American Society of Hematology

Figure 5.

$SLAMF7-mediated fratricide spares a fraction of functional CMV-specific CD8+T cells. (A) SLAMF7 expression on primary CD8+CD45RA–CD45RO+ memory T cells specific for the CMV pp65 A2/NLV epitope in 3 HLA-A*02+ healthy donors. Overlay histograms show staining with anti-SLAMF7 mAb 162.1 (red) and isotype control (blue). (B) CMV-specific CD8+ T-cell lines (CMV-CTL) were prepared from CMV-specific memory T cells and the expression of SLAMF7 was analyzed (2 top left dot plots). CMV-CTL was labeled with eFluor670 and cocultured for 4 hours with autologous SLAMF7-CAR or control CD19-CAR T cells. The expression of SLAMF7 on residual live (ie, 7-ADD–) CMV-CTL was reanalyzed at the end of the coculture (top right dot plot). Residual live CMV-CTL was then stimulated with pp65 NLV peptide-loaded K562/HLA-A2 cells, and IFN-γ production in the SLAMF7+/high and SLAMF7–/low fraction was analyzed by intracellular cytokine staining (2 bottom right dot plots). IFN-γ production in SLAMF7+/high and SLAMF7–/low CMV-CTL before the fratricide assay was analyzed for comparison (2 bottom left dot plots). (C) Bar diagrams show the mean percentage of live (7-AAD–) (left) and IFN-γ–secreting (right diagram) SLAMF7+/high and SLAMF7–/low CMV-CTL after coculture with SLAMF7-CAR and control CD19-CAR T cells. CMV-CTL alone is provided for comparison (target cells only). (D) CMV-CTL were transduced with the SLAMF7-CAR (or CD19-CAR for comparison), and CAR-transduced T cells were enriched using the EGFRt marker. The CAR-transduced CMV-CTL was then stimulated with pp65 NLV peptide–loaded K562/HLA-A2 cells. The diagram shows the fold expansion of CMV-CTL within 14 days of culture. (C-D) The data shown are representative of the results obtained in 2 independent experiments with CAR-transduced CMV-CTL from different donors.$

SLAMF7-mediated fratricide spares a fraction of functional CMV-specific CD8⁺T cells. (A) SLAMF7 expression on primary CD8⁺CD45RA^–CD45RO⁺ memory T cells specific for the CMV pp65 A2/NLV epitope in 3 HLA-A*02⁺ healthy donors. Overlay histograms show staining with anti-SLAMF7 mAb 162.1 (red) and isotype control (blue). (B) CMV-specific CD8⁺ T-cell lines (CMV-CTL) were prepared from CMV-specific memory T cells and the expression of SLAMF7 was analyzed (2 top left dot plots). CMV-CTL was labeled with eFluor670 and cocultured for 4 hours with autologous SLAMF7-CAR or control CD19-CAR T cells. The expression of SLAMF7 on residual live (ie, 7-ADD^–) CMV-CTL was reanalyzed at the end of the coculture (top right dot plot). Residual live CMV-CTL was then stimulated with pp65 NLV peptide-loaded K562/HLA-A2 cells, and IFN-γ production in the SLAMF7^+/high and SLAMF7^–/low fraction was analyzed by intracellular cytokine staining (2 bottom right dot plots). IFN-γ production in SLAMF7^+/high and SLAMF7^–/low CMV-CTL before the fratricide assay was analyzed for comparison (2 bottom left dot plots). (C) Bar diagrams show the mean percentage of live (7-AAD^–) (left) and IFN-γ–secreting (right diagram) SLAMF7^+/high and SLAMF7^–/low CMV-CTL after coculture with SLAMF7-CAR and control CD19-CAR T cells. CMV-CTL alone is provided for comparison (target cells only). (D) CMV-CTL were transduced with the SLAMF7-CAR (or CD19-CAR for comparison), and CAR-transduced T cells were enriched using the EGFRt marker. The CAR-transduced CMV-CTL was then stimulated with pp65 NLV peptide–loaded K562/HLA-A2 cells. The diagram shows the fold expansion of CMV-CTL within 14 days of culture. (C-D) The data shown are representative of the results obtained in 2 independent experiments with CAR-transduced CMV-CTL from different donors.

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