PD-1: CD28 chimeric receptors enhance phenotypic and functional features of T cell subsets

The PD-1/PD-L1 immune checkpoint pathway inhibits T cell function, allowing tumors to escape immune responses. This study aimed to reprogram this inhibitory signaling using a PD- 1:CD28 switch receptor (SR) to convert inhibitory signaling into a co-stimulatory signal and to assess its effects on the function of CD4⁺ and CD8⁺ T cells.

PBMCs isolated from both sources were retrovirally transduced with PD-1:CD28 SR. Intracellular expression of cytokines, including IFN-γ, IL-4, IL-10, as well as the surface CD25 in CD4⁺ and CD8⁺ T cells, and the rate of cell apoptosis during engineering and exposure to target cells were assessed.

Both buffy coat (BC) and whole blood showed comparable transduction efficiency (21.4% versus 18.5%, respectively), but BC yielded nearly double the number of viable PBMCs per 50 mL. Upon stimulation and co-culture with target cells, CD4⁺ SR T cells produced significantly higher levels of IFN-γ, IL-4, and IL-10 compared to CD8⁺ SR T cells. These cytokines also significantly increased in co-culture supernatants exposed to PD-L1⁺ cells. No significant differences were found in CD4/CD8 ratios, CD25 expression, or cytokine profiles between PBMC sources. However, BC-derived T cells showed higher apoptosis rates during co-culture.

BC can be a practical and effective source of PBMCs for T cell engineering. These findings underscore the SR signaling in the activation of CD4⁺ T cells that may impact the activation of CD8⁺ T cells, which is essential for effective tumor eradication.