• 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 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-DRB1*9.1 mice. The exon 2 of mouse I-Eb1 gene that encodes the extracellular domain of I-Eb1 was replaced by human HLA-DRB1 encoding the β1 domain in B-HLA-DRB1*9.1 mice. The  mouse I-Aa, I-Ab1 and I-Eb2 were knocked out in B-HLA-DRB1*9.1 mice.
• Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-DRB1*9.1 mice, but not in wild-type C57BL/6JNifdc mice.
• B-HLA-DRB1*9.1 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-DRB1*9.1 mice. 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-DRB1*9.1 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-DRB1*9.1 mice. The  mouse I-Aa, I-Ab1 and I-Eb2 were knocked out in B-HLA-DRB1*9.1 mice.

Strain specific HLA-DRB1 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-HLA-DRB1*9.1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-DRB1*9.1 mice (H/H), 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-DRB1*9.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-HLA-DRB1*9.1 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous humanized B-HLA-DRB1*9.1 mice (H/H), 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-DRB1*9.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-HLA-DRB1*9.1 mice by flow cytometry. Bone marrow cells were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous humanized B-HLA-DRB1*9.1 mice (H/H), 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-DRB1*9.1 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 (male, n=3, 8-week-old) and homozygous B-HLA-DRB1*9.1 mice (male, n=3, 8-week-old). 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, and Tregs in B-HLA-DRB1*9.1 mice were similar to those in C57BL/6JNifdc mice. The frequency of neutrophils in B-HLA-DRB1*9.1 mice were higher than that in C57BL/6JNifdc mice. The frequency of CD8+ T cells in B-HLA-DRB1*9.1 mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-HLA-DRB1*9.1 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 cells were isolated from wild-type C57BL/6JNifdc mice (male, n=3, 8-week-old) and homozygous B-HLA-DRB1*9.1 mice (male, n=3, 8-week-old). A. Flow cytometry analysis of the blood leukocytes 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-DRB1*9.1 mice were similar to those in C57BL/6JNifdc mice. The frequencies of B cells and CD8+ T cells in B-HLA-DRB1*9.1 mice were lower than that in C57BL/6JNifdc mice, whereas the frequencies of neutrophils and CD4+ T cells in B-HLA-DRB1*9.1 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. Note: The frequency of neutrophils in the B-HLA-DRB1*9.1 mice was somewhat abnormal. We suspect that this was caused by the experiment itself rather than an abnormality in the mice.

Frequency of leukocyte subpopulations in lymph node by flow cytometry. Lymph node cells were isolated from wild-type C57BL/6JNifdc mice (male, n=3, 8-week-old) and homozygous B-HLA-DRB1*9.1 mice (male, n=3, 8-week-old). A. Flow cytometry analysis of the leukocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, NK cells, and Tregs in B-HLA-DRB1*9.1 mice were similar to those in C57BL/6JNifdc mice. The frequency of CD8+ T cells in B-HLA-DRB1*9.1 mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-HLA-DRB1*9.1 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.