Characterization of the Human Ig Heavy Chain Antigen Binding Complementarity Determining Region 3 Using a Newly Developed Software Algorithm, JOINSOLVER

Abstract

We analyzed 77 nonproductive and 574 productive human VHDJH rearrangements with a newly developed program, JOINSOLVER. In the productive repertoire, the H chain complementarity determining region 3 (CDR3H) was significantly shorter (46.7 ± 0.5 nucleotides) than in the nonproductive repertoire (53.8 ± 1.9 nucleotides) because of the tendency to select rearrangements with less TdT activity and shorter D segments. Using criteria established by Monte Carlo simulations, D segments could be identified in 71.4% of nonproductive and 64.4% of productive rearrangements, with a mean of 17.6 ± 0.7 and 14.6 ± 0.2 retained germline nucleotides, respectively. Eight of 27 D segments were used more frequently than expected in the nonproductive repertoire, whereas 3 D segments were positively selected and 3 were negatively selected, indicating that both molecular mechanisms and selection biased the D segment usage. There was no bias for D segment reading frame (RF) use in the nonproductive repertoire, whereas negative selection of the RFs encoding stop codons and positive selection of RF2 that frequently encodes hydrophilic amino acids were noted in the productive repertoire. Except for serine, there was no consistent selection or expression of hydrophilic amino acids. A bias toward the pairing of 5′ D segments with 3′ JH segments was observed in the nonproductive but not the productive repertoire, whereas VH usage was random. Rearrangements using inverted D segments, DIR family segments, chromosome 15 D segments and multiple D segments were found infrequently. Analysis of the human CDR3H with JOINSOLVER has provided comprehensive information on the influences that shape this important Ag binding region of VH chains.

Delayed acquisition of somatic hypermutations in repopulated IGD+CD27+ memory B cell receptors after rituximab treatment

“…Gel-extracted Ig V H 3 family products were further amplified using BigDye Terminator Cycle Sequencing Ready Reaction kit (PerkinElmer Applied Biosystems, Weiterstadt, Germany) and 5Ј V H 3 internal primer followed by sequencing in an ABI Prism 310 automated genetic analyzer (Applied Biosystems). Sequences were analyzed by matching their closest germline counterparts using the online program Joinsolver (27).…”

Humans with chronic granulomatous disease maintain humoral immunologic memory despite low frequencies of circulating memory B cells

“…Sequences were analyzed with IgBLAST (http://www.ncbi.nlm.nih.gov/ igblast/) and JOINSOLVER (http://joinsolver.niaid.nih.gov) programs, as described previously. 11,16,17 Statistics Median values were compared by Mann-Whitney or Wilcoxon matchedpairs signed rank test where appropriate, using Prism Version 6 (GraphPad). Three-way analyses were compared simultaneously by either KruskalWallis or Friedman test and, if significant, prompted appropriate pairwise comparisons.…”

Paucity of V-D-D-J Rearrangements and VH Replacement Events in Lupus Prone and Nonautoimmune TdT−/− and TdT+/+ Mice

“…Nonetheless, analyses of CDR3 regions in both mice and humans have occasionally shown two stretches of nucleotides that show identity to different D H genes, and a few reports have claimed to observe very high frequencies of CDR3 with two D H genes in lupus prone mice (5)(6)(7). Hence, the issue of whether D-D rearrangements actually occur in CDR3 of Ig H chains in vivo, and their frequency, is quite controversial (7)(8)(9)(10)(11)(12)(13)(14). Rearrangements with two apparent D segments in CDR3 have been observed in ϳ5-15% of human CDR3 peripheral blood sequences and in pre-B cells from human bone marrow (15)(16)(17).…”

“…Investigators who analyze mutated sequences sometimes gave allowances for a mismatch in identifying D segments (21,65,66). Thus, these analyses are likely to inadvertently identify a significant number of false positive D-D rearrangements (8,14). One way to avoid this uncertainty is to analyze unmutated CDR3 sequences.…”