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. Author manuscript; available in PMC: 2017 Mar 15.
Published in final edited form as: Cancer Res. 2016 Mar 15;76(6):1578–1590. doi: 10.1158/0008-5472.CAN-15-2524

Figure 3. Increased cytokine production by T-cells co-expressing SS1BBz CAR and PD1CD28 switch-receptor.

Figure 3

FACS analysis of CD4 and CD8 bulk T-cells activated via anti-CD3/CD28 microbeads at a ratio of 3:1 bead:T-cell and transduced with lentivirus led to successful co-expression of both SS1BBz CAR and PD1CD28 switch-receptor (~40%). The CAR expression was detected using an anti-mouse IgG Fab antibody and PD1CD28 was detected with anti-PD1 antibody (A, dot plot). FACS analysis of EMP, EMMESO, and EMMESO-PDL1 tumor cells was performed to confirm high expression of mesothelin and PDL1. (A, histograms) T-cells co-expressing SS1BBz and PD1CD28 (SS1BBz/PD1CD28) or SS1BBz alone (SS1BBz) were co-cultured with EMMESO or EMMESO-PDL1, at different E:T ratios x 18hrs. ELISA was performed to measure the levels of IFNγ (B) and IL-2 (C) present in the supernatants of the cocultures. % specific lysis of both EMMESO and EMMESO-PDL1 by SS1BBz and SS1BBz/PD1CD28 was also calculated after 18hrs of coculture (D). Bar charts show results from a representative experiment (values represent the average ± SE of triplicates)