Targeting the HLA-B15.1 Axis: Biological Roles and Therapeutic Strategies

•  Gene Information: The HLA-B gene, located on chromosome 6p21.3 within the Major Histocompatibility Complex (MHC) locus, encodes the highly polymorphic HLA-B15.1 alpha chain, a crucial component of the human leukocyte antigen class I system. 
• Protein Expression: HLA-B15.1 is a codominantly and ubiquitously expressed cell-surface glycoprotein found on almost all nucleated cells, where it forms a heterotrimer with β2-microglobulin and an 8-to-10 amino acid endogenous peptide. 
• Signaling Pathway: It presents intracellular antigenic peptides directly to CD8+ T-cell receptors (TCRs), triggering a downstream signaling cascade that drives cytotoxic cell lysis. 
• Therapeutic Inhibition: Rather than blocking its function, therapies exploit HLA-B15.1 by developing cancer vaccines, TCR-like antibodies and T-cell engagers that selectively target the specific tumor-peptide/HLA-B15.1 complex.

HLA-B15.1 

• The B2M gene (Exon1 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS and HLA-B*1501 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 in B-HLA-B15.1 mice.

Strain specific B2M and HLA-B15.1 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-HLA-B15.1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-HLA-B15.1 mice (H/H), respectively, and analyzed by flow cytometry with species-specific anti-HLA-ABC antibody (Biolegend, 311406), anti-H-2D^b antibody (Biolegend, 111513), anti-hB2M antibody (Biolegend, 395712), and anti-mB2M antibody (BD Biosciences, 744802) antibody.

Strain specific B2M and HLA-B15.1 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-HLA-B15.1 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-HLA-B15.1 mice (H/H), respectively, and analyzed by flow cytometry with species-specific anti-HLA-ABC antibody (Biolegend, 311406), anti-H-2Db antibody (Biolegend, 111513), anti-hB2M antibody (Biolegend, 395712), and anti-mB2M antibody (BD Biosciences, 744802) antibody.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6JNifdc and B-HLA-B15.1 mice (female, 14-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6JNifdc and B-HLA-B15.1 mice (female, 14-week-old, n = 3). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Detection of vaccine-induced immune responses in B-HLA-B15.1 mice by IFN-γ ELISpot assay. Female C57BL/6JNifdc mice and B-HLA-B15.1 mice at the age of 7–8 weeks were divided into PBS group and HCMV pp65_340-348 group (n = 3), and then inoculated PBS or the peptide at the inside muscle of both legs. Three weeks after the last immunization, mice were sacrificed. The splenocytes were extracted, stimulated with individual peptide or PBS as negative control (NC) or PMA/Ionomycin(R&D, 423302) as positive control(PC), and then measured for IFN-γ secretion. No significant difference in body weight among groups (Data was not shown). (A) Representative results showing stimulation of splenocytes harvested from immunized mice with negative control, the peptide, or positive control in duplicates. (B) Summary of results. NC: negative control, PC: positive control.