112 LightningSeq: a long-read HLA/TCR sequencing platform for scalable profiling of adaptive cellular immunity
Year: 2026
Type: conference-abstract
Abstract: Background Quantitative assessment of T cell immunity remains challenging at scale due to the diversity of T cell receptor (TCR) repertoires, HLA-restricted antigen recognition, and the cost and complexity of existing cellular immune profiling approaches. We developed LightningSeq, a targeted long-read nanopore sequencing platform designed to integrate HLA genotyping with bulk TCR alpha and beta repertoire profiling from low-input clinical specimens. This platform has direct relevance to immuno-oncology, adoptive cell therapy, vaccine response monitoring, and longitudinal tracking of antigen-driven T cell responses.Methods LightningSeq uses targeted amplification of genomic HLA loci and template-switching–based amplification of full V-region TCR alpha and beta transcripts from RNA, followed by sample barcoding and Oxford Nanopore long-read sequencing. Custom bioinformatics pipelines perform HLA allele assignment, TCR clonotype calling, quantitative repertoire analysis, and antigen-associated clonotype tracking. TCR reproducibility was assessed with three independent PBMC donors across two timepoints using 100 ng total RNA input per sample and technical replicate sequencing. Repertoire similarity was evaluated using Top-N clonotype analysis and Bray–Curtis dissimilarity. Biological response detection was evaluated using influenza M1 mRNA–stimulated PBMCs, with matched functional immune assays and sequence-level identification of expanded clonotypes. Preliminary HLA performance was assessed using reference materials, and functional validation capacity was established using a TCR-negative NFAT-luciferase Jurkat reporter system.Results Across six PBMC donors and technical replicates, LightningSeq generated an average of approximately 42,000 alpha-chain reads and 68,000 beta-chain reads per sample, yielding approximately 3,300 unique alpha clonotypes and 4,600 unique beta clonotypes. Technical replicate reproducibility was high, with maximum Bray–Curtis dissimilarity ranging from 0.09–0.32 for TCR alpha and 0.06–0.24 for TCR beta repertoires. Influenza M1 mRNA stimulation produced reproducible antigen-driven expansion of specific alpha and beta clonotypes, including clonotypes matching previously reported influenza M1–specific TCR sequences. Matched functional assays supported the biological relevance of these expanded clonotypes. Preliminary HLA sequencing demonstrated successful locus amplification and allele assignment feasibility, with read-level separation between correct allele alignments (>99% identity) and off-target allele alignments (<95% identity), supporting continued bioinformatic optimization for high-confidence HLA calling.Conclusions LightningSeq enables scalable, low-input, long-read profiling of HLA genotype and TCR repertoire dynamics. These preliminary data demonstrate reproducible quantitative TCR profiling, detection of antigen-driven clonal expansion, and feasibility of integrated HLA-informed immune monitoring. This platform may support high-throughput analysis of adaptive cellular immunity in cancer immunotherapy, vaccine studies, and longitudinal immune surveillance.Ethics Approval The samples in this study were collected by approval from the Arizona State University IRB.
Authors: Oliver Kask, Olivia Roy, Karen S Anderson
Institution: Arizona State University
Language: English
Topic: T-cell and B-cell Immunology
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,490
OpenAlex ID: W7225153426