CD200R-CD28 constructs promote proliferation, accumulation, and effector function of transduced T cells stimulated by CD200⁺tumor target cells in vitro. Splenocytes from naive TCR_gag mice were stimulated in vitro with anti-CD3 (1 μg/mL), anti-CD28 (1 μg/mL), and recombinant human IL-2 (rhIL-2, 100 U/mL) and transduced with retroviral supernatant for 2 days. Cells were restimulated every 7 days with irradiated FBL and splenocytes and cultured with rhIL-2 (50 U/mL) for ≤3 stimulations. T cells were used for assays 5 to 7 days after the last stimulation. (A) Proliferation of CD200R-CD28 (red lines) and GFP empty vector control (blue lines) TCR_gag T cells as measured by CTV dilution after stimulation with CD200^– FBL (upper panels) or CD200⁺ FBL (lower panels) at a low E:T ratio of 25:1 for 3 days. (B) Cumulative proliferation of cells depicted in panel A. Proliferation was normalized across experiments by assessing the proportion of divided CD200R-CD28⁺ T cells relative to empty vector–transduced T cells (% divided_CD200R⁺/% divided_CD200R^– [n = 3]). (C) Enrichment of transduced TCR_gag T cells in a mixed population including nontransduced TCR_gag T cells after 3 weekly cycles of stimulation with irradiated CD200⁺ FBL and splenocytes (n = 4-5/group). **P < .01 (Student t test). (D) Carboxyfluorescein diacetate succinimidyl ester cytotoxicity assay. TCR_gag T cells were transduced with CD200R_-9aas-CD28_cys (red bars) or mock-transduced cells (black bars). Effector TCR_gag T cells were incubated at the indicated effector to target ratio with a 1:1 mix of CD200⁺ FBL and nonspecific EL4 control targets for 4 hours. The relative frequency of FBL vs control tumor cells was determined by flow cytometry, and the percentage of specific lysis was determined by the frequency of FBL cells after T-cell culture relative to FBL incubated without T cells; cumulative of 3 independent experiments. *P < .05 (Student t test). (E) Cytokine production of TCR_gag T cells transduced with GFP control (black lines) or CD200R IFP (red lines) relative to unstimulated T cells (gray filled) after coculture at a 1:1 ratio with CD200^– (upper histograms) or CD200⁺ (lower histograms) FBL for 4 hours in the presence of GolgiPlug (BD Biosciences). Cells were fixed, permeabilized, stained for ic cytokines, and assessed by flow cytometry. (F) Summary of panel E. Stacked bar charts of cytokine production in TCR_gag T cells in response to CD200^– (left) or CD200⁺ (right) FBL stimulation at a 1:1 ratio. Data are presented as no cytokine (white), 1 cytokine (light gray), or 2+ cytokine (dark gray) production; cumulative results of 3 independent experiments. (G) Visualization of CD200R localization within T cell-FBL conjugates. TCR_gag in vitro expanded effector T cells transduced with CD200R_-9aas-CD28_cys (upper panels) or CD200R-CD28_cys (lower panels) were cocultured with CD200⁺ FBL at an E:T ratio of 10:1 at 37°C for 20 minutes to allow conjugate formation. Conjugates were loaded on a μ-Slide VI 0.4 chamber (Ibidi) for an additional 15 minutes. Cells were fixed and stained for CD200R (first panels), CD200 (second panels), and lipid rafts by CTxB (third panels; overlay in fourth panels). Conjugates were imaged by microscopy on the DeltaVision Elite (60×) and analyzed using ImageJ software. (H) Quantification of conjugates that exhibit CD200R staining within the synapse, as in panel G, expressed as the percentage of total T cell-FBL conjugates. Data are represented as mean ± standard deviation from 2 separate experiments for a total of 50 conjugates assessed. (I) pLCK Y394 expression of TCRgag T cells. T cells were transduced with CD200R_-9aas-CD28_cys (red line), CD200R-CD28_cys (blue line), or GFP control (black line) and were left unstimulated (upper left histogram and gray filled in other histograms) or stimulated for 10 minutes with PMA/ionomycin, CD200^– FBL, or CD200⁺ FBL, as indicated. FBL stimulation was performed at an E:T ratio of 10:1; representative of 2 independent experiments.