Cell, Vol. 52, 831-841.
March
25, 1988. CopyrIght
0 1988 by Cell Press
In Transgenic Mice the Introduced
Functional T Cell Receptor p Gene
Prevents Expression of Endogenous
Yasushi
Uematsu;
Stefan
Ryser,*t
Peter
Borgulya,’
Paul Krimpenfort,*
Harald
von Boehmer,*§
and Michael
* Easel
Institute
for Immunology
CH-4005
Basel,
Switzerland
r Central
Research
Units
F. Hoffmann-La
Roche
& Co. Ltd.
CH-4002
Basel,
Switzerland
*Division
of Molecular
Genetics,
The Netherlands
Cancer
Institute
and Department
of Biochemistry,
University
of Amsterdam
Plesmanlaan
121
1066 CX Amsterdam,
The Netherlands
Zlatko
Dembid,‘t
Anton
Berns,l
Steinmetz’t
Summary
Transgenic
mice were constructed
with a functional
T
cell receptor
B gene.
Transcription
of the introduced
gene
is largely
confined
to T cells,
but low levels
of
transcripts
are also seen
in B cells
and in other
tissues.
Serological
analyses
show
that most,
if not all,
of the T lymphocytes
express
the transgenic
p chain
on the cell surface
and lack p chains
encoded
by endogenous
p genes.
Molecular
genetic
analyses
of uncloned
and cloned
T lymphocytes
demonstrate
that
rearrangement
of endogenous
p genes
is incomplete.
Partial
D~l.l~l
rearrangements
are found
preferentially,
while
complete
VDJ
rearrangements
are not
seen.
These
findings
show
that
expression
of the
transgene
regulates
the rearrangement
of endogenous
b genes.
Although
the af3 T cell receptors
of the
transgenic
mice are homogeneous
with respect
to the
b chain,
they are fully functional,
at least
in a variety
of allogeneic
responses.
Introduction
The T cell receptor
expressed
on the cell surface
of the
majority
of T lymphocytes
is composed
of a and 8 glycoproteins,
The disulfide-linked
a8 heterodimer
is noncovalently associated
with at least four different
additional
proteins forming
the CD3 complex
(for review, see Allison and
Lanier, 1987a). With gene transfer
experiments
it has been
shown
(Dembic
et al., 1986; Gabert
et al., 1987; Saito et
al., 1987) that the a and 8 chains
of a given T cell receptor
are necessary
and sufficient
for the recognition
of its
ligand,
usually
a peptide
associated
on the surface
of the
antigen-presenting
cell with a class I or class
II molecule
of the major histocompatibility
complex.
The CD3 components require
the a8 heterodimer
for cell-surface
expres§On sabbatical
leave at the Massachusetts
lnstltute of Technology,
Center of Cancer Research,
Cambridge,
Massachusetts.
p Genes
sion and may be involved
in signal
transduction
(for review, see Weiss
et al., 1986). The a8 heterodimer
forms
the receptor
on helper and cytotoxic
T lymphocytes.
It has
recently
been shown
that less than 5% of peripheral
T
lymphocytes
in mice and humans
do not express
the a(3
heterodimer,
but contain
a distinct
y6 heterodimer.
The antigen specificity
of these cells is not known (for review, see
Allison
and Lanier,
1987b).
T cell receptor
a and 8 chains
are members
of the immunoglobulin
superfamily
and are encoded
by genes
that
are formed
by DNA rearrangements
during
T cell differentiation (for review,
see Kronenberg
et al., 1986). Modes
of
DNA
rearrangement,
gene
segment
organization,
and
signal sequences
important
for rearrangement
are similar
for T cell receptor
and immunoglobulin
genes.
It is believed that both gene families
share
a common
ancestry.
In the mouse,
the T cell receptor
a gene family
is composed
of about
100 variable
(Va), about 50 joining
(Ja),
and a single
constant
(Ca) gene segment.
The murine
8
gene family
contains
at least 21 Vg, 2 diversity
(08),
12
functional
Jo, and two C8 gene segments
spread
out over
at least 450 kb of DNA (Lai et al., 1987; Chou et al., 1987).
The D8, Jf3, and Co gene segments
are organized
into two
clusters
containing
the D81; 6 functional
J81, C81, and
D82; 6 functional
J82; and the C82 gene segment,
in this
order.
During
T cell differentiation
in the thymus,
8 chain
gene segments
appear
to be rearranged
before
a (for review, see Born et al., 1987). Assembly
of a functional
8
chain gene involves
08 to J8 joining
followed
by V8 gene
segment
rearrangement
and deletion
of intramolecular
DNA sequences
(Okazaki
et al., 1987). Partially
(DJ) and
fully (VDJ) rearranged
8 genes
are transcribed
to yield 1.0
and 1.3 kb RNA molecules,
respectively.
Because
the formation
of an open
reading
frame
by rearrangement
is a
stochastic
event,
T lymphocytes
will often contain
nonfunctionally
rearranged
T cell receptor
a and 8 chain
genes.
T cells containing
two functional
a or 8 alleles have
not been found,
and it is therefore
assumed
that like immunoglobulin
genes,
T cell receptor
genes
are subject
to
allelic exclusion.
Transgenic
mice, obtained
by experimental
integration
of foreign
genetic
material
into germ-line
DNA, provide
an
excellent
system
for studying
gene regulation
and function (for review,
see Palmiter
and Brinster,
1986).
More
specifically,
transgenic
mice produced
with immunoglobulin light and heavy chain genes
have been used to study
mechanisms
of gene segment
rearrangement
and allelic
exclusion,
tissue
specificity
of expression,
somatic
hypermutation,
and repertoire
development
(for review,
see
Storb, 1987). It has been found that immunoglobulin
transgenes
are specifically
expressed
in B cells, with the exception
of w heavy
chain transgenes,
which
are also expressed
in T lymphocytes.
The introduction
of functional
immunoglobulin
genes
into transgenic
mice clearly
affects the formation
of functional
endogenous
immunoglobulin
genes,
although
a complete
inhibition
of the syn-
Cell
832
0A
Figure
1 Structure
of the T Cell Receptor
D
APIGAGGACTTTCCCACACCcAGcccTATTTAcTc~TAcAGccATcTccccTTcTATTGA
Transgene
(A) Nucleotlde sequence of the variable region
G~GG-GGGCTGAGACTAAGGTCCAGTACTACAGAGAGCCTAGTGGGAGT~CAGTA
of the transgene
as determmed
by sequencing
TCAACAGATCAC,GACcTcAG~TcTGAcATcAcAGGc~TGATGATAGGAGGAG~GG
5’“T
-
I ~ LS.1
AGGAGCTGACTCCTGCTcTcTcAcc~GAGAccATATccTAGAGG~GcATGTcT~cA
CTGCCTTcCCTGACCCCGCCTGG~cAccAcccTGcTATcTTGGGTTGcTcTcTTTcTcc
- I _ “LIB.2
TGGG~CAAAACACATGG*GGCTGCAGTCACCCAA
ATCGGCAGGACAcGGGGCATGGGcTGAGGcTGATccATTATTcATATGGTGcTGGcAGcA
CCCTCATTCTGGAGTTGGCTACCCCCTCTCAGACATCAGTGTACTTCTGTGCCAGCGGTG
- I
082
a full-length cDNA clone. The 5’ untranslated
region (5’UT). VI)5 1 leader sequence
(L5 1).
VP8 2 gene segment,
D112 sequence
with assoclated N regions, and the first nucleotldes
of
the J[12 3 gene segment are shown The complementary
strand of the VDJ ollgonucleotlde
used as a specific hybridization
probe IS underlined (B) Organuabon
of cosmld clone cos
HY119-1 14-5 containing
the transgene.
The
wsmld clone was constructed
by using DNA
fragments
dewed from the 862 16 clone i.9
and the BALB/c liver clone cos H-2dll-1 14T as
descrtbed
in Expertmental
Procedures
T cell
receptor Ii gene segments that are known to be
located on the cosmld clone are Indicated
IJb2.3
ATAACAGTGCAGMACGCTGT
0B
thesis of endogenous
immunoglobulin
molecules
has not
been found.
Levels
of immunoglobulin
polypeptides
encoded
by the transgenes
appear
to influence
the extent of
allelic
exclusion.
Whether
immunoglobulin
molecules
block rearrangement
directly
or indirectly
via intracellular
or intercellular
interaction
1s not clear.
In our experiments,
we address
the question
of whether
transgenic
mice with homogeneous
expression
of defined
T cell receptor
polypeptides
can be produced.
Using
a
functionally
rearranged
fi gene, we demonstrate
that this
is the case, and we analyze
the transgenic
mice for tissue
specificity
of transgene
expression,
endogenous
11 gene
rearrangements,
and T cell receptor
function.
Results
A Functionally
Rearranged
[J Gene
from
a Male
Antigen-Specific,
H-2Db-Restricted
Cytotoxic
T Cell Clone
The source
of the functionally
rearranged
T cell receptor
[1 gene used In this study is a cytotoxic
T cell clone, called
B6.2.16. Clone B6.2.16
lyses male but not female C57BL/6
cells and IS restricted
by the H-2Db molecule.
This clone
was chosen
because
its T cell receptor
fl chain
can be
identified
by the monoclonal
antibody
F23.1 (Staerz
et al.,
1985)
which
binds to antigenic
determinants
on all three
members
of the Vii8 family
(Behlke
et al., 1987).
Screening
of cDNA
and genomic
lrbraries
with a V()
probe
yielded
a full-length
6 chain
cDNA
clone
and
genomic
clones
containing
two different
fi alleles.
DNA sequence
determination
of the cDNA
clone
(Figure
1A)
showed
that it was derived
from a functionally
rearranged
I) gene composed
of the V(15.1 leader,
V(18.2; a short portion of the 0112 segment,
J(i2.3;
and the Cl12 gene segment. One of the two 1) alleles,
isolated
In clones
1,8 and
h9, corresponds
to the cDNA
clone.
Characterizatron
of
clone
h8 by restriction
mapping
and DNA sequencing
showed
that it contains
the rearranged
Vh8.2 gene about
100 bp downstream
of an apparently
inactive
Vf18.2 leader
exon,
while the V[E.l
leader
sequence
is located
about
2.5 kb farther
upstream
(Figure
1B). Splicing
of the Vfj5.1
leader
exon to the Vfj8.2
exon
in T cells derived
from
C57BL16
mice has been described
by Chou et al. (1987).
The second
11 allele in B6.2.16
contains
an Incomplete
Df%l-Ji12.5
rearrangement
(not shown).
For the production
of transgenic
mice, we wanted
to In-
Transgenlc
833
Mice with a T Cell Receptor
(I Gene
Table 1 Transgemc
Mica
Founder
OffsprIng
SEX”
COPY
Numb&
Transgene
TranscriptIon
T Cell Surface
ExpresslonC
93.1
93.2
93.4
93.9
m
f
f
f
m
m
m
f
m
f
m
m
1
20
10
5
0
20
2
20
3
2
2
2
ND
ND
+
ND
+
+
+
+
+
+
ND
1ova-20%
l OV-20%
ND
>98%
ND
>98%
>98%
>98%
ND
>98%
ND
ND
90
91
92
93
94
95
95.39
95 40
a Sex is indicated by m (male) and f (female).
b Copy number of the transgene was estimated from signal lntensltles on Southern blots with Pvull-dlgested
tall DNA hybridized
c Surface expression
of F23.1.positive
p chains on lymph node T cells was quantitated
by FACS analysis.
ND, not determined.
ject the functionally
rearranged
j3 gene with long 5’ and 3’
flanking
sequences,
because
initial
experiments
(not
shown)
with clone 18 containing
only 1.2 kb of 5’ and 3 kb
of 3’ flanking
sequence
failed to give expression
in nine
independently
obtained
transgenic
mice. We speculated
that lack of expression
could be due to a missing
regulatory element.
Therefore,
clone h9 containing
the functionally rearranged
Vj3 gene together
with 9 kb of 5’ flanking
sequence
was fused
at the unique
Sacll site in the J(32
gene cluster
to a cosmid
clone derived
from BALBlc
liver
DNA containing
the C8 locus
together
with 18 kb of 3’
flanking
sequence.
Thus a new cosmid
clone (cos HY891.145)
for the functionally
rearranged
8 gene was constructed,
as shown
in Figure
1B.
Transgenic
Mice Express
the Introduced
T Cell Receptor
p Gene
The insertion
of this cosmid
clone
was excised
and injected
into pronuclei
or fertilized
eggs obtained
after mating of (CBAIBrA
x C57BL/LiA)F,
mice.
Thirteen
pups
were born that contained
from 1 to about 50 copies
of the
transgene,
according
to Southern
blot analysis
of tail
DNA. Of these
mice, seven were analyzed
after splenectomy for transcription
with an oligonucleotide
probe
that
covered
the VDJ joining
region
and that was therefore
specific
for the transgene.
Six of the seven
mice showed
a 1.3 kb full-length
transcript.
As observed
before for other
transgenes,
no simple
correlation
between
copy number
and transcriptional
activity
was seen.
T lymphocytes
obtained
from lymph
nodes
of four transgenic
mice were
subsequently
analyzed
for cell-surface
expression
of the
transgenic
6 chain on a fluorescence-activated
cell sorter
(FACS)
by using
F23.1 antibodies
(Table
1). While
about
lo%-20%
of T cells were F23.1-positive
in mice 90 and
91, practically
all of the T cells from mice 93 and 95 were
stained
with the F23.1 antibody.
This indicates
that most,
if not all, of the T cells in mice 93 and 95 express
the transgenie 6 chain on the cell surface.
Since a background
of
about
lo%-20%
F23.1-positive
T lymphocytes
is ex-
with the Jp2 probe.
kb
JP2
Figure 2. Southern
Blot Analysis of Offspring
Mice Dewed
Cross of the Female Transgenic
Mouse 93 wth C57L Males
from
a
From 18 pups born, this blot shows the analysis of 9 mice It demonstrates the inheritance
of the transgene
(indicated by the 7.7 kb band)
m either 2 (mice 93.3, 93.4, 93.7) or 20 (mice 93.2, 93.9) copies. Mice
93.5, 93.6, 93.8, and 93.10 did not inherit the transgene. Addittonal offspring mice 93.1, 93.12, 93 13, 93.16, 93 18 did not; 93.14 and 93.15 inherited 2; and 93.11 inherlted 20 copies (not shown). The 5.5 kb band
represents
the endogenous
b locus. Southern blot analysis of Pvulldigested tall DNA was carried out as described In Experimental
Procedures, with a Ja2 probe.
petted
for these
mouse
strains
(Staerz
et al., 1985)
it is
unlikely
that the transgenic
6 chain
is expressed
in mice
90 and 91. In agreement
with the FACS analysis
and our
interpretation,
Northern
blot analysis
did not show
any
transgene
transcription
in T lymphocytes
of mouse
90 (Table 1).
The male founder
mouse
93 was subsequently
crossed
with female
C57L
mice (a F23.1-negative
mouse
strain;
Behlke
et al., 1986). Eighteen
offspring
mice from the first
generation
were analyzed
for the inheritance
of the transgene by Southern
blot hybridization.
Three
(1 male, 2 females)
contained
about 20 copies
of the transgene,
5 (4
males,
1 female)
contained
about 2 copies,
and 10 lacked
the transgene
(Figure
2). The segregation
of the transgene into 2 and 20 copies
could
be due to integration
of
injected
DNA into two unlinked
chromosomal
loci, or could
reflect a deletion
or amplification
event after integration
at
Cd
634
F23 1
am-CD3
c57eL’6
c9
kb
VDJ
dig0
934
Figure 3. Surface Expression
from Transgemc
and Normal
of F23.1.Posltlve
Mice
1) Chains
on T Cells
Lymph node T cells shmulated in vitro by lrradlated allogenelc DEAR
spleen cells were Incubated
with VP&specific
F231 antibodtes
followed by FITC-labeled
sheep antl-mouse
lmmunoglobulin
antibodles,
and were analyzed on FACS. In parallel, the expression
of CD3 molecules was momtored by using the 145 Xl1 monoclonal antibody (Leo
et al., 1987) followed by FITC-labeled
goat anti-hamster
ImmunoglobuIin antlbody.
kb
1.51.3-
VDJ
oligo
a single site. Analysis
of second
generation
offspring
mice
shows
that the two different
forms
of the transgene
are
stably
inherited.
The transgene
is therefore
present
in
germ-line
DNA and transmitted
in a Mendelian
fashion
to
both males
and females.
We analyzed
mice with 2 or 20
copies
of the transgene
for cell-surface
expression
with
F23.1 antibodies.
As shown
in Figure
3, most, if not all, of
the peripheral
T lymphocytes
analyzed
in mice 93.2 (20
copies)
and 93.4 (2 copies)
stain with the Vu8specific
monoclonal
antibody.
The staining
intensity
is similar
in
both T lymphocyte
populations,
indicating
that despite
the
difference
in copy number,
these
mice express
the transgene to the same
extent.
Lymph
node cells from mice with 2 copies
of the transgene were analyzed
for the ratio of CD4- and CD8-positive
T cells. Lymph
node cells from a transgenic
male mouse
were 45% CD4+ and 18% CD8+,
and from a transgenic
female
mouse,
35% CD4’
and 29% CD8*;
control
cells
from a C57BL/6
mouse
were 45% CD4+ and 32% CD8’.
These
numbers
do not deviate
significantly
from those
normally
seen. Thus, although
the transgenic
p chain, derived from a class l-restricted
cytotoxic
T cell clone, is expressed
in most, if not all, of the T lymphocytes,
tt does
not affect
the normal
ratio of CD4- and CD8-positive
T
cells.
Transcription
of the Transgene
Is
Largely
T Cell-Specific
We analyzed
mouse
93.2, containing
20 copies,
for tissuespecific
expression
of the transgene.
Northern
blot analysis using
the VDJ oligonucleotide
as a probe
reveals
strong
transcription
in the thymus,
while little or no tran-
Figure
4. Tissue
Speclfuty
Cg
of Transgene
Ca
Transcription
(A) Northern blot analysts of transgenlc
mouse 93.2. Total RNA was
derived from the tissues Indicated. and hybrldlzed with the VDJ ollgonucleotlde speck
for the transgene
RNA derived from the 86.2.16
cytotoxlc T cell clone was run as a control. (B) A blot with T and B cell
RNA from transgemc
mouse 93.2 and 86.2.16 RNA as a control was
hybrldtzed sequentially
with the VDJ oligonucleotide.
a CD probe. and
a Ca probe. Between hybrldtzatlons.
radIoactIve
probes were stripped
from the falter by lncubatlon in 0 lx SSC, 0.1% SDS, at 95% Sizes of
hybrldlzlng
T cell receptor a and P RNA molecules were determined
by comparison
with 16 and 28 S rlbosomal RNA. B lymphocytes
from
mouse 93 2 were obtalned by treatment of spleen cells with antI-Thy-l
antIbodIes and complement,
followed by stimulation with Ilpopolysaccharade for 5 days. T lymphocytes
were obtalned from lymph nodes
and stimulated in vitro with DBAIZ cells
scription
is seen In the other tissues
analyzed
(Figure
4A).
Similar
results
were obtained
for transgenic
mice 93.4 and
95.39, containing
2 copies
of the transgene
(not shown).
The small amounts
of transcripts
we see in some nonlymphoid tissues
could be due to infrltrating
T lymphocytes
or
incomplete
inactivation
of the transgene.
Several
findings
show
that some
aberrant
transcription
of the transgene
does indeed
occur.
First, a longer
exposure
of Figure
4A
reveals
aberrantly
sized
transcripts
In muscle
cells that
are not found in T cells. Second,
chimeric
mice obtained
by transferring
bone
marrow
cells from transgenic
93.2
offspring
mice into irradiated
C57L
recrpients
were analyzed
for T cell infiltratron
(not shown).
While
transcripts
of the transgene
are detected
in bone
marrow,
kidney,
lung, and liver, no such transcripts
are seen in brain and
muscle.
Thus, at least in muscle
and brain. the transgene
is aberrantly
transcribed
in transgenic
mice. Third,
B lym-
Transgenlc
835
Mice with a T Cell Receptor
Ii Gene
antWCD3
F23 1
L
0
Figure 5 Gel Electrophoretic
Analysis
Chains from Transgemc
T Lymphocytes
with F23 1 Monoclonal AntIbodIes
..
loo
200
irl
0
100
200
of lmmunopreclpltated
13
before and after Preclearlng
Lymph node T cells from transgene
mouse 93 2 were surface labeled
with ‘751, and T cell receptor [i chams were precipitated
with or without
precleatlng
with F23.1 anttbodles as Indicated II- the figure and as described
II- Experimental
Procedures.
Lymph
node T cells from
C57BL/6 mice and clone B6 2.16 were analyzed as controls
phocytes
also show a low level of transgene
transcription.
Both the VDJ oligonucleotide
and the Cf3 probe
identrfy
8 chain transcripts
in B lymphocytes,
while ct chain
transcripts,
even after a longer
exposure,
are not seen (Figure
4B). The observed
transcripts
of the transgene
are therefore not due to residual
T cell contamination
of the B-cell
preparation,
but reflect an about 20.fold lower level of transgene transcription
in B as compared
to T lymphocytes.
Transgenic
T Lymphocytes
Express
Only
F23.GPositive
fl Chains,
Which
Participate
in the Formation
of Functional
Receptors
The F23.1 staining
experiments
indicate
that the transgene is expressed
in the vast majority
of T lymphocytes.
It is possible
that additional
8 chains
are encoded
by endogenous
genes.
Several
independent
approaches
were
used to check
this possibility.
In a first kind of experiment,
membrane
extracts
from T cells of transgenic
mice were
“precleared”
of V88 proteins
by using
F23.1 antibodies
coupled
to Sepharose
beads.
As shown
in Figure
5, after
preclearing,
no further
I$ chains
were precipitated
by a
panspecific
antiserum
(Traunecker
et al., 1986) for mouse
(3 chains.
When
the original
male-specific
clone
B6.2.16
and T cells from C57BL/6
mice were analyzed
for controls,
the panspecific
antiserum
precipitated
I), chains
from the
latter but not from the former
cells after preclearing
with
F23.1 antibodies.
In a second
kind of experiment,
T cell
receptors
were modulated
(by capping
and endocytosis)
by F23.1 antibodies
and rabbit anti-mouse
immunoglobulin at 37°C. Modulated
and nonmodulated
cells were then
stained
with anti-CD3
antibodies
(Leo et al., 1987). As
shown
in Figure
6, modulation
with F23.1 antibodies
re-
Figure 6 Modulation of Surface Expression
of CD3 Molecules
IOUS T Cells by V[W-Spectfic
F23.1 Monoclonal
AntIbodIes
on Var-
Lymph node T cells from transgenic
mouse 93.2 and from normal
C57BL/6 mice as well as cells from the donor clone 86.2.16 were I”cubated with an excess of F23.1 antlbody and with rabbit anti-mouse
lmmunoglobulm
antlbody for 24 hr at 37°C. Then the viable cells were
separated
and statned with antIbodIes
as indicated in the figure.
moved
all T cell receptor-associated
CD3 molecules
from
cell surfaces
of the transgenic
mice and from the 86.2.16
clone but not from other T cells. A third kind of experiment
showed
that the activity
of cytolytic
T cells from transgenic
mice on allogeneic
DBA/2
and C3H/HeJ
target cells was
completely
blocked
by F23.1 antibodies,
whereas
those
from normal
C57BL/6
mice were not inhibited
significantly
when
used on the same
targets
(Figure
7). Lysis of allogeneic
target cells after in vitro stimulation
was specific.
Cytolytic
T cells
from transgenic
mice stimulated
with
DBA12 cells
lysed
DBA/2
but not C3H/HeJ
target
cells,
Killer to target ratto
Figure 7. lnhibltlon of Cytolytlc Activity
Specific F23.1 Monoclonal
AntIbodies
of Transgenic
T Cells by V1$8-
Lymph node T cells from transgemc
mouse 93.2 or from normal
C57BL/6
mice were stimulated
in vitro with Irradiated
DBA/P or
C3H/HeJ spleen cells, and after 4 days were tested for cytolytic actlvlty
on mdlcated targets II- the presence
or absence of F23.1 antIbodIes.
Cell
836
Figure
kb
kb
9.4-
9.4-
6. Endogenous
B Genes
in Transgemc
Mouse
kb
93.2 Show
Predominantly
Partial
Dal-J6
Rearrangements
(A) Hrndlll-drgested
DNA from the cells Indicated were analyzed with a Cg probe by Southern blot hybrrdrzatron
The 3 kb band represents
the CfQ
gene, which gives a strong signal rn cells from mouse 93.2 because of the presence
of the transgene.
The 9.4 kb band is derrved from the Cpl
locus This fragment disappears
(at least partially) in T cells because of rearrangements
to J61 gene segments.
T cell DNA from mouse 93.1 (not
containing the transgene;
see Table I), C57L liver DNA, and T and B cell DNA from mouse 93 2 were used as controls. (8) The filter shown rn (A)
was stripped of the CD probe and subsequently
hybridized with a D61 probe. Note that the 9.4 kb germ-line DNA fragment containing the C51 locus
is picked up rn C57L liver and transgenrc
mouse 93.2 B cell DNA. It shows evidence for heterogeneous
rearrangements
in C57L and mouse 93 1
T cell DNA, as expected
A limited set of rearranged
fragments
is rdentrfied, however, rn thymus and T cell DNA from transgenic
mouse 932 (C)
Wrth a De2 probe, a 5.3 kb Hindlll fragment containing the Jb2 and 052 gene segments in germ-line confrguration
is identrfied as expected in mouse
93.2 brain and B cell DNA, while rearrangements
are evrdent from Its drsappearance
rn mouse 93.1 T cell DNA Surpnsrngly,
the 5.3 kb band remains
mostly rn germ-line confrguratron
also in 93.2 thymus and T cell DNA
and stimulation
with C3H/HeJ
cells gave rise to C3H/HeJdirected,
but not DBA/2directed,
killer cells (not shown).
Taken
together,
these
experiments
indicate
that only 8
chains
encoded
by the transgene
are expressed
on T lymphocytes
of the transgenic
mice and that they are used to
form functional
T cell receptors.
Endogenous
0 Genes
Are Incompletely
Rearranged
Next we wanted
to find out whether
the lack of endogenous 8 chains
on the cell surfaces
of transgenic
T lymphocytes was due to incomplete
rearrangements
of endogenous genes.
Rearrangement
of endogenous
6 genes
can
be distinguished
from the rearranged
transgene
by using
specific
hybridization
probes.
With a C6 probe,
rearrangements at the Cf31 locus can be separated
from those at the
C62 locus
(which
is present
in the transgene)
if Hindllldigested
T cell DNA is analyzed.
This analysis
clearly
shows
that the 9.4 kb germ-line
Hindlll
fragment
containing the C61 locus is rearranged
in transgenic
T lymphocytes from mouse
93.2 (Figure
8A). The rearrangements,
however,
appear
to be qualitatively
different
from those
seen in normal
C57L T lymphocytes
used as a control.
While
no discrete
rearranged
fragments
appear
in C57L
T cell DNA, a series of fragments
(not seen in a 6 cell control) is evident
in T cell and thymus
DNA from transgenic
mouse
93.2. Also, mouse
93.1, which
did not inherit
the
transgene,
looks
like the C57L control.
With a probe
derived
from the 5’ flanking
region
of the
Df31 gene segment,
a series
of discrete
rearranged
fragments is again seen in transgenic
but not in control
T cell,
nor in transgenic
B cell, DNA (Figure
8B). This result
shows
that partial
D(11 rearrangements,
presumably
to
J/31 and J82 gene
segments,
are present
in unusually
high frequencies
in the transgenic
mouse.
Indeed,
the
sizes of the observed
rearranged
fragments
are in agreement with this assumption.
Since
these
fragments
are
identified
with a 5’ flanking
probe of D81, they represent
partial
rearrangements
that do not involve
V8 gene segments.
Inversion
of the V814
gene
segment,
located
downstream
of Cg, would
not delete
the 5’ flanking
sequence
of 06 gene segments
(Malissen
et al., 1986). Analysis of T cell clones
from transgenic
mice (see below) with
a Vf314 probe,
however,
shows
that endogenous
V614
genes
have not been rearranged
to Df3 gene segments.
In
agreement
with the Southern
blot analysis,
a Northern
blot analysis
of thymus
and peripheral
T cell RNA with a
mixture
of 3zP-labeled
V67, Vf39, V811, V612, V814, Vf315,
and V816 gene segments
did not reveal
V8 gene transcripts
in transgenic
mouse
93.2, while a clear signal was
obtained
with a C57BL16
control
(not shown).
In contrast
to the D61 gene segment,
the D62 gene segment does not undergo
frequent
rearrangements
in transgenie mouse
93.2. As shown
in Figure
8C, it rather seems
that most of the D82 gene segments
remain
in germ-line
configuration
in 93.2 thymocytes
and T cells. Mouse
93.4,
containing
only 2 rather than 20 copies
of the transgene,
shows
a similar
high frequency
of partial
D61 rearrangements
(not shown).
Furthermore,
mouse
95.39, independently
generated,
and a transgenic
mouse
obtained
by
coinjection
of the same
8 gene
together
with a T cell
Transgenrc
837
Mrce wrth a T Cell Receptor
Table 2. T Cell Clones
fi Gene
Analyzed
Mouse
Clone”
DO1 to J(11
93.2
2
8
14
16
17
18
19
2oe
2
1
1
1
2"
2
1
2
1
2
93.4
Dfil
to Jfi2
Df12 to Jf12
Dfil to Dff2C
1
1
1
1
1
2
a Rearrangements
of the two homologous
(3 IOCI rn each T cell clone were analyzed wrth CfI. Df11. and Dfi2 probes
b T cell clones were obtarned as described
rn Expertmental
Procedures.
c The sizes of these rearranged
fragments
do not correspond
to those expected
for DJ rearrangements
* Indicates that DJ rearrangements
have occurred
on both homologous
chromosomes.
c Clone 20 contarns one 1) locus rn germ-tine confrguratton.
receptor
a gene show a predominance
of partial
Djil rearrangements
(not shown).
Thus
neither
the high copy
number
of the transgene
in mouse
93.2 nor tts peculiar
chromosomal
location
causes
this unusual
pattern
of rearrangement.
The results
presented
so far do not exclude
a low but
still significant
number
of complete
endogenous
VDJ rearrangements.
To obtain
a more quantitative
estimate,
we
analyzed
nine T cell clones
obtained
from mice 93.2 and
93.4 with the same
CD, D81, and Df52 probes.
Of the 18
endogenous
6 loci screened,
13 rearranged
Dpl to Jhi,
2 fused D[il with J62, 3 joined
D62 with J62, and 2 show
unusual
rearrangements
of both Dpl and Dj12 gene segments
that were not further
characterized
(Table
2). No
complete
VDJ rearrangements
were found,
Indicating
that
they occur
very rarely
If at all.
The partially
rearranged
endogenous
jI genes
are transcribed
into a series
of RNA
molecules
of different
lengths.
A Cjjl-specific
probe,
which
does
not crosshybridize
wrth C1)2 and therefore
does not prck up RNA
transcripts
from
the transgene,
identifies
a number
of
RNA molecules
that range
in size from
1.0 to 1.5 kb in
length
(Figure
9A). RNA
molecules
derived
from completely
rearranged
[1 genes
are 1.3 kb in length
(see
C57BL/6
control),
and It IS evident
from Figure
9A that 1.3
kb CjIl transcripts
are practically
mrssing
in T cells from
transgenic
mouse
93.2. As shown
in Figure
9B, a similar
set of aberrantly
sized RNA molecules
is identified
when
the D81 probe is used,
as expected
from the preponderance of partial
D(31-JjI
rearrangements.
The exact structural
basrs
of these
heterogeneous
transcripts
is not
known.
Discussion
The j1 gene injected
with 9 kb of 5’ and 18 kb of 3’flankrng
sequence
is expressed
in transgenic
mice at high levels
and, to a large extent,
in a tissue-specific
fashion.
Low levels of 8 chain
transcripts,
derived
from the transgene,
were found
in nonlymphoid
tissues
as well as in B cells.
While
the observed
transgene
transcripts
in some
nonlymphoid
tissues
could
be due to T cell infiltration,
it is
clear that B cells,
muscle
cells, and brain cells do show
a low level of aberrant
transcriptron
of the transgene.
kb
1.5IO-
Figure
93.2
9
Transcriptron
of Endogenous
fi Genes
rn Transgenic
Mouse
(A) A C()l-spectftc
probe was used to analyze T cells for Cftl transcripts by Northern blot hybndizatron
RNA from the cytotoxrc
T cell
clone 86.2.16 does not hybrrdtze, rn agreement
with the organrratron
of Its (I alleles (see text). Cfll transcnpts
present rn mouse 93.2 T cells
are clearly different rn we from those seen rn T cells from a C57BL/6
control mouse (B) Northern blot hybndrzatron
with a D(11 probe rdentrfres aberrantly
wed transcripts
rn the 862.16 cytotoxrc
T cell clone
(presumably
dewed from the nonfunctronal
allele) and also rn mouse
93.2 T cells.
Expression
of the Transgene
Regulates
Rearrangement
of Endogenous
[I Genes
The majority
of endogenous
ji genes
in the transgenic
mice show rearrangements
of D(31 to Jpl gene segments.
No endogenous
VDJ
rearrangements
are seen.
The
preponderance
of incomplete
D(11 to Jgl rearrangements
is highly unusual,
and is independent
of the copy number
of the transgene
and Its chromosomal
location.
It can be
deduced
from the analysrs
of T cells and T cell clones
that
approximately
half of the nonproductive
[I rearrangements
Cdl
838
in normal
T cells represent
VDJ rearrangements
(Kronenberg et al., 1985). Of eight nonfunctional
alleles that have
been characterized
in T cell clones,
five show
partial
DJ
rearrangements
(at least three of which are Dpl-Jfl2)
and
three show
aberrant
VDJ rearrangements
(Goverman
et
al., 1985; Chou et al., 1986; Dembih
et al., 1986; Fink et
al., 1986; Malissen
et al., 1986; this paper).
Expression
of
the introduced
p gene in transgenic
mice therefore
blocks
endogenous
f3 gene
rearrangement
between
D-J
and
V-DJ
joining.
The molecular
mechanism
that regulates
rearrangement of endogenous
p genes
needs
to be investigated.
For immunoglobulin
heavy
chain
genes
it has been
shown that rearrangements
are coupled
to transcription
of
unrearranged
VH genes,
presumably
due to an accessible chromatin
structure
(Yancopoulos
and
Alt, 1985,
1986). Trans-acting
factors
might play a role, and could be
subject
to regulation
by heavy
chain
proteins.
A similar
mechanism
could
regulate
rearrangement
of [), chain
genes.
In this case, transgene
expression
during
thymocyte development
could
block
a transacting
factor
that
would otherwise
activate
rearrangement
of VP genes.
On
the other
hand,
a less direct
mechanism
cannot
be excluded.
Early expression
of the p transgene
could lead to
a more rapid differentiation
of thymocytes,
allowing
less
time for p rearrangements
to occur. Whatever
the mechanism, our findings
provide
clear evidence
that DJ and VDJ
rearrangements
of p genes
are separately
regulated.
The
preponderance
of partial
Dbl-JBl
over DPl-JP2
and
DP2-J(12
rearrangements
in transgenic
T cells could
result from a tracking
mechanism
of the recombinase
preferentially
joining
upstream
gene segments.
Analysis
of fetal thymocytes
and thymocytes
from early stages
of adult
T cell differentiation
has also revealed
a predominance
of
partial
Dbl over D[32 rearrangements
(Born et al., 1985;
Haars et al., 1986; Lindsten
et al., 1987). It remains
to be
seen
whether
the inhibition
of endogenous
D chain
production
is dependent
on the amount
of transgenic
b
chain synthesized,
as seems
to be the case for immunoglobulin
K chains
(Rusconi
and Kiihler, 1985; Storb, 1987);
on the presence
of its transmembrane
region,
as for immunoglobulin
)1 chains
(Storb
et al., 1986; Nussenzweig
et al., 1987); and/or
on its association
with other proteins,
as suggested
for immunoglobulin
p and K chains
(for review, see Storb et al., 1986).
Molecular
genetic
analysis
of the transgenic
mice indicates that rearrangements
of T cell receptor
y genes
(Hayday et al., 1985) are found in total T lymphocytes
with little
difference
from those
seen in normal
mice (not shown).
Rearrangement
of 6 genes
(Chien
et al., 1987) in transgenie thymocytes
has not been studied.
Functionality
of the Transgenic
up T Cell Receptor
Serologic
and molecular
genetic
analyses
indicate
that
only one 0 chain,
encoded
by the transgene,
participates
in the formation
of the T cell receptor
repertoire
in the
transgenic
mice. Preliminary
analyses
indicate
that these
mice are heterogeneous
for the a chain,
since
distinct
a
gene rearrangements
were observed
in T cell clones
(not
shown).
Despite
the fact that most, if not all, of the afi T
cell receptors
are homogeneous
with respect
to the [$
chain,
the ratio of CD4- and CD8-positive
T lymphocytes
is normal
in the transgenic
mice and they show normal
allogeneic
responses.
It has been calculated
that variability
of the b chain
enlarges
the T cell receptor
repertoire
by
a factor
of about
2000 (Kronenberg
et al., 1986). Further
experiments
will show whether
the T cell receptor
repertoire in these
transgenic
mice, with variability
only in the
a chain,
IS still capable
of recognizing
a large number
of
different
antigens
in association
with class
I or class
II
molecules
of the major histocompatibility
complex.
Experimental
Procedures
Establishment
of the Male-Specific
86.2.16 Clone
C57BL16 female mice were lmmumzed with lo7 male spleen cells injected lntraperltoneally
After 14 days, 2 x 10’ female spleen cells
were cultured with 2 x 10’ x-lrradtated
male cells in 8 ml of culture
media. After 12 days, lo6 viable female responder cells were restlmulated with 10’ x-irradiated
male cells in 1 ml of medium containing
Interleukln-2
(IL-2) Cells were cloned by culturing 0.3 cells with stlmulators as described
below Clones were tested for cytolytlc activity as
described below, and clone 86 2 16 was selected after surface stalnlng
of various clones with the F23.1 antlbody (“on Boehmer and Haas,
1986).
Isolation
and CharacterizaOon
of fl Chain
cDNA and Genomic
Clones
Total RNA and high molecular weight DNA were Isolated from cytotoxic
T cell clone 86.2 16 and used to construct
cDNA and genomic DNA
llbranes In hgtll and EMBLS, respectively,
followlng published procedures (Young and Davis, 1983; Fnschauf
et al 1983). Both libraries
were screened with a Cl, probe. Posltlve clones were characterized
by
mapplng with restrictlon endonucleases
and hybndlzation
with probes
specific
for Dpl and Jb gene segments.
A full-length
cDNA and
genomlc clones 18 for the functlonal and i,3 for the nonfunctlonal
allele
were subcloned
into M13mp18 for DNA sequence
determination
Sequencing primers were the 17.mer universal Ml3 primer (Amersham)
and 15. to 1Fmer ollgonucleotldes
synthesized
to extend DNA sequence mformatlon
beyond what was determined
with Ml3 primers.
Transgene
Reconstitution
From the genomlc clone h9, a 13 kb Sall-Sacll
fragment, contalnmg
the productively
rearranged
V gene with 9 kb of 5’flanklng
sequence.
was mixed with a 23 kb Sacll-Sal1 fragment contalnmg Jp2 and C[%2
gene segments together with 18 kb of 3’flanking
sequence
The latter
fragment
was Isolated from cosmld clone cos H-2”ll-1.14T
derived
from a EALB/c liver library, previously
described
(Stemmetr
et al
1986). Both fragments
were ligated m the presence
of Sall-digested
pTCF cosmld vector DNA arms. Llgatlon was checked by agarose gel
electrophoresls.
In vitro packaging
and transformahon
of E. co11 stram
490A was carried out as previously
described (Steinmetr
et al., 1985).
Several clones were picked and clone cos HY1)9-1.14-5, containing the
reconstituted
transgene
as shown In Figure 16, was ldentlfled by restrIctIon endonuclease
mapping.
Transgenic
Mice
The 36 kb Insert of cos HY[IS-1.14-5 was released by Sal1 digestion and
Isolated by preparative
agarose gel electrophoresls
and electroelutlon
The DNA was extracted twice with phenol-chloroform
and preclpltated
with ethanol. The DNA pellet was dissolved m ultrapure water and dlalyzed against 10 mM Tris-HCI,
0 1 mM EDTA (pH 7.4). The DNA was
adjusted to a fmal concentration
of 4 wglml Fertilized mouse eggs
were recovered
m cumulus
from the oviducts
of superovulated
(CBA/BrA x C57BL:LiA)F,
females that had mated with F, males several hours earlier. Approximately
100 copies of the 1)gene were InJected
in the most accessible
pronucleus of each fertilized egg, as described
by Hogan et al. (1986). Microinjected
eggs were Implanted into the
oviducts of one-day pseudopregnant
(CBA/BrA
x C57EL/LiA)F,
foster mothers
and carned to term Several weeks after birth, total
genomlc DNAwas Isolated from tail blopsles of the pups. Mice that had
Transgentc
839
mcorporated
breeding.
Mice with a T Cell Receptor
the Injected
1) Gene
DNA in thetr genome
were
used for further
Southern
and Northern
Blot Analyses
Tall DNA was purified from the terminal quarter of tails of 4-week-old
mice. The skin was separated
from the bone and homogenized
in 1
ml of 1% NaCI, 10 mM EDTA (pH 8.0), on ice by using a Polytron with
a PTA7 blade. DNA was isolated by phenol-chloroform
extraction and
dialyzed against 10 mM Trls, 1 mM EDTA (pH 8.0) (TE). One milliliter
of 6% p-aminosalicylate
was added to the first phenol extraction.
Mouse organs were chopped in Ice-cold PBS and homogenized
in 4
M guanidlnium
isothlocyanate,
0.5 mM sodium citrate (pH 7.0). 0.1 M
p-mercaptoethanol,
0.5% Sarkosyl,
using a Polytron as above. From
the homogenate,
RNA and DNA were isolated by differenbal centrtfugatlon as described
previously (Maniatis et al., 1982). DNA-containing
fractions
were dialyzed against TE buffer for 6 hr, extracted
with
phenol-chloroform
three times, and dialyzed against TE overnight
Total cellular RNA (5 to 10 wg) was separated on 1.5% formaldehydeagarose gels according
to Rave et al. (1979) and transferred
to Zeta
probe membranes
(BloRad) or BA85 nltrocellulose
filters (Schlelcher
& Schiill). Genomlc DNAs (10 pg) were digested to completion
with restriction endonuclease,
separated
on 0.6% agarose gels, and transferred to Zeta probe membranes
according
to Reed and Mann (1985).
Northern
blot hybrtdizatlon
v&h labeled restriction
fragments
was
performed
by usmg the same conditions as for Southern blot hybrldlzation described by Steinmetz et al. (1986). Hybridization
with the 30.
mer ollgonucleotide
containing the VDJ folning region of the transgene
was done in 900 mM NaCI, 90 mM Tris-HCI (pH 7.2), 6 mM EDTA. 10x
Denhardt’s solution, 1% SDS, and 500 ug/ml E. coli DNA at 42% overnight. After hybridization
the filter was washed twice in 6x SSC, 0.1%
SDS, at room temperature
and twice in 3x SSC, 0 1% SDS, at 60%
for 10 mln each. Southern blot hybridization
was carried out as described by Uematsu et al. (1988). Falters were exposed
with Kodak
X-Omat S or X-Omat AR films at -70% for 2 hr to 1 month using Intensifying screens.
For hybridization
we used previously
described
Cn and Co probes
(DemblC et al., 1985; Snodgrass
et al.. 1985) The J1)2 probe IS a 1.2
kb Clal-EcoRI
fragment located Immediately
downstream
of the J(j2
gene segments (Chlen et al., 1984). The Dgl probe IS a 1.4 kb Pstl fragment extending 1 25 kb upstream of the Opt gene segment, and the
D()2 probe IS a 2.5 kb Htndlll-EcoRI
fragment extending 2 kb upstream
of the DP2 gene segment (Siu et al., 1984). The Cpl -specific probe was
derived from the 3’ untranslated
region as described
by Gascolgne
et
al. (1984). The transgene-speclflc
VDJ probe IS an oligonucleotlde
of
30 residues
in length ldentlcal to the complementary
strand of the
cDNA sequence
shown in Figure 1A from position 581 to 610. V1)7,
VB9, V[$ll, Vp12. VB14. Vb15, and VP16 probes have been described
by La1 et al (1987)
Surface
Staining of Lymphocytes
Smgle-cell suspensions
were prepared from lymph node or spleen.
Cells werre Incubated
with various monoclonal
antibodies
purlfled
from supernatants
of antibody-producing
hybridoma
cells Cells (106)
were Incubated with a saturating dose of antlbody at O°C for 20 min.
washed. and Incubated
agam with fluorescein
lsothlocyanate
(FITC)
coupled goat antl-mouse
immunoglobulln
anttbodles
In some expenmerits, antIbodIes
coupled with blotin were used it- the first step followed by Incubation with FITC-avidine
(Klsielow et al.. 1984).
Modulation
and lmmunoprecipitation
of Surface
Antigens
For modulation, cells were Incubated with an excess of F23.1 antlbodles (50 pglml) and rabbit antl-mouse
immunoglobulln
antIbodIes for 24
hr at 37°C. Viable cells were obtalned by splnnlng the suspension
through F~coll. For Immunoprecipltatlon,
10’ 86.2.16 cloned T cells.
C57BL16 T cells, and 93.2 T cells were labeled with 0.5 mCI of Nal
(Amersham)
by the lactoperoxldaseiglucoseoxldase
method (Godmg.
1980) for 20 mln at room temperature
in PBS. The cells were washed
five times with ice-cold PBS contalmng 0 1% NaN3 and 17 mg/ml KCI.
and were then lysed for 30 mln on Ice with a buffer contalmng 2% Tnton
X-100, 20 mM Trls (pH a), 150 mM NaCI, 3 mM MgCI. 0.1 mM PMSF,
and 5 mM lodoacetamlde.
The lysate was centrifuged
for 5 mln I” an
Eppendorf
centrifuge.
Half of each sample was precleared
four ttmes
wtth 100 ~1 of F23.1 anttbodies
coupled to Sepharose
beads Pre-
cleared and untreated lysates were then Incubated with 5 wg of an ant,.
fi panspeclfic
antibody (Traunecker
et at., 1986) for 2 hr on Ice, and subsequently incubated with 35 ~11of 50% protein A beads. All samples
were washed with high salt (lysis buffer containing
1 mg/ml ovalbum,”
and 0.65 M NaCI) and low salt (lysls buffer contalning
0.15 M NaCt)
buffers. Samples were reduced with 2-mercaptoethanol
and analyzed
on a 12.5% polyacrylamlde
gel. Autoradiography
was accomplished
with Kodak X-Omat AR film and Intensifying
screens
Generation
of Cytolytic
T Cells and Cytolytic
Assay
Spleen (107) or lymph node (IO’) cells were cultured with lo7 x-lrradiated (2000 rads) allogeneic stimulator cells for 5 days in 4 ml of culture
medium SICr-labeled target cells were prepared by stimulating spleen
cells from various mouse strains with concanavalin
A (Con A) (2.5
rlglml) for 48 hr at lo6 cells per ml. Con A blasts were purified by centrifugatlon over Ficoll, and viable cells were “Cr-labeled
by Incuba.
tion in 5’Cr for 1 hr at 37°C. In the cytotytic assay. lo4 “Cr-labeled
targets were incubated with various numbers of cytolytic T cells for 3.5
hr at 37% in serial bottom wells in 200 )II of medium The plates were
centrifuged
and 100 ml of supernatant
removed to determine released
51Cr (Pohllt et al., 1979). For blocking of activity. cytotytic T cells were
Incubated for 30 min at 37% with F23.1 antlbodies
(50 pglml) Then
S’Cr-labeled targets were added and the cytolytic assay was performed
as described
above.
Establishment
of Cell Lines and T Cell Clones from Transgenic
Mice
Cell lines were made by stimulation of spleen cells with either Con A
or allogeneic x-Irradiated
stimulator cells in medium containing
IL-~.
After 10 to 14 days, the cells were washed and restimulated
(IO6 responder
cells, 10’ x-irradiated
stimulator
cells) in IL-2.containing
medium. Viable cells were obtained by CentrifugatlOn over Ficoll. t-10”.
rng was carried out by seeding 0.3 cells per well ln 96-well mlcrotiter
plates contalntng lo6 x-irradiated
feeder spleen cells per well and 200
~11of IL-2-contaming
medium. After 1 to2 weeks. growing colonies were
transferred
together with 10’ x-irradiated
feeder Cells into 2 ml of IL.2.
contaimng medium in 24.well Costar plates. From them on, restlmuta.
tions were camed out at 7.day intervals with lo6 cloned cells and 107
x-irradiated
stimulators
per 1 ml of IL-2-COntalnlng
culture medium.
Acknowledgments
We thank WIIII Bannwarth.
Hansruedl Klefer. and Relnhard Schulze for
oligonucleotides,
Klaus Karjalamen
for the pansPecIfic
B antlserum:
Dennis Loh for V(, probes: Uwe Staerz for F23 1 antIbodIes, Anrleloes
Beenders. Gholam Reza DastoorNikoo.
Kathrln Hafen. Esther Koorn.
neef. Ollvla Nlkkanen.
and Vreni Stauffer for technical
help, Horst
Bluthmann. Pawel Klsle!ow. and UWe Staerz for comments;
and Anne
Iff for typing the manuscript.
P Krlmpenfort
was Supported by the
Netherlands
Organlzatlon
for the Advancement
of Pure Research
(ZWO) through the Foundation for Medical Research (MEDIGoN).
The
Base1 lnstltute for Immunology
was founded and 1s Supported by F.
Hoffmann-La
Roche & Co. LImited, Basel. Swltrerland.
The costs of publlcatlon of this article were defrayed in part by the
payment
of page charges.
This article must therefore
be hereby
marked "advertfsement"
in accordance
with 18 USC. Sectton 1734
solely to Indicate this fact
Received
November
9. 1987, revised
January
4. 1988
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