Two major challenges have made studying individualized immunorepertoire very difficult: (1) Too many B or T cells: In addition to VDJ recombination, mechanisms such as somatic hypermutation and N region addition contribute to the level of diversity. Each B or T cell clone thus has a unique VDJ that could respond to a specific antigen. Overall, humans are capable of generating 25 million different kinds of B or T cells. However, no one has accurately measured how many different B or T cells are actually present in an individual. (2) Sampling difficulty: A 10 cc peripheral blood sample may include 5 million and 20 million B and T cells respectively. Among this sample, each particular VDJ will be represented by only a few cells. The signal of our study is the particular VDJs and their distribution; however, the noise from the genomic DNA and cellular RNA is tremendous.
Therefore, for studying the individualized immunorepertoire comprehensively, a method to amplify the VDJ signal from the limited amount of sample is crucial. Even more challenging is the requirement for such an amplification to be inclusive and semi-quantitative, so that the samples original VDJ diversity and distribution can be evaluated without any bias associated with the amplification process.
We have successfully developed a technology for studying an individualized immunorepertoire. our method combines advanced technologies in immunology (monoclonal antibody, flow sorting), molecular biology (multiplex PCR and high throughput sequencing), and bioinformatics (individualized immunorepertoire distribution map and database) to study the diversity and distribution of immunoglobulin superfamily molecules (IgSF, including B and T cell receptors VDJ rearrangements and MHC class I and II genes).
We have developed a multiplex PCR technology, called amplicon rescued multiplex PCR (or arm-PCR, patent pending), that uses hundreds of VDJ specific primers in one reaction, semi-quantitatively amplifying all the expressed VDJs in B and T cells from an RNA sample. In a parallel reaction, the genomic DNA from the MHC loci are also amplified with arm-PCR for sequencing. To conduct the study B and T cells as well as subsets of B and T cells (such as naive, mature and memory B and T cells, and Tc, Th, Tnk, Treg etc) are obtained by flow cytometry or magnetic-bead purification methods from peripheral blood, and RNA is extracted from each of the cell populations. The VDJs are amplified by reverse transcription PCR with our arm-PCR method. The amplicon mixture is then subjected to high-throughput sequencing with the next-generation Roche 454 sequencing platform.
Disease as a phenotype, is determined by both genetic and environmental factors. By studying mutations and polymorphism, the classical genetic approach is very efficient to identify those phenotypic changes directly linked to specific mutations. However it is less powerful to isolate environmental influences.
Immunorepertoire studies, on the other hand, provided us a good opportunity to evaluate environmental impact. Immunorepertoire analysis can be used to uncover possible associations between common diseases and abnormal immunorepertoires, to evaluate vaccine efficiency, to identify new biomarkers, and to develop new diagnostics and therapeutics.
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