Coexpression of CD200R-CD28 enhances function in WT1-specific TCR-transduced human primary T cells. (A) Expression of CD200 on CD45^dimCD34⁺ cells (black lines) from a healthy donor leukapheresis sample (left panel) or leukemic blasts from 2 separate donors (center and right panels) in relation to matched FMO control (gray shaded). (B) Schematic representation of CD200R-CD28 constructs. CD200R_tm-CD28 contains CD200R ec and tm domains and a CD28 ic signaling domain. CD200R-CD28_tm contains the ec domain of CD200R and the tm and ic domains of CD28. The remaining 3 constructs also incorporate 12 aas of the ec domain of CD28 to the tm-proximal cysteine to promote multimerization and enhance CD28 signaling. To account for the extra CD28 residues, CD200R_-9aas-CD28_cys has a truncated portion of CD200R that removes the 9-aa membrane-proximal stem region, and CD200R_-12aas-CD28_cys has a truncated portion of CD200R that removes 12 membrane-proximal residues that include additional amino acids beyond the stem region. The first, second, and fifth constructs are predicted to approximate the spatial distance between the T cell and an APC, as indicated by the dashed line. (C-H) CD8⁺ T cells were enriched by magnetic beads from PBMCs harvested from healthy HLA-A2⁺ donors. CD8⁺ T cells were stimulated with anti-CD3/CD28 Dynabeads and transduced with lentiviral supernatant for 2 days. Transduced T cells were isolated by fluorescence-activated cell sorting and restimulated by rapid expansion protocol every 10 to 14 days in the presence of IL-2. (C) Diagram of construct combining IFP, TCRα, and TCRβ chains. The IFP constructs were inserted into single lentiviral vectors with the β and α chains of the HLA-A2–restricted WT1₁₂₆-specific TCR_C4. The first P2A sequence was codon optimized (CO P2A) to prevent genetic recombination with the second P2A sequence (P2A). Flow cytometry plots show expression of TCR_C4 only (left plot) or TCR_C4 + CD200R-CD28 (right plot) in primary human T cells as detected by anti-CD200R antibody and WT1₁₂₆ HLA-A2 tetramer binding. (D) Proliferation of T cells as detected by dilution of CTV. Primary human T cells transduced with TCR_C4 or TCR_C4 and CD200R IFP were stained with CTV and stimulated with WT1₁₂₆-pulsed T2 cells at an E:T ratio of 25:1 in the absence of IL-2 for 6 days. The percentage of cells that diluted CTV (proliferated) was determined by FlowJo proliferation analysis. Cumulative of 3 independent T-cell donors (**P < .01). (E) Representative histogram of CTV dilution in unstimulated (gray filled), TCR_C4-transduced (black line), or TCR_C4- and CD200R_-9aas-CD28_cys-transduced (orange line) T cells. (F) Intracellular cytokine production of CD8⁺ T cells. Primary human T cells transduced with TCR_C4 only (upper panels) or TCR_C4 and CD200R_-9aas-CD28_cys-transduced (lower panels) were unstimulated or stimulated with WT1₁₂₆-pulsed T2 cells for 6 hours in the presence of GolgiPlug (BD Biosciences). T cells were fixed, permeabilized, and stained for ic cytokines and assessed by flow cytometry. (G) Summary of cytokine production in panel F (E:T ratio, 1:1). Data are presented as no cytokine (white), 1 cytokine (light gray), or 2+ cytokine (dark gray) production; cumulative results of 3 independent T-cell donors. (H) Cytotoxicity assay. Primary AML blasts were cocultured with primary human T cells transduced with TCR_C4 alone (black symbols) or TCR_C4 and the CD200R_-9aas-CD28_cys IFP (orange symbols) for 24 hours. Remaining viable blasts were quantified by flow cytometry and the percentage of lysis was determined after normalization with the tumor-only control well (assayed in triplicate; *P < .05, **P < .01).