Figure 3.
Asct2 overexpression enhances Bcma-CAR T-cell activity. (A) Schematic representation of second-generation Bcma-CAR and PSMA-CAR constructs (upper). Schematic representation of second-generation of Asct2 Bcma-CAR construct (lower). (B) Proliferation and IFN-γ production of Bcma and PSMA-CAR T cells in response to Bcma-, Taci-, or ovalbumin-coated plates (as an irrelevant protein). (C) Bcma expression in MM cell lines 5080 and 9275 and Ctrl lymphoma cell line LY5026. (D) IFN-γ production and percentage of lysis induced by Bcma and PSMA-CAR T cells in response to these MM cell lines (5080 and 9275) or to B16OVA melanoma cells. (E) CAR expression in T lymphocytes transduced with retrovirus expressing Bcma or Asct2 BCMA-CAR constructs. Representative flow cytometry plots are shown together with histograms representing the mean fluorescence intensity in RV-transduced CD4+ and CD8+ T cells. (F) Asct2 expression measured by flow cytometry. (G) 3H-labeled Gln uptake by CD4+ and CD8+Bcma-CAR T cells and Asct2 Bcma-CAR T cells (purified by flow cytometric cell sorting). (H) IFN-γ secretion of CD4+ or CD8+Bcma-CAR T cells and Asct2 Bcma-CAR T cells in response to Bcma-coated plates under different Gln concentrations. (I) Specific lysis of 5080 MM cells by Bcma-CAR T cells and Asct2 Bcma-CAR T cells under different Gln concentrations. (J-K) Seahorse metabolic assay was used to measure the OCR and the ECAR of Ctrl and Asct2-overexpressing CD4+ (J) and CD8+ (K) Bcma-CAR T cells. Data are representative of 3 independent experiments. Data represent mean ± SEM, and were analyzed using 2-way ANOVA and 1-way ANOVA with Bonferroni multiple comparisons test (∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05). Ab, antibody; AntiA/Rote, antimycin A/rotenone; APC, allophycocyanin; cpm, counts per minute; Ctrl, control; FSC-A, forward scatter area; H&L, heavy and light; IgG, immunoglobulin G; LTR, long terminal repeat; MFI, mean fluoresecence intensity; Ova, ovalbumin; SC, subcutaneous; stim, stimulated; Taci, transmembrane activator and CAML interactor; unstim, unstimulated.

Asct2 overexpression enhances Bcma-CAR T-cell activity. (A) Schematic representation of second-generation Bcma-CAR and PSMA-CAR constructs (upper). Schematic representation of second-generation of Asct2 Bcma-CAR construct (lower). (B) Proliferation and IFN-γ production of Bcma and PSMA-CAR T cells in response to Bcma-, Taci-, or ovalbumin-coated plates (as an irrelevant protein). (C) Bcma expression in MM cell lines 5080 and 9275 and Ctrl lymphoma cell line LY5026. (D) IFN-γ production and percentage of lysis induced by Bcma and PSMA-CAR T cells in response to these MM cell lines (5080 and 9275) or to B16OVA melanoma cells. (E) CAR expression in T lymphocytes transduced with retrovirus expressing Bcma or Asct2 BCMA-CAR constructs. Representative flow cytometry plots are shown together with histograms representing the mean fluorescence intensity in RV-transduced CD4+ and CD8+ T cells. (F) Asct2 expression measured by flow cytometry. (G) 3H-labeled Gln uptake by CD4+ and CD8+Bcma-CAR T cells and Asct2 Bcma-CAR T cells (purified by flow cytometric cell sorting). (H) IFN-γ secretion of CD4+ or CD8+Bcma-CAR T cells and Asct2 Bcma-CAR T cells in response to Bcma-coated plates under different Gln concentrations. (I) Specific lysis of 5080 MM cells by Bcma-CAR T cells and Asct2 Bcma-CAR T cells under different Gln concentrations. (J-K) Seahorse metabolic assay was used to measure the OCR and the ECAR of Ctrl and Asct2-overexpressing CD4+ (J) and CD8+ (K) Bcma-CAR T cells. Data are representative of 3 independent experiments. Data represent mean ± SEM, and were analyzed using 2-way ANOVA and 1-way ANOVA with Bonferroni multiple comparisons test (∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05). Ab, antibody; AntiA/Rote, antimycin A/rotenone; APC, allophycocyanin; cpm, counts per minute; Ctrl, control; FSC-A, forward scatter area; H&L, heavy and light; IgG, immunoglobulin G; LTR, long terminal repeat; MFI, mean fluoresecence intensity; Ova, ovalbumin; SC, subcutaneous; stim, stimulated; Taci, transmembrane activator and CAML interactor; unstim, unstimulated.

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