Figure 2.

CTLs re-directed with CD20p/HLA-A2-reactive TCR display exquisite antigen specificity and mediate similar degranulation responses to antigen-positive target cells as CD19 CAR-transduced T cells. (A) PBMC from one donor were retrovirally transduced with three different TCRs and expanded. Each TCR-transduced population was subsequently color-coded to allow identification by flow cytometry; A94^mod CTLs with CTV, A23^mod CTLs with CFSE and DMF5 CTLs with CTV/CFSE, and combined into one sample, as shown in the left dot plot, gated on CD8^pos T cells. Degranulation responses (mobilization of CD107a,b) were measured in the CTLs following incubation with indicated target cells; HaCaT cells transfected with HLA-A2 mRNA (+A2), either loaded or not with CD20 peptide, and SupT1 cells induced to express SCT/MART-1p^hc. (B) Summary of degranulation responses measured in T cells expressing A94^mod or A23^mod that were treated and analyzed as described in A following incubation with a panel of target cells (left to right); two HLA-A2^negCD20^pos B cell lines, 10 different HLA-A2^posCD20^neg cell lines of various tissue origins in the absence or presence of externally loaded peptide (CD20p), and four HLA-A2^pos B-cell lines endogenously expressing CD20. HLA-A2 (A2^pos) was either naturally expressed or induced (+A2), as indicated. Bars represent mean frequencies of CD107a,b^pos events among stimulated CD8^pos CTLs following subtraction of values for CTLs alone. Error bars indicate SD of duplicate samples from n = 3 experiments for all conditions, except for CD20p (n = 4) and CD20 mRNA (n = 2). The p values were calculated using Student’s t-test (one star p < 0.05; two stars p < 0.01; three stars p < 0.001). (C) Degranulation of expanded PBMCs retrovirally transduced with A94^mod or a CD19 CAR was measured following co-incubation with indicated target cell lines: Raji (HLA-A2^negCD20^posCD19^pos); K562 transduced with HLA-A2 (HLA-A2^posCD20^negCD19^neg); or HLA-A2^pos cell lines endogenously expressing CD19 and CD20 (Raji+A2 (transduced with HLA-A2), DLCL, JVM-2, HLA-A2^pos EBV-LCL), or expressing CD19 and loaded with CD20p (T2 cells), as indicated. Bars represent mean frequencies of CD107a,b^pos events among CD8^pos CTLs following subtraction of values for CTLs alone and normalized according to the transduction efficiency. Error bars indicate SD of triplicate samples from n = 3 experiments for JVM-2, Raji and T2; n = 2 experiments for DLCL, Raji+A2 and EBV-LCL; n = 1 for K562+A2.