Immunoglobulin Analysis Tool: A Novel Tool for the Analysis of Human and Mouse Heavy and Light Chain Transcripts

Sequence analysis of immunoglobulin (Ig) heavy and light chain transcripts can refine categorization of B cell subpopulations and can shed light on the selective forces that act during immune responses or immune dysregulation, such as autoimmunity, allergy, and B cell malignancy. High-throughput sequencing yields Ig transcript collections of unprecedented size. The authoritative web-based IMGT/HighV-QUEST program is capable of analyzing large collections of transcripts and provides annotated output files to describe many key properties of Ig transcripts. However, additional processing of these flat files is required to create figures, or to facilitate analysis of additional features and comparisons between sequence sets. We present an easy-to-use Microsoft® Excel® based software, named Immunoglobulin Analysis Tool (IgAT), for the summary, interrogation, and further processing of IMGT/HighV-QUEST output files. IgAT generates descriptive statistics and high-quality figures for collections of murine or human Ig heavy or light chain transcripts ranging from 1 to 150,000 sequences. In addition to traditionally studied properties of Ig transcripts – such as the usage of germline gene segments, or the length and composition of the CDR-3 region – IgAT also uses published algorithms to calculate the probability of antigen selection based on somatic mutational patterns, the average hydrophobicity of the antigen-binding sites, and predictable structural properties of the CDR-H3 loop according to Shirai’s H3-rules. These refined analyses provide in-depth information about the selective forces acting upon Ig repertoires and allow the statistical and graphical comparison of two or more sequence sets. IgAT is easy to use on any computer running Excel® 2003 or higher. Thus, IgAT is a useful tool to gain insights into the selective forces and functional properties of small to extremely large collections of Ig transcripts, thereby assisting a researcher to mine a data set to its fullest.

[1]  Andrew D. Ellington,et al.  Antibody Repertoires in Humanized NOD-scid-IL2Rγnull Mice and Human B Cells Reveals Human-Like Diversification and Tolerance Checkpoints in the Mouse , 2012, PloS one.

[2]  Emanuela M. Ghia,et al.  Recombinant antibodies encoded by IGHV1-69 react with pUL32, a phosphoprotein of cytomegalovirus and B-cell superantigen. , 2012, Blood.

[3]  U. Lopes,et al.  A rapid and quantitative method for the evaluation of V gene usage, specificities and the clonal size of B cell repertoires. , 2012, Journal of immunological methods.

[4]  P. Allen,et al.  How the TCR balances sensitivity and specificity for the recognition of self and pathogens , 2012, Nature Immunology.

[5]  D. Dimitrov,et al.  Expressed antibody repertoires in human cord blood cells: 454 sequencing and IMGT/HighV-QUEST analysis of germline gene usage, junctional diversity, and somatic mutations , 2011, Immunogenetics.

[6]  James L. Zehnder,et al.  High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment , 2011, Proceedings of the National Academy of Sciences.

[7]  U. Sack,et al.  Vaccination-induced changes in human B-cell repertoire and pneumococcal IgM and IgA antibody at different ages , 2011, Aging cell.

[8]  Stephen L. Hauser,et al.  Naive antibody gene-segment frequencies are heritable and unaltered by chronic lymphocyte ablation , 2011, Proceedings of the National Academy of Sciences.

[9]  J. Mullikin,et al.  Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing , 2011, Science.

[10]  C. Nusbaum,et al.  High-Resolution Description of Antibody Heavy-Chain Repertoires in Humans , 2011, PloS one.

[11]  Charles P. Lin,et al.  Immune recognition and rejection of allogeneic skin grafts. , 2011, Immunotherapy.

[12]  Steven H. Kleinstein,et al.  Detecting selection in immunoglobulin sequences , 2011, Nucleic Acids Res..

[13]  Véronique Giudicelli,et al.  IMGT/V-QUEST: IMGT standardized analysis of the immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences. , 2011, Cold Spring Harbor protocols.

[14]  M. Lefranc.  IMGT, the International ImMunoGeneTics Information System. , 2011, Cold Spring Harbor protocols.

[15]  W. Meng,et al.  Alternative mechanisms of receptor editing in autoreactive B cells , 2011, Proceedings of the National Academy of Sciences.

[16]  L. Penland,et al.  Determinism and stochasticity during maturation of the zebrafish antibody repertoire , 2011, Proceedings of the National Academy of Sciences.

[17]  Michal Barak,et al.  Somatic hypermutation and antigen-driven selection of B cells are altered in autoimmune diseases. , 2010, Journal of autoimmunity.

[18]  Seung Hyun Kang,et al.  Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells , 2010, Nature Biotechnology.

[19]  P. Lipsky,et al.  Secondary Immunization Generates Clonally Related Antigen-Specific Plasma Cells and Memory B Cells , 2010, The Journal of Immunology.

[20]  David Kipling,et al.  High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations. , 2010, Blood.

[21]  T. Rogosch,et al.  IgE Transcripts in the Circulation of Allergic Children Reflect a Classical Antigen-Driven B Cell Response and Not a Superantigen-Like Activation , 2010, The Journal of Immunology.

[22]  P. Molenaar,et al.  The auto-antigen repertoire in myasthenia gravis , 2010, Autoimmunity.

[23]  T. Rogosch,et al.  Plasma Cells and Nonplasma B Cells Express Differing IgE Repertoires in Allergic Sensitization , 2010, The Journal of Immunology.

[24]  S. Spicuglia,et al.  Epigenetic regulation of antigen receptor gene rearrangement , 2010, F1000 biology reports.

[25]  Yoshihiro Baba,et al.  B cell signaling and fate decision. , 2010, Annual review of immunology.

[26]  Jan Berka,et al.  Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire , 2009, Proceedings of the National Academy of Sciences.

[27]  Haruki Nakamura,et al.  Structural classification of CDR‐H3 revisited: A lesson in antibody modeling , 2008, Proteins.

[28]  Marie-Paule Lefranc,et al.  IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis , 2008, Nucleic Acids Res..

[29]  Uri Hershberg,et al.  Improved methods for detecting selection by mutation analysis of Ig V region sequences. , 2008, International immunology.

[30]  R. Schelonka,et al.  Categorical selection of the antibody repertoire in splenic B cells , 2007, European journal of immunology.

[31]  C. Berek,et al.  The Postnatal Maturation of the Immunoglobulin Heavy Chain IgG Repertoire in Human Preterm Neonates Is Slower than in Term Neonates1 , 2007, The Journal of Immunology.

[32]  K. Rajewsky,et al.  Forced usage of positively charged amino acids in immunoglobulin CDR-H3 impairs B cell development and antibody production , 2006, The Journal of experimental medicine.

[33]  D. Nott,et al.  Antigen selection in the IgE response of allergic and nonallergic individuals. , 2006, The Journal of allergy and clinical immunology.

[34]  P. Lipsky,et al.  Developmental Changes in the Human Heavy Chain CDR3 , 2005, The Journal of Immunology.

[35]  S. Durham,et al.  Biased use of VH5 IgE-positive B cells in the nasal mucosa in allergic rhinitis. , 2005, The Journal of allergy and clinical immunology.

[36]  P. Lipsky,et al.  Molecular basis of immunoglobulin variable region gene usage in systemic autoimmunity , 2005, Clinical and Experimental Medicine.

[37]  Marie-Paule Lefranc,et al.  IMGT, the international ImMunoGeneTics information system® , 2004, Nucleic Acids Res..

[38]  D. Rossi,et al.  Human fetal, cord blood, and adult lymphocyte progenitors have similar potential for generating B cells with a diverse immunoglobulin repertoire. , 2004, Blood.

[39]  J. Engler,et al.  Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures. , 2003, Journal of molecular biology.

[40]  S. L. Bridges,et al.  Contribution of Vh gene replacement to the primary B cell repertoire. , 2003, Immunity.

[41]  Andrew C. R. Martin,et al.  Analysis of the antigen combining site: correlations between length and sequence composition of the hypervariable loops and the nature of the antigen. , 2003, Journal of molecular biology.

[42]  J. Marchalonis,et al.  Incorporation of Long CDR3s into V Domains: Implications for the Structural Evolution of the Antibody-Combining Site , 2002, Experimental and Clinical Immunogenetics.

[43]  H. Schroeder,et al.  Slow, programmed maturation of the immunoglobulin HCDR3 repertoire during the third trimester of fetal life. , 2001, Blood.

[44]  R. Tarone,et al.  Third complementarity‐determining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than non‐mutated genes , 2001, Immunology.

[45]  H. Versmold,et al.  The diversity of rearranged immunoglobulin heavy chain variable region genes in peripheral blood B cells of preterm infants is restricted by short third complementarity-determining regions but not by limited gene segment usage. , 2001, Blood.

[46]  R. Tibshirani,et al.  The Inference of Antigen Selection on Ig Genes1 , 2000, The Journal of Immunology.

[47]  J. Xu,et al.  Diversity in the CDR3 region of V(H) is sufficient for most antibody specificities. , 2000, Immunity.

[48]  Haruki Nakamura,et al.  H3‐rules: identification of CDR‐H3 structures in antibodies , 1999, FEBS letters.

[49]  S. Holgate,et al.  Clonally related IgE and IgG4 transcripts in blood lymphocytes of patients with asthma reveal differing patterns of somatic mutation , 1998, European journal of immunology.

[50]  P. Lipsky,et al.  Immunoglobulin kappa chain receptor editing in systemic lupus erythematosus. , 1998, The Journal of clinical investigation.

[51]  R. Brezinschek,et al.  Delineation of selective influences shaping the mutated expressed human Ig heavy chain repertoire. , 1998, Journal of immunology.

[52]  A Tramontano,et al.  Conformations of the third hypervariable region in the VH domain of immunoglobulins. , 1998, Journal of molecular biology.

[53]  C E Shannon,et al.  The mathematical theory of communication. 1963. , 1997, M.D. computing : computers in medical practice.

[54]  R. Brezinschek,et al.  Analysis of the human VH gene repertoire. Differential effects of selection and somatic hypermutation on human peripheral CD5(+)/IgM+ and CD5(-)/IgM+ B cells. , 1997, The Journal of clinical investigation.

[55]  Haruki Nakamura,et al.  Structural classification of CDR‐H3 in antibodies , 1996, FEBS letters.

[56]  M. Radic,et al.  Receptor editing, immune diversification, and self-tolerance. , 1996, Immunity.

[57]  T. Marion,et al.  Correlation between the amino acid position of arginine in VH-CDR3 and specificity for native DNA among autoimmune antibodies. , 1996, Journal of immunology.

[58]  K. Rajewsky.  Clonal selection and learning in the antibody system , 1996, Nature.

[59]  A. Feeney,et al.  A defective Vkappa A2 allele in Navajos which may play a role in increased susceptibility to haemophilus influenzae type b disease. , 1996, The Journal of clinical investigation.

[60]  M. Zouali.  B-cell superantigens: implications for selection of the human antibody repertoire. , 1995, Immunology today.

[61]  P. Casali,et al.  The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement. , 1994, Immunology today.

[62]  E. Padlan,et al.  Anatomy of the antibody molecule. , 1994, Molecular immunology.

[63]  C. Tonnelle,et al.  Mechanisms that generate human immunoglobulin diversity operate from the 8th week of gestation in fetal liver , 1993, European journal of immunology.

[64]  E. Kabat,et al.  Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. , 1991, Journal of immunology.

[65]  K. Rajewsky,et al.  B cell development regulated by gene rearrangement: Arrest of maturation by membrane-bound Dμ protein and selection of DH element reading frames , 1991, Cell.

[66]  Y. Kurosawa,et al.  Only dfl16, dsp2, and dq52 gene families exist in mouse immunoglobulin heavy chain diversity gene loci, of which dfl16 and dsp2 originate from the same primordial dh gene , 1989, European journal of immunology.

[67]  E. Sasso,et al.  Human IgM molecules that bind staphylococcal protein A contain VHIII H chains. , 1989, Journal of immunology.

[68]  R. Perlmutter,et al.  Early restriction of the human antibody repertoire. , 1987, Science.

[69]  White,et al.  Precise determination of , 1986, Physical review letters.

[70]  G. M. Griffiths,et al.  Molecular events during maturation of the immune response to oxazolone , 1985, Nature.

[71]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[72]  S. Tonegawa,et al.  Somatic generation of antibody diversity. , 1976, Nature.

[73]  Patrice Duroux,et al.  IMGT/HIGHV-QUEST: THE IMGT® WEB PORTAL FOR IMMUNOGLOBULIN (IG) OR ANTIBODY AND T CELL RECEPTOR (TR) ANALYSIS FROM NGS HIGH THROUGHPUT AND DEEP SEQUENCING , 2012 .

[74]  R. Schelonka,et al.  Genetic control of DH reading frame and its effect on B-cell development and antigen-specifc antibody production. , 2010, Critical reviews in immunology.

[75]  P. Lipsky,et al.  The lambda gene immunoglobulin repertoire of human neonatal B cells. , 2008, Molecular immunology.

[76]  K. Lager,et al.  Porcine Reproductive and Respiratory Syndrome Virus Subverts Repertoire Development by Proliferation of Germline-Encoded B Cells of All Isotypes Bearing Hydrophobic Heavy Chain CDR3 , 2008 .

[77]  Stephen R Durham,et al.  Class switch recombination to IgE in the bronchial mucosa of atopic and nonatopic patients with asthma. , 2007, The Journal of allergy and clinical immunology.

[78]  H. Schroeder.  Similarity and divergence in the development and expression of the mouse and human antibody repertoires. , 2006, Developmental and comparative immunology.

[79]  Sang Joon Kim,et al.  A Mathematical Theory of Communication , 2006 .

[80]  Quality Antibodies·,et al.  Recombinant Antibodies , 2001 .

[81]  George Johnson,et al.  Kabat Database and its applications: 30 years after the first variability plot , 2000, Nucleic Acids Res..

[82]  D. Eisenberg.  Three-dimensional structure of membrane and surface proteins. , 1984, Annual review of biochemistry.

[83]  Seiler Fr.  [Structure and function of immunoglobulins]. , 1982, Beitrage zu Infusionstherapie und klinische Ernahrung.