CD200R-CD28 constructs are expressed at high levels on primary murine CD8⁺T cells. (A) 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 a portion of the ec domain of CD28 to the tm-proximal cysteine to promote multimerization and enhance CD28 signaling. To account for the extra 9 ec aas, CD200R_-3aas-CD28_cys has a truncated portion of CD200R that removes 3 membrane-proximal aas, but preserves an N-linked glycosylation site. CD200R_-9aas-CD28_cys has an extracellular portion of CD200R that is truncated 9 membrane-proximal aas. The first, second, and last constructs are predicted to approximate the spatial distance between the T cell and an APC, as indicated by the dashed line. (B) Transgenic expression of CD200R-CD28 constructs on TCR_gag T cells as detected by anti-CD200R antibody. (C) Expression of CD200R in transduced TCR_gag T cells as in panel B, expressed as mean fluorescence intensity (MFI). Cumulative results of 6 independent experiments (P = not significant).