Download The Mouse T Cell Receptor: Structural Heterogeneity of Molecules

Cell, Vol. 34, 739-746,
October
1983, Copyright
0092-8674/83/100739-08$02.00/O
CD1983 by MIT
The Mouse T Cell Receptor:
Structural Heterogeneity of Molecules
of Normal T Cells Defined by Xenoantiserum
Bradley W. McIntyre and James P. Allison
The University
of Texas System Cancer Center
Science Park - Research Division
Smithville, Texas 78957
Summary
We have previously demonstrated that T lymphomas
may express clonally specific epitopes that are carried by a T-cell-restricted, disulfide-bonded heterodimeric glycoprotein. We have used a monoclonal
antibody, 124-40, to isolate the lymphoma-specific
antigen and raise a xenoantiserum to the molecule.
This antiserum immunoprecipitates a family of disulfide-bonded dimers from normal thymocytes and
T cells, but is unreactive with B cells. Peptide maps
prepared after limited proteolytic digestion indicate
that the molecules from the different cell populations
have homologous primary structures. Comparison of
two-dimensional tryptic peptide maps indicate that,
in addition to several common peptides, the molecules exhibit considerable structural heterogeneity.
Taken together, these data indicate that the T-cellspecific heteroduplex has regions of constant and
variable structure consistent with the properties expected for the T cell antigen receptor.
Introduction
T cells (thymus-derived lymphocytes) are responsible for
a variety of cell-mediated
immune reactions, including
cytotoxicity and regulation of other aspects of the immune
response. Functional assays have demonstrated that T
cells have exquisite specificity for foreign antigens, which
are recognized in the context of Class I or Class II products
of the major histocompatibility complex (Zinkernagel and
Doherty, 1979; Klein et al., 1981). However, while the
discriminatory power of T cells requires that they express
specific cell-surface receptors for antigen, the biochemical
nature of the receptor remains a matter of some controversy. On the assumption that nature would not have
solved the antigen-specific receptor problem twice, considerable effort has been directed toward determining
whether T cells, like B cells, might use immunoglobulin
genes, or at least V domains, as components
of the
receptor. The results have been contradictory, and the
balance of evidence at the protein and nucleic acid level
indicates that T cells do not express antibody genes (see
Jensenius
and Williams, 1982, for review and discussion).
In another approach to the problem, soluble antigen-binding factors released by suppressor T cells have been
purified and characterized (Taniguchi and Takei, 1980;
Rosenstein et al., 1981; Fresno et al., 1982; Krupen et al.,
1982). While the structure of these soluble factors is of
considerable interest, their relevance to the T cell surface
receptor is not clear, particularly since these factors, unlike
cytotoxic or helper T cells, do not recognize antigen in the
context of major histocompatibility complex products.
The strategy that we have employed in attempting to
define the T cell receptor is based on the assumption that
different T cell clones should express on their cell surfaces
unique epitopes related to the recognition structure. Since
lymphoid tumors appear to arise by clonal proliferation
from single cells (Canaani and Aaronson, 1979), such
epitopes should be presented by T lymphomas as tumorspecific antigens. We have previously described (Allison
et al., 1982) the production of a monoclonal antibody, 12440, which identifies a tumor-specific epitope expressed by
the T lymphoma line CGXL. The molecule reactive with the
antibody was shown to be a glycoprotein composed of
disulfide-bonded
39 kd and 41 kd subunits. Electrophoretic
analysis of whole lysates of surface-labeled
normal lymphocytes, which were unreactive with the monoclonal
antibody, revealed that similar heterodimers
were expressed by normal T cells and thymocytes, but not by B
cells. This observation raised the possibility that the lymphoma-specific monoclonal antibody was detecting a clonally expressed epitope of a normal T-cell-specific surface
molecule, and that the molecule might be the T cell antigen
receptor. This possibility is supported by recent studies of
clonotypic structures on functional T cell lines. Haskins et
al. (1983) have described the production of a clonotypic
monoclonal
antibody to an antigen-specific
murine T
hybridoma, which blocked binding of the hybrids to antigen-presenting
cells as well as inhibiting antigen-specific
IL-2 release. In addition, Meur et al. (1983) and Reinherz
et al. (1983) have described clonotypic monoclonal antibodies to alloreactive human T cell clones that specifically
inhibited cytotoxic effector function and blocked antigenspecific proliferation.
More recently, Samelson
and
Schwartz (1983) have produced monoclonal antibodies to
a T cell hybridoma, which specifically blocked IL-2 release.
A common observation in all these studies was that the
clonotypic antibodies were found to immunoprecipitate
similar disulfide-bonded
heterodimers with intact molecular
weights of approximately
85 kd and subunit molecular
weights of 40-50 kd.
The structural similarity of the molecules detected in
these reports provides strong evidence that the disulfidebonded heterodimer is indeed the T cell receptor. However, additional structural and functional studies on normal
T cells and other T cell lines has been hampered by the
fact that the antibodies described to date have all been
clonotypic and consequently are useful only when applied
to the cell lines against which they were raised. In this
report, we describe the production of a xenoantiserum to
the CGXL lymphoma antigen. This antiserum reacts with a
family of disulfide-linked heterodimers found on normal T
cells. Structural analysis of the antigens isolated from
lymphoma cells and normal T cells indicates that the
molecules are homologous but display considerable heterogeneity. Comparison of peptide maps indicates that the
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740
molecules have regions of constant and variable structure
as expected for the T cell antigen-specific receptor.
Results
Preparation of Rabbit Antiserum to CGXL
Lymphoma Antigen
Monoclonal antibody 124-40, produced by a hybridoma
constructed from spleen cells of a BALB/c mouse immunized with CGXL, a T cell lymphoma derived from C57BL/
Ka mice, has been shown to be specific for the immunizing
cells and is unreactive with normal lymphoid cells or other
T lymphoma lines (Allison et al., 1982). The reactive antigen
has been isolated by radioimmunoprecipitation
and has
been shown to be a disulfide-linked heterodimer. Diagonal
mapping of CGXL cell-surface molecules using diagonal
SDS-PAGE (a two-dimensional technique in which proteins
are first separated according to intact size under nonreducing conditions, followed by separation by subunit size
under reducing conditions) revealed that the antigen defined by monoclonal antibody 124-40 was the major disulfide-linked molecule on the surface of the lymphoma cells.
Using the diagonal technique we (Allison et al., 1982) as
well as others (Goding and Harris, 1981) have shown that
the major disulfide-linked surface components of B cells
are the immunoglobulin
molecules, while the major disulfide-linked components
on the surface of T cells are
molecules similar in intact and subunit size to the CGXL
lymphoma antigen. This observation suggested that these
molecules represent a related family of T-cell-specific surface proteins that might express crossreactive framework
determinants, as well as clonotypic antigenic and structural
features. To determine whether this might be the case, we
first sought to produce a xenoantiserum
to the CGXL
lymphoma antigen with the goal of obtaining antibodies to
the putative framework determinants with which to isolate
the molecules from normal T cells for structural studies.
In order to raise a xenoantiserum to the CGXL lymphoma
antigen, rabbit #8177 was immunized with complexes
formed by incubation of 0.5% Nonidet P-40 (NP-40) lysates
of CGXL lymphoma cells with antibody from culture supernatants of 124-40 hybridoma cells adsorbed onto rabbit
anti-mouse immunoglobulin-coated
Staphylococcus
aureus cells. After absorption with insolubilized bovine serum
albumin and mouse immunoglobulin
to remove contaminating antibodies, the antiserum was tested for reactivity
with the CGXL lymphoma antigen by immunoprecipitation.
As shown in Figure 1, SDS-PAGE revealed that immunoprecipitates obtained with antiserum 8177 from NP-40
lysates of radioiodinated CGXL cells contained protein spe.
ties with the same mobility as those obtained with the
lymphoma-specific
monoclonal
antibody 124-40 under
both reducing and nonreducing conditions. Under nonreducing conditions, the mobility of the bands indicated an
apparent molecular weight of approximately 75 kd, which
decreased to approximately 40 kd following reduction. No
precipitating activity was present in the preimmune serum
of the rabbit used for immunization. The T-cell-specific
Figure 1. Comparison of CGXL Lymphoma Antigens Isolated by Immunoprecipitation with Clonotypic Monoclonal Antibody and Xenoantiserum 8177
Cell-surface proteins of CGXL lymphoma cells were radiolabeled by lactoperoxidase-catalyzed
iodination, solubilized with NP-40, isolated by immunopracipitation. and analyzed by SDS-PAGE on 10% gels under nonreducing (left) and reducing (right) conditions. lmmunoprecipitations
were performed using monoclonal antibody 124-46 (lanes 1 and 4) and preimmune
(lanes 2 and 5) or immune serum (lanes 3 and 6) from rabbit 8177.
band was not obtained with rabbit anti-mouse immunoglobulin.
To prove that antiserum 8177 was reactive with the
same molecule detected by monoclonal antibody 124-40,
we subjected extracts of radioiodinated
CGXL cells to
exhaustive sequential immunoprecipitation
with 8177, and
tested the resulting supernatants for residual antigen reactive with 124-40. As shown in Figure 2, all antigen
reactive with 124-40 was removed by prior precipitation
with 8177. Prior precipitation with preimmune serum had
no effect on subsequent precipitation of the antigen reactive with 124-40. These results indicate that the rabbit
antiserum and the monoclonal antibody are detecting determinants carried by the same molecule. Antiserum 8177
defines a family of antigenically and structurally related Tcell-specific, disulfide-linked dimers.
To determine whether antiserum 8177 was reactive with
determinants shared by the CGXL lymphoma antigen and
the disulfide-linked dimers expressed by normal T cells,
NP-40 lysates of various lymphoid cell populations labeled
by radioiodination were subjected to immunoprecipitation
and analyzed by SDS-PAGE under reducing and nonreducing conditions. A diffuse band with mobility indicating
an apparent molecular weight of 75-85 kd was obtained
from normal C57BL/6 thymocytes,
splenocytes,
and
splenic T cells under nonreducing conditions (Figure 3B,
lanes 4-9). Under reducing conditions, each of these
populations yielded three bands with molecular weights of
40, 43, and 46 kd. (Figure 3A, lanes 4-9). The mobility of
the CGXL lymphoma antigen was identical to the 40 kd
component of the normal cells (lanes 1-3). Identical results
Murine T Cell Antigen Receptor
741
4-2ME
12345
Figure 2. lmmunodepletion Analysis of CGXL Antrgens
otypic Monoclonal Antibody and Xenoantiserum
Reactive
with Clon-
Fifty ~1 of CGXL extract was subjected to four sequential rmmunoprecrprtations with 10 pl antiserum 8177, and the resulting supematant was assayed
for residual antigen reactrve wrth monoclonal antibody 124-40. Precipitated
antrgens were analyzed by SDS-PAGE on 10% gels.
were obtained with thymocytes of AKR and BALB/C mice
(Figures 3A and 38, lanes 12-15) indicating that the
determinants detected by the xenoantiserum
were not
strain-specific. No bands were obtained from splenic B
cells (Figures 3A and 38, lanes 10, 11). These results
clearly indicate that the lymphoma antigen and the T cell
family of disulfide-bonded
dimers are antigenically related.
The observation that the molecules were not only similar
in gross structure, but were also related antigenically,
strengthened the possibility that these proteins were related in primary structure as well. To determine if this was
indeed the case, one-dimensional
peptide maps were
prepared. The lymphoma antigen and the normal T cell
proteins were isolated from radioiodinated cells by immunoprecipitation
and purified by SDS-PAGE under nonreducing conditions. The purified antigens, which contained
both subunits, were then reduced, subjected to limited
proteolysis by Staph. aureus V8 protease (Cleveland et al.,
1977) and the resulting peptides were resolved by SDSPAGE. As shown in Figure 4, the fragments generated by
digestion of the CGXL lymphoma antigen, isolated with the
monoclonal antibody or antiserum 8177, were virtually
identical with fragments obtained from the thymocyte or
splenic T cell populations. These results indicate that the
amino acid sequences are highly related, and suggest that
the molecules are the products of homologous genes.
Heterodimer
Subunits Exhibit Charge and Size
Heterogeneity.
We have previously demonstrated that the CGXL lymphoma antigen yields a single, relatively diffuse band of
2ME
1 2
3 '4
Figure 3. Identification
serum 8177
5
6'7
8
il m 11 12 13 14 15
of Normal T Cell Antigens
Reactive
with Xenoanti-
Antigens isolated by immunoprecipitation
from NP-40 extracts of radioiodinated cells were analyzed by SDS-PAGE under reducing (A) and nonreducing (6) conditrons. Precipitates were obtained with monoclonal antibody
124-40 (lane 1, Indicated by ‘Mab” at top of figure); preimmune 8177 serum
(even numbered lanes, Indicated by “0” at top of figure); or antiserum
obtained at the seventh bleeding after immunization (lanes 3, 5, 7, 9, 11,
13,15, identified by ‘7” at top of figure). Extracts were from CGXL lymphoma
cells (lanes l-3) C57BL/6 thymocytes
(lanes 4, 5) C57BL/6 splenocytes
(lanes 6, 7). C57BL/6 splenic T cells (lanes 8. 9) C57BL/6 splenic B cells
(lanes 10, 1 l), AKR thymocytes
(lanes 12, 13) and BALB/c thymocytes
(lanes 14, 15).
about 40 kd upon SDS-PAGE under reducing conditions,
but can be resolved by two-dimensional
electrophoresis
into two distinct subunits. Both of these exhibit microheterogeneity, with average isoelectric points of 5.5 and 7.0
(Allison et al., 1982). The observation that at least three
species were evident upon SDS-PAGE analysis of the
homologous proteins obtained from normal T cells (Figure
3) indicated a considerable range of structural heterogeneity in the molecules. To further assess this heterogeneity,
isolated antigens were analyzed by two-dimensional electrophoresis employing pH-dependent
separation in the
horizontal dimension, followed by SDS-PAGE in the vertical
dimensional (O’Farrell et al., 1977). A representative anal-
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Figure 5. Two-Dimensional
Comparison of CGXL Lymphoma
Normal T Cell Antigens Isolated with Xenoantiserum 8177
Antigen and
Antigens isolated by immunoprecipitation
with antiserum 8177 from extracts
of radioiodinated CGXL cells (A) or normal spfenic T cells (B) were analyzed
by pH-dependent
electrophoresis
in the horizontal dimension, followed by
SDS-PAGE in the vertical dimension.
Figure 4. Peptide Maps of T Cell Antigens Digested
with V8 Protease
Cell surface antigens isolated from NP40 extracts of radioicdinated cells
were purified by SDS-PAGE under nonreducing conditions, reduced with
dithiothreitol. and subjected to limited protedysis with 5 pg (lanes 1, 3, 5,
7) or 25 Ag (lanes 2, 4, 6, 8) Staphylococcus
aureus V8 protease. and the
resultant fragments resolved by SDS-PAGE on 11.25% gels. Extracts were
from CGXL lymphoma cells (lanes l-4) C57BL/6 thymocytes (lanes 5. 6)
or C57BL/6 splenic T cells (lanes 7. 6). Monoclonal antibody 12440 was
used to isolate the C6XL antigen for the digestions in lanes 1 and 2.
Xenoantiserum 8177 was used to isolate antigens for the digestions in
lanes 3-8.
ysis of the CGXL lymphoma antigen and the antigens
isolated from normal T cells is shown in Figure 5. The
subunits of the molecule isolated from the lymphoma cells
are clearly resolved into acidic and basic components,
with considerable microheterogeneity
evident in the basic
component. By comparison, the basic components of the
normal T cell structures display considerable
size and
charge heterogeneity, while the acidic components exhibit
only minimal charge heterogeneity. It should be pointed
out that resolution in the acid end of the gel is relatively
poor, and may preclude detection of charge microheterogeneity in the acidic components. Nonetheless, it is evident
that structural heterogeneity is much more pronounced in
the subunits migrating toward the basic end of the firstdimension gel.
Peptide Maps Suggest Regions of Constant and
Variable Structure
One of the structural features expected for the T cell
antigen receptor is the existence of regions of variable
structure related to the antigen-combining
site, in addition
to regions of constant structure related to the molecular
framework. From the results presented above, it is evident
that antiserum 8177 defines, as required, a family of highly
related, but heterogeneous, T-cell-specific proteins. How-
ever, since both subunits of the dimer appear to be
glycosylated (Allison et al., 1982) it is likely that to a
considerable degree the heterogeneity observed in the
one- and two-dimensional electrophoretic analyses results
from posttranslational modification rather than from variability in amino acid sequence. The virtual identity of the V8
protease maps of the lymphoma antigen and the normal
T cell proteins suggested that the molecules are highly
related in amino acid sequence, but failed to reveal any
evidence of variability in primary sequence. However, the
technique measures only the relative spacing of aspartic
and glutamic acid residues within the primary sequence,
and is of insufficient resolving power to allow detection of
regions of variable structure superimposed on a constantframework background. We therefore prepared high-resolution, two-dimensional maps of tryptic tyrosine-containing
peptides of antigens isolated from radioiodinated
cells.
Representative maps are presented in Figure 6 and summarized diagrammatically
in Figure 7. As can be clearly
seen, the overall patterns obtained from the lymphoma
antigen and the normal T cell products are very similar,
with at least seven major peptides common to all preparations (Figure 7, dark spots). Examination of lighter exposures of the maps (not shown) reveals that the large
central spot is composed of at least four additional peptides that are common to all of the cell preparations. Also
evident in the maps are additional unique peptides, including five found only in the lymphoma antigen (Figure 7,
shaded spots) and seven found only in the normal T cell
preparations (Figure 7, open spots). It is also evident upon
examination of the maps that the patterns obtained from
the normal cell preparations contain areas of diffuse, poorly
resolved spots, particularly in the center of the maps, in
contrast to the relatively discrete spots reproducibly obtained from CGXL antigen isolated with the monoclonal
antibody or antiserum 8177. An interpretation consistent
with this observation is that the material isolated from the
normal T cell preparations is composed of a mixture of
polyclonal products that share major peptides but also
have clone-specific differences in other peptides. Taken
Wine
743
T Cell Antigen Receptor
Figure 6. Two-Dimensional
Maps of Tyrosine-Containing Tryptic Peptides of T Cell Antigens Isolated
with Xenoantiserum 6177
Antigens were isolated from extracts of radioiodinated cells by immunoprecipitation,
purified by
SDSPAGE, digested with trypsin, and resolved by
a two-dimensional
procedure employing thin-layer
electrcphoresis
in the first dimension, followed by
chromatography
in the second dimension. The
cells and antibodies used for the isolation are
indicated on the panels.
:TROPHORESIS
together, these data strongly argue that the family of
proteins defined by antiserum 8177 have regions of variable, as well as common structure, consistent with the
requirements of molecules with antigen-specific
receptor
activity.
Discussion
A consideration of the structural features required of an
antigen-specific receptor allows the prediction of several
fundamental characteristics that must be displayed by
molecules considered as candidates for the T cell receptor.
First, the molecules should be restricted to T cells, and
should be expressed by all immunocompetent
T cells. The
mechanism of generating the T cell repertoire should
operate on the same or closely related genes in different
clones, resulting in a high degree of structural homology
and the expression of crossreactive framework determinants related to the common portions of the molecule.
Finally, the molecules from different clones should express
unique antigenic determinants and regions of variable
structure related to the specific antigen recognition site. In
this report, we have described the production of a xenoantiserum to a T lymphoma antigen isolated with a clonotypic monoclonal antibody. The antiserum defines a heterogeneous family of homologous T-cell-specific disulfidebonded dimers that have regions of constant and variable
structure consistent with the expected properties of the T
cell antigen receptor.
The overall structure of the molecule isolated from normal T cells with the xenoantiserum is very similar to that of
molecules isolated from murine and human T cell clones
with clonotypic monoclonal antibodies demonstrated
to
affect specific T cell functions (Haskins et al., 1983; Meuer
et al., 1983; Reinherz et al., 1983; Samelson and Schwartz,
1983). In each case the putative receptor was shown to
be a disulfide-bonded
heterodimer. There are, however,
significant differences in the subunit size of the heterodimers obtained from different murine T cell clones. The
lymphoma-specific
antigen isolated from CGXL cells was
shown to have subunits of 39 kd and 41 kd (Allison et al.,
1982). The receptor isolated from an ovalbumin-specific
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0
l
Figure 7. Comparison
Specific Dimers
0
of Two-Dimensional
Tryptic Peptide Maps of T-Cell-
Comparison of the maps presented in Frgure 6 reveals common peptides
present in all cell preparations (filled spots), peptides unique to CGXL
lyphoma cells (shaded spots), and peptides unique to normal T cells (open
spots).
murine T hybridoma was found to have subunits of 43 kd
(Haskins et al., 1983) while the molecule isolated from a
T hybridoma with specificity for pigeon cytochrome-c was
shown to be composed of subunits of 40-44 kd and 4850 kd (Samelson and Schwartz, 1983). lmmunoprecipitates
obtained from normal T ceils with antiserum 8177 contained major bands at 40, 43, and 46 kd, suggesting that
the variability in subunit size observed in the clones is
reflected in the polyclonal splenic T cell preparations.
While it is not possible at present to rule out the possibility that the variability in subunit size is the product of
posttranslational modification, it is tempting to speculate
that different T cells might draw upon any one of a group
of different, but related, genes in the generation of antigenspecific receptors. An observation that would support this
possibility is the fact that the two-dimensional tryptic maps
of the normal T cell populations contained, in addition to
the major common peptides shared with CGXL lymphoma
cells, at least seven additional discrete spots not found in
the lymphoma maps. One explanation is that the normal T
cell preparation contains a significant population of clones
expressing molecules antigenically related to the CGXL
lymphoma molecule, but having additional framework
structures not present in the lymphoma antigen. Structural
studies of additional T cell clones, particularly those belonging to different functional subclasses, should resolve
the question of whether there are isotypic variations in the
T cell receptor.
The observation that the more basic subunit of the Tcell-specific heterodimer is more heterogeneous than the
acidic subunit is in agreement with the finding that the
basic subunit of the clonotypic antigens from two human
clones were different in charge and showed differences at
the peptide level, while the acidic subunits were identical
(Reinherz et al., 1983). More extensive studies of the
primary structure of individual subunits isolated from different clones should reveal whether one or both subunits
have regions of variable structure.
The availability of an antiserum that can be used to
isolate the putative receptor molecules from diverse clones
should greatly facilitate several areas of experimental work,
which will ultimately contribute to delineation of the genetic
origin of T cell receptors and provide insight into their
structural and functional organization. By reversing the
strategy we used in production of the xenoantiserum, it
should be possible to immunize mice with immune complexes prepared from desired clones and more easily
obtain clonotypic monoclonal
antibodies for functional
studies. Determination of the amino acid sequence of the
isolated subunits and comparison of these sequences with
those of other immunologically
relevant proteins should
reveal evolutionary relationships. By comparing the primary
structure of subunits isolated from clones with specificity
for different antigens, it should be possible to determine
whether variable structure in one or both subunits contributes to antigen specificity. Similarly, comparison of the
primary structure of the subunits from clones of identical
antigen specificity, but differing in MHC restriction, might
reveal whether either or both of the subunits are involved
in recognition of the restriction element. Finally, the antiserum should prove more useful than monoclonal antibodies, which are notoriously conformation-dependent,
in
the screening of cDNA libraries for the molecular cloning
of the genes encoding the T cell receptor.
Experimental
Procedures
Cells and Cell Lines
CGXL, a T cell lymphoma originally induced in C57BL/Ka mice (Lieberman
and Kaplan, 1959) was maintained by serial passage in C57BL/6 mice.
The studies described here were carried out on a subline established in
vitro by the method of Hiai et al. (1961) cloned in soft agar, and maintained
in DME supplemented
with 10% fetal calf serum, 0.11 mg/ml sodium
pyruvate, and 5 x 10m5 M 2-mercaptoethanol.
Normal thymocytes
and splenocytes were prepared by standard methods B and T lymphocytes
were separated by panning radioiodinated
splenocytes
on petri dishes coated with affinity-purified goat anti-mouse
immunoglobulin by a modification (Allison et al.. 1962) of the method of
Wysocki and Sato (1976).
Preparation
of Antiserum
Antiserum was obtained from a male New Zealand White rabbi, x8177,
immunized with immune complexes containing the CGXL lymphoma antigen
prepared by a three-step procedure. Rabbit anti-mouse immunoglobulin
(50 pl) was added to 1 .O ml of 10% (v/v) formafin-fixed Staph. aureus cells
(Kessler, 1975) and incubated for 1 hr at 4°C. The bacteria were pelleted
by centrifugation and washed twfce with 0.01 M Tris-HCI (pH 8.0) containing
0.15 M NaCI, 0.5% NP-40, and 26 KIU/ml aprotinin (SACI buffer). The
washed bacteria were resuspended in 5.0 ml culture supematant from 12440 hybridoma cells and incubated 4-6 hr. The bacteria were then washed
three times with SACI buffer and resuspended
to 1.0 ml. The antibodycoated bacteria were added to 1 .O ml of CGXL lysate prepared by solubilizing 5 x 10’ cells in SACI buffer (Allison et at., 1962). After incubation at
4°C overnight, the bacteria were washed three times with SACI buffer,
twrce with Dulbecco’s phosphate-buffered
sakne (D-PBS), and finally resuspended rn 0.5 ml D-PBS. The rabbrt received an inrtial immunization at five
Munne T Cell Antigen Receptor
745
subcutaneous
sites with 0.5 ml of antigen preparation emulsified in 0.5 ml
incomplete Freund’s adjuvant. Thts was followed in 2 weeks by subcutaneous injection of antigen, without adjuvant, at five additional sites. At 2
week intervals, the rabbit was bled and boosted by injection into the
granulomas at the original injection sites. By the fifth bleeding, antibodies
reactive with the heterodimer were detectable by radioimmune precipitation
of CGXL lymphoma cells. SDS-PAGE analysis of immunoprecrpitates
obtained from radioiodinated splenocytes
and tissue culture lines revealed
that the antiserum also contained antibodies to immunoglobulin and bovine
serum albumin. These were removed by adsorption on columns of Sepharose-conjugated
bovine serum albumin and mouse IgG. The analyses
described rn the report were performed using the absorbed antiserum.
Radiolabeling
of Cells
Cell surface proteins were labeled by lactoperoxidase-catalyzed
nation by a modification (Allison et al., 1982) of the procedure
et al. (1977).
radioiodiof Keski-Oja
Immunoprecipitatfon
Radrolabeled cells were solubrlized and lmmunopreciprtations
performed
usrng Staph. aureus (Kessler, 1975) as previously described (Allrson et al..
1982). For each analysis, 0.1 ml of extract (2 5 x 106 cell equivalents)
precleared with Staph. aureus was immunoprecipitated
with 5 pl 124-40
ascrtes ffuid or antiserum 8177.
Polyacrylamide
Gel Electrophoresis
One-dimensional
SDS-PAGE was carried out on 10% gels according to the
method of Laemmli (1970). Two-dimensronal
electrophoresis
employing
nonequilibrium, pl--dependent
electrophoresis
in the first dtmensron. followed by SDS-PAGE on 10% gels in the second dimension, was performed
as described
by O’Farrell et al. (1977). The sample buffer used for
solubilization and the frrst-dimension gels contained 2% pH 3-10 ampholytes (Bra-Rad). Other details of electrophoresis
and autoradiography
were
as previously described (Allison et al.. 1982).
Peptide Mapping
Staph. aureus V8 protease maps were prepared by the method of Cleveland
et al. (1977) as modrfied by Handman et al. (1981). Radioiodinated antigen
was isolated by rmmunoprecipitation,
subjected to SDS-PAGE under nonreducing conditrons, and visualized by autoradiography
of the dried gels.
The areas of the gel containrng the drsulfide-linked dimer were cut out,
rehydrated for 30 mtn In 0.0625 M Tris-HCI. pH 8.0, containing 10% glycerol,
50 mm dithiothreitol, and 0.2% SDS, and placed in individual sample wells
of a 3% stacking gel containing 4 M urea. The gel slices were overlaid with
0.02 ml buffer contatning 5% glycerol, 50 mM drthrothrettol, 0.02% bromophenol blue, and 5 or 25 ag V8 protease (Sigma). Current was applied (25
mA) until the dye front reached the interface between the stacking gel and
the 10% separating gel, and was then interrupted. After 1 hr, current was
reapplied and electrophoresis
carried out until the dye front reached the
bottom of the gel. The resolved peptides were visualtzed by autoradiography.
Two-dimensional
maps of tyrosine-containing
tryptic peptides were prepared by a modificatron of the method of Elder et al. (1977). Antrgen was
isolated by immunoprecipitation
and purified by SDS-PAGE under nonreducrng conditions. The unfixed gels were frozen and areas containing
radroactlve antrgen located by autoradiography
and cut out. Antigen was
collected from the gel skces by electroelution In 0.0625 M Tris-HCI (pH 6.8)
containrng 0.1% SDS and 10% glycerol. The eluted antigen was lyophilized,
redrssolved rn 0.5 ml of 0.4 M Tris-HCI (pH 8.0) containrng 0.5 mg human
IgG. The sample was then reduced by addition of 5 mg dithiothreitol.
incubated for 4 hr at 37°C and alkylated by addition of 12 mg iodoacetamrde. After 1 hr. the sample was precipitated by additron of trichloroacetrc
acid (TCA) to a final concentrakon
of 20%, washed twice with 15% TCA.
and finally washed twice with acetone. The sample was then dried under
nitrogen and redissolved in 0.2 ml 0.05 M ammonium bicarbonate (pH 8.0)
and 100 pg TPCK-trypsin
(Worthington)
was added. After incubation at
37°C for 18 hr, an additional 100 pg of trypsrn was added, and digestion
was allowed to proceed for 4 more hr. The digested sample was lyophilized,
redissolved in 0.03 ml electrophoresis
buffer (5% formic acid, 15% acetic
acid), and spotted on a thin-layer chromatography
plate (Eastman Chromagram). The plate was then moistened with buffer and electrophoresis
carried out at 560 V
chromatography
tank
acetic acid, pyridine,
and the resulting map
for 1.5 hr. After drying, the plate was placed in a
and developed with a buffer composed of butanol,
and water (58.5:9:45:36). The plate was then dried
visualized by autoradiography.
Acknowledgments
We wish to thank Joanne Lund and David Walker for technical assistance,
and Judy Ing for help with preparation of the art work. We also thank Dr.
Mike Gallatin and Dr. John Carlson for helpful discussions
and critical
reading of the manuscript. This work was supported by grant CA 26321
from the Nattonal Institutes of Health. B. W. McIntyre is the recipient of the
J. S. Abercrombie Foundation predoctoral fellowship.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement”
in accordance
with 18 USC. Section 1734 solely to
indicate this fact.
Received
August
3, 1983
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