Cloning and characterization ofa recombinant gene for generation of CAR-iTreg harnessing CD40-CD154 interaction

Treg;CAR;610.72

의과대학 의과학과 ;

University:서울대학교 대학원

关键词: Treg;    CAR;    610.72;   

Others  :  http://s-space.snu.ac.kr/bitstream/10371/161540/1/000000158015.pdf

美国|英语

来源: Seoul National University Open Repository

【 摘 要 】

Introduction: The regulatory T cells (Treg) are thymus-derived suppressor cells which play a critical role in maintaining immunological tolerance to selfantigens. Here, I describe a new approach to generating potent specific Tregs, using a chimeric antigen receptor (CAR) targeting CD40L. CARs are fusion proteins composed of an extracellular portion that is usually derived from antibodies and intracellular signaling portion derived from T cell signaling proteins. I generated a recombinant gene encoding a full length of the cytoplasmic domain of CD28, IL-2Rβ, TGF-βR1, and CD3ζ fused with a scFv of the MR1 mAb specific to CD40L as extracellular domain. Functionality of the cloned CAR was confirmed in EL4 cells by stimulation with HEK293 cells expressing CD40L.Methods: Mouse CD4+ T cell was isolated by magnetic-activated cell sorting (MACS) separation system according to the manufacturer’s instructions. And this gene was used to compose the CAR construct. Each gene was obtained and extended using specific oligonucleotides by PCR. The synthesized CAR construct was inserted into a lentiviral expression vector. Using this vector and packaging vectors, the CAR was transfected and resulted in the production of lentivirus. The harvested virus was transduced in EL4 cell.Results: The CAR construct was obtained and elongated from mouse cDNA. And, synthesized CAR constructs cloned into a lentiviral expression vector. The expression of CAR was confirmed by ZsGreen1 fluorescence in pLVX-IRESZsGreen1 vector. Also, the cell line expressing CD40L of antigen to CAR was generated and confirmed by fluorescence of mCherry. The co-culture of EL4 cell expressing CAR on the response of HEK293FT cell expressing CD40L was conducted. The activation of EL4 cells was examined by CD69 expression level.Conclusions: I describe a unique strategy to generate CAR. This novel CAR retains an identical costimulatory and CD3ζ signaling regions but also include an intracellular domain of IL-2Rβand TGF-β R1. The inclusion of the intracellular domain of IL2-Rβ and TGF-βR1 enabled conversion of naïve T cells to iTreg in an antigen-dependent manner. Therefore, I expect this CAR construct would generate significant suppression effect in transplantation or autoimmune disease model. This CAR with CD40L specificity might provide a novel strategy to achieve tolerance in various immunopathologic conditions.