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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
cell
Somatic hypermutation creates the autoreactive B cell in a germinal center from a previously normal B cell(Escapes late tolerance checkpoint ?)
XMutation createsautoreactivity
XX
X
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
X
XX
Autoreactive lineage (clone)
XX
X
3; 2r 3; 1r1r
J7.13J2.8
2
17; 15r
75
47
41 11
6
6
66*
4
2*33
J5.8
1
J2.7
J10.7
J10.13
J3.8J7.18
K5.14
J1.1
J3.47
J9.7
J3.1
K6.23
J1.4J4.2
4
7; 4r
J5.5J6.1
6; 5r8; 5r
5
J3.9
J3.10
11r
11; 5r6; 3r
34; 20r
16; 12r
6; 4r
J3.7
K6.18
3
+7 individuals
Six lineages, each with unanimously-shared somatic mutations.
Dendrograms of lineages
9;7r 11; 8r
12,9r
N3.18
6
N12.6
+ 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.
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.
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
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.
CD
R1&
2
FR1,
2&3
CD
R1&
2
FR1,
2&3
CD
R1&
2
FR1,
2&3
CD
R1&
2
FR1,
2&3
CD
R1&
2
FR1,
2&3
CD
R1&
2
FR1,
2&3
VH Vk VlVH Vk Vl
Mouse germline IgV genesAGY Observed/Expected
Human germline IgV genesAGY Observed/Expected
Rat
io
..............................................................................................
_____ _______________ __________
BA
Expected Freq.= 0.016 or 1/61(random codon usage)
Expected Freq. from codonuse tables
0
2
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
0
2
4
6
8
VH Vk Vl
AGYTCN
Rat
io (
Obs
erve
d/E
xpec
ted)
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
0
2
4
6
8
VH Vk Vl
TCNAGY
Rat
io (
Obs
erve
d/E
xpec
ted)
H. sapiens IgV genes
0
2
4
6
8 *** p=0.024
VH Vk Vl
***
Obs
erve
d / C
DR
1&2
M. musculus IgV genes
0
2
4
6
8
******
p=0.157
VH Vk VlO
bser
ved
/ CD
R1&
2
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.
-4-2
0
2
4
6
8
H. sapiens
Vln=39
VHn=54
Vkn=46
n=44 n=36 n=22
(AG
Y S
er)
- (T
CN
Ser
) / C
DR
1&2
-4-2
0
2
4
6
8
Vln=3
VHn=108
Vkn=95
n=70 n=56 n=3
M. musculus
(AG
Y S
er)
- (T
CN
Ser
) / C
DR
1&2
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.
0
2
4
6
8
Rat
io(O
bser
ved/
Exp
ecte
d)
0
2
4
6
8p=0.0004
Num
ber /
VH
CD
R1&
2
TCNAGY TCNAGY
Cartilaginous fishes IgVH CDR1&2
B
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
2
4
6
8AGYTCN
Rat
io (
Obs
erve
d/E
xpec
ted)
0
2
4
6
8AGYTCN
p=0.08 ***
TCRV TCRV
Num
ber
/ CD
R1&
2
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.
H. sapiens IgV genes
0
2
4
6
8
*** ***p=0.002
VH Vk Vl
Num
ber
/ CD
R1&
2
M. musculus IgV genes
0
2
4
6
8
*** ***
VH Vk Vl
p=1
Num
ber
/ CD
R1&
2
Ser-AGC nonSer-AGC
AGT triplets are also enriched in the Ser reading-frame among CDRs.
H. sapiens IgV genes
0
2
4
6
8
******
VH Vk Vl
***
Num
ber
/ CD
R1&
2
M. musculus IgV genes
0
2
4
6
8
p=0.47 ***
VH Vk Vl
p=0.02
Num
ber
/ CD
R1&
2
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.
0
1
2
3
4
*** p=0.0258
VH Vk Vl
p=0.002
Obs
erve
d / C
DR
1&2
0
1
2
3
4
***
p=0.346
VH Vk Vl
***
Obs
erve
d / C
DR
1&2
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.
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.
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
47
32
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.
Influ
enza
#1
Influ
enza
#2
Rhi
novi
rus
Avi
anIn
fluen
za
Wes
tNile
Den
gue
Hep
atiti
sA
, B&
C
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)
#of
amin
o ac
idre
plac
emen
ts/
germ
line-
enco
ded
IgV
gen
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.
Tyr
Arg
Asn
Asp
Gly
Ser
Thr
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
Asn
Asp
Gly
AGY Ser
Thr
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)
Met
Thr
Asn Ly
s
Ser
(AG
Y)
Ser
(Tot
al)
Arg Va
l
Ala
Asp Glu
Gly
Phe Le
u
Tyr
Cys Trp
Pro His
Gln Ile
0
2
4
6
8
Rat
io (
Obs
erve
d/E
xpec
ted)
HumanMouse
AGY-Ser is the predominant contact residue target during SHM
Antibody-Antigen Contact Residues (H+L)
Met
-->A
ny
Thr--
>Any
Asn
-->A
ny
Lys-
->A
ny
Ser
-->A
ny
Arg
-->A
ny
Val--
>Any
Ala
-->A
ny
Asp
-->A
ny
Glu
-->A
ny
Gly
-->A
ny
Phe
-->A
ny
Leu-
->A
ny
Tyr--
>Any
Cys
-->A
ny
Trp-
->A
ny
Pro
-->A
ny
His
-->A
ny
Gln
-->A
ny
Ile-->
Any
0
10
20
30
40
50
M. musculusH. sapiens
Ger
mlin
e co
don
x100
/To
tal S
HM
con
tact
s
AGY-Ser and Asn easily mutate to polar residues by one nucleotide change.
Function Mutability Index
Asn
Ser
(AG
Y)
Ala
Asp Pro
Cys Trp
Tyr
Gly
Arg
(CG
N)
Lys
Thr
Arg
(AG
R)
Gln
Glu Ile Val
Leu
(CTN
)
Met
Phe
Ser
(TC
N)
Leu
(TTR
)0
2
4
6
8
Rat
io [P
olar
/(no
nPol
ar+s
top)
]
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
LoxP
LoxP
Surprisingly KS+ B cells express both reporter genes at low levels.
mCherry GFP
B220+ gate
wt
KS+
wt
KS+
Excision of the NEO gene by FLP increases expression of reporter genes.
mCh
erry
B220
GFP
wt KS+Neo+ KS+FLP+
Acknowledgments
Wyscoki Lab:• Larry Wysocki• Katja Aviszus
Kappler/Marrack Lab:• Mathew Phillips
Funding:R01AI093822R21AI113122