Journal: Frontiers in Microbiology

Article Title: Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner

doi: 10.3389/fmicb.2016.00844

Figure Lengend Snippet: Lysis of HCMV-infected HFF by CAR-T cells is strongly reduced. (A) The histograms shows the expression of the different CARs in αCD3/αCD28-activated or EBV-specific T cells, respectively, 20 h after mRNA electroporation of a representative donor (EBV-specific T cells n = 6; 4 donors; αCD3/αCD28-activated n = 3; 3 donors). (B) Flow cytometric analysis of the gB-expression in 293T cells, gB-expressing 293T (293T-gB) cells, non-infected and HCMV-infected HFF. Shown is a representative histogram of one experiment ( n = 3). (C,D) The diagrams show the lytic potential of redirected αCD3/αCD28-activated or EBV-specific T cells targeting HCMV-infected HFF (C) or gB-expressing 293T (293T-gB; (D) analyzed in a 4 h Eu release assay (E:T-ratio referring to CD8 pos T cells = 25:1). 293T cells, non-infected HFF were used as target cell controls, CEA-CAR-expressing EBV-specific T cells were used as controls for CAR-T cells. (B–D) HCMV-infected HFF in all shown experiments were used at 4 days after infection (AD169, MOI 5).

Article Snippet: HFF were detached with Trypsin/EDTA and resuspended at a density of 0.5 × 10 6 /400 μl in GenePulser ® Electroporation Buffer Reagent (Bio-Rad).

Techniques: Lysis, Infection, Expressing, Electroporation, Release Assay

Journal: Frontiers in Microbiology

Article Title: Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner

doi: 10.3389/fmicb.2016.00844

Figure Lengend Snippet: UL36 and UL37x1 inhibit CAR-T cell-mediated apoptosis in HFF . The anti-apoptotic HCMV-encoded proteins UL36 and UL37x1 were expressed in HFF by either mRNA electroporation or retroviral transduction, respectively. (A) The anti-apoptotic function of the exogenously expressed proteins was confirmed by overnight incubation of the HFF with cycloheximide (CHX, 10 μg/ml) plus an agonistic anti-Fas-antibody (0.2 μg/ml). A GFP encoding vector or mock-electroporation were used as controls. The diagram shows the fraction of apoptotic HFF determined by Annexin V staining in a flow cytometric analysis ( n = 3). (B) The diagram shows the induction of cell death in HFF expressing UL36 and/or UL37x1 after 4 h of co-incubation with chNKG2D-expressing αCD3/αCD28-activated T cells ( n = 3, 3 different T cell donors). The percentages of apoptotic HFF obtained in the co-cultures of CAR-T cells plus HFF without UL36/UL37x1 expression were set to 100%. Concanamycin A (CMA, 100 nM) was used to block perforin-induced apoptosis. (C) The diagram displays the percentage of CAR-T cell-mediated apoptosis in HFF that was inhibited by the combined expression of UL36 and UL37x1 (gray bars; obtained by subtraction of the respective values shown in (B) from 100%). The black bars show the proportion of CAR-T cell induced apoptosis in HFF, which was blocked by CMA (B) i.e., the cell death that was not mediated by perforin/granzyme. If these values were a result of incomplete CMA blockade, then the black bars (non-perforin/granzyme mediated cell death) would be even lower. The respective differences between the gray and the black bars correspond to the proportion of the perforin/granzyme-mediated cell death in the HFF, which was inhibited by the combined expression of UL36 and UL37x1.

Article Snippet: HFF were detached with Trypsin/EDTA and resuspended at a density of 0.5 × 10 6 /400 μl in GenePulser ® Electroporation Buffer Reagent (Bio-Rad).

Techniques: Electroporation, Retroviral, Transduction, Incubation, Plasmid Preparation, Staining, Expressing, Blocking Assay