Fig 1: In vitro comparison of M-CSFR-targeted pCARs and linear CARs(A) Explanation of nomenclature. M, macrophage colony stimulating factor; 34, interleukin (IL)-34. Parallel CAR (pCAR) names are italicized throughout the text.(B) Cartoon structures illustrating M-CSFR-specific CARs, pCARs, and controls as follows: (1) an alternative binary configuration in which a 1G CAR (CD3? endodomain) was co-expressed with a CCR that contains fused CD28+41BB-signaling domains (1G CAR/dual CCR) and (2) an endodomain truncated pCAR that lacks 28? signaling sequences (trunc. CAR in pCAR).(C) The indicated engineered T cells (1 × 106) were co-cultivated with T47D FMS cells without cytokine support. Each week, 1 × 106 T cells were transferred to a new confluent monolayer. Spider plots enumerate T cell number over time (n = 5–20). Since data were not normally distributed, significance was determined using the Kruskal-Wallis test. Comparison with pCAR-M/34 is shown.(D) Number of re-stimulation cycles in (C) from which T cells were retrieved. Statistical analysis was performed using one-way ANOVA, making comparison with pCAR-M/34.(E) “Mid-range” representative single-donor example in which 1 × 106 M-CSFR re-targeted CAR or pCAR T cells were re-stimulated on T47D FMS tumor monolayers as described in (C) (mean ± SEM, n = 3 technical replicates).(F) Re-stimulation cultures in (C) were evaluated for tumor cell viability at 24 h (mean ± SEM), comparing pCAR-M/34 (n = 10) with 2G-M (CD28) (n = 8), 2G-M (41BB) (n = 7), 3G-M (n = 8), 1G CAR/dual CCR (n = 5), 2G-34 (CD28) (n = 3), 3G-34 (n = 3), or Trunc. CAR in pCAR (n = 9).(G) IL-2 concentration in supernatants of re-stimulation cultures in (C) harvested 24 h after each stimulation (mean ± SEM, n = 5 [1G CAR/dual CCR], 6 [trunc. CAR in pCAR], 2 [2G-M (41BB)], 8 [2G-M (CD28), 3G-M], 10 [pCAR-M/34], and 3 [2G-34 (CD28), 3G-34]). Statistical analysis was performed using two-way ANOVA.(H) IFN? concentration in supernatants of re-stimulation cultures in (C) harvested 24 h after each stimulation (mean ± SEM, n = 3 [1G CAR/dual CCR], 3 [2G-M (41BB)], 4 [2G-M (CD28), 3G-M], 4 [pCAR-M/34], and 3 [2G-34 (CD28), 3G-34]). Statistical analysis was performed using two-way ANOVA. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.See Figures S1 and S2 and Table S1 for additional data.