Activation induced deaminase: How much and where?

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Seminars
in
Immunology
24 (2012) 246–
254
Contents
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at
SciVerse
ScienceDirect
Seminars
in
Immunology
j
ourna
l
ho
me
page:
www.elsevier.com/locate/ysmim
Review
Activation
induced
deaminase:
How
much
and
where?
Alexandre
Orthweina,b,
Javier
M.
Di
Noiaa,b,c,d,∗
aInstitut
de
Recherches
Cliniques
de
Montréal,
Montréal,
Québec,
H2W
1R7,
Canada
bDepartment
of
Microbiology
and
Immunology,
Université
de
Montréal,
Montréal,
Québec,
H3C
3J7,
Canada
cDepartment
of
Medicine,
Faculty
of
Medicine,
Université
de
Montréal,
Montréal,
Québec,
H3C
3J7,
Canada
dDepartment
of
Medicine,
McGill
University,
Montréal,
Québec,
Canada
a
r
t
i
c
l
e
i
n
f
o
Keywords:
Activation-induced
deaminase
(AID)
B-lymphocyte
Antibody
gene
diversification
Somatic
hypermutation
Class
switch
recombination
Humoral
immunity
Subcellular
localization
Protein
stability
a
b
s
t
r
a
c
t
Activation
induced
deaminase
(AID)
plays
a
central
role
in
adaptive
immunity
by
initiating
the
processes
of
somatic
hypermutation
(SHM)
and
class
switch
recombination
(CSR).
On
the
other
hand,
AID
also
pre-
disposes
to
lymphoma
and
plays
a
role
in
some
autoimmune
diseases,
for
which
reasons
AID
expression
and
activity
are
regulated
at
various
levels.
Post-translational
mechanisms
regulating
the
amount
and
subcellular
localization
of
AID
are
prominent
in
balancing
AID
physiological
and
pathological
functions
in
B
cells.
Mechanisms
regulating
AID
protein
levels
include
stabilizing
chaperones
in
the
cytoplasm
and
proteins
efficiently
targeting
AID
to
the
proteasome
within
the
nucleus.
Nuclear
export
and
cytoplasmic
retention
contribute
to
limit
the
amount
of
AID
accessing
the
genome.
Additionally,
a
number
of
factors
have
been
implicated
in
AID
active
nuclear
import.
We
review
these
intertwined
mechanisms
proposing
two
scenarios
in
which
they
could
interact
as
a
network
or
as
a
cycle
for
defining
the
optimal
amount
of
AID
protein.
We
also
comparatively
review
the
expression
levels
of
AID
necessary
for
its
function
during
the
immune
response,
present
in
different
cancers
as
well
as
in
those
tissues
in
which
AID
has
been
implicated
in
epigenetic
remodeling
of
the
genome
by
demethylating
DNA.
© 2012 Elsevier Ltd. All rights reserved.
1.
Antibody
diversification
in
germinal
center
B
cells
V(D)J
recombination
assembles
the
primary
repertoire
of
anti-
body
genes
during
B
cell
development.
B
cells
that
have
successfully
rearranged
their
immunoglobulin
(Ig)
genes
express
membrane
IgM
and/or
IgD
and
migrate
to
the
periphery
where
they
will
be
exposed
to
foreign
antigens.
The
first
cognate
antibody–antigen
recognition
of
naïve
B
cells
is
usually
not
of
high
affinity.
This
endows
the
system
with
enough
flexibility
to
interact
with
almost
any
possible
antigen;
however,
high
affinity
antibody–antigen
interactions
are
critical
for
neutralizing
or
disposing
of
antigens.
Thus,
there
are
mechanisms
to
further
change
the
antibodies.
B
cells
activated
by
cognate
antigen
initiate
the
germinal
center
reaction
[1].
Germinal
center
B
cells
divide
rapidly
while
diversifying
the
genes
encoding
for
the
heavy
and
light
antibody
chains.
Diversifi-
cation
occurs
through
the
introduction
of
point
mutations
in
the
variable
regions
of
the
IgH
and
IgL
by
the
mechanism
of
somatic
hypermutation
(SHM).
Antibody
variants
produced
by
SHM
are
selected
for
improved
antigen
recognition
by
antigen
presenting
Abbreviations:
AID,
activation
induced
deaminase;
ALL,
acute
lymphoblastic
leukemia;
CML,
chronic
myeloid
leukemia;
CSR,
class
switch
recombination;
DLBCL,
diffuse
large
B
cell
lymphoma;
Ph+,
Philadelphia
chromosome-positive;
NES,
nuclear
export
signal;
NLS,
nuclear
localization
signal;
SHM,
somatic
hypermutation.
∗Corresponding
author
at:
IRCM,
110
Avenue
des
Pins
Ouest,
Montréal,
Québec,
H2W
1R7,
Canada.
Tel.:
+1
514
987
5642;
fax:
+1
514
987
5528.
E-mail
address:
javier.di.noia@ircm.qc.ca
(J.M.
Di
Noia).
cells
and
T
cells,
which
leads
to
affinity
maturation
of
the
antibody
response.
At
the
same
time,
the
constant
exons
of
the
IgH
encod-
ing
for
IgM
and
IgD
are
exchanged
by
exons
encoding
IgG,
IgE,
or
IgA
isotypes
through
the
mechanism
of
class
switch
recombina-
tion
(CSR);
thereby
leading
to
the
production
of
antibodies
with
conserved
specificity
but
different
biological
properties.
Both,
SHM
and
CSR
are
mutagenic
mechanisms
that
despite
having
different
end
results
(i.e.
single
point
mutations
versus
a
chromosomal
dele-
tion
of
several
tens
of
kbp),
share
several
molecular
steps.
The
most
important
step
common
to
both
SHM
and
CSR
is
their
initiation
by
the
enzyme
Activation
induced
deaminase
(AID)
[2,3].
AID
is
part
of
the
AID/APOBEC
family
of
cytosine
deaminase-
related
enzymes,
most
of
which
have
the
unique
capacity
of
deaminating
deoxycytidine
in
single
stranded
DNA
thus
converting
it
into
deoxyuridine
[4].
This
is
already
a
mutagenic
lesion
caus-
ing
a
C:G
to
T:A
base
change
after
replication.
Processing
of
the
uracil
by
base
excision
and
mismatch
repair
enzymes
leads
to
the
broader
spectrum
of
point
mutations
characterizing
SHM,
and
to
DNA
double
strand
breaks,
which
are
necessary
intermediates
in
CSR
(reviewed
in
[5–7]).
2.
AID
levels
and
disease
2.1.
AID
deficiency
and
haploinsufficiency
As
expected
from
its
central
role
in
antibody
diversification,
loss
of
function
mutations
of
AID
cause
an
immunodeficiency
syndrome
1044-5323/$
–
see
front
matter ©
2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.smim.2012.05.001

A.
Orthwein,
J.M.
Di
Noia
/
Seminars
in
Immunology
24 (2012) 246–
254 247
characterized
by
the
absence
of
switched
isotypes,
recurrent
infec-
tions,
and
lymphoid
tissue
hyperplasia
[3],
which
is
recapitulated
in
Aicda−/−
mice
[2].
Aicda+/−
mice
show
an
intermediate
degree
of
lymphoid
hyperplasia,
between
that
of
wt
and
Aicda−/−
mice
[8,9].
AID
haploinsufficient
mice
have
∼40%
of
AID
mRNA
and
pro-
tein
levels
compared
to
wt
[9,10]
and
several
groups
have
found
reduced
CSR
and
SHM
in
Aicda+/−
mice
[8–11].
The
decrease
in
CSR
is
roughly
proportional
to
the
decrease
in
AID
mRNA
and
protein
levels
although
there
are
some
discrepancies
among
the
studies,
which
could
originate
from
using
different
mouse
strains
and/or
different
in
vitro
experimental
conditions
[12,13].
There
is
consis-
tency
between
studies
in
that
SHM
is
reduced
to
∼30%
of
the
wt
in
Peyer
patches
of
Aicda+/−
[8,9],
with
some
more
variability
when
measured
elsewhere,
ranging
from
70%
of
wt
in
lymph
nodes
JH4
[8]
to
only
15%
of
wt
in
S!
region
of
B
cells
activated
ex
vivo
[10].
These
observations
suggest
that
AID
is
limiting
for
antibody
diversifica-
tion
although
it
is
not
the
only
factor
determining
the
efficiency
of
CSR
and
SHM.
In
vivo,
the
effect
is
highly
compensated
for
by
selection
[9,10]
and
it
is
unclear
how
compromised
the
immune
system
of
an
individual
with
reduced
AID
levels
would
be.
Judging
from
the
lack
of
clinical
symptoms
in
humans
carrying
only
one
functional
AID
allele
[3,14],
this
is
probably
not
a
major
issue
for
human
health.
However,
it
would
have
in
all
likelihood
been
disad-
vantageous
during
evolution.
The
previous
considerations
suggest
a
mechanism
by
which
the
minimal
levels
of
AID
were
selected.
The
pathological
side
effects
of
AID
probably
set
the
upper
limit
of
AID
expression.
2.2.
AID
levels
in
antibody
diversification
and
cancer
Consistent
with
the
view
that
AID
levels
are
limiting
for
anti-
body
diversification,
higher
levels
of
AID
protein
generally
translate
into
more
CSR
and
SHM,
and
are
accompanied
by
an
increased
inci-
dence
of
potentially
transforming
genomic
lesions.
Mouse
models
with
modified
AID
expression
levels
nicely
provided
evidence
for
this.
First,
several
transgenic
mice
overexpressing
AID
have
been
made,
all
of
which
show
increased
CSR
and
SHM
[15–18].
There
are
differences
in
the
magnitude
of
the
effect,
which
might
be
related
to
the
transgene
design.
The
two
transgenic
lines
in
which
AID
was
expressed
from
a
ubiquitous
promoter
showed
a
signifi-
cant
but
modest
increase
[15,16,18].
In
these
mice,
B
cells
express
high
levels
of
AID
throughout
their
development
and
some
counter-
selection
is
possible,
which
could
dampen
the
extent
of
the
effect.
In
contrast,
transgenic
AID
under
the
control
of
the
Ig"
enhancer
showed
maximal
expression
in
germinal
center
B
cells
and
in
this
case
there
was
a
very
high
increase
in
CSR
and
SHM
levels
[17].
Secondly,
boosting
AID
expression
in
B
cells
by
removing
the
neg-
ative
post-transcriptional
regulation
of
AID
by
miR-155
also
leads
to
higher
levels
of
CSR
[19,20].
Yet,
none
of
these
mice
showed
increased
levels
of
switched
serum
Ig,
but
this
is
probably
regulated
at
another
level
(selection,
homeostatic
proliferation),
and
does
not
necessarily
reflect
the
intrinsic
CSR
capacity
of
the
B
cells.
This
lack
of
correlation
between
in
vitro
CSR
and
serum
Ig
levels,
has
also
been
observed
in
MSH2-deficient
and
UNG-deficient
mice
[21–24].
Interestingly,
mice
in
which
higher
levels
of
AID
were
achieved
by
removing
the
miR-155
binding
sites
from
its
mRNA
showed
reduced
affinity
maturation,
but
no
differences
in
the
quantity
or
quality
of
SHM
at
the
Ig
variable
regions
[19].
This
would
suggest
that
expression
of
AID
above
physiological
levels
could
compro-
mise
B
cell
viability,
in
line
with
the
evidence
that
AID
limits
the
size
of
the
germinal
center
by
causing
B
cell
apoptosis
[25].
It
might
be
that
the
upper
limit
for
AID
physiological
expression
levels
could
be
influenced
by
the
increased
apoptosis
that
occurs
with
elevated
AID.
AID
also
contributes
to
the
development
of
cancer,
but
whether
AID
levels
correlate
proportionally
with
the
risk
of
developing
cancer
is
unclear.
AID
oncogenicity
is
most
likely
a
consequence
of
its
capacity
to
mutate
and
produce
DNA
breaks,
thus
initiating
chromosomal
translocations
affecting
a
number
of
loci
in
normal
B
cells
[26–30].
Increased
levels
of
AID
in
B
cells
correlates
with
more
IgH-cMyc
translocations
[17,20]
and
increased
mutations
in
some
non-Ig
targets
in
vivo
and
in
vitro
[17,19,31].
However,
this
does
not
always
have
oncogenic
consequences
[16,20]
(see
below).
Mice
overexpressing
AID
have
conclusively
demonstrated
the
oncogenic
capacity
of
AID.
Ubiquitous
transgenic
overexpression
of
AID
leads
to
T
cell
lymphomas
as
well
as
lung
adenomas
and
adeno-
carcinomas,
but
not
B
cell
malignancies
[18].
B
cells,
in
comparison
to
other
cell
types
that
do
not
express
AID,
may
have
evolved
pro-
tective
mechanisms
against
transformation,
thus
explaining
this
observation.
Indeed,
the
apoptotic
control
that
normally
eliminates
cells
with
AID-induced
translocations
[26],
prevented
B
cell
lym-
phomas
in
an
AID-Ig"transgenic
model
[17].
In
the
absence
of
p53,
AID-Ig"developed
predominantly
mature
B
cell
lymphomas,
even
outpacing
the
T
cell
lymphomas
that
usually
kill
p53-deficient
mice
[17].
Curiously,
germinal
center
B
cells
have
reduced
levels
of
p53
to
allow
for
the
necessary
DNA
damage
that
accompanies
antibody
gene
diversification
[32],
which
suggests
the
presence
of
additional
mechanisms
to
prevent
AID-initiated
lymphomagenesis
(for
DNA
repair
regulation
see
Saribasak
and
Gearhart,
this
issue).
For
instance,
off-target
AID
mutations
are
more
frequent
in
the
absence
of
a
number
of
DNA
repair
pathways,
even
with
normal
endogenous
AID
levels
[31,33,34].
Endogenous
levels
of
AID
do
initiate
B
cell
transformation,
albeit
infrequently.
The
etiological
role
of
AID
in
B
cell
lymphomas
orig-
inating
from
germinal
center
B
cells
was
demonstrated
using
the
I!HABCL6
transgenic
oncogene
model,
which
deregulates
BCL6
in
B
cells
and
results
in
frequent
mature
B
cell
lymphomas.
When
crossed
with
Aicda−/−
mice,
I!HABCL6
transgenic
mice
did
not
develop
mature
B
cell
lymphomas
[35].
I!HABCL6
lymphomas
are
akin
to
human
DLBCL
(diffuse
large
B
cell
lymphoma),
which
displays
a
high
prevalence
of
AID
expression
[36–40],
strongly
sug-
gesting
an
etiological
role
for
endogenous
AID
in
this
lymphoma.
In
addition,
endogenous
AID
levels
contribute
to
chromosomal
translocations
of
multiple
partners
with
the
Ig
locus,
such
as
the
IgH-Ig#
[41]
or
IgH-Pax5
[29,30],
and
even
between
two
non-Ig
genes
[17,29,30].
Indeed,
most
of
the
translocations
occurring
in
a
p53-deficient
background
occur
between
c-Myc
and
miR-142
[17],
a
molecular
hallmark
of
acute
prolymphocytic
leukemia
[42].
Although
a
few
DLBCL
samples
show
higher
than
normal
AID
levels,
in
general
DLBCL
and
follicular
lymphoma
samples
show
similar
or
lower
AID
levels
than
germinal
center
B
cells
[36–38,40].
Although
lymphoma
biopsies
contain
normal
and
non-B
cells,
where
only
AID
mRNA
has
been
measured
in
many
cases,
it
does
not
seem
that
AID
overexpression
is
necessary
for
lymphomagenesis.
The
latter
was
demonstrated
in
the
Balb/Bcl-xL
mouse
model
of
plasmacytoma.
In
this
model,
AID
is
required
for
most
pristane-induced
plasmacy-
tomas,
where
it
underpins
the
oncogenic
IgH-cMyc
translocation
[34,39].
AID
haploinsufficient
mice
show
reduced
incidence
of
lym-
phoma
compared
to
Aicda+/+,
but
still
significantly
higher
than
the
Aicda−/−
[10].
Thus,
significantly
lower
than
normal
levels
of
AID
are
sufficient
to
cause
plasmacytomas
when
they
are
combined
with
another
predisposing
condition.
The
IgH-cMyc
translocation,
which
is
caused
by
AID
[26,28,43,44],
is
a
hallmark
of
Burkitt’s
lym-
phoma
in
humans.
AID
is
expressed
at
near
normal
levels
in
this
type
of
lymphoma
[36].
Near
to
normal
AID
levels
are
also
expressed
in
Ph+
ALL
(Philadelphia
chromosome-positive
B
cell
acute
lym-
phoblastic
leukemia)
and
CML
(chronic
myelogenous
leukemia)
[45–47].
In
ALL
and
CML,
AID
can
be
a
disease
progression
factor
by
accelerating
leukemia
clonal
evolution
[46]
and/or
by
mutat-
ing
the
BCR-ABL1
oncogene,
thus
underpinning
resistance
to
the
therapeutic
drug
imatinib
[47].
Chronic
lymphocytic
leukemia
is
another
B
cell
malignancy
expressing
variable
levels
of
AID
due

248 A.
Orthwein,
J.M.
Di
Noia
/
Seminars
in
Immunology
24 (2012) 246–
254
to
population
heterogeneity,
where
only
a
defined
subpopulation
of
cells
expresses
most
of
AID
mRNA
[48,49].
The
presence
of
this
subpopulation
correlates
with
a
worst
prognosis,
but
AID
mRNA
in
these
cells
is
not
higher
than
in
normal
B
cells
[48,49].
Finally,
AID
is
also
expressed
in
a
number
of
tumor
cell
lines
and
primary
tumors
of
non-lymphoid
origin
including
breast
[50],
prostate
[51],
stomach
[52],
liver
[53,54]
and
lung
[55],
where
AID
is
normally
not
expressed
or
expressed
at
much
lower
levels
than
in
B
cells
[56].
2.3.
AID
and
autoimmunity
AID
levels
also
influence
antibody-mediated
autoimmune
dis-
eases.
AID
is
important
in
determining
the
severity
of
the
pathology,
as
demonstrated
in
mouse
models
of
lupus
and
arthritis
in
which
AID
deficiency,
or
even
haploinsufficiency,
results
in
a
less
severe
disease
[11,57–59].
In
keeping
with
this,
increased
levels
of
AID
cor-
relate
with
higher
levels
of
autoantibodies
in
the
MRL/faslpr/lpr and
BXD2
mouse
models
of
lupus
and
rheumatoid
arthritis,
respectively
[57,60–62].
Interestingly,
AID-deficient
mice
also
have
autoim-
mune
disorders
albeit
of
a
different
nature
[63].
This
fits
well
with
the
predisposition
to
autoimmune
disorders
noted
in
AID-deficient
human
patients
[64].
The
recent
finding
that
low
AID
expression
levels
during
B
cell
development
play
a
role
in
establishing
B
cell
tolerance
could
explain
these
findings
[65,66].
Although,
like
in
can-
cer,
there
are
predisposing
conditions
beyond
AID
for
developing
autoimmunity,
here
again
we
find
that
AID
levels
are
important
in
balancing
an
efficient
immune
response
with
disease.
This
equilib-
rium
may
be
altered
by
factors
inducing
AID,
such
as
estrogens
[67],
and
could
in
part
explain
the
higher
susceptibility
to
autoimmunity
in
women.
3.
Regulating
AID
The
data
summarized
above
illustrates
how
the
optimal
expres-
sion
levels
of
AID
in
germinal
center
B
cells
was
selected
during
vertebrate
evolution
as
a
compromise
between
being
able
to
mount
an
effective
adaptive
immune
response
and
delaying
the
onset
of
cancer
or
autoimmune
diseases.
A
strikingly
complex
network
of
regulatory
mechanisms
exists
to
ensure
that
the
optimal
amount
of
biologically
active
AID
protein
gets
to
the
Ig
locus.
Any
alteration
in
the
efficacy
of
these
mechanisms
could
predispose
to
immunodefi-
ciency,
autoimmunity
or
B
cell
lymphomas.
Increased
longevity
and
reduced
selective
pressure
in
humans
have
turned
evolutionary
irrelevant
side
effects
of
AID
into
medical
problems.
Understanding
AID
regulation
and
being
able
to
manipulate
its
levels
could
pro-
vide
ways
of
targeting
AID
to
prevent
or
ameliorate
some
of
these
pathologies.
3.1.
Aicda
expression
in
and
outside
B
cells
3.1.1.
Aicda
in
germinal
center
B
cells
and
cancer
Aicda
transcription
is
induced
by
cytokines
and
cell–cell
interac-
tions
in
the
context
of
antigen-triggered
B
cell
activation
during
an
immune
response
(reviewed
in
[68]
and
see
Vuong
and
Chaudhuri
this
issue).
Thus,
Aicda
is
highly
transcribed
in
germinal
center
cells
but
stringently
repressed
in
plasma
and
memory
B
cells
[69,70].
Measurements
of
AID
mRNA
in
the
B
cell
lineage
agree
well
with
western
blot
and
immunohistochemistry
data,
as
well
as
with
the
analysis
of
an
AID-GFP
reporter
mouse
[40,69,71,72].
A
combi-
nation
of
promoter,
enhancers
and
silencers
located
within
four
evolutionary-conserved
regulatory
regions
determines
AID
expres-
sion
in
germinal
center
B
cells
of
the
spleen
and
mucosal-associated
lymphoid
tissues
(Peyer’s
patches,
tonsils,
lymph
nodes)
[68,73].
However,
the
original
belief
of
AID
being
exclusively
expressed
in
germinal
center
B
cells
has
been
revised,
as
AID
expression
is
also
found
in
other
tissues,
not
only
in
several
pathological
situations
but
also
in
normal
tissues.
It
is
conceivable
that
the
expression
of
AID
in
B
lymphoid
malig-
nancies
can
be
induced
by
similar
stimuli
than
in
mature
B
cells,
or
remains
as
a
relict
of
the
physiological
counterpart
of
the
neo-
plasia.
In
other
cases,
such
as
leukemia
originating
from
progenitor
B
cells
(like
in
Ph+
ALL)
or
myeloid
cells
(like
CML),
where
AID
has
been
convincingly
detected
[45,47],
it
is
less
clear
but
the
BCR-ABL1
oncogene
itself
could
be
involved
in
inducing
Aicda
[45].
It
is
also
unclear
how
AID
expression
is
induced
in
malignant
epithelial
cells
from
solid
tumors.
Ectopic
AID
expression
in
these
tissues
is
likely
to
be
caused
by
the
transcription
factor
Nf-"B,
a
critical
transcrip-
tion
factor
in
orchestrating
inflammatory
and
innate
immunity
responses.
In
fact,
Nf-"B
participates
in
Aicda
induction
in
nor-
mal
B
cells
[44,74]
and
was
shown
to
mediate
AID
expression
in
hepatocytes
[52].
Furthemore,
epithelial
cancers
with
AID
expres-
sion
are
often
associated
to
chronic
inflammation,
sometimes
with
an
underlying
infection
such
as
Hepatitis
C
virus
[52,75],
Heli-
cobacter
pylori
[76,77]
or
human
immunodeficiency
virus
[78,79].
Also,
other
infectious
agents
like
Epstein-Barr
virus
[78],
Abelson
murine
leukemia
virus
[80]
and
Moloney
murine
leukemia
virus
[81],
or
stimulation
of
innate
immune
signaling
through
toll-like
receptors
[44]
can
induce
AID,
probably
through
Nf-"B
[81].
In
summary,
it
is
difficult
to
generalize
the
reasons
behind
AID
expres-
sion
in
cancer
cells,
let
alone
its
relevance
for
the
pathology.
It
is
likely
that
the
clinical
history
and
the
selection
acting
in
each
case
influence
the
variations
in
AID
expression
levels
we
see
between
patients.
3.1.2.
Aicda
transcription
in
other
normal
tissues
AID
is
expressed
outside
of
the
B
cell
compartment
under
nor-
mal
conditions.
The
most
notable
example
is
the
ovary,
in
which
basal
AID
mRNA
levels
are
∼50–70%
of
those
found
in
the
spleen
[56,82].
Furthermore,
Aicda
is
induced
by
estrogen
which
increase
AID
mRNA
a
few
fold
in
spleen
but
by
>20-fold
in
the
ovary
[67].
Breast
tissue
also
expresses
AID
when
stimulated
with
estrogens
[67],
and
several
human
breast
cancer
cells
express
AID
mRNA
[50],
but
we
do
not
know
the
basal
levels
in
normal
breast
tis-
sue.
Other
examples
of
AID
expression
in
normal
non-lymphoid
cells
include
prostate,
heart
and
lung,
although
the
estimates
of
AID
levels
in
these
cases
are
less
precise
[51,56,82].
The
presence
of
AID
would
suggest
a
physiological
role
for
it
in
these
tissues,
espe-
cially
in
the
ovary
where
levels
are
quite
high,
but
it
is
unclear
what
it
is.
Evidence
is
accumulating
to
suggest
that
AID
could
be
part
of
a
mechanism
to
demethylate
5meC
at
CpG
sites
in
the
genome,
thereby
influencing
gene
expression
[56,83,84].
A
role
in
epigenetic
reprograming
during
development
has
been
proposed,
which
could
explain
the
presence
of
AID
in
cell
types
such
as
oocytes,
spermato-
cytes,
primordial
germ
cells
or
embryonic
stem
cells
[56,67,82–86].
As
mentioned,
oocytes
express
AID
mRNA
to
comparable
levels
of
B
cells,
although
protein
expression
has
not
been
tested
[56,82].
AID
mRNA
is
very
low
in
testis
[56,69,82],
which
contrasts
with
the
fact
that
the
protein
has
been
detected
by
IF
in
spermatocytes
[86].
There
is
discrepancy
about
the
timing
when
AID
is
expressed
in
primordial
germ
cells
although
all
reports
find
mRNA
levels
sub-
stantially
lower
than
in
B
cells
(5–10%)
[56,84,85].
Similarly
AID
mRNA
levels
in
stem
cells
are
∼5–10%
of
those
found
in
B
cells
[56,83].
Thus,
the
amount
of
AID
required
for
its
proposed
role
in
epigenetic
reprogramming
seems
to
be
considerably
lower
than
that
required
for
antibody
diversification.
Even
lower
AID
mRNA
levels
than
in
stem
cells
have
been
measured
during
B
cell
develop-
ment
[65,66],
so
low
that
it
cannot
be
detected
by
using
an
AID-GFP
reporter
gene
[69].
And
yet,
convincing
evidence
suggests
that
it
is
enough
to
play
a
role
in
B
cell
tolerance
[65,66].
The
possibility
of
quite
low
levels
of
AID,
which
would
be
ineffective
for
antibody

A.
Orthwein,
J.M.
Di
Noia
/
Seminars
in
Immunology
24 (2012) 246–
254 249
diversification
in
a
normal
immune
response,
being
functional
is
even
more
striking
when
one
considers
the
post-translational
reg-
ulatory
mechanisms
restraining
AID
in
activated
B
cells.
3.2.
Post-translational
regulation
of
AID
in
B
cells
3.2.1.
AID
subcellular
localization
AID
is
predominantly
cytoplasmic
(see
Häsler,
Rada,
and
Neu-
berger,
this
issue)
in
steady-state
as
first
reported
for
AID-GFP
in
Ramos
B
cells
[87],
and
confirmed
by
subcellular
fractionation
and
IHC
in
primary
B
cells
[40,71,88].
The
deletion
or
mutation
of
the
C-terminal
10
amino
acids
of
AID
led
to
its
nuclear
accumu-
lation,
identifying
this
region
as
a
nuclear
export
signal
(NES)
and
demonstrating
that
AID
is
a
nuclear-cytoplasmic
shuttling
protein
[89–91].
This
leucine-rich
NES
is
a
typical
recognition
motif
for
the
exportin
CRM1,
as
was
demonstrated
by
using
the
CRM1-specific
inhibitor
leptomycin
B
[90,91]
and
by
coimmunoprecipitation
[92,93].
AID
gains
access
to
the
nucleus
by
active,
energy-dependent
transport
[93].
Due
to
its
small
size
(24
kDa),
AID
could
in
principle
diffuse
passively
through
the
nuclear
pores.
However,
it
is
actively
imported
into
the
nucleus,
as
demonstrated
by
its
capacity
to
con-
fer
nuclear
localization
to
large
proteins
that
are
well
above
the
nuclear
pore
passive
diffusion
cut-off
[93].
The
nuclear
localization
signal
(NLS)
of
AID
has
not
been
completely
defined.
AID
N-terminal
region
(roughly
residues
5–50)
contains
multiple
basic
residues,
a
characteristic
of
many
NLS,
and
it
is
clearly
a
major
part
of
it
[90,93].
However,
by
itself
it
is
not
sufficient
to
mediate
nuclear
import
of
heterologous
proteins,
which
requires
the
first
181
out
of
the
198
AID
amino
acids
[93].
There
are
other
residues
elsewhere
in
the
pro-
tein
that
form
part
of
and/or
are
critical
in
displaying
the
NLS,
which
prompted
the
suggestion
that
AID
has
a
conformational
NLS
[93].
The
factors
mediating
AID
nuclear
import
are
also
not
well
defined.
AID
binds
in
vitro
to
several
karyopherin-$
importins,
which
are
dedicated
nuclear
import
factors,
but
their
functionality
has
been
only
indirectly
inferred
because
oxidative
stress,
which
inhibits
karyopherin-$-mediated
nuclear
import
[94,95],
also
inhibits
AID
import
[93].
However,
AID
also
interacts
with
CTNNBL1,
a
nuclear
protein
presumed
to
work
in
mRNA
splicing,
which
binds
to
NLS
through
an
armadillo-like
domain
[96,97].
Nuclear
accumulation
of
an
AID
variant
with
a
mutated
NES
is
partially
compromised
in
DT40
CTNNBL1−/−
cells,
suggesting
a
role
for
CTNNBL1
in
AID
nuclear
import
[97].
Still,
CH12
CTNNBL1−/−
cells
did
not
show
any
reduction
in
CSR
demonstrating
that
it
is
at
least
redundant
with
another
mechanism
for
importing
AID
into
the
nucleus
[98].
Since
CTNNBL1
interacts
with
importin-$s
[97],
they
could
be
part
of
the
same
pathway.
GANP,
a
protein
associated
with
the
RNA
shut-
tling
machinery
[99],
has
also
been
proposed
to
mediate
AID
active
nuclear
import,
largely
based
on
the
observation
that
its
overex-
pression
increases
the
nuclear
fraction
of
AID
[100].
Regardless
of
the
pathway,
it
is
clear
that
AID
is
actively
imported
into
the
nucleus
while
its
small
size
would
allow
it
to
passively
diffuse,
which
raises
the
question
of
why
this
would
be
necessary.
The
explanation
could
be
that
there
is
a
need
to
counteract
cytoplasmic
retention,
which
prevents
its
diffusion
[93].
Indeed,
a
motif
in
AID
C-terminal
region,
overlapping
with,
but
being
distinct
from
the
NES,
is
able
to
limit
the
passive
diffusion
of
GFP
into
the
nucleus
[93].
Separation
of
function
mutations
exist
to
corroborate
that
these
two
sig-
nals
are
distinct
and
mediate
different
protein–protein
interactions
[93].
The
translation
factor
eEF1$
could
participate
in
retention,
at
least
in
part,
since
it
is
stoichiometrically
associated
with
AID
in
the
cytoplasm
and
mutating
the
proposed
AID
cytoplasmic
reten-
tion
motif
can
disrupt
this
interaction
[93,101].
However,
genetic
confirmation
has
not
been
possible
since
eEF1$is
an
essential
factor.
AID
subcellular
localization
is
dynamic
and
reflects
the
equi-
librium
between
nuclear
export
and
cytoplasmic
retention,
and
the
opposing
active
nuclear
import.
The
fraction
of
steady
state
nuclear
AID
resulting
from
this
equilibrium
has
been
consistently
estimated
to
be
around
<10%
[40,88,91].
Whether
this
equilibrium
is
regulated
(such
as
to
increase
diversification
after
some
signal-
ing
event,
at
a
particular
cell
cycle
stage
[102],
etc.),
or
whether
the
proportion
of
AID
in
the
nucleus
is
constant
at
all
times
[68,103],
is
unresolved.
Drastic
alterations
or
truncation
of
the
AID
NES
abolish
CSR,
most
likely
because
this
region
also
contains
a
domain
medi-
ating
interactions
required
for
CSR
[90,91,104,105].
Nevertheless,
the
relocalization
of
AID
into
the
nucleus
by
mutating
or
delet-
ing
the
NES
increases
SHM
and
immunoglobulin
gene
conversion
in
overexpression
studies
in
vitro
[90,104,105].
Similarly,
mutating
the
C-terminal
cytoplasmic
retention
signal
of
AID
leads
to
faster
nuclear
import
and
higher
SHM
and
CSR
[93,105].
The
interpretation
of
this
data
at
face
value,
indicates
that
alterations
in
the
mechanisms
of
AID
subcellular
localization
have
consequences
on
antibody
diversification
and
that
nuclear
exclu-
sion
limits
the
biological
activity
of
AID.
However,
there
are
caveats
in
this
interpretation
as
most
AID
nuclear
variants
tend
to
show
higher
catalytic
activity
[91,104,106]
and
lower
expression
lev-
els
[91,105,107].
The
contribution
of
these
characteristics
to
the
observed
effect
on
antibody
diversification
is
unknown
and
we
could
be
over
or
underestimating
the
magnitude
of
the
effect.
3.2.2.
AID
stability
The
stability
of
AID
is
directly
linked
to
its
subcellular
localiza-
tion
with
cytoplasmic
AID
being
much
more
stable
than
nuclear
AID
[107].
The
∼8
h
half-life
of
AID
in
B
cells
represents
in
fact
a
rough
average
of
its
∼2.5
h
nuclear
and
18–20
h
cytoplasmic
half-
life
[107].
Indeed,
inhibition
of
AID
nuclear
export
with
leptomycin
B
and
mutation
or
deletion
of
the
NES,
as
well
as
disruption
of
AID
cytoplasmic
retention,
increase
the
nuclear
fraction
of
AID
and
cor-
relate
with
a
decrease
in
its
half-life
[93,101,105,107,108].
On
the
other
hand,
restricting
AID
to
the
cytoplasm
resulted
in
increased
AID
half-life
[93,107].
There
are
some
mechanistic
explanations
for
this
difference
in
stability.
HSP90
interacts
with
AID
in
the
cytoplasm
and
prevents
its
polyubiquitination
and
therefore
its
proteasomal
degradation
[108].
The
first
step
in
the
HSP90
molec-
ular
chaperoning
pathway
and
stabilization
is
the
interaction
of
AID
with
the
HSP40
and
HSP70
system
[109].
In
particular,
there
is
a
specific
dependence
on
the
HSP40
DnaJa1,
since
its
deficiency
results
in
decreased
stability
and
reduced
levels
of
AID,
accompa-
nied
by
loss
of
biological
activity
[109].
Simultaneous
inhibition
of
HSP90
and
the
proteasome,
but
not
inhibiting
only
the
proteasome,
results
in
massive
accumulation
of
polyubiquitinated
AID
[108].
Thus,
there
seems
to
be
very
little
turnover
of
cytoplasmic
AID,
stabilization
being
the
default
pathway.
HSP90
is
critical
for
this
stabilization,
most
likely
just
after
AID
protein
synthesis,
but
does
not
exclude
the
possibility
that
later
complexes
with
eEF1$
[101]
or
importins
[93],
could
also
be
stabilizing.
It
has
also
been
proposed
that
some
maturation
step
such
as
post-translational
modification
or
oligomerization
could
stabilize
AID
[103,108].
Once
inside
the
nucleus,
AID
is
much
less
stable,
either
because
it
looses
stabilizing
interactions
and/or
it
is
actively
destabilized.
Unlike
cytoplasmic
AID,
nuclear
AID
seems
to
be
constantly
tar-
geted
to
the
proteasome
by
ubiquitin-dependent
and
-independent
pathways
[107,110].
Proteasomal
inhibition
is
sufficient
for
sub-
stantial
accumulation
of
polyubiquitinated
nuclear
AID
[107].
The
E3
ubiquitin
ligases
modifying
nuclear
AID
are
unknown.
MDM2
interacts
with
AID
and
could
be
one
of
them
[111].
However,
DT40
cells
deficient
in,
or
overexpressing,
MDM2
show
a
very
modest
increase
and
decrease
in
Ig
gene
conversion,
respectively
[111].
Thus,
MDM2
is
either
redundant
with
some
other
ubiquitin
lig-
ase
or
not
relevant
for
AID.
Interestingly,
AID
with
no
internal

250 A.
Orthwein,
J.M.
Di
Noia
/
Seminars
in
Immunology
24 (2012) 246–
254
lysine
residues
is
still
significantly
polyubiquitinated
[107],
sug-
gesting
either
N-terminal
ligation
or
a
non-canonical
pathway
of
polyubiquitination
for
AID.
A
proportion
of
AID
interacts
with,
and
is
targeted
for
degradation
by,
the
nuclear
protein
REG-%,
which
brings
proteins
for
proteasomal
degradation
in
an
ubiquitin-
and
ATP-independent
fashion
[110]
(see
below).
The
cytoplasmic
stabilization
by
the
DnaJa1–HSP90
pathway
is
critical
for,
and
limiting
in,
producing
AID.
Inhibiting
HSP90,
or
DnaJa1
farnesylation
(which
prevents
binding
to
AID),
as
well
as
DnaJa1
knockdown
or
knockout,
all
result
in
lower
endogenous
AID
levels
and
a
nearly
proportional
decrease
in
antibody
diversifica-
tion
by
SHM
and
CSR
[108,109].
This
data
is
in
keeping
with
the
results
from
AID
haploinsufficient
mice
[9,10];
supporting
the
view
that
AID
protein
is
limiting.
Also
consistent
with
this
view,
DnaJa1
overexpression
increases
AID
levels
and
antibody
diversification
in
vitro
[109].
Similarly,
REG-%deficiency
results
in
higher
AID
steady-state
levels,
increased
nuclear
AID
stability
and
CSR
in
mice
[110].
Thus,
modulating
AID
stability
seems
to
be
one
more
way
of
limiting
AID
expression
and
yet
another
mechanism
restraining
antibody
diversification.
In
the
absence
of
AID-specific
repressors,
HSP90
inhibitors
derived
from
geldanamycin,
which
are
already
in
clinical
trials,
or
farnesyltransferase
inhibitors
to
inactivate
DnaJa1,
could
be
exploited
to
indirectly
target
AID
[108,109].
They
offer
the
possi-
bility
to
treat
some
of
the
pathologies
associated
with
exacerbated
antibody
diversification,
like
autoimmune
diseases,
or
derived
from
AID
side
effects
such
as
imatinib
resistance
in
CML
or
tumor
clonal
evolution.
The
challenge
here
is
to
identify
a
disease
where
AID
implication
and
onset
can
be
predicted
to
use
these
inhibitors
at
the
right
time.
3.2.3.
AID
phosphorylation
In
addition
to
AID
protein
levels
and
subcellular
localization,
phosphorylation
regulates
AID
activity.
Five
evolutionary
con-
served
AID
residues,
have
been
found
to
be
phosphorylated
in
B
cells:
Ser3,
Thr27,
Ser38,
Thr140
and
Tyr184
[8,112–116].
Despite
Tyr184
being
very
close
to
the
NES
and
the
cytoplasmic
retention
signal,
and
Ser3,
Thr27
and
Ser38
being
close
to
or
immersed
in
the
NLS,
neither
phosphonull
nor
phosphomimicking
mutations
at
any
of
these
sites
seem
to
affect
AID
subcellular
localization
[103,115,117].
Also,
it
is
not
known
whether
phosphorylation
may
contribute
to
AID
stability.
Phosphorylation
of
Thr27
inhibits
AID-induced
deamination
and
CSR
in
vitro,
suggesting
it
modulates
AID
specific
activity
[112,113,117],
but
whether
this
is
used
in
vivo
to
regulate
AID
is
unknown.
Phosphorylation
at
Ser3
reduces
AID
biological
activity
ex
vivo
but
does
not
impact
its
catalytic
activity,
and
its
role
and
importance
in
vivo
are
unknown
[114].
On
the
other
hand,
phos-
phorylation
of
Ser38
and
Thr140
are
not
essential
for
AID
catalytic
activity
in
vitro,
but
both
significantly
increase
AID
biological
activ-
ity
in
vivo
[8,112,115,118,119].
Ser38
is
phosphorylated
by
PKA
on
chromatin,
which
allows
AID
to
interact
with
RPA
and
greatly
facil-
itates
CSR
and
SHM
[8,112,113,115,119,120].
However,
the
effect
of
mutating
Ser38
and
Thr140
in
vivo
was
much
more
pronounced
when
combined
with
AID
haploinsufficiency
[8,118],
thus
suggest-
ing
that
these
modifications
are
not
limiting
for
AID
activity.
4.
Conclusion
and
questions
4.1.
Regulation
of
AID
in
cells
with
high
versus
low
levels
of
AID
expression
The
post-translational
regulation
of
AID
(by
stabilization,
com-
partmentalization,
and
phosphorylation)
greatly
contributes
to
balancing
antibody
gene
diversification
and
widespread
DNA
damage
in
germinal
center
B
cells.
The
expected
(and
in
some
cases
known)
contribution
of
AID
to
cancer
is
by
the
same
mechanisms
it
uses
for
antibody
diversification:
induction
of
mutations
and
DNA
breaks.
Where
the
information
is
available,
the
AID
levels
in
lym-
phoid
malignancies
and
solid
tumors
from
human
patients
tend
to
be
within
the
same
range
as
those
in
B
cells.
In
many
of
these
cancers,
AID
post-translational
regulation
mechanisms
seem
to
be,
for
the
most
part,
functional.
Indeed,
many
B
cell
lymphoma
cell
lines
expressing
AID
have
been
used
to
study
compartmentaliza-
tion,
stability
and
phosphorylation
of
AID,
and
these
findings
were
then
confirmed
in
primary
cells.
In
almost
every
cell
line
tested,
AID
is
cytoplasmic
and
responds
to
leptomycin
B;
is
sensitive
to
HSP90
inhibition,
and
is
phosphorylated.
However,
there
could
be
quantitative
or
qualitative
differences
that
go
unnoticed
because
not
much
work
has
been
done
on
actual
activated
germinal
center
B
cells.
For
instance,
there
could
be
differences
in
the
relative
strength
of
the
different
mechanisms
regulating
AID
subcellular
localization
and/or
stability,
which
may
shift
the
balance
of
AID
regulation
and
promote
DNA
damage.
This
could
partly
explain
why
certain
tis-
sues
can
be
more
sensitive
to
transformation
than
B
cells
in
AID
transgenic
mice
(i.e.
thymus,
lung
or
testis)
[18,121].
Tissues
expressing
very
low
AID
levels
are
quite
a
different
cat-
egory
to
consider.
First
there
is
genetic
evidence
suggesting
that
the
expression
of
AID
during
early
B
cell
development
in
the
bone
marrow
plays
a
physiological
role
by
eliminating
autoreactive
B
cells
[65,66].
It
was
suggested
that
AID
expression
during
B
cell
development
mutates
the
IgV
region
[122,123].
This
is
reminis-
cent
of
what
is
observed
in
sheep,
rabbits
and
cattle
where
SHM
or
Ig
gene
conversion
contributes
to
the
diversity
of
the
primary
antibody
repertoire
[124–126].
In
humans,
AID
expression
during
early
B
cell
development
could
be
an
evolutionary
relic
or
may
con-
tribute
to
tolerance
by
mutating
autoreactive
clones,
thus
changing
their
specificity.
However,
given
the
almost
linear
proportionality
between
AID
protein
levels
and
SHM
in
germinal
center
B
cells,
it
is
difficult
to
imagine
that
the
1000-fold
lower
levels
of
AID
expressed
in
immature
B
cells
when
compared
to
germinal
center
B
cells
[66],
could
do
much
SHM.
The
mechanism
of
AID-mediated
B
cell
tol-
erance
is
unknown
but
one
wonders
whether
the
1000-fold
less
mRNA
linearly
translates
into
1000-fold
less
AID,
or
whether
only
10%
of
this
AID
is
in
the
nucleus
and
how
unstable
is
it.
Simi-
lar
questions
can
be
asked
about
cells
where
AID
is
proposed
to
underpin
epigenetic
reprogramming.
One
might
need
to
invoke
a
much
more
efficient
(and
error
free)
mechanism
for
AID-mediated
demethylation
than
for
SHM.
4.2.
A
network
or
a
cycle
for
AID
protein
regulation?
It
is
likely
that
multiple
mechanisms
were
selected
during
evo-
lution
to
restrain
the
activity
of
AID.
The
alternatives,
widespread
genomic
damage
or
immunodeficiency,
are
highly
deleterious
traits.
Nevertheless,
the
redundancy
of
mechanisms
with,
appar-
ently,
the
same
function
is
striking.
The
total
AID
levels
expressed
in
B
cells
are
much
higher
than
the
amount
of
it
that
can
be
found
in
the
nucleus
at
any
time,
and
there
are
multiple
mechanisms
to
enforce
this
nuclear
exclusion.
These
mechanisms
could
be
seen
as
a
cycle
or
as
a
network
(Fig.
1).
The
first
model
envisions
that
these
mechanisms
actually
form
a
circuit
(f.i.
release
from
HSP90
is
always
followed
by
AID
associated
to
cytoplasmic
retention,
which
precedes
nuclear
import,
etc.).
This
could
allow
mechanisms
with
apparently
the
same
effect
to
play
distinct
roles.
Cytoplasmic
reten-
tion
could
contribute
more
to
AID
nuclear
exclusion
than
nuclear
export
(as
the
largely
cytoplasmic
distribution
of
endogenous
AID
in
Ramos
cells
treated
with
leptomycin
B
could
suggest
[93]).
Nuclear
export
could
contribute
to
the
recycling
of
AID
from
the
nucleus
in
keeping
with
the
decay
observed
when
nuclear
export
is
inhibited.
An
alternative
model
could
be
that
there
are
several

A.
Orthwein,
J.M.
Di
Noia
/
Seminars
in
Immunology
24 (2012) 246–
254 251
AHA-1
Networ
kPharmacologi
cal d
est
abilizat
ion
Proteasome
Hsp90 inhibito
rs
FTI
HSC70 ATP
ADP
CYTOPLASMIC
RETENTION
CYTOPLASMIC
RETENTION
NUCLEAR
IMPORT
NUCLEAR
IMPORT
NUCLEAR
EXPORT
NUCLEAR
DEGRADATION
NUCLEAR
EXPORT
CYTOPLASMIC
DEGRADATION
NUCLEAR
DEGRADATION
TARGETING
TARGETING
STABILIZATION
STABILIZATION
Oligomerization ?
PTM ?
?
CRM1
DnaJa1
HSP90
AID eEF1α
CTNNBL1
GANP
?
Poly Ub REG-γ
Cycle
Proteasome
E3?
Transcription
RNA processing factors
PKA phosphorylation
AID
ATP
ADP
DnaJa1 HSP90
A
CB
E3 (?)
P
Importin
α
Fig.
1.
Major
post-translational
regulation
steps
affecting
AID
levels.
(A)
Schematic
representation
of
steps
participating
in
stability
and
subcellular
localization
regulation
represented
within
a
cycle.
Only
selected
AID
interacting
factors
are
shown.
AID
is
synthesized
in
the
cytoplasm,
where
unfolded
AID
is
met
by
the
HSP40-HSC70
system,
the
specific
action
of
the
HSP40
DnaJa1
allows
transferring
AID
into
the
HSP90
molecular
chaperoning
stabilization
cycle.
After
some
undefined
maturation
step,
or
conformational
change,
AID
is
passed
onto
eEF1$
and/or
other
cytoplasmic
retention
factors
before
active
nuclear
import.
A
number
of
factors
could
be
implicated,
alternatively
or
jointly
in
AID
nuclear
import.
Inside
the
nucleus
AID
is
either
exported
by
CRM1
or
targeted
to
the
Ig
loci
by
interacting
with
a
number
of
RNA
processing
factors,
where
it
is
phosphorylated
by
PKA.
AID
is
(subsequently)
degraded
in
the
nucleus
either
through
ubiquitin-
or
REG%-dependent
proteasomal
degradation.
(B)
Simplified
schematic
representation
of
the
same
steps
as
in
A,
but
in
the
form
of
a
network
in
which
most
pools
of
AID
are
interconnected
(see
text).
(C)
Schematic
representation
of
AID
cytoplasmic
degradation
following
inhibition
of
the
HSP90
molecular
chaperoning
pathway.
HSP90
inhibitors
prevent
the
ATP
hydrolysis
cycle
of
the
chaperone.
FTI,
farnesyltransferase
inhibitors,
prevent
farnesylation
of
DnaJa1,
which
is
required
for
binding
to
and
stabilization
of
AID.
Both
inhibitors
lead
to
polyubiquitination
and
proteasomal
degradation
of
cytoplasmic
AID.
options
after
each
regulatory
point,
with
as
many
possible
destina-
tions
competing
for
AID
(f.i.
release
from
HSP90
could
be
followed
by
cytoplasmic
retention
or
nuclear
import
or
degradation;
nuclear
AID
could
have
a
similar
probability
of
being
exported,
targeted
to
the
Ig
locus
or
degraded,
etc.).
Of
course,
a
third
possibility
would
be
a
mixture
of
both
models,
but
this
simplification
could
help
in
postulating
testable
hypothesis.
Much
experimental
work
is
still
needed.
The
germinal
center
is
probably
the
only
normal
tissue
in
which
the
presence
of
AID
pro-
tein
has
been
accurately
measured.
Detection
elsewhere
has
mostly
relied
on
RT-PCR
[56,67,82–84]
or
non-quantitative
immunode-
tection
by
IHC
or
IF.
Comparing
the
relative
levels
of
AID
protein
between
different
tissues;
developing
sensitive
methods
to
fol-
low
AID
in
precursor
B
or
stem
cells,
comparing
the
regulatory

252 A.
Orthwein,
J.M.
Di
Noia
/
Seminars
in
Immunology
24 (2012) 246–
254
mechanisms
of
AID
between
germinal
center
and
transformed
B
cells
or
non
B
cell
expression
sites,
would
all
contribute
to
under-
standing
AID.
Acknowledgments
We
thank
Stephen
Methot
for
reading
the
manuscript.
The
work
in
our
laboratory
was
funded
by
operating
grants
from
the
Canadian
Institutes
of
health
research
and
the
Cancer
Research
Society
and
an
infrastructure
grant
from
the
Canadian
Fund
for
Innovation,
leaders
opportunity
fund.
JMDN
is
supported
by
a
Canada
Research
Chair
Tier
2
and
AO
by
a
Cole
Foundation
doctoral
fellowship.
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