• The B2M encodes a serum protein found in association with the major histocompatibility complex (MHC) class I heavy chain on the surface of nearly all nucleated cells. The protein has a predominantly beta-pleated sheet structure that can form amyloid fibrils in some pathological conditions. HLA-B belongs to the HLA class I heavy chain paralogues. The heavy chain is anchored in the membrane. Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen so that they can be recognized by cytotoxic T cells. They are expressed in nearly all cells.
• The B2M gene (Exon1 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS and HLA-B*3501 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2D^b gene containing the α3, transmembrane and cytoplasmic domains.
• Human B2M and HLA-B35.1 were exclusively detectable in homozygous B-HLA-B35.1 mice.
• B-HLA-B35.1 mice provide a powerful preclinical model for in vivo evaluation of vaccines.
• Application: For example, this product is used for pharmacodynamics and safety evaluation of vaccines for cancers.

Gene targeting strategy for B-HLA-B35.1 mice. The B2M gene (Exon1 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS and HLA-B*3501 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2D^b gene containing the α3, transmembrane and cytoplasmic domains.

Strain specific B2M and HLA-B35.1 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-HLA-B35.1 mice by flow cytometry. Splenocytes (A) and blood (B) were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-HLA-B35.1 mice (H/H), respectively, and analyzed by flow cytometry with species-specific anti-HLA-B35.1 (Biolegend, 311406), anti-H-2Db (Biolegend, 111513), anti-hB2M (Biolegend, 395712), and anti-mB2M (BD Biosciences, 744802) antibody. HLA-B35.1 and human B2M were exclusively detectable in homozygous B-HLA-B35.1 mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice and homozygous B-HLA-B35.1 mice (female, 7-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, neutrophils and Tregs in B-HLA-B35.1 mice were similar to those in C57BL/6JNifdc mice. The frequency of CD8+ T cells in B-HLA-B35.1 mice were lower than in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-HLA-B35.1 mice were higher than in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.0001.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood were isolated from wild-type C57BL/6JNifdc mice and homozygous B-HLA-B35.1 mice (female, 7-week-old, n=3). A. Flow cytometry analysis of the blood was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, neutrophils and Tregs in B-HLA-B35.1 mice were similar to those in C57BL/6JNifdc mice. The frequency of CD8+ T cells in B-HLA-B35.1 mice were lower than in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-HLA-B35.1 mice were higher than in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.0001.

Frequency of leukocyte subpopulations in lymph node by flow cytometry. Lymph node cells were isolated from wild-type C57BL/6JNifdc mice and homozygous B-HLA-B35.1 mice (female, 7-week-old, n=3). A. Flow cytometry analysis of the lymph node was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, NK cells and Tregs in B-HLA-B35.1 mice were similar to those in C57BL/6JNifdc mice. The frequency of CD8+ T cells in B-HLA-B35.1 mice were lower than in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-HLA-B35.1 mice were higher than in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.0001.

Detection of vaccine-induced immune responses in B-HLA-B35.1 mice by IFN-γ ELISpot assay. Female C57BL/6JNifdc mice and B-HLA-B35.1 mice at the age of 16 weeks were divided into PBS group and gp471-480 group (n = 3), and then immunized with PBS or the peptide at the inside muscle of both legs twice. One week after the last immunization, mice were sacrificed. The splenocytes were collected, and respectively stimulated with the peptide, PBS as negative control (NC) and  PMA/Ionomycin (R&D, 423302) as positive control (PC). IFN-γ secretion was measured 20 hours after the stimulation. There was no significant difference in the body weight among the groups (Data was not shown). (A) Representative results of IFN-γ secretion. (B) Summary of results. The results demonstrate that B-HLA-B35.1 mice provide a powerful preclinical model for in vivo evaluation of vaccines. NC: negative control, PC: positive control.