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. Author manuscript; available in PMC: 2019 Feb 13.
Published in final edited form as: Nat Biotechnol. 2018 Aug 13;36(9):847–856. doi: 10.1038/nbt.4195

Fig. 4. Co-expression of CAR and E27 scFv protects proliferative and lytic capacity of T cells in the context of PD-L1+ tumor cells.

Fig. 4.

(a) Representative flow cytometry dot plots demonstrating lysis of Raji-PDL1 tumor cells, as determined by flow cytometry following 72 hour co-culture. Data shown is representative of 3 independent donors and experiments. (b). 1928z-E27 T cells lyse significantly more Raji-PDL1 tumor cells compared to 1928z T cells, data shown the mean +/− SEM from 5 independent experiments, *p=0.03 by a one-tailed paired t test. (c) CAR-T cells expansion numbers following co-culture with Raji-PDL1 or NALM6-PDL1 tumor cells as determined by flow cytometry, data shown is the average total number of T cells +/− SEM from 4 independent experiments, *p=0.05 for Raji experiment and *p=0.02 for Nalm6 experiment, both by a two-tailed paired t test. Representative flow cytometry plot (d) and quantification (e) showing increased PD-1 detection on 1928z T-cells compared to 1928z-E27 T-cells following 7 days co-culture with Raji-PDL1 tumor cells. Data shown in the mean +/− SEM from 3 independent experiments. *p=0.03 for percent positive CAR-T cell and MFI of staining, both by two-tailed paired test. (f) 1928z and 1928z-E27 T-cells were co-cultured with human T cells transduced to overexpress PD-1 and after 4 days stimulation with CD3/CD28 beads, the cells were sorted by flow cytometry to separate CAR+ and CAR- cells. Western blot was performed on the sorted populations and probed with anti-HA mAb. Data shown is representative of 3 independent donors and experiments. (g) Representative example of 1928z and 1928z-E27 T cells fold expansion when cultured with 3T3-empty or 3T3-PDL1 cells and stimulated with CD3/CD28 beads. Data shown is representative of 3 independent donors and experiments. (h) Cells were enumerated and re-plated on new 3T3 cells on days 3, 6, 9 and 12. 1928z T cells had reduced expansion when cultured with 3T3-PDL1 cells compared to 3T3- empty cells. 1928z-E27 cells had equivalent expansion when cultured on 3T3- empty or 3T3-PDL1 cells. Data shown is the mean fold expansion +/− SEM from 4 independent experiments, *p<0.05 by two-tailed paired t test. (i) Expansion of 1928z-E27 T cells on 3T3-PDL1 cells was due to an increase in both CAR+ and CAR- cells, comparing populations on day 0 and following expansion on 3T3-PDL1 cells at day 12. Data shown is representative of 3 independent experiments.