High throughput sequence analysis of the TCR repertoire in glioma-associated immune dysregulation. B. Grinshpun1, J. Sims2, Y. Feng1,5, P. Canoll3, P. Sims1,4, J. Bruce2, Y. Shen1,5 1) Columbia Initiative in Systems Biology, Columbia University, New York, NY; 2) Department of Neurological Surgery, Columbia University, New York, NY; 3) Department of Pathology and Cell Biology, Columbia University, New York, NY; 4) Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY; 5) Department of Biomedical Informatics, Columbia University, New York, NY.
The adaptive immune system plays both causal and reactionary roles in the diseases of humans and other vertebrates. While overreaction leads to autoimmunity and allergy, and insufficiency leads to infection and occurs concomitantly with the development of many cancers, these dysfunctions stem from the ability to recognize and respond to specific self and non-self antigens. Diversity in the binding specificity of T cell receptors (TCRs) is generated by recombination of germline-encoded V, D, and J cassettes and the addition of random nucleotides between them. We have utilized high-throughput sequencing, combined with commercially-available systems for amplification of the complementarity-determining region 3 (CDR3) of the TCR-alpha and TCR-beta chains from the T cells, to examine the sequence repertoires of whole T cell populations. We have developed a computational pipeline for mapping TCR cassettes, in silico translation, and error analysis, and applied these tools to the development of glioblastoma (GBM), the most common and deadly brain tumor. We quantitatively compare the diversity of T cell populations between subjects and between the periphery and tumor tissue, assess clonal expansions, and investigate the relative contributions of recombination vs. junctional nucleotide diversity on the properties of the repertoires. We are interested in quantifying CDR3 diversity as a way to assess immune potential, and measuring clonal expansion to identify tumor specific TCRs. Recent advances in read length of the paired-end the Illumina MiSeq platform, allow us use sequence length-dependent alignment to reduce and in most cases eliminate uncertainty in V,J cassette mapping while leveraging read pairs to correct for errors in sequencing. Furthermore, we applied methods from ecology theory and information theory to describe CDR3 population size and select sequences prominent in GBM tissue. Combined with the preprocessing pipeline described above, this allows us to not only identify CDR3 sequences associated with or expanded in the tumor, but also to provide metrics for the contribution of cassette usage to the diversity of the CDR3 population. Quantitative, sequence-based characterization of the whole TCR repertoire, as well as the ability to tie specific CDR3s to the functional changes observed among T cells during disease states, promises new insights into the nature of T cell dysregulation and potential targets for vaccine and immunotherapy.
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