Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire

Abstract

Antibody repertoire diversity, potentially as high as 10 11 unique molecules in a single individual, confounds characterization by conventional sequence analyses. In this study, we present a general method for assessing human antibody sequence diversity displayed on phage using massively parallel pyrosequencing, a novel application of Kabat column-labeled profile Hidden Markov Models, and translated complementarity determining region (CDR) capture-recapture analysis. Pyrosequencing of domain amplicon and RCA PCR products generated 1.5 ؋ 10 6 reads, including more than 1.9 ؋ 10 5 high quality, full-length sequences of antibody variable fragment (Fv) variable domains. Novel methods for germline and CDR classification and fine characterization of sequence diversity in the 6 CDRs are presented. Diverse germline contributions to the repertoire with random heavy and light chain pairing are observed. All germline families were found to be represented in 1.7 ؋ 10 4 sequences obtained from repeated panning of the library. While the most variable CDR (CDR-H3) presents significant length and sequence variability, we find a substantial contribution to total diversity from somatically mutated germline encoded CDRs 1 and 2. Using a capture-recapture method, the total diversity of the antibody library obtained from a human donor Immunoglobulin M (IgM) pool was determined to be at least 3.5 ؋ 10 10 . The results provide insights into the role of IgM diversification, display library construction, and productive germline usages in antibody libraries and the humoral repertoire.HMM ͉ phage display ͉ pyrosequencing ͉ CDRs

CNS Aquaporin‐4‐specific B cells connect with multiple B‐cell compartments in neuromyelitis optica spectrum disorder

“…The VH family distribution of the naive B cells approximated the expected germline frequency with a slight overrepresentation of the VH5 family across the different populations (average distribution of VH1 19%, VH2 5%, VH3 34%, VH4: 23%, VH5: 12%). VH family distributions were similar to those reported by Glanville et al14, 15 and demonstrated no significant differences between peripheral blood B‐cell populations (Fig. S1).…”

Rearrangement and expression of the immunoglobulin μ-chain gene in human myeloid cells

“…23 In a single-cell PCR analysis of peripheral blood lymphocytes from a normal adult, VH3-23 was found in approximately 17% of all functional rearrangements, 24 and VH3-48 has been reported as an unusual gene in circulating B-cell repertoires with a high homology to VH3-23. 25 Our results also revealed that VH3-23 accounted for 47.5% (29 of 61 clones; Tables 2 and 5) of all rearrangements in AML cell lines and primary AML cells, which was higher than that in patients with non-hematopoietic neoplasms (26.0%, 7 of 27 clones) and healthy individuals (26.0%, 7 of 27 clones). Furthermore, although our results showed an over-representation of VH3-23 and VH3-48 similar to that in B cells, it was surprising that VH4-61 and VH6-1 occurred at a much higher frequency than expected when compared to CD19 1 B-cell-derived VH.…”

Structural Elucidation of the Mechanistic Basis of Degeneracy in the Primary Humoral Response

“…Dlw interactions include 135 van der Waals contacts with the Ab residues. The common interacting residue Trp 91L makes strong hydrophobic interactions with Leu 2 and Trp 3 , whereas Trp 33H contacts only Trp 3 . In addition to this, all three common interacting residues make several van der Waals contacts with different peptide residues, as shown in Table IV.…”

“…This conundrum was elegantly answered many years ago by an expansion of the primary Ab repertoire through genetic recombination of VDJ gene segments and by resultant combinatorial diversity arising from rearranged heterodimers of light and heavy chains (1). Recent high-throughput sequencing data in zebra fish and humans have provided further insights into the extent of the expressed Ab repertoire, as envisaged by Tonegawa et al (2,3) However, quantitative assessment, even after considering other possible mechanisms, proves this diversity to be inadequate for recognition of infinite Ags (4).…”