Pecha Kucha:

Integrating immune repertoire data for biomedical research and patient care

Jamie K. Scott, MD, PhDProfessor, MBB & FHS

Simon Fraser University

10 April 2015

IRMACS Theatre

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B and T cells develop from hematopoietic stem-cell 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

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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.

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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.

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Antibodies and T-cell receptors bind specifically to the antigens that initially selected them.

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Combinatorial diversity plus imprecise joining create the vast genetic diversity within the antibody and T-cell receptor repertoires

Gene fragments (GFs) encoding the front-end and back-end of a gene.

Front-end GFs Back-end GFs

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.

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Different recombination and joining events create clonal diversity among T and B cells

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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 differentiationInto memory and effector cells.(Mutation of antibody genes)

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Memory B and T cells produced after initial contact with antigen will respond to a new introduction of antigen.

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The reason for booster shots: Clonal expansion of memory B and T cells leads to larger, faster responses on second exposure.

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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.

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B-cell and T-cell repertoires are now being characterized by massive parallel sequencing.

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Cancer treatment: Immune receptor deep sequencing is more sensitive than flow

cytometry in detecting residual leukemia

Red dots indicate leukemic T-cells

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Vaccine Discovery: Reconstruction of the Evolution of an HIV-Neutralizing Antibody

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Autoimmune Disease: Phylogenetic reconstruction identifies “cross-talk” between CNS lesions and draining lymph nodes in MS

Stern et al. Science Transl. Med. 2014

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Steps in Immune Repertoire Sequencing & Analysis:Getting the Data

Obtain tissue (e.g., white blood cells)

Sort Cellular Subsets

mRNA -> cDNA -> PCR amplifyPrepare immune receptor library

Sequence immune receptor library

Protocols for sorting cellular subsets

Protocols for library preparation

Standard primer setsSequencing platforms and parameters

Protocols for obtaining tissue samples

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Steps in Immune Repertoire Analysis (cont.):Analysing the data

Analyze V-gene usage, clonal lineage frequency and expansion (VDJFasta, AbMining, Change-O, etc)

Need for a common data-base formatBioinformatic platforms(iReceptor, VDJServer, GigaGen)Ethical/Privacy considerationsLegal/IP considerations

Annotate germline V, D, J genes, junctions, somatic mutations(IMGT, IgBlast, SODA, JoinSolver, iHMMmune-Align, pRESTO, etc.)

Germline gene reference setOntology of gene regionsAssignment of somatic mutationsInfererence of new germline allelesStatistical uncertainty

Definition and determination of clonal lineage

Comparison to other immune repertoire data bases

Sequence quality control filters Minimum sequence lengthFASTQ quality score cutoffs

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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.

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iReceptor Architecture

iReceptor Gateway

Annotation ToolsIMGT V-Quest

IgBlast ...

Analysis Pipeline ToolsVDJFasta

AbMining ... (Compute Canada Resources)

...

...

Data FederationData ExplorationAnalysis Results

IReceptor Data BaseSimon Fraser University

IReceptor Data BaseUniversity of Toronto

IReceptor Public Data Base Common RepositoryCompute Canada

VDJServer Public Data BaseCommon RepositoryUT Southwest Medical Center

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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.

Thank you!

Happy Anniversaries to

The IRMACS Center

and SFU!