The CD200R-CD28 switch receptor shows improved benefit compared with CD200RDN or CD200R knockout approaches. (A) CD200R knockout was measured by flow cytometry on day 3. The results shown are the averages of 3 independent experiments on samples from different healthy donors. (B) BCMA CAR T cell groups as indicated were tested for their cytotoxicity at an E:T ratio of 0.3 for 3 days. The results shown are from a representative experiment (values represent the average ± SD of triplicates). (C) CAE for CAR T-cell groups as indicated were cocultured with RPMI-CD200 cells for 10 days at an E:T ratio of 0.3. Flow cytometry cell counting for CD45⁺ T cells was performed at indicated time points. The results show a representative experiment of repeats with CAR T cells made using samples from 2 different healthy donors. (D) Supernatants collected from the CAE shown in panel C at the indicated time points were assayed in the IsoPlexis CodePlex 32-plex chip and all cytokines and chemokines elevated above background in at least 1 group were plotted. (E) CAR T cells from the CAE shown in panel C were assayed using the SeaHorse platform at baseline and after the first target cell incubation on day 4, to measure the oxygen consumption rate and extracellular acidification rate (ECAR) in order to assess facets of T-cell metabolism. Data are shown as mean ± SD, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001, by either Student t test with Welch correction (A), unpaired t test with Welch correction and multicomparison correction by false discovery rate (FDR) (B), and 1-way ANOVA with Šidák multiple comparison test (E).