Next generation sequencing (also known as deep sequencing) of antibody repertoires is being widely used now for basic repertoire studies and for many clinical studies also. The main approaches that are used are amplification of genomic DNA with a pool of V and J gene primers or amplification of cDNA. However, there are significant disadvantages of both. Amplification of cDNA has a major pitfall in that activated B cells produce far more immunoglobulin mRNA than resting B cells, so that most resting B cells in peripheral populations cannot be measured. For genomic DNA, a major advantage is that every cell has either 1 or 2 VhDJh rearrangements, and all rearrangements will be equally available for amplification, thus potentially permitting a truly quantitative reliable repertoire analysis. However, a significant bias is imposed on amplifications of rearrangements from DNA due to the necessity of using a pool of multiple V and J gene primers to attempt to amplify the many V regions. There are ~ 100 functional Vh and V? genes in the mouse, and in the human there are ~50 functional Vh genes, and ~45 functional V? genes, with significant polymorphism among the Vh genes. There is no location in either Vh or Jh that is identical in all V or J genes, respectively. Clearly, there are so many V genes that it is impossible to design a consensus V primer, even if one tries to design family- specific primers. In this proposal, we describe our approach to devise an innovative new technique to amplify genomic DNA in a completely unbiased manner. In our opinion, this will be a major improvement over all current techniques, and will be widely applicable for antibody repertoire studies in humans as well as mice, and especially useful for mixed populations of activated and resting B cells. Since next generation sequencing of antibodies has become an ever increasingly commonly used technique in clinical studies, this new technique will have wide significance.
Development of the optimal protocols to perform accurate deep sequencing of VDJ sequences from high throughput sequencing platforms will be of general use to all investigators interested in any area of antibody repertoire analyses. Importantly, once analyses have been made of the normal repertoire, this technology can be expanded to examine potential perturbations of the repertoire in disease states such autoimmunity (e.g., lupus, rheumatoid arthritis, diabetes), or to follow the fate of certain clonotypes (identified by CDR3 and V gene usage) following immunization or infection with a variety of pathogens/pathogenic antigens. This technology that we are developing will be highly useful to assess the antibody repertoire in populations of cells in which resting and activated B cells are both present, since they produce such vastly different levels of antibody mRNA.
| Kleiman, Eden; Loguercio, Salvatore; Feeney, Ann J (2018) Epigenetic Enhancer Marks and Transcription Factor Binding Influence V? Gene Rearrangement in Pre-B Cells and Pro-B Cells. Front Immunol 9:2074 |
