Measurement and Clinical Monitoring of Human Lymphocyte Clonality by Massively Parallel V-D-J Pyrosequencing

Abstract

The complex repertoire of immune receptors generated by B and T cells enables recognition of diverse threats to the host organism. Here, we show that massively parallel DNA sequencing of rearranged immune receptor loci can provide direct detection and tracking of immune diversity and expanded clonal lymphocyte populations in physiological and pathological contexts. DNA was isolated from blood and tissue samples, a series of redundant primers was used to amplify diverse DNA rearrangements, and the resulting mixtures of bar-coded amplicons were sequenced with long-read ultradeep sequencing. Individual DNA molecules were then characterized on the basis of DNA segments that had been joined to make a functional (or nonfunctional) immune effector. Current experimental designs can accommodate up to 150 samples in a single sequence run, with the depth of sequencing sufficient to identify stable and dynamic aspects of the immune repertoire in both normal and diseased circumstances. These data provide a high-resolution picture of immune spectra in normal individuals and in patients with hematological malignancies, illuminating, in the latter case, both the initial behavior of clonal tumor populations and the later suppression or reemergence of such populations after treatment.

High-throughput antibody sequencing reveals genetic evidence of global regulation of the naïve and memory repertoires that extends across individuals

“…In all three peripheral blood subsets, either V H 3-23 (naïve and IgM memory) or V H 3-30 (IgG memory) was the most commonly used variable gene, which is consistent with previous data. [11][12][13][14] Joining gene use was consistent between our sample and the previously published cord blood repertoire, however, with J H 4 being the most commonly used joining gene in all peripheral blood subsets and cord blood.…”

“…The primers were selected for their ability to produce accurate, reproducible amplification of both naïve and mutated antibody repertoires, 12,13 and the variable gene use of our repertoire closely matched repertoire analysis in which amplification was performed on single B cells. 9 prominence of each V(D)J recombination within the repertoire of each cell subset (Figures 1a-c).…”

“…11 Recently, there have been several studies that leveraged high-throughput amplicon sequencing to perform in-depth analysis of human antibody repertoires. [11][12][13][14][15] These previous studies have been limited in scope, however, owing to analysis of the use of V, D or J genes in isolation. In the peripheral blood antibody repertoire, individual V, D and J genes are not expressed in isolation, but are linked by recombination.…”

Using synthetic templates to design an unbiased multiplex PCR assay

“…In extreme cases, such bias can result in undetectable levels of specific under-amplifying target templates. A bias-free assay is critical for studies aiming to quantitatively measure the frequency of specific immune receptor rearrangements, such as minimal residual disease (MRD) monitoring in leukaemia [9][10][11][12] , following exposure-specific immune repertoires over time 13,14 and research to study basic B-and T-cell biology 15,16 .…”

Antigen Receptor Galaxy: A User-Friendly, Web-Based Tool for Analysis and Visualization of T and B Cell Receptor Repertoire Data

“…When multiple replicates are performed the number of sequences that overlap between the replicates can be determined ("determine the number of sequences that share the same clonal type between the replicates"). When three or more replicates are analyzed the "determine clonality of the donor" option allows the calculation of the number of overlapping sequence and the clonality score as described by Boyd et al (25). Data visualization.…”