A Stochastic Event in the Development of Anti-Nuclear Antibodies in Systemic Autoimmunity

The role of somatic hypermutation and AGY serine codons in (auto)immunity

Thiago DetanicoWysocki Lab

The Germinal Center Reaction

Talk OutlineSomatic hypermutation plays a predominant role in generating anti-nucleosome antibodies.

IgV genes are prone to acquire CDR-Arg mutations and autoreactivity.

AGY Ser codons are plastic and can easily mutate to other key antigen-contact residues.

Systemic Lupus ErythematosusIs a systemic autoimmune disease.

Is characterized by the presence of high-avidity, IgG anti-nuclear antibodies, especially nucleosome (breach in B cell tolerance).

Insert slide with why nucleos talk abouy tg stuff and when do they brake tolerance they look like tdependent responses and binding to the nucleos4

The Origin of Autoimmune B cells

Development in BMB cells born with autoreactive BCR participate in T-cell dependent autoantibody responses.Autoreactive B celltraverses all tolerancecheckpointsGermline founder hypothesisV/D/J recombination

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The Origin of Autoimmune B cellsMutation founder hypothesisNonautoreactiveB cell stimulated byimmunogen

Somatichypermutation{Autoreactive B cell

Somatic hypermutation creates the autoreactive B cell in a germinal center from a previously normal B cell(Escapes late tolerance checkpoint ?)

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XMutation createsautoreactivity

XXX

Predictions of mutation founder hypothesisB cells (hybridomas) belonging to a common lineage should share at least one somatic mutation producing an amino acid change conferring autoreactivity

Reverting ALL somatic mutations to germline sequence should eliminateautoreactivityUnmutated precursorAdditional mutationsmay enhance affinityfor self-AgXXXAutoreactive lineage (clone)XXX

Mixed and not entirely satisfactory resultsSeveral groups have addressedwith mixed and not entirely satisfactoryThomas Winklers groupThe problem has always been identifying mutations unambiguously

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3+7 individualsSix lineages, each with unanimously-shared somatic mutations.

Dendrograms of lineages

9;7r 11; 8r12,9r

N3.186

N12.6+ 1 individualGuo et al, J. Exp. Med. 2010

8Keep in mind that there are probably more members because we sequenced only about half of recovered hybridomas.This illustrates universally shared mutationsHere is summary of sequence data. Based on shared mutation, we build lineage tree. 15 hybridomas in one mouse derived from one clone, they share15 mutations, indicate they derived the same mutated

Supporting evidence for the Mutation Founder HypothesisIn a system where every somatic mutation could be identified (TdT-/-), we have shown that the majority of anti-chromatin antibodies were generated in the periphery.

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Only two out of thirty-three auto-Abs were derived from a B cell that was born autoreactive

Guo et al, J. Exp. Med. 2010Mutated mAbs

Revertant mAbs R

Supporting evidence for the Mutation Founder HypothesisAID deficiency (no somatic hypermutation) severely delays the development of anti-nuclear antibodies in the B6.Nba2 lupus-prone mouse.

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Only 2 of 8 B6.Nba2 AID-/- mice develop high-titers of the prototypical anti-nucleosome Abs

Anti-NucleosomeDetanico et al, J. Autoimm. 2015

Summary I

In a system where every somatic mutation could be identified (Tdt -/-), we have shown that reversion of somatic mutations to a germline sequence, eliminated detectable autoreactivity in a majority of clones.

In an autoimmune mouse model, where no somatic hypermutation was observed (AID deficiency), we found that anti-nuclear responses were delayed and severely diminished.

Together these support the view that most IgG+ anti-nuclear clones arise from nonautoreactive precursors via somatic hypermutation.

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Talk OutlineSomatic hypermutation plays a predominant role in generating anti-nucleosome antibodies.

IgV genes are prone to acquire CDR-Arg mutations and autoreactivity.

AGY Ser codons are plastic and can easily mutate to other key antigen-contact residues.

BackgroundIs the creation of an ANA clone by SHMa rare or frequent event?

Arginine residues contribute substantially to antibody affinity for nuclear antigens.

Arginine residues often arise by somatic hypermutation of AGC and AGT (AGY) serine codons.

AGY Ser codons are prone to mutate to Arg codons.

AGY Ser codons can mutate to Arg residues by three different nucleotide replacements. AGY Ser codons(X)GY=Arg, if X=C

AG(X)=Arg, if X=A or G

Arg codons

SHM

Do IgV genes have high frequencies of AGY Ser codons?Rationale: High frequencies of AGY codons may lead to a high frequency of somatically generated Arg residues and autoreactivity.

High frequencies of AGY Ser codons among IgV CDR sequences.

Do IgV genes have high frequencies of Ser codons?Rationale: If the high frequencies of AGY Ser codons use in CDRs were merely due to a selection pressure favoring Ser residues among germline-encoded CDR sequences, we would expect equally high frequencies of four other serine codons (TCN).

High frequencies of AGY, but not TCN Ser codons among germline-encoded human IgV-CDR sequences.

High frequencies of AGY, but not TCN Ser codons among germline-encoded mouse IgV-CDR sequences.

High frequencies of AGY over TCN Ser codons among germline-encoded IgV-CDR sequences.

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High frequencies of AGY over TCN Ser codons among germline-encoded IgV-CDR sequences.

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How early in evolution did the CDR AGY Ser codon bias appear? Approach: Analyze IgV-CDR sequences from cartilaginous fishes.

The AGY Ser bias was established early in evolution in IgVH-CDR.

Is the AGY Ser codon bias restricted to IgV genes? Approach: Compare mouse TCRV genes with IgV genes.

Mouse TCRV genes lack the AGY over TCN Ser codon CDR bias.

M. musculus TCRV CDR1&2

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Is CDR AGY abundance driven by selection pressure to focus somatic mutation on CDRs?Rationale: AGY triplets are a preferential AID target motif.

Approach: If AGY triplets were selected only on the basis of mutation then other possible reading-frames should also be enriched.

AGY triplets are enriched in the Ser reading-frame among CDR sequences.

Approach: If AGY triplets were selected only on the basis of mutation then other possible reading-frames should be enriched too.

AGC triplets are enriched in the Ser reading-frame among CDR sequences.

Move Ser-AGC and nonSer AGC up30

AGT triplets are also enriched in the Ser reading-frame among CDRs.

Is the highly mutable AGCT sequence also selectively enriched in the Ser reading frame?Rationale: AGCT sequence is often target by AID during somatic hypermutation.

Approach: If AGCT sequences were selected only on the basis of mutation then other possible reading-frames should be enriched too.

AGCT palindromic sequence is enriched in the Ser reading-frame among CDRs.Approach: If AGCT sequences were selected only on the basis of mutation then other possible reading-frames should be enriched too.

AGCT palindromic sequence is enriched in the Ser reading-frame among CDRs.

H. sapiens IgV genes M. musculus IgV genes

Summary IIEvidence for evolutionary selection pressure to have AGY Ser codons among IgV-CDR sequences

CDR, but not FR sequences from IgV genes have high frequencies of AGY Ser codons.

AGY triplets in IgV genes were enriched in the Ser codon reading-frame

This phenomenon is restricted to IgV genes (not seen in TCRV genes), and occurred early in evolution.

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Talk OutlineSomatic hypermutation plays a predominant role in generating anti-nucleosome antibodies.

IgV genes are prone to acquire CDR-Arg mutations and autoreactivity.

AGY Ser codons are plastic and can easily mutate to other key antigen-contact residues.

RationaleSeveral groups have shown that polyreactive/autoreactive Abs play an important role in immune responses to enveloped viruses.

BackgroundAGY Ser codons are enriched among IgV-CDR sequences and are a preferential target for AID.

AGY readily mutate to Arg codons.

CDR-Arg codons are often a signature of anti-nuclear antibodies.

HIV broadly neutralizing antibodies are often autoreactive.

Are mutations that create Arg codons associated with HIV broadly neutralizing antibodies?Approach: Analyze published HIV broadly neutralizing antibody sequences for the presence of somatically generated arginine codons.

Arg residues often arise by somatic mutation of AGY Ser codon in HIV broadly neutralizing Abs.*Database: http://bnaber.org/

Somatically generated Arg codon

Do somatically generated Arg residues frequently occur in antibody responses against other viruses?Approach: Analyze other available antibody sequences for the presence of somatically generated Arg residues.

Arg residues arise often by somatic hypermutation of CDR-AGY Ser codons.

Do AGY Ser codons preferentially mutate only to Arg codons in anti-viral Abs?Approach: Analyze available antibody sequences for the presence of replacement mutations in AGY Ser codons.

Germline-encoded AGY Ser codons often mutate into Asn, Arg, Gly and Thr during viral immune responses.

VirusesAsnGlyThrArgOthers#mut CDR-AGYInfluenza122%11%21%10%36%146Influenza230%11%25%16%18%126West Nile20%0%60%20%0%5Dengue14%0%43%29%14%7Rhinovirus7%0%15%26%52%27Avian Influenza50%0%17%33%0%13Hep. A, B & C22%18%18%20%22%72Average23.6%5.7%28.4%22%20%

Analysis of many crystal structures of antigen-antibody interactions have shown that 7 amino acids are often observed as contact residues.

Tyr

Arg

Asn

Asp

Gly

Ser

ThrRaghunathan G. at all, J. Mol. Recognition. 2012; 25:103-113

AGY Ser codons can easily mutate into 4 out of the 7 residues by one nucleotide change, or stay unchanged (5 out of 7). AGY SerA(X)Y=Asn, if X=A

A(X)Y=Thr, if X=C(X)GY=Gly, if X=G

(X)GY=Arg, if X=C

AG(X)=Arg, if X=G or A

AG(X)=Ser, if X=C or T

Preferential target for the 2nd and 3rd nucleotide bases in germline-encoded AGY Ser triplets.

Viruses(X)GYA(X)YAG(X)2 changes3 changes#mut CDR-AGYInfluenza112%53%11%20%4%146Influenza211%52%15%20%2%126West Nile0%80%20%0%0%5Dengue15%57%14%14%0%7Rhinovirus0%22%19%52%7%27Avian Influenza0%67%33%0%0%13Hep. A, B & C12%35%18%35%0%72Average7.1%52.3%18.6%20.1%1.9%

Summary IIIThe autoreactive potential of AGY Ser codons may be a small cost to pay for such plastic codons.

Somatic hypermutation of AGY Ser codons often creates Arg codons, but also creates Asn, Gly and Thr residues.

AGY Ser codons easily mutate into Arg, Asn, Gly and Thr codons by one nucleotide change.

Crystal structures of antibody-antigen interactions have shown that Arg, Asn, Asp, Gly, Ser, Thr and Tyr antibody residues are often contact residues.

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Conclusions

Anti-nuclear specificity arises frequently by somatic hypermutation of AGY Ser codons.

Autoreactivity may be the price of having such a plastic codon in the CDRs.

Sometimes weak autoreactivity may be beneficial to immune responses.

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Model: Mutational plasticity of AGY Ser codons supports affinity maturation.

Tyr

Arg

Asn

Asp

Gly

AGY Ser

Thr

Acknowledgments

Wyscoki Lab:Larry WysockiKatja Aviszus

Kappler/Marrack Lab:Mathew Phillips

Funding:R01AI093822R21AI113122

Key contact amino acids are often observed at germline-encoded IgV CDR sequences.

AGY-Ser is the predominant contact residue target during SHM

AGY-Ser and Asn easily mutate to polar residues by one nucleotide change.

Why autoimmunity is the exception?

Somatic hypermutation of the BCR cannot account solely to the stochastic nature of autoimmunity *We think that autoreactive B cells are often created by somatic hypermutation in the Germinal Center.

Similar kinetics between Tumor and Lupus-like models

p53-/-

(NZBxNZW)F1Age (weeks)Jacks et al, Current Biology 1994Ishida et al, J Exp Med 1994

P53 knock out and slide with acquiring several mutation order 4-757

The delayed kinetics in p53-/- is due to the requirement of several mutations in other genes prior to tumor clonal expansion.

non-tumor cells from p53-/-

1st mutation2nd mutation

several mutationstumor growth/survival

Hypothesis:To overcome the several tiers of tolerance, an autoimmune B cell must first acquire several mutations in immunoregulatory genes.

Make a slide with an autoimmune B cell and several checkpoints59

The Stochastic Event Model

non-autoimmune B cells

autoimmune B celltolerance checkpoints

autoimmune B cell is eliminated from the B cell repertory.

autoimmune B celltolerance checkpoints

somatic mutationsin regulatory pathwayssurvival/growth of autoimmune B cellsnon-autoimmune B cellsThe Stochastic Event Model

So how can we test it? Requires to isolate an anti-nuclear B cell.

Requires to expand a single anti-nuclear B cell.

Sequence the whole genome for mutations.

Caveat: So far we were unable to isolate an anti-nuclear B cell.

So we decided to develop a BCR heavy and light-chain knock in mouse, where we can track and control the fate of an anti-nuclear B cell.

The kappa switch (KS) knock in mouse Non autoreactive light-chainAutoreactive light-chainB cells are born with a non autoreactive light-chain, however upon Cre-expression, the autoreactive light-chain substitutes the non autoreactive light-chain, creating an anti-nuclear B cell when paired with a particular heavy-chain.

NEOVkJkCk-IRES-mCherryVkJkCk-IRES-GFP

L

FRTFRTLoxPLoxP

Surprisingly KS+ B cells express both reporter genes at low levels.

mCherryGFPB220+ gatewtKS+wtKS+

Excision of the NEO gene by FLP increases expression of reporter genes.

mCherryB220GFPwtKS+Neo+KS+FLP+

Acknowledgments

Wyscoki Lab:Larry WysockiKatja Aviszus

Kappler/Marrack Lab:Mathew Phillips

Funding:R01AI093822R21AI113122