Targeting the HLA-A24.2 Axis: Biological Roles and Therapeutic Strategies 

• Gene Information: The HLA-A gene, located on chromosome 6p21.3 within the Major Histocompatibility Complex (MHC) locus, encodes the highly polymorphic HLA-A24.2 alpha chain, a crucial component of the human leukocyte antigen class I system. 
• Protein Expression: HLA-A24.2 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-A24.2 by developing cancer vaccines, TCR-like antibodies and T-cell engagers that selectively target the specific tumor-peptide/HLA-A24.2 complex.

HLA-A24.2 

• The B2M gene (Exon1 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS and HLA-A*2402 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-A24.2 mice.

Strain specific B2M and HLA-A expression analysis in homozygous B-HLA-A24.2 mice by flow cytometry. Splenocytes were collected from both wild-type C57BL/6 (+/+) and homozygous B-HLA-A24.2 mice (H/H) were analyzed by flow cytometry. B2M expression was analyzed by flow cytometry using species-specific anti-mouse B2M antibody (Biolegend, 154503) and anti-human B2M (Biolegend, 316305). HLA expression was analyzed by flow cytometry using species-specific anti-H-2D^b antibody (Biolegend, 114607) and anti-HLA-A,B,C antibody (Biolegend, 311406).

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6 and B-HLA-A24.2 mice (n=4, 2 female and 2 male, 8-week-old). 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/6 and B-HLA-A24.2 mice (n=4, 2 female and 2 male, 8-week-old). 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-A24.2 mice by IFN-γ ELISpot assay. Female B-HLA-A24.2 mice at the age of 9-10 weeks were divided into PBS group, Group 2 and Group 3 (n = 2), and then inoculated PBS or vaccines 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 target-unrelated polypeptide as negative control (NC) or anti-CD3 as positive control, 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, or peptide vaccines, or positive control in duplicates. (B) ELISpot Quantification: Statistical summary of IFN-γ-secreting cells.