Cutting Edge: Ig H Chains Are Sufficient to Determine Most B Cell Clonal Relationships Julian Q. Zhou and Steven H. Kleinstein This information is current as J Immunol published online 4 September 2019 of September 23, 2021. http://www.jimmunol.org/content/early/2019/09/03/jimmun ol.1900666 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/09/03/jimmunol.190066 Material 6.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 23, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 4, 2019, doi:10.4049/jimmunol.1900666 Cutting Edge: Ig H Chains Are Sufficient to Determine Most B Cell Clonal Relationships Julian Q. Zhou* and Steven H. Kleinstein*,†,‡ B cell clonal expansion is vital for adaptive immunity. largely unique, with recent estimates suggesting 1 3 1016–1018 High-throughput BCR sequencing enables investigat- unique paired Abs in the circulating repertoire (2). ing this process but requires computational inference Adaptive immune repertoire receptor sequencing allows to identify clonal relationships. This inference usually for high-throughput profiling of the diverse BCR repertoire relies on only the BCR H chain, as most current pro- via full-length V(D)J sequencing in bulk (3). An ensuing tocols do not preserve H:L chain pairing. The extent to challenge is to computationally infer B cell clonal relation- which paired L chains aids inference is unknown. Us- ships (4). This step is of great importance as the assessment ing human single-cell paired BCR datasets, we assessed of repertoire properties such as diversity (5) depends on the Downloaded from the ability of H chain–based clonal clustering to iden- proper identification of clones, as does the reconstruction of tify clones. Of the expanded clones identified, ,20% B cell clonal lineages (6) for tracing isotype switching (7) and grouped cells expressing inconsistent L chains. H chains Ag-specific (8) Abs. To infer clones, differences at the se- from these misclustered clones contained more distant quence nucleotide level, especially the high diversity in the junction sequences and shared fewer V segment muta- CDR3 region, can serve as “fingerprints” (9). Likelihood- tions than the accurate clones. This suggests that addi- based (10) and distance-based (11–14) approaches exist. For http://www.jimmunol.org/ tional H chain information could be leveraged to refine instance, cells sharing the same IGHV and IGHJ genes and clonal relationships. Conversely, L chains were in- whose H chain junctional sequences are sufficiently similar sufficient to refine H chain–based clonal clusters. based on a fixed (11–13) or adaptive (14) distance threshold Overall, the BCR H chain alone is sufficient to identify may be clustered as clones. For validation, existing methods clonal relationships with confidence. The Journal of used simulated and experimental H chain sequences (10, 13, Immunology, 2019, 203: 000–000. 14), measuring the fractions of sequences inferred to be clonally unrelated and related of being, respectively, truly un- related and related (specificity and sensitivity). Recently, Nouri by guest on September 23, 2021 cell–mediated immunity relies on Ig Abs produced and Kleinstein (14) reported both metrics at over 96% based as a result of B cell clonal expansion. A BCR is the on simulated data. B membrane-bound form of an Ab and is made up of The majority of current BCR repertoire studies utilize bulk H and L chains paired in a heterodimeric fashion. Each chain sequencing (15), during which VH:VL pairing is lost (16). contains a variable (V) region, and together, the V regions In the absence of VH:VL pairing, computational methods from the H and L chains form the Ag-binding sites. The V for identifying clones have focused on H chain BCR data. regions are formed via V(D)J recombination. In human, This is justified under the assumption that H chain junc- this shuffling process brings together one gene each from tional diversity alone should be sufficiently high such that, numerous IGHV, IGHD, and IGHJ genes for the H chain even without L chains, the likelihood of clonally unrelated V (VH) region and one gene each from either IGKV and cells being clustered together will be negligibly small (13). IGKJ genes or IGLV and IGLJ genes for, respectively, the This reasoning has yet to be rigorously tested with experimental k or the l L chain V (VL) region. Enzyme-mediated editing data. Recent breakthroughs in single-cell BCR sequencing of the V(D)J junctions and the pairing of H and L chains technology have enabled the recovery of native VH:VL pair- inject additional diversity (1). During adaptive immune re- ing (17, 18). We now have the opportunity to investigate the sponses, B cells proliferate and further diversify via somatic extent to which the inclusion of L chains impacts the ability to hypermutation (SHM), forming clones consisting of cells that accurately detect B cell clonal relationships. originated from the same V(D)J recombinant events yet whose Using single-cell VH:VL paired BCR data, we assessed the BCRs differ at the nucleotide level. As a result, each BCR is performance of H chain–based computational methods for *Interdepartmental Program in Computational Biology and Bioinformatics, Yale Address correspondence and reprint requests to Dr. Steven H. Kleinstein, Yale School University, New Haven, CT 06511; †Department of Pathology, Yale School of Medi- of Medicine, 300 George Street, Suite 505, New Haven, CT 06511. E-mail address: cine, New Haven, CT 06520; and ‡Department of Immunobiology, Yale School of [email protected] Medicine, New Haven, CT 06520 The online version of this article contains supplemental material. ORCIDs: 0000-0001-9602-2092 (J.Q.Z.); 0000-0003-4957-1544 (S.H.K.). Abbreviations used in this article: NSCLC, non–small cell lung carcinoma; SHM, Received for publication June 18, 2019. Accepted for publication August 2, 2019. somatic hypermutation; V, variable; VH, H chain V; VJL, V-J-junction-length; VL, L chain V. This work was supported in part by the National Institutes of Health under Award R01AI104739. Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900666 2 CUTTING EDGE: H CHAINS CAN DETERMINE CLONES WITH CONFIDENCE identifying clones by measuring the extent to which the in- Calculation of the number and frequency of nucleotide mutations ferred clonal members expressed consistent L chains sharing The number and frequency of nucleotide mutations were calculated based the same V and J genes and junction length. We conclude that on IGH/K/LV positions leading up to the junction region using the clonal members of the majority of the inferred clones exhibited “calcObservedMutations” function from SHazaM (v0.1.10) (22). To calculate the number of IGHV mutations shared pairwise between cells L chain consistency. For the majority of the accurately inferred from the same clone, we counted the number of positions at which mutations H chain–based clones, L chain information did not lead to involving the same nucleotide change were observed in both cells. further clonal clustering with greater granularity. At least some of the information gained from paired L chain data were Results apparent when considering the pattern of shared mutations in We performed clonal relationship inference for five single-cell, the H chain V segment, which is not considered in current VH:VL paired, human BCR datasets, using only the H chain distance–based clonal clustering methods, thus offering the sequence from each cell. The datasets included four publicly potential for further improvements in H chain–based clonal available ones from 10x Genomics and one described by (19) inference. (Materials and Methods). Of these, the B lymphoblastoid GM12878 cell line dataset served as a positive control, as Materials and Methods any cells present in a cell line culture can be expected to Single-cell immune profiling datasets comprise genetically identical clonal members. A distance- Four human datasets (Supplemental Fig. 1A) published by 10x Genomics for based spectral clustering method (14) was applied to identify public use on August 1, 2018 were accessed (https://support.10xgenomics.com/ clones for each dataset. The datasets contained between 3 and Downloaded from single-cell-vdj/datasets) on November 3, 2018. Two datasets were sorted and produced by direct Ig enrichment of, respectively, CD19+ B cells isolated from 157 nonsingleton clones (i.e., clones containing at least two PBMCs from a healthy donor, and GM12878 B lymphoblastoid cell line. They cells) (Table I). Because of the small number of clones in each contain VH:VL paired reads for individual cells. The other two datasets were of the six food-allergic individuals in the dataset from (19), we unsorted and produced by V(D)J+59 gene expression profiling of, respectively, aggregated those results for display after performing analysis PBMCs from a healthy donor and squamous non–small cell lung carcinoma (NSCLC) cells from a fresh surgical resection. These contain gene expres- on a per-subject basis.