Figure S3.
Impaired T cell responses and NK cell expansion toward LCL of the patient or P815 expressing CD137LV140G. (A) FACS histograms of HLA-A02 and CD70 expression on control HLA-A02* LCL (Ctr. LCL), CD137L-deficient/patient LCL (Pat. LCL), LCL from a patient with a DiGeorge syndrome (DGS LCL), LCL of the patient transfected with an empty vector (Pat. LCL + empty) or a vector containing cDNA coding CD137L (Pat. LCL + CD137L). Anti-CD137L and isotype control staining corresponding to histograms with black line with deep gray and gray line with pale gray, respectively. (B) FACS dot plots of IFN-γ and Granzyme B expression of EBV-specific T cell expansions at day 25 (from co-cultures in Fig. 5) that have been re-stimulated with irradiated Ctr. LCL or Pat. LCL before intracellular co-staining with anti–IFN-γ and Granzyme B antibodies. Double IFN-γ+ GranzymeB+ cells are shown in gates and numbers indicating proportions of cells in gates. (C) Graphs of cell lysis assays of EBV-specific T cell expansions at day 28 (from co-cultures in Fig. 5) as effector cells and control LCLs (Ctr. LCL) or LCLs of the patient (Pat. LCL) as target cells that were mixed at the indicated target cells/effector cells ratio in the longitudinal axis. (D) FACS histograms of mCherry (upper panel) or CD137L (lower panel) in P815 cells ectopically expressing WT CD137L, CD137L-V140G, V140A, A97V mutants, or infected with an empty vector. (E) FACS dot-plots of proliferation assays of PBMCs labeled with the CTV dye and cocultured or not (no P815) for 6 d with irradiated P815 cell lines expressing CD137L V140G, V140A, A97V (as positive control), or an empty vector in the presence of anti-CD3 antibody. Staining with anti-CD25 antibody as activation marker and gating on CD3+ cells. Proliferating T cells in the gate with numbers corresponding to the proportions of cells. (F) FACS dot-plots of proliferation assays of PBMCs labeled with the CTV dye and cocultured or not (no P815) for 6 d with irradiated P815 cell lines expressing CD137L, CD137L V140G and V140A, or an empty vector. Staining with anti-CD25 antibody. CD25 as activation marker. Cells gated on CD3−/CD56+ cells. Proliferating NK cells in the gate with numbers corresponding to the proportions of cells. (A) One representative experiment of two. (D) One representative experiment of four. (E) One representative experiment of three. (F) Data representative of three experiments.

Impaired T cell responses and NK cell expansion toward LCL of the patient or P815 expressing CD137L V140G . (A) FACS histograms of HLA-A02 and CD70 expression on control HLA-A02* LCL (Ctr. LCL), CD137L-deficient/patient LCL (Pat. LCL), LCL from a patient with a DiGeorge syndrome (DGS LCL), LCL of the patient transfected with an empty vector (Pat. LCL + empty) or a vector containing cDNA coding CD137L (Pat. LCL + CD137L). Anti-CD137L and isotype control staining corresponding to histograms with black line with deep gray and gray line with pale gray, respectively. (B) FACS dot plots of IFN-γ and Granzyme B expression of EBV-specific T cell expansions at day 25 (from co-cultures in Fig. 5) that have been re-stimulated with irradiated Ctr. LCL or Pat. LCL before intracellular co-staining with anti–IFN-γ and Granzyme B antibodies. Double IFN-γ+ GranzymeB+ cells are shown in gates and numbers indicating proportions of cells in gates. (C) Graphs of cell lysis assays of EBV-specific T cell expansions at day 28 (from co-cultures in Fig. 5) as effector cells and control LCLs (Ctr. LCL) or LCLs of the patient (Pat. LCL) as target cells that were mixed at the indicated target cells/effector cells ratio in the longitudinal axis. (D) FACS histograms of mCherry (upper panel) or CD137L (lower panel) in P815 cells ectopically expressing WT CD137L, CD137L-V140G, V140A, A97V mutants, or infected with an empty vector. (E) FACS dot-plots of proliferation assays of PBMCs labeled with the CTV dye and cocultured or not (no P815) for 6 d with irradiated P815 cell lines expressing CD137L V140G, V140A, A97V (as positive control), or an empty vector in the presence of anti-CD3 antibody. Staining with anti-CD25 antibody as activation marker and gating on CD3+ cells. Proliferating T cells in the gate with numbers corresponding to the proportions of cells. (F) FACS dot-plots of proliferation assays of PBMCs labeled with the CTV dye and cocultured or not (no P815) for 6 d with irradiated P815 cell lines expressing CD137L, CD137L V140G and V140A, or an empty vector. Staining with anti-CD25 antibody. CD25 as activation marker. Cells gated on CD3−/CD56+ cells. Proliferating NK cells in the gate with numbers corresponding to the proportions of cells. (A) One representative experiment of two. (D) One representative experiment of four. (E) One representative experiment of three. (F) Data representative of three experiments.

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