Universal Transplant-Specific Chimeric Antigen Receptor Regulatory T cells Save Transplantation from Immune Rejection

Introduction: Transplantation serves as a promising way to restore vital functions in patients with cell, tissue, or organ failure, such as  cells in Type I diabetes. The malfunction of the transplantation is largely due to the acute and chronic immune rejection upon the surgery. Regulatory T cells (Tregs) function as an essential immune suppressor. However, the specificity of Tregs could only be improved by endogenous transplant-specific antigens identified individually. To boost Tregs specificity in the transplant while minimizing the waiting period for identifying the endogenous antigens, here, we introduce a strategy to develop universal transplant-specific chimeric antigen receptor (CAR) Tregs. Dinitrophenyl (DNP)-sugars can be metabolically labeled in  cells and recognized by anti-DNP CAR Tregs in vitro and in vivo. The anti-CAR Tregs is expected to suppress CD8+ T cells and dendritic cells (DCs) upon stimulated by the DNP labeled  cells and prolong the  cells transplantation and pancreas transplantation survival.

Materials and

Methods:  cells were incubated with DNP-sugars for 72h in culture. Flow cytometry was then used to test DNP presence on the cell surface. Primary Tregs was isolated from female C57BL/6 mice using CD4+CD25+ Regulatory T cells isolation kit (Miltenyi Biotec). Lentivirus containing DNP component was packaged in HEK293T. Primary Tregs was transduced by the above lentivirus and positively selected by antibiotic and cell sorting. Labeled  cells will be coculture with primary CD8+ T cells or primary DCs isolated from C57BL/6 with the presence of anti-DNP CAR Tregs or a control CAR Tregs. Finally, the  cells or the whole DNP-sugars perfused pancreas will be transplanted in the mouse recipient along with the injection of CAR Tregs and the graft survival will be monitored from then on.

Results, Conclusions, and Discussions: Multiple types of DNP-sugars were synthesized, and different concentrations of DNP-sugars were selected to label the  cells. Currently, the  cells successfully labeling with DNP-sugars without affecting the viability was achieved at different sugar concentrations. Anti-DNP and control CAR Tregs were successfully constructed. The coculture studies are currently ongoing.

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