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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 Cell 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- Cell 742 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 Cell 744 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 Allison, J. P., McIntyre, of munne T lymphoma 129.2293-2300 B. W., and Bloch. D. (1982). Tumor-specific antigen cells defined with monoclonal antibody. J. Immunol. Canaani. E.. and Aaronson, S. A. (1979). Restrictton enzyme analysis of mouse cellular type C viral DNA: emergence of new viral sequences in spontaneous AKR/J lymphomas. Proc. Nat. Acad. SCI. USA 76, 16771681. Cleveland, D. W., Fischer, S. G.. Kirschner, M. W., and Laemmli, U. K. (1977). 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