Discovering my research interest by eric garson sheffield university 2017
RIP 24Feb2016v4
1. The role of somatic hypermutation and
AGY serine codons in (auto)immunity
Thiago Detanico
Wysocki Lab
2. The Germinal Center Reaction
Bone Marrow
Thymus
LN/Spleen
B cells
CD4T cells
T-B border
Germinal center,
affinity maturation and
somatic hypermutation
Plasma cells
3. 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.
4. 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).
5. The Origin of Autoimmune B cells
Development in BM
B cells “born” with autoreactive BCR participate in T-cell
dependent autoantibody responses.
Autoreactive B cell
traverses all tolerance
checkpoints
“Germline founder hypothesis”
V/D/J recombination
6. The Origin of Autoimmune B cells
“Mutation founder hypothesis”
Nonautoreactive
B cell stimulated by
immunogen
Somatic
hypermutation { Autoreactive B
cell
Somatic hypermutation creates the autoreactive B cell in a
germinal center from a previously normal B cell
(Escapes late tolerance checkpoint ?)
7. XMutation creates
autoreactivity
X
X
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 eliminate
autoreactivity
Unmutated precursor
Additional mutations
may enhance affinity
for self-Ag
X
X
X
Autoreactive lineage (clone)
X
X
X
9. 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.
10. 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”
11. 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.
12. 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
13. 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.
14. 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.
15. Background
Is 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.
16. 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
17. 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.
18. High frequencies of AGY Ser codons
among IgV CDR sequences.
CDR1&2
FR1,2&3
CDR1&2
FR1,2&3
CDR1&2
FR1,2&3
CDR1&2
FR1,2&3
CDR1&2
FR1,2&3
CDR1&2
FR1,2&3
VH Vk VlVH Vk Vl
Mouse germline IgV genes
AGY Observed/Expected
Human germline IgV genes
AGY Observed/Expected
Ratio
..............................................................................................
_____ _______________ __________
BA
Expected Freq.= 0.016 or 1/61
(random codon usage)
Expected Freq. from codon
use tables
0
2
4
6
8
19. 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).
20. H . sa p ie n s g e rm line Ig V -C D R se q ue nce s
0
2
4
6
8
V H V k V l
A GY
TC N
Ratio(Observed/Expected)
High frequencies of AGY, but not TCN
Ser codons among germline-encoded
human IgV-CDR sequences.
21. High frequencies of AGY, but not TCN
Ser codons among germline-encoded
mouse IgV-CDR sequences.
M . m u s c u lu s g e rm line Ig V -C D R s e q ue nc e s
0
2
4
6
8
V H V k V l
TC N
A GY
Ratio(Observed/Expected)
22. H. sapiens IgV genes
0
2
4
6
8 *** p=0.024
VH Vk Vl
***
Observed/CDR1&2
M. musculus IgV genes
0
2
4
6
8
***
***
p=0.157
VH Vk Vl
Observed/CDR1&2
Ser-AGY Ser-TCN
High frequencies of AGY over TCN Ser
codons among germline-encoded IgV-
CDR sequences.
23. High frequencies of AGY over TCN Ser
codons among germline-encoded IgV-
CDR sequences.
-4
-2
0
2
4
6
8
H. sapiens
Vl
n=39
VH
n=54
Vk
n=46
n=44 n=36 n=22
(AGYSer)-(TCNSer)/CDR1&2
-4
-2
0
2
4
6
8
Vl
n=3
VH
n=108
Vk
n=95
n=70 n=56 n=3
M. musculus
(AGYSer)-(TCNSer)/CDR1&2
24. How early in evolution did the
CDR AGY Ser codon bias appear?
Approach: Analyze IgV-CDR sequences from cartilaginous fishes.
25. The AGY Ser bias was established early
in evolution in IgVH-CDR.
26. Is the AGY Ser codon bias
restricted to IgV genes?
Approach: Compare mouse TCRV genes with IgV genes.
27. Mouse TCRV genes lack the AGY over
TCN Ser codon CDR bias.
TCRV TCRV
0
2
4
6
8
A G Y
T C N
Ratio(Observ
0
2
4
6
8
AGY
TCN
p=0.08 ***
TCRV TCRV
Number/CDR1&2
M. musculus TCRV CDR1&2
28. 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.
29. 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.
30. 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
Number/CDR1&2
M. musculus IgV genes
0
2
4
6
8
*** ***
VH Vk Vl
p=1
Number/CDR1&2
Ser-AGC nonSer-AGC
31. AGT triplets are also enriched in the
Ser reading-frame among CDRs.
H. sapiens IgV genes
0
2
4
6
8
***
***
VH Vk Vl
***
Number/CDR1&2
M. musculus IgV genes
0
2
4
6
8
p=0.47
***
VH Vk Vl
p=0.02
Number/CDR1&2
Ser-AGT nonSer-AGT
32. 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.
33. 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
34. 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
Observed/CDR1&2
0
1
2
3
4
***
p=0.346
VH Vk Vl
***
Observed/CDR1&2
Ser-AGCT nonSer-AGCT
H. sapiens IgV genes M. musculus IgV genes
35. Summary II
Evidence 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.
36. 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.
37. Rationale
Several groups have shown that
polyreactive/autoreactive Abs play an important
role in immune responses to enveloped
viruses.
38. 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.
39. 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.
40. 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 Arg
HIV bNAbs
Somatically generated Arg codon
41. 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.
42. Arg residues arise often by somatic
hypermutation of CDR-AGY Ser codons.
Influenza#1
Influenza#2
Rhinovirus
AvianInfluenza
WestNile
Dengue
Hepatitis
A,B&C
0.0
0.2
0.4
0.6
0.8
1.0
5
10
15
20
25
Total IgV replacements (CDRs & FRs)
Total Arg by SHM (CDRs & FRs)
AGY-Ser to Arg (CDRs only)
#ofaminoacidreplacements/
germline-encodedIgVgene
43. 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.
44. 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%
45. 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
46. 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 Ser
A(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
47. 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%
48. Summary III
The 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.
49. 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.
52. 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
Lys
Ser(AGY)
Ser(Total)
Arg
Val
Ala
Asp
Glu
Gly
Phe
Leu
Tyr
Cys
Trp
Pro
His
Gln
Ile
0
2
4
6
8
Ratio(Observed/Expected)
Human
Mouse
53. AGY-Ser is the predominant
contact residue target during SHM
A n t ib o d y - A n t ig e n C o n t a c t R e s id u e s ( H + L )
Met-->Any
Thr-->Any
Asn-->Any
Lys-->Any
Ser-->Any
Arg-->Any
Val-->Any
Ala-->Any
Asp-->Any
Glu-->Any
Gly-->Any
Phe-->Any
Leu-->Any
Tyr-->Any
Cys-->Any
Trp-->Any
Pro-->Any
His-->Any
Gln-->Any
Ile-->Any
0
1 0
2 0
3 0
4 0
5 0
M . m u s c u lu s
H . s a p i e n s
Germlinecodonx1
TotalSHMcontac
54. AGY-Ser and Asn easily mutate to polar residues by
one nucleotide change.
Function Mutability Index
Asn
Ser(AGY)
Ala
Asp
Pro
Cys
Trp
Tyr
Gly
Arg(CGN)
Lys
Thr
Arg(AGR)
Gln
Glu
Ile
Val
Leu(CTN)
Met
Phe
Ser(TCN)
Leu(TTR)
0
2
4
6
8
Ratio
[Polar/(nonPolar+stop)]
56. 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.
57. 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
58. 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 mutation
2nd mutation
several mutations
tumor
growth/survival
59. Hypothesis:
• To overcome the several tiers of tolerance, an
autoimmune B cell must first acquire several
mutations in immunoregulatory genes.
60. The Stochastic Event Model
non-autoimmune B cells
autoimmune B cell
tolerance
checkpoints
autoimmune B cell is
eliminated from the B cell
repertory.
62. 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.
63. 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.
64. 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
65. Surprisingly KS+ B cells
express both reporter genes at
low levels.
mCherry GFP
B220+ gate
wt
KS+
wt
KS+
66. Excision of the NEO gene by FLP
increases expression of reporter genes.mCherry
B220
GFP
wt KS+Neo+ KS+FLP+
