Antigen-specific and HLA-A2–restricted anti-MM activities and proliferation of BCMA-specific iPSC-T cells through specific recognition of cognate heteroclitic BCMA72-80 (YLMFLLRKI) peptide. (A) Direct cytotoxic activity of BCMA iPSC–T cells (4-hour calcein-release assay) against U266 MM cells (HLA-A2+ BCMA+) occurred in an effector cell dose-dependent manner (effector-to-target cell ratios 1:1, 5:1, 10:1, and 20:1) but neither against antigen-mismatched MDA-MB231 breast cancer (HLA-A2+ BCMA−) nor against HLA-A2–mismatched RPMI MM (HLA-A2− BCMA+) cells. BCMA iPSC–T cells alone served as an effector cell control. Summary of 3 independent analyses (N = 3; mean ± SD). (B) Direct cytotoxic activity of BCMA iPSC–T cells and parental BCMA-CTLs (4-hour calcein-release assay) against HLA-A2+ BCMA+ U266 MM cells (● ▲) occurred in an effector cell dose-dependent manner (effector: target cell ratios = 1:1, 5:1, 10:1, and 20:1). There was complete inhibition of the cytotoxic activity after culturing U266 MM target cells (○ △) overnight with anti-HLA-A2 mAb (5 μg/mL), demonstrating their specific anti-tumor activities in an HLA-A2–restricted manner. Summary of 3 independent analyses (N = 3; mean ± SD). (C, top) Representative flow cytometric analyses showing higher CD107a degranulation and IFN-γ/IL-2/TNF-α cytokine production by BCMA iPSC–T cells than by parental BCMA-CTL in response to primary HLA-A2+ CD138+ MM cells (6-hour coculture). BCMA iPSC–T cells alone served as a negative control. Effector-to-target cell ratio, 1:1. (C, bottom) Summary of 3 independent analyses (N = 3; mean ± SD). (D, top) Representative flow cytometric analyses showing early apoptosis (annexin V+ PI−; 3 hrs) continuing to late apoptosis (annexin V+ PI+; 6 hrs) in gated U266 MM target cells (CFSE-labeled) induced by the BCMA iPSC–T cells. Effector-to-target cell ratio, 1:1. (D, bottom) Summary of 3 independent analyses (N = 3; mean ± SD). (E, top) Representative flow cytometric analyses showing the cell death (PI+) of primary BMMC and CD138+ cells in BMMC from a patient with HLA-A2+ MM induced by BCMA iPSC–T cells (6-hour assay), but not in control HLA-A2+ T cells. Effector-to-target cell ratio, 1:1. (E, bottom) Summary of 3 independent analyses (N = 3; mean ± SD). (F, top) Representative flow cytometric analyses showing the specific proliferation of BCMA iPSC–T cells (CFSE-low) in response to cognate heteroclitic BCMA72-80 (YLMFLLRKI) peptide, not HLA-A2 irrelevant HIV Gag77-85 peptide–loaded stimulator cells (T2 or K562-A∗0201) on day 6 of coculture. BCMA iPSC–T cells alone or BCMA iPSC–T cells stimulated with T2 or K562-A∗0201 cells (no peptide loaded) served as controls. Responder-to-stimulator cell ratio, 1:1. (F, bottom) Summary of 3 independent analyses (N = 3; mean ± SD). (G, top) Representative flow cytometric analyses demonstrating a time-dependent increase in BCMA iPSC–T-cell proliferation (CFSE-low) in response to heteroclitic BCMA72-80 (YLMFLLRKI) peptide–loaded stimulator cells (T2, U266) on day 5, 6 or 7 of coculture. BCMA-iPSC–T cells stimulated with T2 or U266 cells (no peptide loaded) served as controls. Responder-to-stimulator cell ratio, 1:1. (G, bottom) Summary of 3 independent analyses (N = 3; mean ± SD). (H, top) Representative flow cytometric analyses showing the downregulation of Ki-67, a cellular proliferation marker, in U266 MM cells (CFSE-labeled) upon 4 or 16 hours of coculture with BCMA iPSC–T cells in a time-dependent and effector cell dose-dependent (effector-to-target cell ratios 1:5, 1:1, and 5:1) manner. (H, bottom) Summary of results (N = 3; mean ± SD).

Antigen-specific and HLA-A2–restricted anti-MM activities and proliferation of BCMA-specific iPSC-T cells through specific recognition of cognate heteroclitic BCMA72-80 (YLMFLLRKI) peptide. (A) Direct cytotoxic activity of BCMA iPSC–T cells (4-hour calcein-release assay) against U266 MM cells (HLA-A2+ BCMA+) occurred in an effector cell dose-dependent manner (effector-to-target cell ratios 1:1, 5:1, 10:1, and 20:1) but neither against antigen-mismatched MDA-MB231 breast cancer (HLA-A2+ BCMA−) nor against HLA-A2–mismatched RPMI MM (HLA-A2− BCMA+) cells. BCMA iPSC–T cells alone served as an effector cell control. Summary of 3 independent analyses (N = 3; mean ± SD). (B) Direct cytotoxic activity of BCMA iPSC–T cells and parental BCMA-CTLs (4-hour calcein-release assay) against HLA-A2+ BCMA+ U266 MM cells (● ▲) occurred in an effector cell dose-dependent manner (effector: target cell ratios = 1:1, 5:1, 10:1, and 20:1). There was complete inhibition of the cytotoxic activity after culturing U266 MM target cells (○ △) overnight with anti-HLA-A2 mAb (5 μg/mL), demonstrating their specific anti-tumor activities in an HLA-A2–restricted manner. Summary of 3 independent analyses (N = 3; mean ± SD). (C, top) Representative flow cytometric analyses showing higher CD107a degranulation and IFN-γ/IL-2/TNF-α cytokine production by BCMA iPSC–T cells than by parental BCMA-CTL in response to primary HLA-A2+ CD138+ MM cells (6-hour coculture). BCMA iPSC–T cells alone served as a negative control. Effector-to-target cell ratio, 1:1. (C, bottom) Summary of 3 independent analyses (N = 3; mean ± SD). (D, top) Representative flow cytometric analyses showing early apoptosis (annexin V+ PI−; 3 hrs) continuing to late apoptosis (annexin V+ PI+; 6 hrs) in gated U266 MM target cells (CFSE-labeled) induced by the BCMA iPSC–T cells. Effector-to-target cell ratio, 1:1. (D, bottom) Summary of 3 independent analyses (N = 3; mean ± SD). (E, top) Representative flow cytometric analyses showing the cell death (PI+) of primary BMMC and CD138+ cells in BMMC from a patient with HLA-A2+ MM induced by BCMA iPSC–T cells (6-hour assay), but not in control HLA-A2+ T cells. Effector-to-target cell ratio, 1:1. (E, bottom) Summary of 3 independent analyses (N = 3; mean ± SD). (F, top) Representative flow cytometric analyses showing the specific proliferation of BCMA iPSC–T cells (CFSE-low) in response to cognate heteroclitic BCMA72-80 (YLMFLLRKI) peptide, not HLA-A2 irrelevant HIV Gag77-85 peptide–loaded stimulator cells (T2 or K562-A∗0201) on day 6 of coculture. BCMA iPSC–T cells alone or BCMA iPSC–T cells stimulated with T2 or K562-A∗0201 cells (no peptide loaded) served as controls. Responder-to-stimulator cell ratio, 1:1. (F, bottom) Summary of 3 independent analyses (N = 3; mean ± SD). (G, top) Representative flow cytometric analyses demonstrating a time-dependent increase in BCMA iPSC–T-cell proliferation (CFSE-low) in response to heteroclitic BCMA72-80 (YLMFLLRKI) peptide–loaded stimulator cells (T2, U266) on day 5, 6 or 7 of coculture. BCMA-iPSC–T cells stimulated with T2 or U266 cells (no peptide loaded) served as controls. Responder-to-stimulator cell ratio, 1:1. (G, bottom) Summary of 3 independent analyses (N = 3; mean ± SD). (H, top) Representative flow cytometric analyses showing the downregulation of Ki-67, a cellular proliferation marker, in U266 MM cells (CFSE-labeled) upon 4 or 16 hours of coculture with BCMA iPSC–T cells in a time-dependent and effector cell dose-dependent (effector-to-target cell ratios 1:5, 1:1, and 5:1) manner. (H, bottom) Summary of results (N = 3; mean ± SD).

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