RIP 24Feb2016v4

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

Thiago DetanicoWysocki Lab

The Germinal Center ReactionBone Marrow

Thymus

LN/ SpleenB cells

CD4T cells T-B border

Germinal center,affi nity maturation andsomatic hypermutation

Plasma cells

Talk Outline• Somatic 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 Erythematosus

• Is a systemic autoimmune disease.

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

The Origin of Autoimmune B cells

Development in BM

B cells “born” with autoreactive BCR participate in T-cell dependent autoantibody responses.

Autoreactive B celltraverses all tolerancecheckpoints

“Germline founder hypothesis”

V/D/J recombination

The Origin of Autoimmune B cells“Mutation founder hypothesis”

NonautoreactiveB cell stimulated byimmunogen

Somatichypermutation { Autoreactive B

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

XMutation createsautoreactivity

Predictions of mutation founder hypothesis

B 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 eliminateautoreactivity

Unmutated precursor

Additional mutationsmay enhance affinityfor self-Ag

Autoreactive lineage (clone)

3; 2r 3; 1r1r

J7.13J2.8

17; 15r

J10.13

J3.8J7.18

J1.4J4.2

J5.5J6.1

6; 5r8; 5r

11; 5r6; 3r

34; 20r

16; 12r

+7 individuals

Six lineages, each with unanimously-shared somatic mutations.

Dendrograms of lineages

9;7r 11; 8r

+ 1 individual

Guo et al, J. Exp. Med. 2010

Supporting evidence for the “Mutation Founder Hypothesis”

• In 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.

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

born autoreactive

Guo et al, J. Exp. Med. 2010

Mutated mAbs

Revertant mAbs “R”

Supporting evidence for the “Mutation Founder Hypothesis”

• AID deficiency (no somatic hypermutation) severely delays the development of anti-nuclear antibodies in the B6.Nba2 lupus-prone mouse.

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

anti-nucleosome Abs

Anti-Nucleosome

Detanico 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.

BackgroundIs the creation of an ANA clone by SHM

a 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 GArg codons

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.

VH Vk VlVH Vk Vl

Mouse germline IgV genesAGY Observed/Expected

Human germline IgV genesAGY Observed/Expected

..............................................................................................

_____ _______________ __________

Expected Freq.= 0.016 or 1/61(random codon usage)

Expected Freq. from codonuse tables

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).

H. sapiens germline IgV-CDR sequences

VH Vk Vl

AGYTCN

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.

M. musculus germline IgV-CDR sequences

VH Vk Vl

TCNAGY

H. sapiens IgV genes

8 *** p=0.024

VH Vk Vl

M. musculus IgV genes

******

p=0.157

VH Vk VlO

Ser-AGY Ser-TCN

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

CDR sequences.

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

CDR sequences.

H. sapiens

Vln=39

VHn=54

Vkn=46

n=44 n=36 n=22

VHn=108

Vkn=95

n=70 n=56 n=3

M. musculus

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.

8p=0.0004

TCNAGY TCNAGY

Cartilaginous fishes IgVH CDR1&2

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.

TCRV TCRV0

8AGYTCN

p=0.08 ***

TCRV TCRV

M. musculus TCRV CDR1&2

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.

NNAGYNN

CDR start: Ser-AGY

CDR start: nonSer-AGY

NNAGYNN

CDR end: Ser-AGY

CDR end: nonSer-AGY

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.

*** ***p=0.002

VH Vk Vl

*** ***

VH Vk Vl

Ser-AGC nonSer-AGC

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

******

VH Vk Vl

p=0.47 ***

VH Vk Vl

p=0.02

Ser-AGT nonSer-AGT

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.

NNNAGCTNN

CDR start: Ser-AGCT

CDR start: nonSer-AGCT

CDR start: not counted

NNNAGCTNN

CDR end: Ser-AGCT

CDR end: nonSer-AGCT

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

*** p=0.0258

VH Vk Vl

p=0.002

p=0.346

VH Vk Vl

Ser-AGCT nonSer-AGCT

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.

Rationale

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

Background

• AGY 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/

n=49 n=49

Any Arg Ser ArgHIV bNAbs

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.

0.00.20.40.60.81.0

510152025

Total IgV replacements (CDRs & FRs)Total Arg by SHM (CDRs & FRs)AGY-Ser to Arg (CDRs only)

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.

Viruses Asn Gly Thr Arg Others #mut CDR-AGY

Influenza1 22% 11% 21% 10% 36% 146

Influenza2 30% 11% 25% 16% 18% 126

West Nile 20% 0% 60% 20% 0% 5

Dengue 14% 0% 43% 29% 14% 7

Rhinovirus 7% 0% 15% 26% 52% 27

Avian Influenza 50% 0% 17% 33% 0% 13

Hep. A, B & C 22% 18% 18% 20% 22% 72

Average 23.6% 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.

Raghunathan 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)GY A(X)Y AG(X) 2 changes 3 changes #mut CDR-AGY

Influenza1 12% 53% 11% 20% 4% 146

Influenza2 11% 52% 15% 20% 2% 126

West Nile 0% 80% 20% 0% 0% 5

Dengue 15% 57% 14% 14% 0% 7

Rhinovirus 0% 22% 19% 52% 7% 27

Avian Influenza 0% 67% 33% 0% 0% 13

Hep. A, B & C 12% 35% 18% 35% 0% 72

Average 7.1% 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.

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.

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

TyrArg

AGY Ser

Acknowledgments

Wyscoki Lab:• Larry Wysocki• Katja Aviszus

Kappler/Marrack Lab:• Mathew Phillips

Funding:R01AI093822R21AI113122

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

Germline-encoded CDR1&2 amino acid usage (IgV genes average)

Asn Ly

Arg Va

Asp Glu

Phe Le

Cys Trp

Pro His

Gln Ile

HumanMouse

AGY-Ser is the predominant contact residue target during SHM

Antibody-Antigen Contact Residues (H+L)

Ile-->

M. musculusH. sapiens

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

Function Mutability Index

Asp Pro

Cys Trp

Glu Ile Val

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)F1

Age (weeks)

Jacks et al, Current Biology 1994 Ishida et al, J Exp Med 1994

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 mutations

tumor growth/survival

Hypothesis:

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

The Stochastic Event Model

non-autoimmune B cells

autoimmune B cell

tolerance checkpoints

autoimmune B cell is eliminated from the B cell repertory.

autoimmune B cell

tolerance checkpoints

somatic mutationsin regulatory pathways

survival/growth of autoimmune B cells

non-autoimmune B cells

The 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-chain Autoreactive light-chain

B 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.

NEO VkJkCk-IRES-mCherry VkJkCk-IRES-GFPL

FRT FRT

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

mCherry GFP

B220+ gate

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

wt KS+Neo+ KS+FLP+

Acknowledgments

Wyscoki Lab:• Larry Wysocki• Katja Aviszus

Kappler/Marrack Lab:• Mathew Phillips

Funding:R01AI093822R21AI113122

RIP 24Feb2016v4

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