The T-cell receptor (TCR) is an essential component of the adaptive immune response elicited by vaccines, infectious agents and cancer. As such it is the source of a new class of immunotherapies in the drug development pipeline. An individual’s TCR repertoire represents the vaccinations and disease challenges they have faced over the course of their life. Individuals that survive cancer and infectious disease can reveal a valuable source of antibodies and TCRs for development into immunotherapies. Immune repertoire analysis achieved through next generation sequencing is one step in this drug discovery process. The technique could be particularly useful for analyzing tumor or inflamatory disease biobanks as well as infectious disease.
Unfortunately, current repertoire sequencing approaches are complex and expensive, limiting the ability to screen large numbers of samples. H. Benjamin Larman of Johns Hopkins University School of Medicine, Baltimore, and colleagues sought to develop a simple, quantitative, multiplex PCR-based approach that prioritizes the ultra-efficient analysis of TCR beta chain hypervariable complementarity determining region 3 (CDR3) sequences, revealing T-cell clonal identity.
The author’s Framework Region 3 AmplifiKation Sequencing (FR3AK-seq) comprises maximally compact primer sets, the design of which is automated using a greedy algorithm and a streamlined workflow. PCR amplification bias has been minimized while maintaining high sequencing accuracy. The lower sequence depth requirements, combined with single-end, short-read sequencing, substantially reduced the cost of TCR repertoire analysis.
For development, analysis was performed on T cell RNA extracted from peripheral blood mononuclear cells (PBMCs). FR3AK-seq was benchmarked against the immunoSEQ hsTCRB “Deep Resolution” sequencing service offered by Adaptive Biotechnologies. MiXCR open source software was used to analyze CDR3 sequences from both assays. FR3AK-seq was also compared with ArcherDX’s unique molecular identifier (UMI) based Immunoverse HS TCR assay. In both cases the sequencing results were found to be comparable.
As a proof of concept, at a cost of roughly USD $20/ sample, muscle biopsies from 145 patients with idiopathic inflammatory myopathies and 21 controls were analyzed with FR3AK-seq. The number of non-bystander T cells was shown to be elevated in disease tissues versus the control. GLIPH software was able to identify clusters of related CDR3 sequences.
The authors stated, “By minimizing amplification bias (via reduction of primer number and amplicon length), the resulting sequencing libraries quantitatively capture clonal abundance distributions with an accuracy comparable to both multiplex PCR-based and UMI-based industry standards.”