Pecha Kucha: Integrating immune repertoire data for biomedical research and patient care Jamie K. Scott, MD, PhD Professor, MBB & FHS Simon Fraser University 10 April 2015 IRMACS Theatre B and T cells develop from hematopoietic stemcell precursors in the bone marrow “naïve” B cells and T cells differentiate into antibody-secreting plasma cells and effector T cells after stimulation by antigen/pathogen 2 B and T cells circulate among lymph nodes, scanning them for antigens. Fluids and cells from peripheral tissues drain into lymph nodes, bringing antigens/pathogens with them B and T cells circulate through lymph nodes scanning for antigens/pathogens to recognize and respond to. 3 B-cell and T-cell immune repertoires comprise cell-surface antibodies and T-cell receptors. Antibodies on different B cells and T-cell receptors on different T cells differ from each other in their antigen-binding sites. 4 Antibodies and T-cell receptors bind specifically to the antigens that initially selected them. 5 Combinatorial diversity plus imprecise joining create the vast genetic diversity within the antibody and T-cell receptor repertoires Front-end GFs Back-end GFs Gene fragments (GFs) encoding the front-end and back-end of a gene. In the developing B or T cell, different combinations of front- and back-end GFs pair up at random, with intervening DNA being excised. The final joint is also altered by “imprecise joining” in forming a full antibody or T-cell receptor gene. 6 Different recombination and joining events create clonal diversity among T and B cells 7 Antigen “selects” a B-cell or T-cell clone from an immune repertoire by binding to its antibody or T-cell receptor. A “naïve” immune repertoire of B or T cells. Clonal selection by pathogen/antigen. Clonal amplification and differentiation Into memory and effector cells. (Mutation of antibody genes) 8 Memory B and T cells produced after initial contact with antigen will respond to a new introduction of antigen. 9 The reason for booster shots: Clonal expansion of memory B and T cells leads to larger, faster responses on second exposure. 10 Lymph nodes collect antigens, while naïve and memory B and T cells circulate among them, scanning for antigens that bind their Ab &TcRs Once a B or T cell finds antigen, it will stay in the lymph node to differentiate into effector and memory cells. Effectors will leave and migrate to peripheral tissues where they will act, while memory cells will continue to scan the lymph nodes for antigen. 11 B-cell and T-cell repertoires are now being characterized by massive parallel sequencing. 12 Cancer treatment: Immune receptor deep sequencing is more sensitive than flow cytometry in detecting residual leukemia Red dots indicate leukemic T-cells 13 Vaccine Discovery: Reconstruction of the Evolution of an HIV-Neutralizing Antibody 14 Autoimmune Disease: Phylogenetic reconstruction identifies “cross-talk” between CNS lesions and draining lymph nodes in MS Stern et al. Science Transl. Med. 2014 15 Steps in Immune Repertoire Sequencing & Analysis: Getting the Data Obtain tissue (e.g., white blood cells) Protocols for obtaining tissue samples Sort Cellular Subsets Protocols for sorting cellular subsets mRNA -> cDNA -> PCR amplify Prepare immune receptor library Protocols for library preparation Sequence immune receptor library Standard primer sets Sequencing platforms and parameters 16 Steps in Immune Repertoire Analysis (cont.): Analysing the data Sequence quality control filters Minimum sequence length FASTQ quality score cutoffs Annotate germline V, D, J genes, junctions, somatic mutations (IMGT, IgBlast, SODA, JoinSolver, iHMMmune-Align, pRESTO, etc.) Germline gene reference set Ontology of gene regions Assignment of somatic mutations Infererence of new germline alleles Statistical uncertainty Analyze V-gene usage, clonal lineage frequency and expansion (VDJFasta, AbMining, Change-O, etc) Comparison to other immune repertoire data bases Definition and determination of clonal lineage Need for a common data-base format Bioinformatic platforms (iReceptor, VDJServer, GigaGen) Ethical/Privacy considerations Legal/IP considerations 17 iReceptor - A Distributed Data Management System for Storing and Comparing Immune Repertoires iReceptor is: A platform for integrating immune receptor data bases Distinguished by two main characteristics: - Distributed data base system - Enriched data sets associated with immune repertoire sequence data bases Based on iPlant – TACC Texas Advanced Computing Centre Uses Agave, a open-source scientific gateway webservice architecture. 18 iReceptor Architecture iReceptor Gateway Data Federation Data Exploration Analysis Results IReceptor Data Base Simon Fraser University IReceptor Data Base University of Toronto ... Annotation Tools IMGT V-Quest IgBlast ... IReceptor Public Data Base Common Repository Compute Canada Analysis Pipeline Tools VDJFasta AbMining ... (Compute Canada Resources) VDJServer Public Data Base Common Repository UT Southwest Medical Center ... 19 Community Meeting: Analysis, Storage and Sharing of NGS Data from Antibody and T-cell Receptor Repertoires Purpose: Bring together the researchers producing immune repertoires, legal and ethics experts, funding agencies, human-subject advocates, journal representatives, and others. Goal: Develop recommendations for standards and best practices for: (i) Production of immune repertoire NGS data and associated metadata; (ii) Data analysis and sharing (including software and platforms); (iii) Ethical, legal, and intellectual property considerations. Supported by: Canadian Institutes of Health Research (CIHR), US National Institutes of Health (NIH), The Antibody Society, International Society for Vaccines. SFU, the IRMACS Centre, SFU’s Faculty of Science, Dept. of Molecular Biology & Biochemistry, and Dept. of Biological Sciences, and GenMab. 20 Thank you! Happy Anniversaries to The IRMACS Center and SFU!