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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: Clin Cancer Res. 2015 Apr 2;21(13):2993–3002. doi: 10.1158/1078-0432.CCR-14-2920

Figure 1. Development of clinically feasible platform for derivation of bi-specific T cells.

Figure 1

(A) CMV-specific T cells from CMV immune HLA A2 donors were selected using IFNγ capture after overnight stimulation with cGMP grade CMVpp65 protein. After selection, the cells were stained with antibodies specific to IFNγ, CD4, and CD8. The frequency of each population is presented after exclusion of dead cells with DAPI. (B) The selected cells were transduced with the second generation CD19CAR with a double mutation in the spacer, 24 hours after the IFNγ capture. 7–10 days later, the transduced cells were stimulated with irradiated CD19 expressing NIH3T3 cells at 10:1 ratio (3T3: T cells) and the stimulation was repeated 7 days post the first stimulation. CAR expression was defined by cetuximab-biotin and streptavidin (SA) APC-Cy7 staining. Percentages of CAR+ cells are indicated in each histogram (filled gray), and based on subtraction of that stained with SA- APC-Cy7 alone (black line). (C) Growth of total cell number was determined by Guava Viacount at different time points.