• 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-A 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. HLA-DRA is one of the HLA class II alpha chain paralogues. HLA-DRB1 belongs to the HLA class II beta chain paralogs. This class II molecule is a heterodimer consisting of an alpha (DRA) and a beta (DRB) chain, both anchored in the membrane. It plays a central role in the immune system by presenting peptides derived from extracellular proteins. Class II molecules are expressed in antigen presenting cells (APC: B lymphocytes, dendritic cells, macrophages). The alpha chain is approximately 33-35 kDa and its gene contains 5 exons. The beta chain is approximately 26-28 kDa and its gene contains 6 exons. DRA does not have polymorphisms in the peptide binding part and acts as the sole alpha chain for DRB1, DRB3, DRB4 and DRB5.
• The B2m gene (Exon1 to Exon3) of mouse was replaced by the sequence encompassing the human B2M CDS and HLA-A*0201 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. The exon 2 of mouse I-Ea gene that encodes the extracellular domain of I-Ea was replaced by human HLA-DRA encoding the α1 domain in B-HLA-A2.1/HLA-DRB1*1.1 mice. The exon 2 of mouse I-Eb1 gene that encodes the extracellular domain of I-Eb1 was replaced by human HLA-DRB encoding the β1 domain in B-HLA-A2.1/HLA-DRB1*1.1 mice. The  mouse I-Aa, I-Ab1 and I-Eb2 were knocked out in B-HLA-A2.1/HLA-DRB1*1.1 mice.
• Human B2M, HLA-A2.1, and HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.
• B-HLA-A2.1/HLA-DRB1*1.1 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-A2.1/HLA-DRB1*1.1 mice.

The B2m gene (Exon1 to Exon3) of mouse was replaced by the sequence encompassing the human B2M CDS and HLA-A*0201 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.

The exon 2 of mouse I-Ea gene that encodes the extracellular domain of I-Ea was replaced by human HLA-DRA encoding the α1 domain in B-HLA-A2.1/HLA-DRB1*1.1 mice. The exon 2 of mouse I-Eb1 gene that encodes the extracellular domain of I-Eb1 was replaced by human HLA-DRB encoding the β1 domain in B-HLA-A2.1/HLA-DRB1*1.1 mice. The  mouse I-Aa, I-Ab1 and I-Eb2 were knocked out in B-HLA-A2.1/HLA-DRB1*1.1 mice.

Strain specific B2M and HLA expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, and analyzed by flow cytometry with species-specific anti-mouse B2M antibody (BD, 744802), anti-human B2M (Biolegend, 395712), anti-mouse H-2Db (Biolegend, 111513) and anti-human HLA-ABC (Biolegend, 311406). Mouse B2M and H-2Db were only detectable in wild-type mice. Human B2M and HLA-A2.1 were exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Bone marrow cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Bone marrow cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Bone Marrow cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice and homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice (male, 8-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-A2.1/HLA-DRB1*1.1 mice were similar to those in C57BL/6JNifdc mice. The frequency of CD8+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were higher than that 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-A2.1/HLA-DRB1*1.1 mice (male, 8-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, NK cells, dendritic cells, monocytes, macrophages, and Tregs in B-HLA-A2.1/HLA-DRB1*1.1 mice were similar to those in C57BL/6JNifdc mice. The frequency of neutrophils in B-HLA-A2.1/HLA-DRB1*1.1 mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of B cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were higher than that in C57BL/6JNifdc mice. The frequency of CD8+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were higher than that 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-A2.1/HLA-DRB1*1.1 mice (male, 8-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 NK cells, and Tregs in B-HLA-A2.1/HLA-DRB1*1.1 mice were similar to those in C57BL/6JNifdc mice. The frequency of T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were higher than that in C57BL/6JNifdc mice. The frequency of CD8+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were higher than that 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.