Construction of CAR-Treg cells targeting HLA-A2 and expressing PD-L1 and validation of inhibitory effect on CD4+ T cells

doi:10.3877/cma.j.issn.2095-3232.2024.05.022

Abstract

Abstract:

Objective

To construct regulatory T cells (CAR-Treg) targeting HLA-A2 and expressing PDL1 chimeric antigen receptor, and to validate the inhibitory effect of CAR-Treg on CD4⁺T cells.

Methods

CAR gene was designed and synthesized. CAR-Treg core plasmids targeting HLA-A2 and expressing PDL1 were constructed by lentivirus vector plasmid, which were identified by BamHI/EcoRI dual enzyme digestion. The expression of CAR in T cells transfected with CAR plasmids was detected by flow cytometry. The proliferation of HLA-A2^+/- breast cancer cells was inhibited to confirm that CAR could successfully target HLA-A2. Subsequently, Foxp3, PDL1, CD25, ICOS, CTLA4 and other biomarkers related to Treg cell function were detected by flow cytometry. The inhibitory effect of CAR-Treg upon CD4⁺ T cell proliferation was validated by cell metabolism analysis and in vitro CD4⁺ T cell proliferation inhibition experiment.

Results

Dual enzyme digestion showed that CAR plasmids targeting HLA-A2 were successfully constructed. Flow cytometry of CAR-T/CAR-Treg cells confirmed the stable expression of HLA-A2 CAR on the cell surface. Co-culture of HLA-A2 CAR-T cells and MCF-7(HLA-A2⁺) breast cancer cells confirmed that CAR-T cells expressing HLA-A2 could successfully recognize HLA-A2 targets. Flow cytometry demonstrated that CAR-Treg highly expressed Foxp3 and PDL1, and the number of cell proliferation and passage was significantly increased. Cell metabolism analysis showed that CAR-Treg exhibited a higher basal cell oxygen consumption rate than ordinary Treg cells, and CAR-Treg was more dependent on fatty acid oxidative metabolism. Cell proliferation inhibition experiment revealed that CAR-Treg could effectively inhibit the proliferation of CD4⁺ T cells in vitro.

Conclusions

Successful construction of CAR-Treg targeting HLA-A2 and expressing PDL1 can effectively inhibit the proliferation of CD4⁺ T cells in vitro, which provides experimental evidence for subsequent animal experiments.

Key words: Chimeric antigen receptor (CAR), Regulatory T cell (Treg), HLA-A2, Programmed death ligand-1 (PD-L1), Immunosuppression

Yingxin Zhang, Ting Lin, Jianwen Zhang. Construction of CAR-Treg cells targeting HLA-A2 and expressing PD-L1 and validation of inhibitory effect on CD4⁺ T cells[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2024, 13(05): 719-728.

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References 29

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