Fig. 1: CAR design influences proximal signaling molecule phosphorylation.
a, CD19-CD8H/TM-4-1BBζ and CD19-CD28H/TM-4-1BBζ CAR T cells from two unique donors were stimulated with 5 μg ml−1 anti-CD19 CAR idiotype and goat anti-mouse cross-linking antibodies and incubated at 37 °C for 5, 15 or 90 min. Samples were then processed for proteomic analysis with liquid chromatography–mass spectrometry (LC–MS). PC analysis of quantified phosphopeptides for CD19-CD8H/TM-4-1BBζ and CD19-CD28H/TM-4-1BBζ CAR T cells for the two donors is shown. b, Volcano plots depicting FC (log2 (FC)) and P value (–log10(adjusted P value)) for differentially phosphorylated peptides identified at 5 and 15 min after stimulation. Differential peptide analyses were conducted using an empirical Bayes moderated t-test, and selected proteins with a log2 (FC) of ≥0.5 and Benjamini–Hochberg-adjusted P value of ≤0.05 are labeled. Green indicates TCR-proximal signaling proteins, blue indicates proteins interacting with the LAT signalosome and/or cytoskeleton, and magenta indicates distal signaling proteins and transcription factors. c, Top, schematic of proximal signaling molecules tested for CAR T cell enhancement. Bottom, IL-2 produced by CD19-4-1BBζ CAR T cells with or without overexpression of the indicated proximal signaling molecules after coculture with Nalm6 tumor cells expressing the indicated densities of CD19. Data are shown as mean ± s.d. of three technical replicates and are representative of two independent experiments with different T cell donors.
