Proc. Nati. Acad. Sci. USAVol. 80, pp. 6336-6340, October 1983Immunology

Structural analysis of antigen-specific Ia-bearing regulatory T-cellfactors: Gel electrophoretic analysis of the antigen-specificaugmenting T-cell factor

(T-celi hybridoma/T-cell receptor/antigen-specific T-celi factor/Igh-l-linked alloantigen/I-A subregion)

SEIJI MIYATANI*, KEIICHI HIRAMATSU*t, PAMELA B. NAKAJIMA*, FRANCES L. OWENt, ANDToMio TADA**Department of Immunology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and tDepartment of Pathology and Cancer Research Center, Tufts UniversitySchool of Medicine, Boston, MA 02111

Communicated by Herman N. Eisen, June 29, 1983

ABSTRACT An antigen-specific T-cell factor (TaF) that spe-cifically augments the antibody response was purified and bio-chemically analyzed by NaDodSO4/polyacrylamide gel electro-phoresis and isoelectric focusing. Biosynthetically labeled TaF wasseparated from the Nonidet P-40 extract of T-cell hybridoma FL1O,which produces a keyhole limpet hemocyanin-specific TaF, by af-finity chromatography either with antigen or with monoclonal anti-I-A antibodies. The material thus obtained was composed of twodifferent types of molecules of molecular weights of 67,000 and33,000 under nonreducing conditions. After reduction with di-thiothreitol, all the molecules migrated to the position of molec-ular weight 33,000. The absorption studies with immunoadsor-bents of antigen and antibodies revealed that the intact TaF is aheterodimer of two discrete polypeptide chains, one carrying adeterminant detectable by a monoclonal anti-Tindd directed to anIgh-1-linked allotypic structure of T cells and being associated withthe antigen-binding site and the other expressing a unique deter-minant controlled by the I-A subregion of murine H-2 major his-tocompatibility complex but being different from known class IIpolypeptide chains. The antigen-binding polypeptide has an iso-electric point of pH 5.6, and the I-A polypeptide has an isoelectricpoint of pH 6.3.

Two types of antigen-specific T-cell factors, one able to sup-press (TsF) and the other able to augment (TaF) the antibodyresponse, have been reported from our laboratory (1-3). Thesefactors share certain interesting immunological characteristicsand are considered to play important roles in the regulation ofthe immune response (reviewed in ref. 4). These character-istics are (i) exquisite specificities for the antigen with whichthey were generated as demonstrated by both their antigen-spe-cific regulatory functions and antigen-binding activity, and (ii)possession of determinants controlled by a gene in the im-mune response region of the major histocompatibility complex(MHC)-i.e., I-J subregion for TsF and I-A subregion for TaF.Subsequently, a number of T-cell hybridomas producing theseT-cell factors have been established (5-11), and the functionaland genetic properties of the molecules are being extensivelystudied (12-15).

Although these factors provide important clues for deter-mining the structure of antigen recognition units of T cells, nei-ther factor has been sufficiently characterized biochemically,primarily due to the minute amount of the factor produced byT-cell hybridomas and to the absence of suitable reagents forpurifying the molecules. However, a number of laboratorieshave recently been successful in obtaining monoclonal anti-

bodies reactive with T-cell factors. These include monoclonalanti-I-J (16, i7) and anti-I-A antibodies (18), and those specificfor the allotypic determinants controlled by genes linked to im-munoglobulin heavy chain (Igh) loci (19, 20). Anti-I-A reagentsare particularly interesting, because they adsorb antigen-spe-cific TaF and react with antigen-binding T cells. The reagentsare, however, unable to react with conventional Ia moleculeson B cells and macrophages (18), and thus such a monoclonalanti-I-A antibody may be able to detect a novel type of I regionproduct. In this paper, we will refer to such monoclonal anti-I-A antibodies specific for the T-cell product of H-2k haplotypeas anti-I-AkT. Two monoclonal antibodies specific for the Igh-linked allotypic determinants of T cells-i.e., anti-Tindd andanti-Tsud, were also available for the analysis (21).One of our T-cell hybridomas producing TaF (FL10) was found

to be strongly reactive with monoclonal anti-I-Ak antibodies(18). Functional studies revealed that TaF from FL10 was ab-sorbable by anti-I-Ak and anti-Tindd (22). We have, thus, at-tempted to analyze the biochemical nature of TaF molecule,using monoclonal anti-I-A k and anti-Tindd antibodies. This pa-per describes the detailed immunoprecipitation analysis of TaFwith monoclonal anti-I-Ak antibody in NaDodSO4 and isoelec-tric focusing (IEF) gels.

MATERIALS AND METHODST-Celi Hybridomas. The T-cell hybridoma FL10 was estab-

lished by fusion of thymoma BW5147 cells with antigen-bind-ing T cells of A/J mice (H-2a, Igh-le) immune to keyhole lim-pet hemocyanin (KLH) (9). The methods for the hybridizationand selection were described previously (23). Another T-cellhybridoma, 7C3-13, which was characterized by the expressionof IJk 'determinants and the binding specificity for a hapten 4-hydroxy-3-nitrophenylacetyl, was used as a control (24).

Antigens. KLH was purchased from Calbiochem (lot no.730192). Fowl gamma globulin (FGG) was prepared from chickenserum by precipitation with 45% saturated ammonium sulfate.

Antibodies. Rabbit anti-mouse immunoglobulin (anti-MIg)was raised by hyperimmunizing rabbits with gamma globulinfraction of normal mouse serum and was purified by affinity

Abbreviations: TaF, augmenting T-cell factor; TsF, suppressor T-cellfactor; MHC, major histocompatibility complex; KLH, keyhole limpethemocyanin; MIg, mouse immunoglobulin; anti-MIg, rabbit anti-mouseimmunoglobulin; Igh, immunoglobulin heavy chain gene; FGG, owlgamma globulin; pI, isoelectric point; NP-40, Nonidet P-40; VH, vari-able portion of mouse immunoglobulin heavy chain; VL, variable pQr-tion of mouse immunoglobulin light chain; IEF, isoelectric focusing.t Present address: Massachusetts Institute of Technology, Center forCancer Research, Cambridge, MA 02139.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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chromatography with a MIg column. Conventional anti-Iak andanti-IaS antisera were prepared by repeated reciprocal immu-nizations of A.TH and A.TL spleen cells. Rabbit antibodiesagainst the framework structure of the variable portions of mouseimmunoglobulin heavy and light chains (anti-VH and anti-VL)were kind gifts from D. Givol of the Weizmann Institute ofScience (Rehovot, Israel). Monoclonal anti-Ia.2 and anti-Ia. 17antibodies reactive with conventional Ta antigens on B cells ofH-2k haplotype were provided by L. A. Herzenberg.

Hybridomas producing monoclonal anti-I-A k antibodies wereprepared in our laboratory by fusion of cells from myeloma cellline P3-X63-Ag8-653 with spleen cells from A.TH mice im-munized with A.TL lymphoid cells (18). Ascitic fluids contain-ing monoclonal antibodies against allotypic determinants of pu-tative antigen receptors of T cells-i.e., Tindd and Tsud arethe materials used in a previous publication (19).

Incorporation of Radioactive Amino Acids into TaF. FL10cells were internally labeled for 16 hr with 0.5 mCi (1 Ci = 3.7X 1010 Bq) of [35S]methionine and 20 ACi of ['4C]leucine (bothfrom New England Nuclear) in a 5% CO2 incubator. The la-beled cells were lysed by freezing and thawing in 1 ml of 150mM NaCl/5 mM EDTA/50 mM Tris HCI/0.02% sodium azide(NET buffer), pH 7.4, containing 0.5% Nonidet P40 (NP-40;Shell Chemical, Piscataway, NJ) and 1 mM phenylmethylsul-fonyl fluoride. The lysate was obtained by ultracentrifugationat 100,000 X g for 1 hr.

Immunoprecipitation of TaF with Monoclonal Antibodies.Radiolabeled cell lysate (0.5 ml) was incubated for 2 hr with 10,ul of an immunoglobulin fraction of ascites fluid containingmonoclonal anti-I-Ak (14P) followed by the addition of purifiedanti-MIg at an equivalence at 4°C. The precipitate was pelletedby centrifugation at 5,000 x g for 15 min and washed four timeswith cold 0.05% NP-40/NET buffer. The precipitate was sol-ubilized in 100 t1 of NaDodSO4 sample buffer [10% (vol/vol)glycerol, 2.3% NaDodSO4/62.5 mM Tris HCI, pH 6.8] withheating at 100°C for 3 min.

Purification of TaF with Antigen- or Antibody-Coated Plas-tic Dishes. Antigen (KLH)- or antibody-coated plastic disheswere prepared by incubating the protein solutions (7 ml of Img/ml) in 100 x 15 mm plastic dishes (Fisher, lot no. 8-757-12) overnight at 4°C. The dishes were thoroughly washed withphosphate-buffered saline, pH 7.2, and then with 0.2 M gly-cine HCl buffer, pH 2.8, followed by further repeated washingwith phosphate-buffered saline. The saline extract of FL1O wasapplied to the immunoadsorbent dishes at 4°C for 5 hr withswirling. After washing with phosphate-buffered saline five times,the materials bound to the dishes were eluted with 2 ml of 0.2M glycine HCl buffer, pH 2.8. The eluate thus recovered wasinstantly neutralized with 0.5 M potassium phosphate buffer,pH 8.0, and was dialyzed against phosphate-buffered saline.The material was labeled with "2I by the method of Hunter(25).

Adsorption of Radiolabeled Materials with Immunoadsor-bents. Sepharose CL-4B immunoadsorbents composed of an-tigen and antibodies were prepared by the method of Axen etal. (26). These adsorbents were packed in small columns andthoroughly washed with phosphate-buffered saline. Portions ofthe "2I-labeled soluble materials were passed through the col-umns at 4°C and were subjected to the gel analysis.

Polyacrylamide Gel Electrophoresis in NaDodSO4. Radio-labeled materials were analyzed by NaDodSO4/polyacrylamidegel electrophoresis by the method of Laemmli (27) under re-ducing and nonreducing conditions. Reduction was initiated with10mM dithiothreitol (Eastman-Kodak) for 1 hr at 25°C followedby alkylation with 20 mM iodoacetamide (Eastman-Kodak).Samples were analyzed in NaDodSO4 (Sigma)/10% poly-

acrylamide gels (7 mm diameter x 12 cm length) crosslinkedwith NN,N',N'-tetramethylethylenediamine (TEMED, Nak-arai Chemicals, Kyoto, Japan) in the presence of 0.1% NaDod-S04 and 0.375 M Tris HCl buffer, pH 8.8. Molecular weight(Mr) markers used were bovine serum albumin (68,000), oval-bumin (43,500), and lactoperoxidase (18,000). After completionof electrophoresis the radioactivity in 1-mm gel slices was mea-sured.IEF in Polyacrylamide Gels. The isoelectric point (p1) of ra-

diolabeled TaF molecules was- determined by IEF in glass tubes(130 x 2.4 mm) as described by O'Farrell (28). The gradient ofpH 5.0 to 8.0 was made by mixing Ampholines (pH 5-7 andpH 3-10, LKB) and electrophoresis before loading the sample.The electrophoresis of radiolabeled purified TaF was at 400 Vfor 12 hr and then at 800 V for 1 hr. The gel was dried and ana-lyzed by autoradiography. The autoradiogram was scanned witha densitometer.

RESULTS

Immunoprecipitation and NaDodSO4/Polyacrylamide GelElectrophoretic Analysis of Biosynthetically Labeled TaF. TheNP-40 lysate of FL10 cells metabolically labeled with [3S]me-thionine and [14C]leucine was immunoprecipitated with amonoclonal anti-I-A k antibody (14P). The precipitate was sol-ubilized in NaDodSO4 sample buffer without reduction andsubjected to electrophoresis in a NaDodSO4 gel. Two sharp ra-dioactive peaks with Mr of 65,000-67,000 and 33,000-34,000were invariably observed (Fig. IA). After reduction of the ma-terial with 10 mM dithiothreitol, the Mr 67,000 peak disap-peared, and a single peak of Mr 33,000 was observed (Fig. 1B).This indicates that the Mr 67,000 molecule is composed of twopolypeptide chains bound by disulfide bonds (see below). Noneof the monoclonal antibodies against class II conventional B cellTa antigens (anti-Ia.2 and anti-Ia. 17) were able to precipitateradioactive materials from the FL10 lysate (Fig. 1C). The sameimmunoprecipitation with the NP-40 lysate from I-Jkpositivesuppressor T-cell hybridoma 7C3-13 and the parental lym-phoma BW5147 showed no detectable radioactive peaks withthe monoclonal anti-I-A k antibody.NaDodSO4 Gel Analysis of TaF Purified with Antigen-Coated

Plastic Dishes. The saline-soluble extract from FL10, whichhad been shown to contain TaF activity (9), was used for thepurification of the materials for the gel analysis. The saline ex-tract from FL10 was incubated in KLH-coated plastic dishes,and the materials absorbed to the dishes were carefully re-covered by acid elution. They were then radioiodinated andsubjected to the NaDodSO4 gel analysis.

As shown in Fig. 2A, two radioactive peaks of Mr 67,000 and33,000 were observed under nonreducing conditions, both ofwhich were undetectable in the control material obtained bythe identical procedure from plastic dishes coated with an un-related antigen, FGG. The reduction with dithiothreitol re-sulted in the cleavage of the Mr 67,000 molecule into a Mr 33,000molecule as shown in Fig. 2C. We confirmed that nothing isreleased from KLH-coated dishes by the same treatment withglycine HCl buffer, pH 2.8. The absorption of the radioactivematerial with an immunoadsorbent column of antigen (KLH)resulted in the disappearance of both peaks, whereas the ab-sorption with the monoclonal anti-I-Ak immunoadsorbent elim-inated only the Mr 67,000 peak, leaving the Mr 33,000 peak un-altered (Fig. 2B). The results indicate that molecules elutedfrom KLH dishes are in two different forms under nonreducingconditions: a heterodimer of antigen-binding and I-Akpositivepolypeptide chains of Mr 67,000 and a single antigen-bindingpolypeptide of Mr 33,000 lacking the I-A' determinant. The

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FIG. 1. NaDodSO4/polyacrylamide gel electrophoretic analysis ofTaF from FL10 cells immunoprecipitated with monoclonal anti-I-ATand anti-MIg. T-cell hybridomas FL10 and 7C3-13 (control) were met-abolically labeled with [35S]methionine and [14C]leucine and cell ly-sates in NP-40 were analyzed. (A) Lysates from FL10 and 7C3-13 wereimmunoprecipitatedwith monoclonal anti-I-Ak and anti-MIg and wereelectrophoresed under nonreducing conditions. Note two radioactivepeaks with Mr 67,000 and 33,000 in the precipitate from FL10 (.) butnot from 7C3-13 (0). Markers: 1, bovine serum albumin; 2, ovalbumin;3, lactoperoxidase. (B) The same immunoprecipitate from FL10 withmonoclonal anti-I-AM was analyzed under reducing conditions. The Mr67,000 peak disappeared, and all the radioactive material migrated tothe position of Mr 33,000 (e). (C) FL10 lysates immunoprecipitated withmonoclonal anti-Ia.2 (e) or anti-Ia.17 (0) antibodies and anti-MIg. Noteno detectable radioactivity in either preparation.

mild reduction dissociated the heterodimer into monomers ofMr 33,000.The analysis of the material similarly purified with the anti-

I-Ak immunoadsorbent gave essentially the same gel electro-phoretic pattern, in which Mr 67,000 heterodimer and Mr 33,000I-A polypeptide chains were detected. We tried to absorb thesematerials with rabbit anti-VH and anti-VL. Anti-VH but not anti-VL could absorb the heterodimer, whereas. both were unableto absorb I-Ak polypeptide (data not shown).

Presence of Tindd Determinant on the Antigen-BindingPolypeptide. It has recently been postulated that antigen re-

ceptors of T cells may have determinants controlled by geneslinked to Igh gene cluster (29). Owen (19) produced monoclonalantibodies that react with Igh allotype-linked structures ex-

pressed on functionally different T-cell subsets. These T-cellalloantigens have been designated Tindd (21), Tsud, and Tthyd(30). Because we have recently demonstrated that the Tindddeterminant is present on both the membrane of FL10 and TaFproduced by FL10 (22), we examined the material purified withKLH-coated dishes for the presence of Tindd determinant byabsorption with immunoadsorbent of monoclonal anti-Tindd or

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FIG. 2. NaDodSO4/polyacrylamide gel electrophoretic analysis ofFL10-derived TaF purified with antigen. Cell lysates of FL10 were in-cubated in KLH- or-FGG-coated plastic dishes. After washing, the ma-terials bound to the dishes were eluted with acid and labeled with 1251.(A) The radiolabeled materials purified with KLH- or FGG-coated dishesanalyzed under nonreducing conditions. The materials-purified withKLH-coated dishes show two radioactive peaks of Mr 67,000 and Mr33,000 (e). No radioactive peaks are observed in the eluate from FGG-coated dishes (a). (B) The materials elutedfrom KLH were reabsorbedwith KLH or monoclonal anti-I-Mr antibody under nonreducing con-ditions. Reabsorption with KLH completely removed both radioactivepeaks (e), whereas only the peak of Mr 67,000 was eliminated by ab-sorption with anti-I-AT antibody (o). (C) The radiolabeled materialspurified with KLH-coated dishes analyzed under reducing conditions.All the radioactive material migrated to the position of Mr 33,000 (0).

anti-Tsud (Fig. 3). Whereas the anti-Tindd column completelyremoved both Mr 67,000 and Mr 33,000 peaks from the KLHeluate, the anti-Tsud column was unable to remove either ma-terial. The same anti-Tindd column could absorb most but notall reduced material of Mr 33,000 (not shown). The results in-dicate that the antigen-binding polypeptide chain of TaF is as-

sociated with Tindd but not Tsud determinants.pI of Antigen-Binding and I-AkT-Bearing Polypeptides. An

attempt was made to separate these two polypeptide chains ofthe same molecular weight by IEF in a polyacrylamide gel. TheNP-40 lysate of metabolically labeled FL10 was immunopre-cipitated with anti-I-AkT. The precipitate was dissolved in theIEF lysis buffer and electrophoresed in IEF gel with a pH rangeof 5.0 to 8.0 (Fig. 4). The autoradiogram of the dried geLdem-onstrates two clear radioactive bands with pl5.6 and 6.3. Whenthe NP-40 lysate from FL10 was first reduced by 10 mM di-thiothreitol and alkylated, then immunoprecipitated withmonoclonal anti-I-AT only a single radioactive band with pI 6.3was observed in the IEF gel (Fig. 4, scan B). When the same

material was immunoprecipitated with anti-Tindd, the radio-active band of pI 5.6 was obtained without any radioactivity atpI 6.3 (Fig. 4, scan C). These results indicate that TaF is com-posed of two polypeptides with the same molecular weight but

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FIG. 3. NaDodSO4/polyacrylamide gel electrophoretic analysis of

FL10-derived TaF purified with KLH after-absorption with anti-Tinddand anti-Tsud. The KLH-binding material was labeled with 125I andabsorbed with immunoadsorbent of monoclonal antiTindd or antiTsud.Both radioactive peaks ofMr 67,000 and Mr 33,000 were eliminated byabsorption with the anti-Tindd.column (W). Neither peak was elimi-nated with the anti-Tsud (a).

with different pls: pl 6.3 for I-Ak polypeptide and pl 5.6 forantigen-binding Tindd-bearing polypeptide.

DISCUSSION

The structure utilized for antigen recognition by T cells is cur-

rently one of the central issues in immunology. Although muchphenomenology has been accumulated, no conclusive bio-chemical studies have been reported. The biochemical analysisof antigen-specific T-cell factors that specifically bind to anti-gen and exert specific regulatory functions is one of the prom-ising approaches, and attempts have been made to characterizebiochemically the soluble factors released from T-cell lines andhybridomas. However, such attempts have not been fully suc-

cessful for several reasons: First, the quantity of material pro-

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FIG. 4. IEF gel analysis of FL10-derived TaF immunoprecipitatedwith monoclonal anti-I-A' and antiTindd antibodies. FL10 was met-abolically labeled with [35S]methionine and ['4C]leucine, and the celllysates were prepared with NP-40. Densitometer scan A, FL10 lysatewas immunoprecipitated with a monoclonal anti-I-Ak and anti-MIg and

was electrophoresed under reducing conditions. Two. clear radioactivebands of pI 5.6 and 6.3 were observed in the autoradiogram (top). ScanB, the reduced cell lysates of FL10 were immunoprecipitated.with a

monoclonal anti-I-Ak antibody and anti-MIg. Only a single radioactiveband of pI 6.3 is observed. Scan C, the reduced cell lysate of FL10 was

immunoprecipitated with monoclonal anti-Tindd and anti-MIg. The ra-

dioactive band with pI 5.6 is observed without any radioactivity at pI6.3.

duced by T-cell lines and hybridomas was much too small fordirect biochemical analysis. Although several monoclonal an-tibodies were produced against the putative structures of T-cellfactors (16-18), it has not been possible to purify enough ma-terial for biochemical analysis, probably due to the extremelyunstable expression of corresponding antigens on many of theT-cell hybridomas (5). In order to make a breakthrough of thissituation, we have to have both stable cell lines and suitablereagents.We have previously reported -the derivation of a character-

istic T-cell hybridoma FL10 that produces a KLH-specific TaF(9). The factor was shown to possess KLH-binding activity anddeterminants controlled by the I-A subregion of MHC, whilethe I-A determinants on TaF were different from those on knownclass II antigens. Taking advantage of monoclonal antibodiesreactive with I-A determinants on TaF, we have attempted topurify TaF from a NP40 lysate of biosynthetically labeled FL10.The results reported in this paper indicate that TaF can be

purified by a modified immusaosorbent method or by immu-noprecipitation with anti-I-Ak antibody or with antigen and thatthe molecules purified by the two methods are essentially iden-tical. The purified TaF fraction thus obtained was found to becomposed of two types of molecules as demonstrated byNaDodSO4/polyacrylamide gel electrophoresis; one is a het-erodimer.of.Mr 67,000 composed of two different polypeptideseach of Mr 33,000, and the other is either one of the two chains.After mild reduction, all the molecules migrated to the positionof Mr 33,000. The absorption of the material with immunoad-sorbents composed of antigen and antibodies revealed that onepolypeptide is antigen binding and characterized by two im-portant determinants: the determinant reactive with rabbit an-tibodies against the framework structure of immunoglobulinheavy-chain variable region (anti-VH) (31) and an allotypic de-terminant detectable by a monoclonal antibody against an Igh-linked structure of T cells (anti-Tindd). The polypeptide thatdoes not bind antigen carries determinants controlled by the I-A subregion of MHC but is different from known class II Iapolypeptides. This was demonstrated by the facts that none ofour anti-I-Ak reacted with B cells and macrophages and thattwo of the well-defined anti-class II antibodies specific for pri-vate and public Ia determinants of H-2k-i.e., anti-Ia.2 andanti-Ia.17-were unable to precipitate TaF. These two poly-peptides are different in their isoelectric points: the antigen-binding molecule had a pI of 5.6, and the I-Ak polypeptide hada pI of 6.3.A question arises as to the nature of I-Ak polypeptide as-

sociated with TaF. The molecular weight of this polypeptide isvery similar to that of A,, chain of class II antigen, yet the an-tigenic determinants are entirely different. It is possible thatthere exists a unique gene in the I-A subregion that codes forthe I-Ak- polypeptide associated with TaF. Another possibilityis that Aa chain is severely modified in its tertiary structure whenit is expressed on T cells that lack A,3 expression. However, itis highly unlikely that all the known class II determinants arelost and several new determinants are created on Aa chain whenexpressed on T cells. Such a change may not be attributable tothe association with antigen-binding structure, because themonoclonal antibodies used in the present experiment can reactwith a separate I-Ak polypeptide chain produced by reduction.Other possibilities are the existence of presently unknown ge-netic processes for the expression of I region-controlled poly-.peptides on T cells,.such as gene conversion (32), insertion ofunique small bases, or post-translational modification (33, 34).Thus, at the moment-we think that the I-Ak-positive polypep-tide is not identical to Aa chain but may be a unique moleculeexpressed only on some functional T cells. We have to await the

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complete molecular genetic analyses of the I region or the aminoacid sequence of this polypeptide for the identification of thisinteresting molecule.

Although we have clearly demonstrated the antigen-bindingpolypeptide in NaDodSO4 gel apart from the I-A-controlledpolypeptide, some controversies have to be discussed here. Thispolypeptide has a Mr of 33,000 and a pI of 5.6, and it carriesTindd but not Tsud determinants. Spurll and Owen (21) havereported that the monoclonal anti-Tindd precipitated a groupof polypeptides from normal Lyt-l T cells-i.e., Mr 62,000,45,000, and 17,000, none of which correspond to the M, 33,000polypeptide derived from FL10. They suggested that Mr 45,000and 17,000 molecules are produced by the post-translationalprocessing of the Mr 62,000 molecule. Because they were un-able to demonstrate the association of such molecules with Iregion-controlled determinants, it may be that the Mr 33,000Tindd-positive polypeptide is also a post-translational productthat can interact with I-Ak polypeptide to form the functionalTaF molecule. Again, it is possible that there are multiple an-tigen-binding polypeptides of T cells apparently linked to theIgh-1 locus and carrying the same crossreactive Tindd deter-minant.The multiplicity of antigen-recognition units on T cells has

been suggested by several investigators. For example, TsF, whichspecifically suppresses the immune responses, can be dividedinto suppressor inducer (TsFi) and suppressor effector (TsFe)factors (35, 36). Taniguchi et al. (12, 13) reported that their TsFiderived from suppressor hybridomas is composed of two poly-peptides, one antigen-binding and the other a product of theI-J subregion of MHC. Although these polypeptides were notbiochemically analyzed, Taniguchi et al. have recently shownthat these two functional polypeptides can be translated fromdifferent mRNA fractions (37). Fresno et al. (36) reported a TsFefrom a continuous T-cell line as a single polypeptide of M, 70,000that has no I region determinants. Kapp and co-workers (38,39) established T-cell hybridomas producing TsF specific forpoly(Glu60Ala30Tyr10) (GAT) that is composed of a single poly-peptide of Mr 24,000 possessing antigen-binding site, an I-J de-terminant, and an idiotype shared with anti-GAT antibodies.This molecule can also be produced by an in vitro translationfrom mRNA of the hybridoma, and the active product had a Mrof 19,000.

Because of the- above-mentioned complexity of the findings,we are again alert for the critical comparisons of these func-tionally different T-cell-derived factors on a solid biochemicalbasis. The successful purification and biochemical analysis ofTaF in gels encourages further characterization. As the frame-work structure and antigenic determinants of TaF have beendetermined by the present study, further tools in molecular ge-netics should allow more detailed analysis of the structure andexpression of an antigen-recognition structure of T cells.We thank Ms. YokoYamaguchi, Ms. Taeko Fukuda, and Ms. Mariko

Sato for secretarial and technical assistance. This work was supportedby a grant from the Ministry of Education, Science and Culture of Ja-pan. P.B.N. is a Fellow of the Cancer Research Institute (New York).

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