Figure 5.
Figure 5. CD70 on LCLs is required for patient and control T cell expansion of EBV-specific T cells. (A) FACS histograms showing the expression of CD70 and HLA-A02* on LCLs from an HLA-A02* healthy donor, in which the CD70 gene was knocked out by CRISPR-Cas9–targeting exon 3 with three different RNA guides (CD70-CRISPR1, CD70-CRISPR2, and CD70-CRISPR3). One representative of four independent experiments is shown. (B) Dot plots from FACS analysis showing EBV-specific T cells gated on CD8+CD3+ T cells stained with EBV-specific HLA-A02* pentamers among PBMCs that have been co-cultured for 8 d or not in the presence of irradiated HLA-A02* CD70-expressing LCLs or HLA-A02* CD70-deficient CD70-CRISPR1 LCLs. PBMCs from the patient and four healthy donors (Ctr.1, Crt.2, Ctr.3, and Ctr.4). Control 4 has not been infected by EBV and had a negative EBV serology (EBV neg.). Numbers in the plots indicate the percentage of EBV pentamer–positive cells in the gates. One representative of four independent experiments is shown. (C) Same as B, except that the presence of EBV-specific T cells of healthy donor control 1 was tested in the presence of two other irradiated HLA-A02* CD70-deficient LCLs, CD70-CRISPR2 and CD70-CRISPR3, obtained by CRISPR-Cas9. Numbers in the plots indicate the percentage of EBV-pentamer–positive cells in the gates. One representative of two independent experiments is shown. (D) Analysis of cytolytic activity of T cells expanded from PBMCs of the CD70-deficient patient (Pat. expanded T cells) and one healthy control (Ctr. expanded T cells) for 8–15 d with irradiated autologous CD70-expressing (pLenti-CD70 or empty CRISPR) or CD70-deficient (empty vector or CD70-CRISPR) LCL cells. Cytolytic activity of T cells was then tested against a mixture of autologous CD70-expressing (blue) or CD70-deficient (red) LCL cells as target cells (Target LCLs) at an effector-to-target ratio of 1:1 of 1 for 0, 4, and 12 h. Residual target cells were evaluated by FACS analysis. Data are represented in percentages of cells normalized to the percentages at time 0. One representative of two independent experiments is shown.

CD70 on LCLs is required for patient and control T cell expansion of EBV-specific T cells. (A) FACS histograms showing the expression of CD70 and HLA-A02* on LCLs from an HLA-A02* healthy donor, in which the CD70 gene was knocked out by CRISPR-Cas9–targeting exon 3 with three different RNA guides (CD70-CRISPR1, CD70-CRISPR2, and CD70-CRISPR3). One representative of four independent experiments is shown. (B) Dot plots from FACS analysis showing EBV-specific T cells gated on CD8+CD3+ T cells stained with EBV-specific HLA-A02* pentamers among PBMCs that have been co-cultured for 8 d or not in the presence of irradiated HLA-A02* CD70-expressing LCLs or HLA-A02* CD70-deficient CD70-CRISPR1 LCLs. PBMCs from the patient and four healthy donors (Ctr.1, Crt.2, Ctr.3, and Ctr.4). Control 4 has not been infected by EBV and had a negative EBV serology (EBV neg.). Numbers in the plots indicate the percentage of EBV pentamer–positive cells in the gates. One representative of four independent experiments is shown. (C) Same as B, except that the presence of EBV-specific T cells of healthy donor control 1 was tested in the presence of two other irradiated HLA-A02* CD70-deficient LCLs, CD70-CRISPR2 and CD70-CRISPR3, obtained by CRISPR-Cas9. Numbers in the plots indicate the percentage of EBV-pentamer–positive cells in the gates. One representative of two independent experiments is shown. (D) Analysis of cytolytic activity of T cells expanded from PBMCs of the CD70-deficient patient (Pat. expanded T cells) and one healthy control (Ctr. expanded T cells) for 8–15 d with irradiated autologous CD70-expressing (pLenti-CD70 or empty CRISPR) or CD70-deficient (empty vector or CD70-CRISPR) LCL cells. Cytolytic activity of T cells was then tested against a mixture of autologous CD70-expressing (blue) or CD70-deficient (red) LCL cells as target cells (Target LCLs) at an effector-to-target ratio of 1:1 of 1 for 0, 4, and 12 h. Residual target cells were evaluated by FACS analysis. Data are represented in percentages of cells normalized to the percentages at time 0. One representative of two independent experiments is shown.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal