Expressed antibody repertoires in human cord blood cells: 454 sequencing and IMGT/HighV-QUEST analysis of germline gene usage, junctional diversity, and somatic mutations

Abstract

Human cord blood cell-derived IgM antibodies are important for the neonate immune responses and construction of germline-based immunoglobulin libraries. Several previous studies of a relatively small number of sequences found that they exhibit restrictions in the usage of germline genes and in the diversity of the variable heavy chain complementarity determining region 3 compared to adults. To further characterize such restrictions on a larger scale and to compare the early B-cell diversity to adult IgM repertoires, we performed 454 sequencing and IMGT/HighV-QUEST analysis of cord blood IG libraries from two babies and determined germline gene usage, V-D-J rearrangement, VHCDR3 diversity, and somatic mutations to characterize human neonate repertoire. Most of the germline subgroups were identified with frequencies comparable to those present in the adult IgM repertoire except for the IGHV1-2 gene that was preferentially expressed in the cord blood cells. The gene usage diversity contributed to 1,430 unique IGH V-D-J rearrangement patterns while the exonuclease trimming and N region addition at the V-D-J junctions along with gene diversity created a wide range of VHCDR3 with different lengths and sequence variability. We observed a lower degree of somatic mutations in the CDR and framework regions of antibodies from cord blood cells compared to adults. These results provide insights into the characteristics of human cord blood antibody repertoires, which have gene usage diversity and VHCDR3 lengths similar to that of the adult IgM repertoire but differ significantly in some of the gene usages, V-D-J rearrangements, junctional diversity, and somatic mutations.

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

“…15 In the cord blood repertoire, V H 1-2 was found to be the most commonly used germline gene. In the naïve, IgM memory and IgG memory subsets, not only was V H 1-2 not the most commonly used variable gene, it was not even the most commonly used V H 1 family gene.…”

“…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.…”

De novo identification of VRC01 class HIV-1–neutralizing antibodies by next-generation sequencing of B-cell transcripts

“…Advances in next-generation sequencing (13)(14)(15) allow for the routine determination of millions of antibody heavy and light chain sequences from a sample of donor B cells, providing the potential for a much more detailed analysis of the expressed antibody repertoire-the antibodyome (6,(16)(17)(18). Here we ask whether the information acquired from next-generation sequencing can be combined with recognition/sequence strategies of population metrics, sequence signatures, and evolution to identify VRC01 class antibodies in an individual, donor C38, with no previously characterized antibodies.…”

Sterile DJH Rearrangements Reveal that Distance Between Gene Segments on the Human Ig H Chain Locus Influences Their Ability To Rearrange

“…V H and J H genes are flanked by 23-spacer recombination signal sequence (RSS) and D genes by 12-spacer RSS. Generally, a gene segment flanked by a 12-spacer RSS is only paired with a gene segment flanked by a 23-spacer RSS (1)(2)(3). This rule ensures that the IgH gene is assembled in the correct order.…”