HLA-A11.1/B2M(2)

Fig.1 Expression analysis of hB2M and HLA-ABC on CD45+ cells in blood of HLA-A11.1/B2M humanized mice. 

Leukocytes were collected from wild-type C57BL/6 mice(11 weeks old)  and homozygous HLA-A11.1/B2M humanized mice(15.7 weeks old) , and analyzed by flow cytometry with anti-mB2M, anti-hB2M, anti-mH2D and anti-HLA-ABC. hB2M and HLA-ABC were exclusively detectable in HLA-A11.1/B2M humanized mice, whereas mB2M and mH2D were exclusively detectable in wild-type C57BL/6 mice.

Fig.2 Analysis of immune cell subpopulations in the blood (n=3 per group). 

Leukocytes were collected from the blood of wild-type C57BL/6 mice (female, 8 weeks old; male, 9 weeks) and homozygous HLA-A11.1/B2M humanized mice (9 weeks old), and Flow cytometry analysis was performed to assess leukocyte subpopulations.

Abbr.  WT, wild type; HO, homozygous.

Fig.3 Analysis of immune cell subpopulations in the spleen (n=3 per group). 

Leukocytes were collected from the spleens of wild-type C57BL/6 mice (female, 8 weeks old; male, 9 weeks) and homozygous HLA-A11.1/B2M humanized mice (9 weeks old), and flow cytometry analysis was performed to assess leukocyte subpopulations.

Abbr.  WT, wild type; HO, homozygous.

Fig.4 Detection of KrasG12V vaccine and peptide induced immune responses in HLA-A11.1/B2M mice by ELISpot assay.

Splenocytes from mice immunized with PBS, or KRAS G12V LNP-mRNA were collected and stimulated in vitro with RPMI 1640 medium（Non-stimulation）, NY-ESO-1: 157–165, KRAS G12V: 8–16, or cell stimulation cocktail (PC), respectively. Upon stimulation with KrasG12V: 8-16 peptide, WT mice showed no response, whereas HLA-A11.1/B2M mice after KRAS G12V mRNA-LNP immunization generated a robust cellular immune response.

Fig.5 KrasG12V: 8-16 peptide tetramer+ T cells assays.

Splenocytes from mice immunized with PBS or KRAS G12V LNP-mRNA were collected and stimulated with the hHLA-11.1-restricted KRAS G12V: 8–16 peptide. Flow cytometry analysis revealed that the KRAS G12V mRNA-LNP vaccine predominantly elicited a strong antigen-specific CD8+ T cell expansion (~20%).

Fig.6 T-cell intracellular cytokine staining (ICS) assays.

Splenocytes from groups G1–G5 were collected and stimulated with PBS (NC), NY-ESO-1: 157–165, KRAS G12V: 8–16, or cell stimulation cocktail (PC), respectively and analyzed. Flow cytometry analysis revealed that only the hHLA-11.1/B2M mice immunized with KRAS G12V LNP-mRNA (G4) generated a large number of IFN-γ-positive CD4+ and CD8+ T cells.