-target ratio of 10:1 against peptide or DMSO-loaded HLA-matched target cells or MM cell lines.