Functional characterization of myeloma-associated antigens. (A) Myeloma-associated T-cell epitopes with their corresponding HLA restrictions and frequencies of immune recognition by myeloma patient-derived T cells in IFNγ-ELISPOT assays. (B) Example of myeloma-associated T-cell epitopes evaluated in an IFNγ-ELISPOT using HV PBMCs. An EBV epitope mix containing the frequently recognized peptides BRLF109-117 YVLDHLIVV (A*02) and EBNA3247-255 RPPIFIRRL (B*07 served as positive control). Benign-tissue–derived peptides KLFEKVKEV (HLA-A*02) and KPSEKIQVL (B*07) served as negative control. (C) Examples of myeloma-associated T-cell epitopes evaluated in IFNγ-ELISPOTs using MM patient PBMCs (n = 3). Results are shown only for immunoreactive peptides. An EBV epitope mix containing 5 frequently recognized peptides (BRLF109-117 YVLDHLIVV [A*02], EBNA3471-479 RLRAEAQVK [A*03], EBNA3247-255 RPPIFIRRL [B*07], BZLF1190-197 RAKFKQLL [B*08], EBNA6162-171 AEGGVGWRHW [B*44]) was used as positive control. Benign-tissue–derived peptides KLFEKVKEV (HLA-A*02) and KPSEKIQVL (B*07) served as negative control. (D-E) Tetramer staining of CD8⁺ T cells after 3 cycles of aAPC-based in vitro primings using T cells derived from (D) a healthy individual and (E) a myeloma patient: Leftmost panels, P₂-tetramer staining of CD8⁺ T cells primed with P₂-aAPCs (SLLEQGLVEA, A*02); left middle panels, ex vivo P₂-tetramer staining of CD8⁺ T cells; right middle panels, control staining with A*02-tetramer containing a nonrelevant A*02-restricted control peptide (KAMEAASSL, A*02) on CD8⁺ T cells derived from the same population as T cells depicted in the left panels. Rightmost panels, Positive control: tetramer staining of CD8⁺ T cells primed with CMV-aAPCs (NLVPMVATV, A*02). EBV, Epstein-Barr virus; neg., negative; pos., positive; UPN, uniform patient number.