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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Nat Med. 2015 May 4;21(6):581–590. doi: 10.1038/nm.3838

Figure 3.

Figure 3

Tonic CAR signaling during ex vivo expansion leads to early exhaustion. (a) Western blot evaluating phosphorylation levels of CAR signaling domains versus total CAR signaling domains, using an anti-phospho-CD3ζ and anti-CD3ζ antibody, respectively. Evaluated day 5 after initial activation. “Basal” phosphorylation evaluated without further stimulation. “After crosslinking” evaluated following incubation with OKT3, anti-CD19 CAR idiotype, or anti-GD2 CAR idiotype antibodies. Representative of 3 donors. (b) Nine amino acid point mutations were introduced to eliminate signaling via the GD2.28z CAR (GD2.mut-28.mut-z CAR). (c) Activation and (d) exhaustion marker expression of GD2.mut-28.mut-z CAR T cells 10 days after initial activation. Representative of 3 donors. (e) Ex vivo expansion and (f-g) exhaustion marker expression of CD19.28z CAR T cells cultured ± anti-idiotype antibody (anti-Id; 0.25 ug/ml) and a crosslinking F(ab’)2 (2.5 ug/ml). n=4 replicates/group; representative of 3 donors. (h) Tumor growth curves of NSG mice inoculated with 106 143B-CD19 periosteally on day 0 followed by adoptive transfer of 107 transduced CAR T cells on day 14. Mice received mock-transduced or CD19.28z CAR cultured with or without anti-idiotype antibody. No anti-idiotype antibody was given to mice. n=8 mice/group. (i) CD19 expression on tumors 10 days following adoptive transfer into mice from (h). (j) Quantification of T cells within the blood 9 days following adoptive T cell transfer into mice from (h). n=3 mice/group. In vivo results are representative of two experiments. * = p<0.05 by Student’s T-test. Bar graphs represent mean ± SEM.