针对CD4膜蛋白的CAR-T细胞抗T细胞淋巴瘤的活性研究

OBJECTIVE: To study the specific killing effect of CD4 membrane protein targeted chimeric antigen receptor modified T (CAR-T) cell. METHODS: The second generation CD4 targeted chimeric antigen receptor containing 4-1BB costimulation domain was insert into lentiviral vector through recombinant DNA technology. Lentivirus was prepared and packaged by 293T cells with four plasmids. Beads activated T cells were transduced with lentivirus and the transduction efficiency was checked with Protein L and flow cytometry. T cell subsets and IFN-γ concentrations were detected with probe-tagged antibody and cytometric bead assay. RESULTS: ①The transduction efficiency of activated T cells with prepared lentivirus were 50.0%–70.0%. A subset of CD8(+) T cell acquired dim expression of CD4 membrane protein after activation. CD4(+)T cell and CD8(+)CD4(dim) T cell were gradually killed by CD4 targeted CAR-T post lentivirus transduction. ②The kill efficacy of CD4 targeted CAR-T cell and control T cell toward KARPAS 299 T cell at an E∶T ratio of 8∶1 for 24 h was (96.9±2.1)% and (11.2±3.1)%, CAR-T cell has a higher killing efficacy than control T cell (t=7.137, P=0.028). The IFN-γ concentrations in culture supernatant of CAR-T cell with K562-CD4 cell, CAR-T cell with K562 cell and CAR-T cell alone were (15 648±2 168), (1 978±354) and (1 785±268) pg/ml, CAR-T cell cocultured with K562-CD4 cell produced more IFN-γ than the other two controls (P<0.01). CONCLUSION: CD4 targeted CAR-T has an immunophenotype of CD8(+)CD4(−)T cell. CD4 targeted CAR-T cell has killing efficacy toward normal CD4(+)T cell and CD4(+)T lymphoma cell. CD4 targeted CAR-T cell also has a killing efficacy toward CD4(dim) target cell.