The mouse B2m gene was replaced by human B2M and HLA-G coding sequence in B-hB2M/hHLA-G MC38. Human HLA-G is highly expressed on the surface of B-hB2M/hHLA-G MC38 cells.

Gene targeting strategy for B-hB2M/HLA-G MC38 cells. The exogenous promoter and human B2M and HLA-G coding sequence were inserted to replace part of murine exon 1 and all of exon 2 and 3. The insertion disrupts the endogenous murine B2m gene, resulting in a non-functional transcript.

HLA-G expression analysis in B-hB2M/HLA-G MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hB2M/HLA-G MC38 cells cultures were stained with species-specific anti-HLA-G antibody. Human HLA-G was detected on the surface of B-hB2M/HLA-G MC38 cells, but not on the surface of wild-type MC38 cells. The 2-A08 clone of B-hB2M/HLA-G MC38 cells was used for in vivo experiments.

B-hB2M/HLA-G MC38 cells were subcutaneously transplanted into B-Tg(hLILRB2/hLILRB3) mice (n=3), and on 28 days post inoculation, tumor cells were harvested and assessed for human HLA-G expression by flow cytometry. As shown, human HLA-G was highly expressed on the surface of tumor cells. Therefore, B-hB2M/HLA-G MC38 cells can be used for in vivo efficacy studies of novel HLA-G therapeutics.