Figure 1.
CD7− T cells are predominantly CD4+ memory cells and are resistant to fratricide when expressing CD7-CAR. (A-B) Flow cytometric analysis of surface expression of CD3 and CD7 in (A) bulk peripheral blood mononuclear cells (PBMCs) from healthy donors: CD3+CD7+ (mean, 94.37 ± 4.55%) vs CD3+CD7− (mean, 5.18 ± 4.51%) (N = 50, P < .0001, paired t test) and (B) B-ALL patients: CD3+ CD7+ (mean, 93.4 ± 3.8%) and CD3+ CD7− (mean, 6.5 ± 3.8%) (N = 5, P < .0001, paired t test) and T-ALL patients: CD3+ CD7+ (mean, 95.6 ± 3.6) and CD3+CD7− (mean, 4.4 ± 3.6%) (N = 10, P < .0001 paired t test). (C) Representative dot plots of PBMCs before and after CD7 depletion and CD3 enrichment (left) and efficiency of CD7 selection on day 10 post–activation of nontransduced (NT) T cells (CD7+: 8.27 ± 4.61% and CD7−: 91.36 ± 4.56%, range 78.2-96.4, N = 26, P < .0001, paired t test). (D) Schematic of CD7-CAR construct. (E) Expression of CD7-CAR by flow cytometry using F(ab’)2 on day 7 after transduction (mean transduction efficiency 65.9 ± 16.7%, N = 22, P < .0001, paired t test). Representative dot plot showing all remaining CD7+ T cells were eliminated post–CD7-CAR transduction. (F) Expansion kinetics (absolute cell count) and viability (trypan blue exclusion). CD7-CARCD7− T cells had similar expansion to NT T cells (N = 11, P = ns, 2-way ANOVA). Bulk CD7-CAR T cells did not expand (N = 11, P < .01, 2-way ANOVA) and had decreased viability compared with CD7-CARCD7− and NT T cells (N = 11, P < .01, 2-way ANOVA). (G) Immunophenotype of CD7-CARCD7− T cells on day 7 posttransduction (CD4 vs CD8, effector memory [EM]: CCR7−, CD45RA−; central memory [CM]: CCR7+, CD45RA−; naïve-like: CCR7+CD45RA+; EM T cells reexpress CD45RA, EMRA: CCR7−, CD45RA+) (N = 11, 2-way ANOVA, bulk T cells vs CD7− T cells: CD4 vs 8 P < .0001, EM P < .0001, Naïve P < .0001, EMRA P < .01). (H) Luciferase-based cytotoxicity assay of CD7-CARCD7− T cells against CCRF (CD7+) and control BV173 (CD7−) target cells at 7 different effector:target (E:T) ratios (N = 8, P < .001, 2-way ANOVA at all ratios). (I) CD7-CARCD7− T cells were cocultured with media, BV173 (CD7lo target), CCRF or MOLT3 (CD7+ targets) at a 2:1 E:T ratio. Supernatants were harvested after 24 hours and analyzed for IFN-γ or IL-2 by ELISA (IFN-γ: N = 7-9, IL-2 N = 7-9, 1-way ANOVA, ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001). IgG1 Fc, immunoglobulin 1 fragment crystallizable; scFv, single-chain variable fragment; ELISA, enzyme-linked immunosorbent assay; ns, not significant.

CD7− T cells are predominantly CD4+ memory cells and are resistant to fratricide when expressing CD7-CAR. (A-B) Flow cytometric analysis of surface expression of CD3 and CD7 in (A) bulk peripheral blood mononuclear cells (PBMCs) from healthy donors: CD3+CD7+ (mean, 94.37 ± 4.55%) vs CD3+CD7− (mean, 5.18 ± 4.51%) (N = 50, P < .0001, paired t test) and (B) B-ALL patients: CD3+ CD7+ (mean, 93.4 ± 3.8%) and CD3+ CD7− (mean, 6.5 ± 3.8%) (N = 5, P < .0001, paired t test) and T-ALL patients: CD3+ CD7+ (mean, 95.6 ± 3.6) and CD3+CD7− (mean, 4.4 ± 3.6%) (N = 10, P < .0001 paired t test). (C) Representative dot plots of PBMCs before and after CD7 depletion and CD3 enrichment (left) and efficiency of CD7 selection on day 10 post–activation of nontransduced (NT) T cells (CD7+: 8.27 ± 4.61% and CD7−: 91.36 ± 4.56%, range 78.2-96.4, N = 26, P < .0001, paired t test). (D) Schematic of CD7-CAR construct. (E) Expression of CD7-CAR by flow cytometry using F(ab’)2 on day 7 after transduction (mean transduction efficiency 65.9 ± 16.7%, N = 22, P < .0001, paired t test). Representative dot plot showing all remaining CD7+ T cells were eliminated post–CD7-CAR transduction. (F) Expansion kinetics (absolute cell count) and viability (trypan blue exclusion). CD7-CARCD7− T cells had similar expansion to NT T cells (N = 11, P = ns, 2-way ANOVA). Bulk CD7-CAR T cells did not expand (N = 11, P < .01, 2-way ANOVA) and had decreased viability compared with CD7-CARCD7− and NT T cells (N = 11, P < .01, 2-way ANOVA). (G) Immunophenotype of CD7-CARCD7− T cells on day 7 posttransduction (CD4 vs CD8, effector memory [EM]: CCR7−, CD45RA−; central memory [CM]: CCR7+, CD45RA−; naïve-like: CCR7+CD45RA+; EM T cells reexpress CD45RA, EMRA: CCR7−, CD45RA+) (N = 11, 2-way ANOVA, bulk T cells vs CD7− T cells: CD4 vs 8 P < .0001, EM P < .0001, Naïve P < .0001, EMRA P < .01). (H) Luciferase-based cytotoxicity assay of CD7-CARCD7− T cells against CCRF (CD7+) and control BV173 (CD7−) target cells at 7 different effector:target (E:T) ratios (N = 8, P < .001, 2-way ANOVA at all ratios). (I) CD7-CARCD7− T cells were cocultured with media, BV173 (CD7lo target), CCRF or MOLT3 (CD7+ targets) at a 2:1 E:T ratio. Supernatants were harvested after 24 hours and analyzed for IFN-γ or IL-2 by ELISA (IFN-γ: N = 7-9, IL-2 N = 7-9, 1-way ANOVA, ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001). IgG1 Fc, immunoglobulin 1 fragment crystallizable; scFv, single-chain variable fragment; ELISA, enzyme-linked immunosorbent assay; ns, not significant.

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