Extended Data Fig. 5: Effect of CAR on cell viability and polarization of CARMA and T cell trafficking.
a, b, Proliferation rate of Ki67+/cFMS+ cells (a) and cell viability (b) of the primary macrophage and different CARMA were examined using FACS and MTS. (n = 4 / group). c, Infiltration index of primary macrophage or different CARMA was measured using the boyden chamber. The CARMA were seeded on the upper chamber and H2030BrM cells were seeded on the lower chamber (E/T ratio: 30:1). After 72 h of incubation, the macrophages in the lower chamber were examined by FACS. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 5 / group). d, Expression of lysosome capacity in primary macrophage and CARMA. e, Polarization phenotype in N- and MyD-CARMA before or after CAR insertion. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 4 / group). f, Expressions of TNF-α, IL-1β, and INF-γ in primary macrophage, N-CARMA and MyD-CARMA cells that were co-cultured with H2030BrM were quantified by ELISA. One-way ANOVA with Tukey’s multiple comparisons test: **, p = 0.002; *, p = 0.018; *, p = 0.013. (n = 6 / group). g-i, Expressions of TNF-α, IL-1β, and INF-γ in MyD-CARMA cells before or after CAR insertion and CARMA-H2030BrM co-culture were quantified by ELISA. One-way ANOVA with Tukey’s multiple comparisons test: *, p = 0.03; ***, p = 0.001 (n = 5 / group). j, k, H2030BrM cells were co-cultured with PBMC-derived CARMA (E/T ratio: 30:1) for 72 h. The primary CARMA cells were then isolated from the co-culture and the expressions of CXCL9 (j) and CXCL10 (k) of the isolated CARMA cells were examined by qRT-PCR. One-way ANOVA with Tukey’s multiple comparisons test: (j) *, p = 0.03; **, p = 0.0012; ***, p = 0.0001; (k) ***, p = 0.0001 (n = 5 / group). l, Primary T cells were co-cultured with H2030BrM-pretreated primary CARMA (E/T ratio: 30:1) for 24 h. The T cells were then isolated from the co-culture, and the expression of CXCR3 of the T cells was examined by qRT-PCR. One-way ANOVA with Tukey’s multiple comparisons test: *, p = 0.03; ***, p = 0.0001. (n = 5 / group). m, H2030BrM cells were co-cultured with primary CARMA (E/T ratio: 30:1) for 72 h. CARMA cells were then separated from the co-culture and incubated with primary T cells that were labeled with cell proliferation dye. The T cell proliferation rate was then examined by FACS. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 5 / group). n, H2030BrM cells were co-cultured with primary CARMA (E/T ratio: 30:1) for 24 h. CARMA cells were then separated from the co-culture and seeded in the lower chamber, and the primary T cells were seeded in the upper chamber. After 3 days of co-culture, the primary T cells were isolated from the lower chamber and examined for the chemotactic index. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 5 / group). o, The luciferins-labeled H2030BrM cells were co-cultured with ‘IL-4-activated primary T cell only (Tumor + pri T)’ or ‘CARMA plus IL-4-activated primary T cells (Tumor + CARMA + pri. T)’ for 0, 2, 5 days, and the tumor cell growth was examined by IVIS. One-way ANOVA with Tukey’s multiple comparisons test: Day 2: **, p = 0.015; **, p = 0.014; Day 5: *, p = 0.02; **, p = 0.009; ***, p = 0.0001. (n = 5 / group). The data are presented as the mean ± SD.
