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Fig. 1 | Journal of Hematology & Oncology

Fig. 1

From: Targeting glycosylation of PD-1 to enhance CAR-T cell cytotoxicity

Fig. 1

Mutations of N74 decreased PD-1. a Surface expressions of wild-type PD-1 and its derivate N74A (A74) mutation in 293 T cells. b Potential mutations resulted from single-nucleotide conversions at N74. c Mutations at N74 decreased surface expression of PD-1. PD-1 harboring wild-type or mutated N74 were tandemly linked with self-cleaving P2A and GFP, then transiently expressed in 293 T cells. Surface PD-1 expression was determined in GFP+ cells by FACS assay. d Sanger sequencing of PDCD1 of CAR-T cells expressing scramble or N74-targeted gRNA after base editing. e–j CAR-T cells having comparable rates of GFP+ cells were activated with equal amounts of anti-CD3/CD28 beads without exogenous cytokines. e Western blots of PD-1 in CAR-T cells activated or not. f qRT-PCR detecting PD-1 expressions in resting and activated CAR-T cells. g Surface expressions of PD-1 in CAR-T cells before and after activation. And mean fluorescence intensity (MFI) values were compared. h CAR-T cells were stained with eFluor 670 dyes and then continued to culture with or without beads. Forty-eight hours later, proliferations of CAR-T cells were determined according to eFlour 670 dilution. Activation markers, CD69 (i) and CD27 (j) were detected and compared in different CAR-T cells before and after activation. **P < 0.01 and ****P < 0.001

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