Antibody-encoding repertoires of bone marrow and peripheral blood—a focus on IgE
Authors: Mattias Levin, Fredrik Levander, Robert Palmason, Lennart Greiff, Mats Ohlin
Journal: Journal of Allergy and Clinical Immunology (2016)

Allergic responses are initiated by the interaction between antigen and IgEs carried on effector cells such as mast cells and basophils. However, the detailed molecular nature of IgE is not amendable to analysis using many of the most commonly used methodologies because B cells/plasma cells that produce them are scarce. Lately, next-generation sequencing (NGS) has allowed us to analyze immunoglobulin-encoding genes at a rate unimaginable in the past.1Georgiou G. Ippolito G.C. Beausang J. Busse C.E. Wardemann H. Quake S.R. The promise and challenge of high-throughput sequencing of the antibody repertoire.Nat Biotechnol. 2014; 32: 158-168Crossref PubMed Scopus (447) Google Scholar Although such investigations tend to focus on antibody repertoires other than IgE, the technology has already enhanced our understanding of IgE responses in allergic disease (see Table E1 in this article's Online Repository at www.jacionline.org). Many antibody sequencing studies focus on repertoires encoded by cells in peripheral blood (PB), a readily available source of antibody-producing cells. However, studies have defined differences between repertoires of immunoglobulin-encoding genes of PB and those encoded in various immunologically active tissues.2Briney B.S. Willis J.R. Finn J.A. McKinney B.A. Crowe J.E. Tissue-specific expressed antibody variable gene repertoires.Plos One. 2014; 23: e100839Crossref Scopus (30) Google Scholar Furthermore, a recent study demonstrated that IgE produced by cells in PB poorly represented circulating IgE.3Eckl-Dorna J. Pree I. Reisinger J. Marth K. Chen K.W. Vrtala S. et al.The majority of allergen-specific IgE in the blood of allergic patients does not originate from blood-derived B cells or plasma cells.Clin Exp Allergy. 2012; 42: 1347-1355Crossref PubMed Scopus (37) Google Scholar In all, because much IgE is produced by cells localized to tissues beyond PB, analysis of this disease-causing isotype is lacking in breadth and relevance. To address the nature of immunoglobulin repertoires, including IgE, at a site readily available for analysis (PB) and at a major site of antibody production (bone marrow [BM]), we have now examined the immunoglobulin heavy chain repertoire, a major carrier of antibody specificity,4Xu J.L. Davis M.M. Diversity in the CDR3 region of V-H is sufficient for most antibody specificities.Immunity. 2000; 13: 37-45Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar encoded by cells residing at these sites. Samples were collected from 6 allergic subjects in biological duplicates so as to specifically allow analysis of the IgE repertoire in subjects in whom it actually inflicts disease. These subjects (see Table E2 in this article's Online Repository at www.jacionline.org) suffered from allergic rhinitis but had not undergone specific immunotherapy (SIT). Samples were collected out of season of most seasonal allergens, but stimulation with perennial allergens around the time of sample collection cannot be excluded. At the time of sample collection, these subjects, though, had no symptoms of an ongoing allergic response. Sequencing (MiSeq Technology; Illumina, Inc, San Diego, Calif) and analysis were performed as outlined in the Methods section in the Online Repository at www.jacionline.org (see also Tables E3 and E4 and Figs E1 and E2 in this article's Online Repository at www.jacionline.org). Sequence analysis revealed a large number of clones derived from a diversity of germline genes. Repertoires of IgA, IgG, and IgM of BM and PB showed very similar composition in terms of IGHV gene, IGHJ gene, and IGHD subgroup usage, and CDRH3 length distributions (see Tables E5 and E6 and Figs E3-E6 in this article's Online Repository at www.jacionline.org). IgA, IgG, and IgM repertoires in BM were more mutated than their corresponding repertoires encoded in PB (see Figs E7 and E8 in this article's Online Repository at www.jacionline.org). Analysis of duplicated samples of BM and PB demonstrated an appreciable degree of reproducibility in terms of overlap in these samples' description of the IgA, IgG, and IgM repertoires (Fig 1, C; see Tables E5 and E6 and Figs E3 and E5). Furthermore, a substantial clonal overlap was identified between samples obtained from BM and PB (Fig 2). Both sample types thus appeared to adequately describe a sizable fraction of the available repertoires. Furthermore, a small but detectable fraction of clones from IgA and IgG repertoires, in particular, were also detected in other isotypes' repertoires (except IgE) (Fig 2).Fig 2Frequency of transcripts of IgA, IgE, IgG, and IgM encoded by BM and PB and found as related members in duplicate samples of the other tissue (PB and BM, respectively) and in other isotypes of the same tissue type. The analysis included up to 1000 of the commonest transcript variants of each sample (a variant was defined as sequence originating from the same germline gene irrespective of allelic variant and with identical CDRH3-encoding nucleotide sequences; each analyzed sequence set must be larger than 0.01% of the largest sequence set in the sample, and it must be represented by at least 2 sequence reads).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In stark contrast to the reproducibility of IgA, IgG, and IgM repertoires of BM and PB, we found that samples of PB of a commonly available volume (10 mL) did not adequately describe the IgE repertoire. Very few, entirely different rearrangements were in general found in biological duplicate samples. The sequences' germline gene origin and the length of their encoded third hypervariable loop differed dramatically, and 1 of 6 donors lacked amplifiable IgE-encoding rearrangements in PB altogether. In contrast, the IgE-encoding repertoires found in duplicate BM samples were larger in size, reproducible in composition, and more diverse in terms of germline gene usage and length of the encoded third hypervariable loop (Fig 1; see Tables E5 and E6 and Figs E3 and E9 in this article's Online Repository at www.jacionline.org). Clonally diversified sequences were identified among these sequences (see Fig E10 in this article's Online Repository at www.jacionline.org). However, irrespective of tissue origin, clustering analysis of transcript populations largely failed to associate IgE-encoding transcript populations to the donor's transcriptomes encoding other isotypes (Figs 1, C, and 2), suggesting that IgE's clonal origin is to a large degree different from those of other isotypes. In summary, IgE repertoires of BM samples are more diverse than their corresponding PB samples. Individual PB samples of a commonly available size not only poorly describe the IgE-encoding transcriptome but also substantially underestimate the diversity of IgE encoded by transcriptomes in BM, a major site of antibody production. IgE-encoding transcriptomes defined in PB must consequently be used with caution in studies of IgE. Antigen-specific IgG4 is an antibody associated with prevention of allergic reactions.5Nouri-Aria K.T. Wachholz P.A. Francis J.N. Jacobson M.R. Walker S.M. Wilcock L.K. et al.Grass pollen immunotherapy induces mucosal and peripheral IL-10 responses and blocking IgG activity.J Immunol. 2004; 172: 3252-3259Crossref PubMed Scopus (478) Google Scholar All 6 donors produced IgG4 (see Table E2). The sequencing format allowed us to identify IgG4-encoding transcripts in 5 of 6 donors. IgG4-encoding sequences in general used a larger number of IGHV genes, a wider distribution of CDRH3 lengths, and mostly represented a larger number of independent clones (Fig 1, D and E; see Tables E5 and E6) than did the IgE-encoding repertoires of the same samples. The length distribution of IgG4-encoding CDRH3, IGHV gene usage pattern, and clonal composition of BM were also mostly well replicated between biological duplicate samples (see Figs E3, E9 and E11 in this article's Online Repository at www.jacionline.org), suggesting that each BM sample adequately represented major dominant features of the IgG4 repertoire. In contrast, such features of the IgG4 repertoire were not as well reproduced in duplicate samples of PB (see Figs E3, E9 and E11). To assess the relationship of the IgE and IgG4-encoding transcriptomes, both regulated in similar ways,6Nirula A. Glaser S.M. Kalled S.L. Taylor F.R. What is IgG4? A review of the biology of a unique immunoglobulin subtype.Curr Opin Rheumatol. 2011; 23: 119-124Crossref PubMed Scopus (242) Google Scholar and transcriptomes encoding isotypes other than IgE, we analyzed the clonal overlap between these repertoires. The analysis used the 50 most prominent IgE clones and the entire set of sequences encoding other isotypes. The algorithm used to assess the overlap between different transcriptomes required that matching sequences originated in the same IGHV gene (not considering allelic variation) and had the same CDRH3 length but it allowed for up to 20% diversification of CDRH3 at the nucleotide level, to minimize false negatives caused by somatic hypermutation targeting CDRH3. Despite using these relaxed criteria, a very minor part (<0.2%) of the transcriptomes encoding IgG4 (as well as those encoding IgA, IgG, and IgM) in BM and PB showed evidence of clonal relatedness to the IgE-encoding population. In fact, in most cases, no related sequences were identified at all (see Fig E12 in this article's Online Repository at www.jacionline.org). Altogether these findings indicate that cells of BM, a major site of antibody production, and PB of these subjects who have not undergone SIT mostly produce IgE with no clonal relatedness to clones that encode other major antibody isotypes. SIT induces allergen-specific antibodies, often IgG4,5Nouri-Aria K.T. Wachholz P.A. Francis J.N. Jacobson M.R. Walker S.M. Wilcock L.K. et al.Grass pollen immunotherapy induces mucosal and peripheral IL-10 responses and blocking IgG activity.J Immunol. 2004; 172: 3252-3259Crossref PubMed Scopus (478) Google Scholar only some of which (most likely those that target the same epitopes as those bound by IgE) protect against allergen-mediated reactions.7Shamji M.H. Ljorring C. Francis J.N. Calderon M. Larche M. Kimber I. et al.Functional rather than immunoreactive levels of IgG4 correlate closely with clinical response to grass pollen immunotherapy.Allergy. 2012; 67: 217-226Crossref PubMed Scopus (218) Google Scholar We hypothesize, on the basis of present observations, that immunotherapy that is able to exploit the same clonal rearrangements as those used for production of IgE also for production of protective antibodies (eg, IgG4) will be particularly successful in alleviating symptoms and in providing cure. We therefore hypothesize that sequence-based analysis demonstrating enhanced clonal overlap between IgE and IgG4 (or other antibody isotypes/subclasses) may provide objective biomarkers in the analysis of SIT efficacy. Antibody-encoding gene sequences as such do not per se define the specificity of an encoded antibody. Indeed, the vast diversity of the immune repertoire suggests that the response to a complex antigen composed of multiple epitopes, like a protein, will be diverse in terms of sequence. In several instances, though, specific sequence features (stereotyped rearrangements) are highly enriched in a given antigen-specific antibody response.8Henry Dunand C.J. Wilson P.C. Restricted, canonical, stereotyped and convergent immunoglobulin responses.Philos Trans R Soc Lond B Biol Sci. 2015; 370: 20140238Crossref PubMed Scopus (30) Google Scholar IgEs specific for grass pollen allergen Phl p 2 often display a 10-residue long CDRH3 with glycin as the fifth residue, and are encoded by genes originating from IGHV4-30-4 or IGHV4-31 germline genes.9Persson H. Flicker S. Sadegh M.K. Greiff L. Valenta R. Ohlin M. A common idiotype in IgE and its relation to recognition of the grass pollen allergen Phl p 2.Mol Immunol. 2008; 45: 2715-2720Crossref PubMed Scopus (13) Google Scholar When searching for such sequences in the presently investigated transcriptomes, we identified 3 donors of BM (donors 2, 3, 6) and 1 donor of PB (donor 3) whose samples contained large frequencies of such IgE-encoding transcripts (see Fig E13 in this article's Online Repository at www.jacionline.org). Such sequences were very rarely represented in these subjects' primary repertoires (transcripts encoding unmutated IgM), and in IgG (Fig E13). Importantly, these 3 donors, but not donors 1, 4, and 6, were also seropositive for Phl p 2–specific IgE (see Table E7 in this article's Online Repository at www.jacionline.org). In summary, there was a correspondence between the presence of a particular rearrangement and the presence of allergen-specific, circulating IgE. This finding sets the foundation for using antibody variable domain sequences as objective biomarkers in those instances in which stereotyped rearrangements can be associated with a specific immune response. By greatly expanding our current small set of human allergen-specific IgE,10Gadermaier E. Levin M. Flicker S. Ohlin M. The human IgE repertoire.Int Arch Allergy Immunol. 2014; 163: 77-91Crossref PubMed Scopus (27) Google Scholar we envisage that we will be able to identify additional stereotyped allergen-specific IgE responses, signatures that will find use as biomarkers in the future. In conclusion, IgE repertoires of BM samples are more diverse and more reproducible in their composition than those of commonly sized PB samples. Samples from BM should consequently be preferred over those from PB in future studies of IgE and its sequence-based biomarkers. Studies of allergen-specific antibodies of isotypes other than IgE must not a priori be used to define the character of IgE because the degree of overlap between these repertoires is very small. Furthermore, we propose that there is a window of opportunity for NGS to assist in the assessment of successful SIT, if such treatment is able to induce a high degree of clonal overlap between IgE and other isotypes, and the IgG4 subclass in particular. Finally, we envisage that BM-encoded repertoires, through their larger diversity, may also be an excellent source of IgE diversity for the development of human monoclonal IgE through cellular cloning or combinatorial library approaches,10Gadermaier E. Levin M. Flicker S. Ohlin M. The human IgE repertoire.Int Arch Allergy Immunol. 2014; 163: 77-91Crossref PubMed Scopus (27) Google Scholar antibodies that will enhance our understanding of IgE-allergen interactions and their outcomes. We are grateful to Lena Glantz-Larsson and Charlotte Cervin-Hoberg for management of allergic subjects. We acknowledge support from Science for Life Laboratory, the Knut and Alice Wallenberg Foundation, the National Genomics Infrastructure funded by the Swedish Research Council, and Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with NGS and access to the UPPMAX computational infrastructure, and from Bioinformatics Infrastructure for Life Sciences for bioinformatics analysis. 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