CD200R-CD28 switch receptor enhances the in vitro functions of CAR T cells targeting CD200⁺ MM cell lines in short- and long-term assays. (A) BCMA CAR T cells and BCMA-CD200R-CD28 CAR T cells were tested for their cytotoxicity at indicated E:T ratio of 0.3 for 3 days. The results shown are the averages of 3 independent experiments. (B) BCMA CAR T cells, CD200R-CD28 BCMA CAR T cells, and CD200R BCMA CAR T cells were stained with cell trace violet (CTV) before stimulation with RPMI-8226 and RPMI-8226-CD200 cells, and flow cytometry proliferation assays were performed at day 8. (C) BCMA CAR T cells and CD200R-CD28 BCMA CAR T cells were cocultured with RPMI-8226-CD200 cell at E:T ratio of 1:1, and supernatants were collected at indicated time point while fresh media were added to the cell culture. ELISA cytokine secretion measurements were performed with the culture supernatants. Bar graphs show results from a representative experiment (values represent the average ± standard error [SE] of triplicates) for IL-2 (left), IFN-γ (middle), and tumor necrosis factor α (TNF-α; right). (D) IsoPlexis single-cell 32-plex human adaptive immune secretome analysis of BCMA CAR T cells and CD200R-CD28 BCMA CAR T cells cocultured with CD200⁺ or CD200⁻ tumor cells. Left panel: PSI, defined as the percentage of polyfunctional cells (secreting ≥2 cytokines or chemokines) in each sample multiplied by the average signal intensity for each secreted factor. Stacked bars represent contributions to the total PSI by subpopulations, with secretomes characterized by distinct dominant factors (refer to key inset). Right panel: polyfunctional heat map visualizes the subsets of polyfunctional cells that are secreting cytokines in various combinations, with abundance as indicated (see key inset). (E) CAE assay for BCMA CAR T cells and CD200R-CD28 BCMA CAR T cells. CAR T cells were cocultured with RPMI-CD200 cells for 2 weeks at an E:T ratio of 1:3, replacing the target cells every 3 to 4 days with fresh targets. Flow cytometry cell counting for CD45⁺ T cells were performed at indicated time point (left); Ki67 (middle) and granzyme B (right) levels were also detected by flow cytometry at indicated time point. The results shown are representative of three independent experiments with CAR-T cells made using different healthy donors. (F) TnMUC1- and CD200R-CD28 TnMUC1 CAR-T cells were tested for their cytotoxicity at E:T ratio of 10 for 24 hours. The results shown are representative of three independent experiments. (G) TnMUC1- and CD200R-CD28 TnMUC1 CAR-T cells were cocultured with RPMI-CD200 cells for 24 hours for ELISA cytokine secretion measurement in culture supernatants. Bar graphs show results from a representative experiment for IL-2 (left), IFN-γ (right). (H) CAE assay for TnMUC1- and CD200R-CD28 TnMUC1 CAR-T cells. CAR-T cells were cocultured with RPMI-CD200 cells for 2 weeks at an E:T ratio of 3, replacing the target cells every 3 to 4 days with fresh targets. Flow cytometry-based cell counting were performed at indicated time points (left), Ki67 (middle) and granzyme B (right) levels were also detected by flow cytometry at indicated time points. The results shown are representative of 3 independent experiments with samples from different healthy donors. Data are shown as mean ± standard deviation, ∗P < .05, ∗P < .01, ∗P < .001, and ∗∗∗∗P < .0001, by 1-way analysis of variance (ANOVA) and Tukey multiple comparison test.