B-hHLA-E plus/hPD-L1 MC38

Basic Information

Description

The mouse H2-t23 and Pdl1 gene were replaced by the human HLA-E and PD-L1 coding sequence in B-hHLA-E plus/hPD-L1 MC38 cells. Human HLA-E and PD-L1 are highly expressed by B-hHLA-E plus/hPD-L1 MC38.

Gene targeting strategy

Gene targeting strategy for B-hHLA-E plus/hPD-L1 MC38 cells. The exogenous promoter and human HLA-E coding sequence was inserted to replace part of murine exon 3 and all of exons 4-7. The exogenous promoter and human PD-L1 coding sequence was inserted to replace part of murine exon 3. The insertion disrupts the endogenous murine H2-t23 and Pdl1 gene, resulting in non-functional transcripts.

Protein expression analysis

PD-L1 and HLA-E protein expression analysis in B-hHLA-E plus/hPD-L1 MC38 cells. Supernatant from wild-type MC38 and B-hHLA-E plus/hPD-L1 MC38 cell cultures were stained with species-specific anti-PD-L1 and anti-HLA-E antibodies and analyzed by flow cytometry. Human PD-L1 and HLA-E were detected in B-hHLA-E plus/hPD-L1 MC38 cells but not in that of wild-type MC38 cells. The B-hHLA-E plus/hPD-L1 MC38 1-E09 clone was used for in vivo experiments.

Tumor growth curve in wild-type mice

Subcutaneous homograft tumor growth of B-hHLA-E plus/hPD-L1 MC38 cells. B-hHLA-E plus/hPD-L1 MC38 cells (5×10⁵) and wild-type MC38 cells (5×10⁵) were subcutaneously implanted into C57BL/6N mice (female, 8-week-old, n=6). 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-hHLA-E plus/hPD-L1 MC38 cells were able to establish tumors in vivo, which can be used for efficacy studies.

Individual tumor growth curve

Protein expression analysis post-inoculation

B-hHLA-E plus/hPD-L1 MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=6), 28 days post inoculation, tumor cells were harvested and assessed for human PD-L1 and HLA-E protein expression by flow cytometry. As shown, human PD-L1 and HLA-E  were highly expressed by tumor cells. Therefore, B-hHLA-E plus/hPD-L1 MC38 cells can be used for in vivo efficacy studies.

Tumor growth curve in humanized mice

Subcutaneous homograft tumor growth of B-hHLA-E plus/hPD-L1 MC38 cells. B-hHLA-E plus/hPD-L1 MC38 cells (5×10⁵) were subcutaneously implanted into B-hPD-1/hPD-L1/hCD94/hNKG2A mice (female, 8-week-old, n=6). 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-hHLA-E plus/hPD-L1 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.