Quantifying HLA transcripts by genotype in chimeric mixtures at single-cell resolution

Gene products from the highly variable major histocompatibility locus, including HLA, are essential for self-recognition and immune surveillance of malignancy. Following allogeneic hematopoietic cell transplantation (alloHCT), genetic and epigenetic alterations in HLA can drive disease recurrence, making precise HLA assessment critical for determining future therapy. However, current methods lack the sensitivity to quantify HLA transcripts at the single-cell level, limiting their clinical utility. We introduce scrHLA-typing, a novel technique that accurately identifies and quantifies HLA transcripts in single cells using long-read sequencing. When applied to samples from patients with post-transplant relapse, scrHLA-typing successfully detected HLA allele-specific expression, across a range of levels of donor-recipient chimerism, at clinically actionable levels. By characterizing allele expression in residual leukemia cells, our assay identified differences in expression patterns among patients. This capability highlights scrHLA-typing's potential to improve risk stratification and guide the selection of appropriate salvage therapies, enhancing personalized treatment strategies after relapse.