Profiling Allogeneic HLA-specific B-cell Responses Utilizing a 64-plex Single-HLA Reporter Cell Panel.

Area of Science:

• Immunology
• Transplantation Science
• Computational Biology

Background:

• Identifying allogeneic HLA-specific B cells is crucial for understanding humoral immunity in transplantation but is hindered by their low frequency.
• Current methods face technical challenges in efficiently screening these rare cells.

Purpose of the Study:

• To develop a cost-efficient, high-throughput platform for identifying and characterizing allogeneic HLA-specific B cells in sensitized individuals.
• To enable detailed analysis of B cell phenotype, function, and B cell receptor (BCR) genetics.

Main Methods:

• Generation of a 64-plex single-HLA reporter cell (HLA64-RC) panel for multiplexed B cell screening.
• Development of the companion R package 'HLA64' for automated data analysis and visualization.
• Integration with a high-throughput BCR discovery workflow for comprehensive B cell characterization.

Main Results:

• Successful identification of thirteen HLA-specific B cells from sensitized transplant candidates.
• Characterization of these B cells revealed an IgG+ CD24low phenotype, diverse HLA specificities, and recurrent V-gene usage.
• Analysis of B cell lineages showed divergent binding patterns within clones despite shared clonal origin.

Conclusions:

• The developed HLA64-RC platform and HLA64 R package provide a reliable method for identifying and characterizing allogeneic HLA-specific B cells.
• This approach facilitates deeper understanding of allorecognition, immunodominant epitopes, and immunological risk assessment in transplantation.
• Broader application promises to improve immunological risk stratification and allograft outcomes.