Figure 4.

Down-Modulation of CAR Surface Expression after Antigen Encounter

CAR persistence was evaluated by flow cytometry from the peripheral blood of mice inoculated with NALM-6 ALK^high cells followed by ALK CAR T cells. (A) The number of surface CAR⁺ T cells in the peripheral blood on day 5 and day 12 post T cell transfer are shown (top panel). CAR persistence was also evaluated by qPCR of genomic DNA isolated from peripheral blood (bottom panel). Differences in the number of T cells expressing surface CAR or CAR DNA compared to mock T cells were assessed by a repeated-measures two-way ANOVA followed by a Sidak’s multiple comparison test. Flow cytometry data are representative of three in vivo experiments with different donors, and qPCR data were collected from one experiment at two time points. (B) ALK or CD19 CAR T cells were mixed 1:2 with CD19^KO NALM6-GL (ALK⁻CD19⁻) or NALM6-GL-ALK⁺ (ALK⁺CD19⁺) tumor lines, and CAR expression was evaluated 5 hr later by flow cytometry. (C) Measures of surface CAR mean fluorescent intensity (MFI) and CFP MFI after antigen encounter are expressed as percentages of initial CAR/CFP expression. Differences from initial CAR expression at 0 min were determined by a repeated-measures two-way ANOVA followed by a Dunnett’s multiple comparisons test. (D) T cells expressing a CD19 CAR-CFP fusion protein were exposed to CD19-expressing tumor for 24 hr, and then they were replated with or without tumor. T cells were evaluated for CAR surface expression and total CAR expression (CFP) by flow cytometry at 24 and 40 hr. Downregulation experiments were repeated three times with different PBMC donors.