Affinities in the immune repertoire

Thomas Dupic

CIML, October 2023

Background

  • PhD [Y. Ikhlef and B. Estienne] Conformal Field Theory applied to statistical models
  • Post-Doc [A. Walczak and T. Mora] Statistical properties of the immune repertoire
  • Post-Doc [M. Desai] Binding affinity landscapes of antibodies

Immune repertoire

B-cells
V(D)J recombination
T-cells
B-cell and T-cell receptors share:
  • a similar protein structure
  • near identical generation mechanisms
  • extremely high diversity

    • Immune repertoire diversity

    • Immune repertoires contain more than 10⁹ unique B and T-cell receptors
    • Only a small fraction of these receptors are shared, most are unique
    • This diversity influences the effectiveness of the immune response .
    Dupic et al., PLoS Genetics, 2021

    Immune repertoire diversity

    How is immune receptor diversity generated and regulated?

    What is its impact on immune response?

    To what extent can we predict immune system reactions?

    Outline

    • I. Correlation between α and β chains during T-cell receptor rearrangement
    • II. Evolution of a broadly neutralising antibody
    • III. Affinities in the immune repertoire

    V(D)J recombination

    T-cell receptor diversity is created during T-cell development.

    T-cell receptors are formed by two chains, α and β, both generated by V(D)J recombination.

    V(D)J recombination is well understood statistically, and each chain can be represented by a small number of parameters .
    Germline
    TCRα gene
    V allele
    J allele
    delV
    delJ
    insVJ
    Marcou et al., Nat Comm., 2018
    Sethna, …, Dupic et al., PLoS Comp Bio, 2020

    Multi-TCR T-cells

    T-cells can have more than one receptor.
    α chain
    β chain
    Chromosome #1
    Chromosome #2
    But only one-third of all cells have two functional receptors and nearly none have four.
    • V(D)J recombination often fails.
    • Allelic exclusion on the β chain.
    Petrie et al., JEM, 1993
    Brady et al., J Immunol., 2010

    T-cell Receptor generation

    How much do these different recombination process interact with each other ?
    Dataset with 10⁷ paired sequences:
    • Infer back their recombination process
    • Extract out the effects of thymic selection + clonal expansion
    • Infer correlations
    Howie et al., Sci. Transl. Med., 2015

    Correlation between chains


    β and α chains are largely independent from one another.



    Strong correlation between the two α chains.
    Intra-chain
    Inter-chains
    Dupic et al., Plos Comp. Bio, 2019

    Rescue mechanism

    Dilution of specificity

    For specific antigens, the α and β chains need to be strongly correlated.
    But only a very small fraction of the repertoire reacts to a specific antigen, the "interesting" signal is drowned in the noise.
    Sethna,…, Dupic et al., PLoS Comp Bio, 2020

    Outline

    • I. Correlation between α and β chains during T-cell receptor rearrangement
    • II. Evolution of a broadly neutralising antibody
    • III. Affinities in the immune repertoire

    B-cell affinity maturation

    • B-cell receptors can mutate post-rearrangement.
    • Darwinian selection against specific antigens.
    • Can we infer what were the selection pressure from the end result ?

    Broadly neutralising antibodies

    Broadly neutralizing antibodies offer protection against many variants of a virus but are rarely generated by the immune system.

    How did they evolve ?
    CR9114 and CR6261
    Throsby et al., Plos One, 2008
    Dreyfus et al., Science, 2012

    Combinatorial exploration

    We recreated all potential intermediate sequences between germline and somatic and measured their affinity toward three antigens H1, H3 , fluB

    Adams et al., eLife, 2016
    Phillips,…, et al., eLife, 2021

    CR9114

    Histogram

    Force-Directed Layout
    65536 measurements

    Sequential exposure

    Phillips,…, Dupic et al., eLife, 2021
    Phillips,…, Dupic et al., eLife, 2022

    Conclusion

    Affinity measurements can reveal an antibody's history.
    They go beyond structural data.
    Future directions: in-vitro evolution.
    Generations
    Generations
    Frequency

    Binding affinity in proteins

    How did SARS-CoV-2 variant Omicron emerge ? 30'000+ Spike protein variants tested against cell receptors and antibodies.

    Measurement of binding affinity of germinal center antibodies.
    Moulana*, Dupic* et al., Nat Comm, 2022
    Moulana*, Dupic* et al., eLife, 2023
    Phillips,…,Dupic, eLife, 2023

    Outline

    • I. Correlation between α and β chains during T-cell receptor rearrangement
    • II. Evolution of a broadly neutralising antibody
    • III. Affinities in the immune repertoire

    Project overview

    Immune receptors are crucial for adaptive immunity but their diversity make them challenging to study.

    • Develop tools, both experimental (high-throughput affinity measurements) and theoretical (statistical modelling) to help us study the immune repertoire.
    • Understand the functional diversity of immune repertoires and its impact on immune response.
    • Explore the impact of receptor affinity differences on cell behavior.
    High-Throughput affinity measurements
    1. Select sequences in-silico.
    2. Library creation
      templated V-genes + oligo-pools.
    3. Yeast-display
      single-chain variable fragment (scFv) format.
    4. Fluorescence-Activated Cell Sorting (FACS).
    5. Sequencing of each well.
    6. Repeat for different concentrations of antigens.
    Adams et al., eLife, 2016

    Caveats

    High precision on binding affinity but:
    • Yeast expression pattern / glycosylation
    • Proteins are messy
    T-cells can also be expressed in scFv format.
    Low affinity ⇒ tetramer MHC
    Smith et al., Methods Mol. Biol., 2015
    Altman et al., Curr. Protoc. Immunol., 2016

    B-cell affinity

    Antibody affinity spans 6 orders of magnitude; differences in affinity influence qualitatively B-cell behaviour.

    But current measurements of affinity are low-throughput or binary.

    Can we measure the functional diversity of the B-cell response ?

    Activated B-cells

    activated B-cell are B-cells that have encountered their specific antigen.
    Antigen-reacting cells (flow-cytometry)
    (1‰, 1% of B-cells)
    Cells with activated phenotypes (scRNA-seq)
    (20% of B-cells)
    Expanding clones (bulk sequencing)
    (10% of B-cells)
    Hartley et al., Science, 2020
    Stephenson et al., Nat. Med., 2021
    Horns et al., Cell Reports, 2020

    SARS-CoV-2 vaccination

    • Rapidly evolving virus
    • Important proportion of immunologically naïve individuals (3 years ago)
    • RNA vaccine: one, well-characterized antigen
    • Massive amount of sequencing and characterisation
    Scheid et al., Cell, 2021
    Montague et al., Cell Reports, 2021
    Stephenson et al., Nat. Med., 2021
    Scheid et al., Cell, 2021
    Kotagiri et al., Cell Reports, 2022
    Kim et al., Nature, 2022
    Gregoire et al., Immunity, 2022
    Ortega et al., PLOS Genetics, 2023
    Tong et al., Cell, 2021
    ...

    Better epitopes

    Epitopes play an important role in viral evolution.

    Defined via antibody panel and characterized structurally.

    Yeast-display experiments can test a broader range of antibodies and define these families more precisely.
    Moulana, Dupic et al., Nat. Comm., 2022
    Barnes et al., Cell, 2020

    Measuring T-cell Affinity

    • T-cell receptors generally have lower affinity than antibodies
    • They depend on MHC presentation (hence on HLA type)
    • Collective effects play a larger role: Quorum Sensing

    Neoantigens

    Neoantigen are new or mutated proteins produced by cancer cells that can act as target for the immune system.
    In pancreatic cancer repertoire-neoantigen correspondence seems to be able to predict immunoediting as well as immunotherapy effectiveness.
    Łuksza et al., Nature, 2022
    Barnes et al., Cell, 2020

    Predicting T-cell / peptide affinity

    T-cells receptors / peptide-MHC interaction is simpler than antibody/antigen. But models lack quantitative data.
    Collaboration with computational biologists and physicists (Deep Neural Network, Large Language Models, Restricted Boltzmann Machine)
    Sethna,…,Dupic et al., Plos Comp. Bio, 2020
    Bravi et al., Plos Comp. Bio, 2021
    Shugai et al., Nucleic Acids Res., 2018

    Conclusion

    Quantitative approach with a different experimental toolset.

    • To what extent do receptors' affinities influence cell phenotype, immune response and immunoprotection ?
    • What is needed to predict receptor affinity in-silico ?
    • How much functional variation is there between repertoires ?


    Thanks!