The mouse Pdl1 gene was replaced by human PD-L1 coding sequence in B-hPD-L1 MC38 cells. Human PD-L1 is highly expressed on the surface of B-hPD-L1 MC38 plus cells.

Gene targeting strategy for B-hPD-L1 MC38 plus 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 MC38 plus cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hPD-L1 MC38 plus cultures were stained with species-specific anti-PD-L1 antibody. Human PD-L1 was detected on the surface of B-hPD-L1 MC38 plus cells but not wild-type MC38 cells. The 42 clone of B-hPD-L1 MC38 plus cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hPD-L1 MC38 plus cells. B-hPD-L1 MC38 plus cells (2x10⁶ and 5x10⁵) were subcutaneously implanted into C57BL/6 mice (female, n=4). 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 MC38 plus cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hPD-L1 MC38 plus cells. B-hPD-L1 MC38 plus cells (5x10⁵,1x10⁶ and 5x10⁶) were subcutaneously implanted into C57BL/6J mice and homozygous B-hTIM3 mice. 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 MC38 plus were able to establish tumors in vivo and can be used for efficacy studies.