The mouse Pdl1 gene was replaced by human PD-L1 coding sequence in B-hPD-L1 EL4 cells. Human PD-L1 is highly expressed on the surface of B-hPD-L1 EL4 cells.

Gene targeting strategy for B-hPD-L1 EL4 cells. The exogenous promoter and human PD-L1 coding sequence was inserted to replace part of murine exon 3. The insertion disrupts the endogenous murine Pdl1 gene, resulting in a non-functional transcript.

PD-L1 expression analysis in B-hPD-L1 EL4 cells by flow cytometry. Single cell suspensions from wild-type EL4 and B-hPD-L1 EL4 cultures were stained with species-specific anti-PD-L1 antibody. Human PD-L1 was detected on the surface of B-hPD-L1 EL4 cells but not wild-type EL4 cells. The 6-G08 clone of B-hPD-L1 EL4 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hPD-L1 EL4 cells. B-hPD-L1 EL4 cells (2x10⁵) and wild-type EL4 cells (2x10⁵) were subcutaneously implanted into C57BL/6 mice (female, 7-week-old, n=5). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B) Body weight (Mean± SEM). Volume was expressed in mm³ using the formula: V=0.5 X long diameter X short diameter². As shown in panel A, B-hPD-L1 EL4 cells were able to establish tumors in vivo and can be used for efficacy studies. Animals with tumor volume over 3000mm³ in the control group were euthanized.