Thymoma-associated myasthenia gravis: Transplantation of thymoma and extrathymomal thymic tissue into SCID mice

Abstract

To study the possible role of thymomas and of extrathymomal thymic tissue in the development and maintenance of myasthenia gravis, we transplanted fragments of either tissue into SCID mice and monitored the production of anti-acetylcholine receptor antibodies in the recipients. Furthermore, the transplants were characterized by immunohistochemistry. Unlike after transplantation of thymus with lymphofollicular hyperplasia that induced high titers of anti-acetylcholine receptor antibodies, thymoma transplants never produced autoantibodies. Mice transplanted with extrathymomal thymic tissue also failed to produce anti-acetylcholine receptor antibodies except one group that received transplants containing hyperplastic extrathymomal tissue. These findings may explain the refractoriness of thymomatous myasthenia to thymectomy.

Full text

American
Journal
of
Pathology,
Vol.
148,
No.
5,
May
1996
Copyrigbt
()
Amencan
Society
for
Investigative
Patbology
Short
Communication
Thymoma-Associated
Myasthenia
Gravis
Transplantation
of
Thymoma
and
Extrathymomal
Thymic
Tissue
into
SCID
Mice
Simone
Spuler,*t
Andreas
Sarropoulos,*
Alexander
Marx,*
Reinhard
Hohlfeld,*t
and
Hartmut Wekerle*
From
the
Department
of
Neuroimmunology,*
Max-Planck-
Institute,
Martinsried,
Department
of
Neurology,t
University
of
Munich,
and
Department
of
Pathology,t
University
of
Wurzburg,
Wurzburg,
Germany
To
study
the
possible
role
of
thymomas
and
of
extrathymomal
thymic
tissue
in
the
develop-
ment
and
maintenance
of
myasthenia
gravis,
we
transplantedfragments
of
either
tissue
into
SCID
mice
and
monitored
the
production
of
anti-acetylcholine
receptor
antibodies
in
the
recipients.
Furthermore,
the
transplants
were
characterized
by
immunohistochemistry.
Un-
like
after
transplantation
of
thymus
with
lym-
phofollicular
hyperplasia
that
induced
high
titers
of
anti-acetylcholine
receptor
antibodies,
thymoma
transplants
never
produced
autoan-
tibodies.
Mice
transplanted
with
extrathy-
momal
thymic
tissue
also
failed
to
produce
anti-acetylcholine
receptor
antibodies
except
one
group
that
received
transplants
containing
hyperplastic
extrathymomal
tissue.
These
find-
ings
may
explain
the
refractoriness
of
thy-
momatous
myasthenia
to
thymectomy.
(Am
J
Pathol
1996,
148:1359
-1365)
Thymic
abnormalities
occur
in
80
to
90%
of
patients
with
myasthenia
gravis
(MG).
The
vast
majority
of
patients
have
lymphofollicular
hyperplasia
of
the
thy-
mus,
but
approximately
10%
have
a
thymoma.1-3
The
two
groups
of
patients
differ
in
the
average
age
at
onset,
the
presence
of
anti-striated
muscle
anti-
bodies4
and
in
the
poor
clinical
response
to
thymec-
tomy5'6
of
patients
with
thymomas.
These
differences
may
reflect
different
pathogenic
events
that
lead
to
the
clinical
picture
of
MG.
In
MG
with
lymphofollicular
thymic
hyperplasia,
it
has
been
proposed
that
the
thymus
is
the
primary
site
of
au-
tosensitization
against
the
acetylcholine
receptor
(AchR).7
All
elements
necessary
for
the
initiation
of
an
immune
response
against
the
AchR
are
present
in
the
thymus,
ie,
AchR
on
thymic
myoid
cells,
which
are
often
surrounded
by
professional
antigen-pre-
senting
interdigitating
cells,8
9
AchR-specific
CD4-
positive
T
cells,10'11
and
anti-AchR-antibody-pro-
ducing
B
cells.12
Indeed,
transplantation
of
small
pieces
of
hyperplastic
thymus
under
the
kidney
cap-
sule
of
immunodeficient
SCID
mice1314
is
sufficient
to
induce
a
long-lasting
production
of
anti-AchR
an-
tibodies."51
We
investigated
the
possible
role
of
thymoma
in
the
pathogenesis
of
thymoma-associated
MG.
Pieces
of
thymoma
or
extrathymomal
thymic
tissue
were
transplanted
into
SCID
mice.
Serum
antibody
titers
of
total
immunoglobulin
(1g)G,
anti-AchR
anti-
bodies,
and
anti-striated
muscle
antibodies
were
measured
biweekly,
and
all
transplants
were
char-
acterized
by
immunocytochemistry.
This
work
was
reported
in
part
at
the
meeting
of
the
European
Neurological
Society
(ENS)
in
Barcelona,
July
1994
(J
Neurol,
1994,
241
(suppl
1):S100.
This
work
is
part
of
Andreas
Sarropoulos
med-
ical
thesis.
Supported
by
Max-Planck-Society,
Germany
and
Mus-
cular
Dystrophy
Association,
U.S.A.
Accepted
for
publication
January
15,
1996.
Address
reprint
requests
to
Dr.
Simone
Spuler,
Department
of
Neurology,
Guggenheim
Building
G
801,
Mayo
Clinic,
Rochester,
MN
55905.
1359

1360
Spuler
et
al
AJP
May
1996,
Vol.
148,
No.
5
Table
1.
Characteristics
of
Patients
with
Thymoma-Associated
MG
Age
Patient
(years)
Anti-AchR
Start
of
antibody
titer
Sex
Type
disease
(nmol/L)
WD
32
Male
Generalized
1980
PT
44
Male
Generalized
1993
WS
74
Male
Generalized
1992
AM
54
Female
Generalized
1990
ND,
not
done.
Materials
and
Methods
Patients
The
patients
had
typical
clinical
and
electrophysio-
logical
signs
of
generalized
MG.
The
anti-AchR
an-
tibody
titers
ranged
between
34
and
96
nmol/L.
Tests
for
anti-striated
muscle
antibodies
were
posi-
tive
in
two
patients,
negative
in
one,
and
not
deter-
mined
in
one.
The
clinical
data
of
the
patients
are
summarized
in
Table
1.
Thymectomies
were
per-
formed
at
the
Department
of
Surgery
of
the
Martha
Maria
Hospital
or
at
the
Department
of
Surgery,
Grosshadern
Medical
Center,
Munich,
and
at
the
Department
of
Thoracic
Surgery,
University
of
Ulm.
Among
the
four
histologically
studied
thymuses,
three
were
cortical
thymomas
and
one
was
a
well
differentiated
thymic
carcinoma.
The
extrathymomal
tissue
was
normal
in
three
specimens
and
showed
lymphofollicular
hyperplasia
in
one.
Animals
C.B-17
scid/scid
(SCID)
mice
were
kindly
supplied
by
Professor
St.
Thierfelder
(Gesellschaft
fOr
Strahl-
enforschung,
Munich,
Germany).
The
mice
were
kept
under
specific
pathogen-free
conditions
and
used
between
5
and
8
weeks
of
age.
Leaky
mice
with
serum
titers
of
murine
Ig
greater
than
50
ng/ml
were
excluded
from
the
experiments.18
Preparation
of
Thymus
and
Operation
A
total
of
41
SCID
mice
were
transplanted,
24
with
pieces
of
thymoma
and
17
with
extrathymomal
thy-
mus
as
previously
described.15
Briefly,
SCID
mice
were
anesthetized
with
ketamine,
0.05
mg/g
intra-
peritoneal
(Ketanest,
Parke-Davis,
Freiburg,
Ger-
many)
and
diazepam,
0.15
p.g/g
intraperitoneal
(Va-
lium,
Hoffmann
La
Roche,
Basel,
Switzerland).
The
kidney
was
exteriorized,
and
a
small
piece
of
thymus
>20
ND
Cortical
thymoma
96
Negative
Cortical
thymoma
and
lymphofollicular
hyperplasia
in
the
thymus
34
Positive
Well
differentiated
thymic
carcinoma
56
Positive
Cortical
thymoma
(1
mm3)
was
placed
beneath
the
renal
capsule.
The
wound
was
closed
with
5-0
vicryl
sutures.
All
procedures
were
performed
under
sterile
con-
ditions.
Blood
was
taken
from
the
lateral
tail
veins.
Reagents
The
following
anti-human
monoclonal
antibodies
(MAbs)
were
used
for
immunohistochemistry:
OKT-4
(anti-CD4)
and
L243
(anti-HLA-DR;
both
from
the
American
Type
Culture
Collection,
Rockville,
MD),
T8
(anti-CD8,
Coulter
Electronics,
Krefeld,
Germany),
Leu-14
(anti-CD22,
Becton
Dickinson,
Heidelberg,
Germany),
KS
1A3
(anti-cytokeratin
peptide
13,
Sigma,
Deisenhofen,
Germany).
The
MAb
155
is
a
rat
MAb
recognizing
the
cyto-
plasmic
epitope
371
to
378
of
the
a-subunit
of
the
AchR.19
This
antibody
cross-reacts
with
a
153-kd
protein
expressed
on
thymoma
epithelial
cells
and
also
with
fast
troponin
1.20,21
Unlabeled
control
mouse
IgGl
and
mouse
IgG
2a/b
were
purchased
from
Becton
Dickinson.
Polyclonal
goat
anti-mouse,
goat
anti-human,
and
mouse
anti-
goat
antibodies
(unlabeled
or
peroxidase
labeled)
were
obtained
from
Dianova
(Hamburg,
Germany).
Immunoperoxidase
Staining
The
tissue
was
mounted
in
OCT
compound
(Tissue-
Tek
Miles,
Elkhart,
IN),
and
10-p.m
cryosections
were
cut
using
a
Jung-Frigocutt
2800E
Kryostat
(Leica,
Germany).
Acetone
fixation
was
performed
for
5
min-
utes
at
-200C.
For
hematoxylin
and
eosin
(H&E)
stains,
the
tissue
was
fixed
in
paraformaldehyde
for
10
minutes.
The
engrafted
kidney
was
cut
serially
and
the
transplants
were
located
in
stained
sections
at
150-p.m
intervals.
Fixed
sections
were
blocked
with
a
solution
con-
taining
phosphate-buffered
saline,
pH
7.2,
2%
bo-
vine
serum
albumin,
10%
heat-inactivated
serum
Anti-striated
muscle
antibodies
Histology

Thymomas
Transplanted
in
SCID
Mice
1361
AJP
May
1996,
Vol.
148,
No.
5
C.
Figure
1.
Overview
of
a
kidney
with
a
MG
thymoma
graftplaced
under
the
kidney
capsule
of
SCID
mice.
All
stainings
were
performed
on
sequential
sectionsfrom
the
same
kidney
5
weeks
after
transplanitation.
a:
H&E
stain
(magnification,
X35).
b:
Anti-HLA
ABCstaining(X
60).
C:
Anti-cytokeratin
peptide
13(X
60).
d:
Anti-CD4(X
60).
e:
Anti-CD22(X
60).
f:
MAb
155(X
60).
from
AB'
donors,
and
5%
heat-inactivated
horse
serum.
Subsequently,
MAbs
were
added
at
the
op-
timal
concentration
in
the
same
solution
for
30
min-
utes.
Thereafter,
a
three-step
peroxidase
staining
was
done
as
follows.
Peroxidase-labeled
rabbit
anti-
mouse
antibody
was
added
at
1:50,
and
then
per-
oxidase-labeled
donkey
anti-rabbit
antibody
was
ap-
plied
at
1:700.
The
signal
was
further
enhanced
by
adding
peroxidase-anti-peroxidase
complex
(Boeh-
ringer,
Mannheim,
Germany).
The
final
reaction
product
was
developed
with
diaminobenzidine.
The
sections
were
counterstained
with
hematoxylin,
dehydrated,
cleared,
and
mounted
in
Protex
(Lerner
Laboratories,
Pittsburg,
PA).
Detection
of
Human
IgG
and
Specific
Anti-AchR
Antibodies
Human
and
mouse
IgM
and
IgG
levels
were
mea-
sured
using
a
two-site
enzyme-linked
immunosor-
bent
assay.
The
plates
were
read
at
OD405
using
a
Titertek
Multiskan
(Flow
Laboratories,
Meckenheim,
Germany).
Ig
standards
were
obtained
from
Dianova
(ChromPure).
The
assay
was
sensitive
to
an
Ig
con-
centration
of
10
ng/ml.
Antibodies
against
human
AchR
were
measured
with
a
radioimmunoprecipitation
assay.22
Briefly,
hu-
man
AchR
was
labeled
with
[1251]a-Bungarotoxin
and
incubated
with
appropriate
serum
dilutions.
An-
tibodies
were
precipitated
with
anti-human
IgG,
and
the
concentration
of
anti-AchR
antibodies
was
cal-
culated
from
the
amount
of
precipitated
radioactivity.
Detection
of
Anti-Striated
Muscle
Antibodies
Antibodies
against
striated
muscle
components
in
the
SCID
sera
were
determined by
an
immunofluo-
rescence
technique.
The
sera
were
incubated
with
rat
muscle,
and
binding
of
anti-striated
muscle
anti-
body
was
detected
by
staining
with
fluorescein-
isothiocyanate-labeled
goat
anti-human
IgG.
Results
Histological
and
Immunocytochemical
Characterization
of
MG
Thymus
Grafts
in
SCID
Mice
Transplants
of
Thymoma
Tissue
The
H&E
stain
demonstrated
the
demarcation
be-
tween
the
thymoma
transplant
and
the
surrounding
renal
tissue.
As
can
be
seen
in
Figure
la,
the
thy-
moma
did
not
infiltrate
the
mouse
tissue
but
re-
mained
separated
from
the
kidney
by
a
connective

1362
Spuler
et
al
AJP
May
1996,
Vol.
148,
No.
5
a
Figure
2.
Transplants
of
extrathymomal
thymic
tissue
under
the
kidney
capsule
of
SCID
mice.
a
to
c
and
d
toe:
Obtained,
respectively,
from
sequential
sections
from
the
same
kidney
6
weeks
after
transplantation.
a:
Thymic
tissue
with
lymphofollictular
hyperplasia.
H&E
stain;
magnification,
x
50.
b:
71Tymic
tissue
with
Iymphofollicular
hyperplasia.
Anti-CD22;
X300.
C:
7Tymic
tissue
with
Iymphofollicular
hyperplasia.
MAb
155;
X
1200.
d:
Thymic
tissue
with
atrophy.
Anti-HLA
ABC
stain;
X
600.
e:
Thymic
tissue
with
atrophy.
Anti-CD22
stain;
X
600.
tissue
capsule.
This
capsule
became
detectable
2
weeks
after
transplantation.
The
non-infiltrating
growth
of
the
thymoma
was
con-
firmed
immunocytochemically
by
staining
against
HLA-ABC
(Figure
1
b).
Furthermore,
expression
of
HLA-DR
was
demonstrated
in
serial
sections.
Figure
lc
demonstrates
staining
for
cytokine
p13.
CD4-
(Fig-
ure
ld)
and
CD8-positive
T
cells
were
abundantly
present
in
thymoma
transplants,
whereas
B
cells
were
never
found
(Figure
le).
The
immunostain
with
MAb
155
was
positive
in
all
thymoma
transplants
(Figure
1f).
No
myoid
cells
were
present
in
the
transplants.
Transplants
with
Extrathymomal
Thymic
Tissue
Transplants
of
the
extrathymomal
thymic
tissue
were
also
examined
on
H&E-stained
sections
(Figure
2a).
Although
the
transplants
obtained
from
a
given
pa-
tient
were
similar
in
appearance,
there
were
marked
differences
between
the
specimens
in
the
degree
of
fatty
involution.
In
all
transplants
from
the
thymic
tissue
with
lymphofollicular
hyperplasia,
cytokeratin,
CD4+
and
CD8+
T
cells,
and
CD22+
B
cells
were
readily
detectable
(Figure
2b).
Staining
with
the
MAb
155
revealed
typical
myoid
cells
(Figure
2c).
Fewer
lymphocytes
were
found
in
the
involuted
than
in
the
hyperplastic
thymic
transplants,
but
they
also
contained
CD22+
B
cells
in
areas
positive
for
anti-HLA
ABC
(Figure
2,
d
and
e).
Human
Anti-AchR
Antibody
Measurements
and
Whole
IgG
in
the
Serum
of
Transplanted
SCID
Mice
Anti-AchR
antibodies
could
not
be
detected
in
SCID
mice
transplanted
with
thymoma
tissue
(n
=
24).
Fur-
thermore,
anti-AchR
antibodies
were
also
absent
in
the
sera
of
mice
that
had
received
involuted
thymic
tissue
(n
=
11).
However,
consistent
with
previous
findings,
transplants
obtained
from
hyperplastic
thymuses
(n
=
6)
produced
high
titers
of
anti-AchR
antibodies
for
pe-
riods
of
3
to
4
months
(Figure
3a).15
Anti-striated
mus-
cle
antibodies
were
never
detected
in
thymoma
nor
in
extrathymomatous
thymic
transplants.
Assays
of
whole
human
IgG
showed
no
human
antibodies
in
SCID
sera
tested
from
thymoma-trans-
planted
mice.
In
contrast,
human
IgG
was
easily
detected
in
all
sera
from
SCID
mice
that
had
re-
ceived
extrathymomal
thymus
transplants
regard-
less
of
whether
these
transplants
were
hyperplastic
or
involuted
(Figure
3b).
None
of
the
mice
showed
clinical
signs
of
MG.

Thymomas
Transplanted
in
SCID
Mice
1363
AJP
May
1996,
Vol.
148,
No.
5
-
E
Ll
43
.0
1
c
S
ID
C!,
:3
o)
70
60
50
40
30
20
in
6
8
10
12 14
weeks
after
transplantation
e,B
Control.
Thymoma
1
n=10
transplants
f
n-12
Figure
3.
A:
Anti-AchR
antibody
titers
(nanomoles
per
liter)
in
sera
from
six
SCID
mnice
transplanted
with
fragments
of
MG
thymic
tissue
nvith
Iymphofollicular
hyperplasia.
B:
Human
IgG
titers
(micrograms
per
milliliter)
in
serafrom
SCID
mice
(
nontransplanted
controls,
trans-
planted
with
thymoma
fragments,
transplanted
with
fragments
of
atrophic
thymus,
transplanted
with
fragments
of
thymus
with
tympho-
follicular
h4perplasia).
The
bars
represent
individual
SCID
sera.
Discussion
Thymectomy
is
considered
to
be
beneficial
in
MG
as-
sociated
with
lymphofollicular
hyperplasia
of
the
thy-
mus.
By
contrast,
the
clinical
response
to
thymectomy
is
usually
disappointing
in
the
presence
of
thymoma.5'6
One
explanation
for
the
superior
clinical
benefit
of
thymectomy
in
MG
patients
with
lymphofollicular
hy-
perplasia
may
be
the
removal
of
a
major
source
of
anti-AchR-antibody-producing
B
cells.
Removal
of
hy-
perplastic
thymus
tissue
commonly
results
in
a
reduc-
tion
of
anti-AchR
antibodies,
but
in
thymoma-associ-
ated
MG,
thymomectomy
rarely
reduces
the
autoantibody
titers.
In
these
cases,
the
numbers
of
autoantibody-producing
B
cells
seems
to
remain
undi-
minished
upon
thymectomy.
Indeed,
in
our
samples,
B
cells
were
completely
absent
from
thymoma
tissue,
and
no
human
IgG
was
produced
by
thymoma
frag-
ments
transplanted
into
SCID
mice.
In
the
extrathymomal
thymic
tissue,
however,
B
cells
are
present
at
variable
frequency
and
they
secrete
antibodies
(IgG)
in
SCID
mice.
But,
although
the
patients
had
very
high
anti-AchR
antibody
titers,
no
or
only
small
amounts
of
autoantibodies
origi-
nated
from
this
site.
This
finding
was
consistent
in
all
thymuses
studied
except
in
one
case
in
which
there
was
lymphofollicular
hyperplasia
with
ample
anti-
AchR
antibody
production.
Secretion
of
anti-AchR
antibodies
and
anti-striated
muscle
antibodies
by
tumor-free
thymic
remnants
was
reported
by
oth-
ers,23
but
these
remnants
may
have
been
hyperplas-
tic
(for
a
summary
of
antibody
production
in
thymic
transplants,
see
Table
2).
The
major
site
of
anti-AchR
antibody
production
in
thymoma-associated
MG
is
still
not
known.
Regional
lymph
nodes
or
the
spleen
are
potential
candidates
and
will
be
investigated
in
future
studies.
Although
the
autoantibodies
in
thymoma-associated
MG
are
produced
at
sites
other
than
the
thymus,
the
thymus
may
still
play
a
central
role
in
the
immunopatho-
genesis
of
thymoma-associated
MG.
Thymomas
con-
stitute
a
heterogeneous
group
of
thymic
epithelial
tu-
mors,
not
all
of
which
are
associated
with
the
development
of
MG.
Recently,
a
classification
of
thy-
momas
was
introduced
that
relates
the
characteristics
of
the
tumor
to
the
histological
structure
of
the
normal
thymus.24
Medullary,
mixed,
and
cortical
thymomas
and
the
well
differentiated
thymic
carcinoma
retain
or-
ganotypic
features.25
Among
thymomas,
the
cortical
and
the
well
differentiated
type
have
the
strongest
as-
sociation
with
MG,
but
immunohistochemistry
and
Northern
blot
analysis
never
showed
expression
of
AchR
in
thymomas.26
Instead,
a
153-kd
protein
(p153)
was
identified
on
thymoma
epithelial
cells
of
those
his-
tological
types
that
are
frequently
associated
with
MG.
This
protein
is
distinct
from
the
AchR26
but
is
recog-
nized
by
the
MAb
155,
originally
raised
against
the
cytoplasmic
epitope
371
to
378
of
the
a-subunit
of
the
AchR.
19'20'27
In
this
study,
p153
could
be
detected
in
the
thymoma
transplants.
The
function
of
p153
is
pres-
ently
unknown.
However,
it
can
be
speculated
that
T
cells
are
sensitized
intrathymically
against
epitopes
of
p153,
which
cross-react
with
other
epitopes
on
the
Table
2.
Summary
of
Antibody
Production
of
MG-
Thymus-Transplanted
SCID
Mice
Antibodies
measured
in
SCID
mice
Transplanted
tissue
Normal
thymus
Hyperplastic
thymus
Thymoma
Atrophic
extrathymomal
tissue
Hyperplastic
extrathymomal
tissue
Human
Anti
AchR
total
IgG
antibody
++
++
++
++

1364
Spuler
et
al
AJP
May
1996,
Vol.
148,
No.
5
AchR.
Such
intrathymically
activated
cross-reactive
T
lymphocytes
could
act
in
the
peripheral
immune
sys-
tem
as
helper
lymphocytes
activating
B
lymphocytes
to
produce
autoantibodies
against
native
AchR.28
Acknowledgments
We
are
grateful
to
Prof.
Spelsberg,
Prof.
Sunder-
Plassmann,
and
Dr.
Dienemann
for
kindly
providing
thymus
tissue
and
Prof.
Thierfelder
for
the
SCID
mice.
We
thank
Dr.
A.
G.
Engel
for
helpful
discussion
of
the
manuscript.
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