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

ABSTRACT Identifying allogeneic HLA-specific B cells in sensitized individuals is essential for defining the cellular basis of allogeneic humoral immunity but remains technically challenging due to their low frequency. To overcome this barrier, we generated a 64-plex single-HLA reporter cell (HLA64-RC) panel that provides a cost-efficient, multiplex, high-throughput platform for screening B-cell specificity. We additionally developed a companion R package, HLA64, for automated data analysis and visualization. Integrated with a streamlined high-throughput BCR discovery workflow, this platform enables reliable identification and characterization of allogeneic HLA-specific B cells from sensitized transplant candidates. In a pilot application, thirteen HLA-specific B cells were identified, enabling linked analyses of phenotype, function, and BCR genetics. These B cells exhibited an IgG+ CD24low phenotype, diverse HLA allele-specificity profiles, and recurrent heavy- and light-chain V-gene usage. In two independent B-cell lineages, clonal members within each lineage displayed divergent binding patterns despite sharing a common clonal origin. Broader application of this approach for systematic profiling of alloreactive B-cell responses will help elucidate the molecular basis of allorecognition, define immunodominant HLA eplets, and ultimately improve immunological risk assessment and allograft outcomes in transplant recipients. Competing Interest Statement The authors have declared no competing interest.