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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Cancer Immunol Res. 2022 Sep 1;10(9):1055–1068. doi: 10.1158/2326-6066.CIR-21-0626

Figure 1. CD19 CAR T-cell treatment induces rapid reduction of CD19 surface protein on B-ALL cells.

Figure 1.

a, Three-dimensional holotomography-based live cell microscopy reveals different types of interactions between CD19 CAR T cells and NALM-6 B-ALL cells (60x magnification). Upper sections show refractive index, lower sections show selected two-dimensional Z-stack slice (slice 0, 0 and 15 respectively) for identification of CFSE-labeled NALM-6 target cells. Top, productive interaction with cell–cell contacts suggesting immunological synapse formation, target-cell blebbing and nuclear condensation (Supplementary Movie S1). Middle, sustained immunological synapse without cytotoxicity (Supplementary Movie S2). Bottom, leukemia cell escape after interaction (Supplementary Movie S3). b-e, CFSE-labeled B-ALL NALM-6 cells (Target, T) were co-cultured with either CD19 CAR T cells or uninfected T cells (Effector, E). Quantification of 7-AAD negative live cells and CD19 expression performed by flow cytometry. Data are representative of at least two independent experiments from two different T-cell donors, n=3. b, Gating strategy to identify CFSE+ (left) and 7-AAD– live (right) target cells in a representative 5E:1T CAR T cell co-culture sample. c, Cell viability of NALM-6 B-ALL cells after 6, 12, and 24h co-culture with CD19 CAR T cells (red) compared to the viability of NALM-6 B-ALL cells co-cultured with uninfected T cells (blue). d, Representative histograms demonstrating CD19 expression on live NALM-6 cells (target cell population) with increasing E:T ratios after 6h of co-culture with CAR T cells (red histogram) or uninfected control T cells (blue histogram). Gray histograms show CD19negative T cells from the same experiment. e, Normalized mean fluorescence intensity (MFI) for CD19 expression on live NALM-6 B-ALL cells co-cultured at the indicated E:T ratios for 1, 2, 4, 12, and 24h (data normalized to triple-stained (CD19-PE, CFSE, 7-AAD) control cells that have not been co-cultured with effector cells). f-g, Reduced CD19 expression on B-ALL cells after exposure to CD4+ CAR T (f) or CAR Jurkat (g) cells is independent of CAR effector killing efficacy. CD4+ CAR T-cells or CAR Jurkat cells co-cultured with NALM-6 B-ALL target cells at an E:T ratio of 5:1 for 24 hours. Left, remaining fraction of live NALM-6 cells; middle, CD19 expression (MFI) on live NALM-6 cells; right, relative mean CD19 surface expression. Data representative of two independent experiments with n=3 each (red=NALM-6 cells from CAR T cell co-culture, blue=NALM-6 cells from uninfected T cell co-culture). P values determined by unpaired two-tailed Student’s t-test. ns=p>0.05 **=p<0.01, ****=p<0.0001. Data are mean ± s.d.