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Comparative Study
. 2012 May;64(5):337-50.
doi: 10.1007/s00251-011-0595-8. Epub 2011 Dec 27.

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

Ponraj Prabakaran  1 , Weizao Chen, Maria G Singarayan, Claudia C Stewart, Emily Streaker, Yang Feng, Dimiter S Dimitrov
Affiliations
Comparative Study

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

Ponraj Prabakaran et al. Immunogenetics. 2012 May.

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.

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Figures

Fig. 1
Fig. 1
Gene usage frequencies observed for the VH and VL domains of IG or antibody cDNA from human cord blood samples of two babies (CB1 and CB2). Gene usage was calculated as the percentage of the total unique population of productive (P) and unproductive (UP) sequences. a–c VH domains: IGHV subgroup, IGHD set, and IGHJ gene utilization observed in productive and unproductive sequences. d–f VL domains: IGKV and IGLV subgroups as well as IGKJ and IGLJ gene utilization in productive and unproductive sequences
Fig. 2
Fig. 2
Length distributions of the complementarity determining regions CDR1 and CDR2 observed in the V regions of the VH, V-KAPPA, and V-LAMBDA domains. CDR lengths and number of sequences in percent are shown in the horizontal and vertical axes, respectively. a Length distributions of VH CDR1 and CDR2. b Length distributions of V-KAPPA CDR1 and CDR2. c Length distributions of V-LAMBDA CDR1 and CDR2
Fig. 3
Fig. 3
Number of amino acid (AA) changes from the closest germline counterparts found in the combined unique V region sequences of 13,888 VH, 12,164 V-KAPPA, and 3,948 V-LAMBDA, obtained from two human cord blood samples. a Total number of AA changes in the V regions of VH and VL domains. b Number of AA changes observed in the CDR1 and CDR2 of VH, V-KAPPA, and V-LAMBDA domains. c The average number of AA changes observed in FR1, CDR1, FR2, CDR2, and FR3 of VH, V-KAPPA, and V-LAMBDA
Fig. 4
Fig. 4
N, P regions and trimming at the variable heavy chain complementarity determining region VH CDR3. The V-D-J junctional diversity created by P region, exonuclease trimming and N region addition significantly contributes to a large VH CDR3 repertoire. The histograms show the average numbers of N and P nucleotides added at the V-D-J junctions, and the average numbers of nucleotides deleted at the 3′V, 5′D, 3′D and 5′J by exonuclease trimming
Fig. 5
Fig. 5
CDR3 lengths in VH and VL domains, and amino acid usage observed in the cord blood-derived VH CDR3 regions. a Length distribution of 2,927 unique V-KAPPA CDR3 regions. b Length distribution of 1,193 unique V-LAMBDA CDR3 regions. c Length distribution of 24,188 unique VH CDR3 regions showing the highly diversified CDR3 repertoire with different lengths. d Average values of AA composition calculated for all unique VH CDR3 sequences in percent shows the preferred AA usage. The amino acids are arranged by relative hydrophobicity values as found in Kyte-Doolittle scale (Pommié et al. 2004). e The frequency of individual amino acids at specific positions for the most prevalent VH CDR3 of 2,963 sequences having the length of 13 amino acids (the basic length of CDR3-IMGT) is shown. CDR3 positions are shown according to the IMGT unique numbering (Lefranc et al. 2003)
Fig. 6
Fig. 6
Frequency of V-D-J unique rearrangements derived from the same IGFCV, IGHD and IGHJ genes and expressed in the cord blood VH repertoire. The A-axis represents the IGFCV genes used in association with pertinent IGFCD and IGHJ genes which are represented by the left and right y-axes, respectively. A total of 1,430 unique V-D-J rearrangement patterns that share the same IGFCV, IGHD and IGFCJ genes are shown in different shapes indicating the frequency count for each V-D-J rearrangement pattern observed in the cord blood VH repertoire
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