Proc. Natl. Acad. Sci. USAVol. 82, pp. 7399-7403, November 1985Immunology

Use of anti-idiotypic antibodies to identify a receptor for the T-cellI-J determinant

(I-J receptor/anti-idiotype/T-cell suppression)

KARIN ZUPKO*, CARL WALTENBAUGHt, AND BErrY DIAMOND*tDepartments of *Microbiology and Immunology and *Medicine and Irvington House Institute for Medical Research, Albert Einstein College of Medicine, 1300Morris Park Avenue, Bronx, NY 10461; and tDepartment of Microbiology and Immunology, Northwestern University Medical School, 303 East ChicagoAvenue, Chicago, IL 60611

Communicated by Harry Eagle, July 8, 1985

ABSTRACT In order to identify the molecule(s) interact-ing with the I-J determinant on suppressor T cells, we havegenerated two anti-idiotypic sera: one to monoclonal anti-I-Jdantibody and one to monoclonal anti-I-PJ antibody. Theseantisera specifically block suppressor T-cell function in agenetically restricted manner and have no effect on helperT-cell activation. Both recognize a marker on primarymonocytes and B cells but not on T cells. A myeloma cell linebearing this marker has been identified. Therefore, theseantisera may recognize a molecule on cells interacting withsuppressor T cells that is involved in mediating suppressorT-cell activity. The relationship between the T-cell I-J deter-minant and the molecule identified by the anti-idiotype may besimilar to the relationship between the receptor on helper Tcells and Ia molecules.

It is now well established that an accessory cell is required foractivation of helper T cells. Recent studies have suggestedthat an accessory cell might also be necessary for inductionof suppressor T cells and that macrophages may perform thisaccessory cell function (1, 2). While I-J antigens both marksuppressor T cells and are intimately involved in theirsuppressive activity, nothing is known about the membranemolecules on accessory cells that interact with I-J. Since ananti-idiotype to anti-insulin antibodies has been shown tobind to the insulin receptor (3), we have used a similarstrategy to produce antibodies to the macrophage I-J recep-tor. In this paper we report the production of polyclonalanti-idiotypic antisera to two monoclonal anti-I-J antibodies.These anti-idiotypic sera react with macrophages and B cellsand inhibit suppressor T-cell activity in a genetically restrict-ed manner.

MATERIALS AND METHODSImmunization and Assay for Production of Anti-Idiotypic

Antibodies. A female rabbit (Dutchland, Denver, PA) wasinjected intradermally at ".25 sites with 10 ,g of purifiedanti-I-Jd antibody in complete Freund's adjuvant (2-ml totalvolume), and boosters were administered 2 and 6 wk later.The anti-I-Jd antibody, an IgG2b,k, was produced by immu-nizing (B10.A x D2.GD)F1 mice five times (biweekly) withB10.D2 spleen cells (107 per injection); 14 days after the finalinjection, spleen cells were fused with the P3x63Ag8.653myeloma by using polyethylene glycol. A single secretinghybridoma, WF18.2B15, was identified by its ability to bothreverse poly(LGlu50LTyr50) [(Glu5 Tyr50)]-specific suppres-sion in BALB/c (H-2d, I-Jd) mice and bind to BALB/cT-cell-derived suppressor factor (TsF) [(Glu50Tyr50),-TsF].

A second female rabbit (Dutchland) was similarly immu-nized with purified anti-I-Jk derived from the hybridomaWF8.C12.8. The production of this IgGl,k hybridoma hasbeen described (4).

Anti-idiotypic antibody activity was determined by usingthe Bio-Rad Immuno-Blot procedure. A positive assay foranti-idiotypic antibody activity was one in which activityagainst anti-I-Jd was present both when immune serum wasincubated with anti-IlJd alone and when incubated in thepresence of an excess of IgG2b protein. A similar protocolwas used to detect anti-idiotypic activity against anti-I-Jk withIgG1 protein.

Preparation of F(ab')2 Fragments from Rabbit Sera. F(ab')2fragments were made from an ammonium sulfate cut ofpreimmune sera, immune sera, and several anti-IgG2 rabbitsera, followed by pepsin digestion at pH 3.5. Samples weretreated with protein A-Sepharose, following which the ab-sence of intact Ig was verified by NaDodSO4/PAGE (5).Samples were then dialyzed against phosphate-buffered sa-line with azide.

In some cases, samples were treated with IgG2b-Seph-arose or IgG1-silicate to remove anti-constant region activity.The IgG1-silicate was prepared by coupling MOPC 21 (IgG1)to a glutardialdehyde-activated matrix (Boehringer Mann-heim). In the case ofthe anti-I-Jk anti-idiotypic serum, F(ab')2fragments of anti-idiotypic antibodies were affinity-purifiedon an anti-I-JkSepharose column following adsorption on anIgG1 column.Immunosuppression Studies. Immunosuppression studies

were carried out as described (6). Single-cell suspensions ofspleen cells from BALB/cAnNCr (H-2d, I-Jd), AKR/NCr(H-2k, I-J), or CBA/JNCr (H-2k, 4-k) mice were prepared.An F(ab')2 preparation of each anti-idiotype, adsorbed on theappropriate Sepharose-IgG column (and in the case of I-Jk,affinity-purified on an anti-I-Jk column), was added from thebeginning ofculture. In all cases, data shown are from a singlerepresentative experiment, but three or four experimentswere performed.

Assays for Antigen Presentation. Assays for antigen pre-sentation by the macrophage-like cell line BAC1 to T-cellhybridomas have been described (7). 3D062.1 (specific forI-Ed alone) and D011.10 (specific for I-Ad and ovalbumin)were gifts of J. Kappler and P. Marrack (National JewishHospital and Research Center, Denver, CO).For blocking studies, MKD6 (anti-I-Ad) or 14.4.4S (anti-

I-Ed) ascites or rabbit antiserum was added at the initiationof culture at a 1:10 dilution.

Abbreviations: (Glu5°Tyr-'°),, poly(LGlu50LTyr50); GAT, (Glu'-Ala30Tyrl0)", poly(LGlU'LAIa3"LTyr1O); (Glu50Tyr50) -MeBSA,poly(LGlu-'LTyr'°)-methylated bovine serum albumin; PFC, plaque-forming cell(s); BSA, bovine serum albumin; IL-2, interleukin 2;TsF, T cell-derived suppressor factor; FACS, fluorescence-activatedcell sorter; FITC, fluorescein isothiocyanate.

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Analysis of Primary Cells by Fluorescence-Activated CellSorter (FACS II). Spleens were removed from femaleBALB/cAnNCr (H-2"), AKR/NCr (H-2, C57BL/6J (H-2b),or A.CA/J (H-I) mice, and single-cell suspensions wereprepared. Cells were incubated for 1-2 hr in a tissue cultureplate at 370C in a CO2 incubator. The nonadherent cells werethen removed. The adherent cell population was contaminat-ed by <3% Thy-i-positive cells and 4% surface Ig-positivecells. The nonadherent cell population contained 50% cellsbearing surface Ig and 50% cells bearing the Thy-1 marker.F(ab')2 fragments of immune serum, preimmune serum, orirrelevant anti-IgG2b serum were added to the cells at aconcentration of 0.3 mg/ml in phosphate-buffered saline with2% bovine serum albumin (BSA) and azide for 30 min at 40C.The samples were washed and incubated with 70,4l of a 1:60dilution of fluorescein isothiocyanate(FITC)-conjugated goatanti-rabbit IgG F(ab')2 fragment (Cappel Laboratories,Cochranville, PA) for 30 min at 4TC. Analysis was on a FACSII (Becton Dickinson) with an argon laser.Thymocytes were obtained from a 6-wk-old BALB/c

mouse or an AKR mouse and prepared as described above.In addition, thymocytes were incubated with a 1:10 dilutionof rabbit anti-mouse Thy-1 antibody (Cedarlane Laborato-ries, Homby, ON).

Analysis of Cell Lines. The macrophage-like cell linesJ774.2, P388D1, and BAC1 were analyzed by the sameprocedure as for primary cells. B-cell lines were also ana-lyzed: MPC11, B50.10.1 (gift of Sherie Morrison), S107, andsome hybridomas made against anti-DNA antibodies (8.12.5,1.12, 3I.14) (8, 9).

Cell lines that were stained with the anti-idiotypic antise-rum to anti-I-Jd were analyzed for Ia expression. The anti-I-Aused was 28.16.8S ascites (1:10) or undiluted 28.16.8S super-natant (hybridoma cells from American Type Culture Col-lection, Rockville, MD), an IgM antibody. An irrelevant IgMwas included as a control. The anti-I-E preparation used wasF(ab')2 fragments (prepared by Ming-der Chang in our lab)from 14.4.4S hybridoma cells (American Type Culture Col-lection). The second antibody was a 1:60 dilution of FITC-conjugated goat anti-mouse IgM (Cappel Laboratories) or a1:30 dilution of FITC-conjugated F(ab')2 rabbit anti-mouseIgG (Jackson ImmunoResearch, Avondale, PA).

RESULTS

Generation of an Anti-Idiotypic Antiserum to a MurineMonoclonal Anti-IWJd Antibody. A rabbit was immunized witha IgG2b,k mouse monoclonal anti-I-Jd. A second rabbit wasimmunized with monoclonal anti-I-Jk. The monoclonal anti-bodies are specific for I-Jd or I-Jk; block activity of suppressorT cells; bind suppressor T-cell factors of the H-2d or H-2khaplotypes, respectively, and do not cross-react with I-Jk orI-Jd, respectively. Preimmune serum from the rabbits showedno reactivity to the anti-I-J antibodies or to irrelevant murineIgGs. Immune serum showed reactivity to both anti-I-Jd andto other murine IgG2b,k proteins or to anti-I-Jk and otherIgGi proteins (Fig. 1). To determine if anti-idiotypic activitywas present in each case, the serum was incubated with themonoclonal anti-I-J antibody in the presence of an excess ofthe appropriate IgG (Fig. 1).The serum of the rabbit immunized with anti-I-Jd continued

to react with the anti-I-Jd, demonstrating the presence ofanti-idiotypic antibodies. Therefore, it was adsorbed on anIgG2b,k affinity column. The adsorbed antiserum had noreactivity with IgG2b,k proteins by dot-blot analysis, whichcan detect nanogram amounts of protein. All functionalstudies were done with this adsorbed antiserum. In addition,a rabbit antiserum made against a mouse IgG2b,k myelomaprotein with anti-IgG2 activity was used as a control in all ofthe experiments reported to ensure that the data do not reflect

A H C (

1

FIG. 1. Dot-blot assay for anti-idiotypic activity. To each squareof lane A, 1 p4 of U148 [purified IgG1 at 0.25 mg/ml in phosphate-buffered saline (Pi/NaCl)] was adsorbed; to each square of lane B,1 /4 of anti-IJk (0.25 mg/ml in Pi/NaCl) was adsorbed; and to laneC, 1 Ml of Pi/NaCl was applied. Dot-blot 1 was incubated in a 1:30dilution of immune serum followed by an enzyme-linked anti-rabbitIgG. Dot-blot 2 was incubated in a 1:30 dilution of immune serum inthe presence of U148 ascites. After development, the positive assayfor anti-idiotypic antibody showed activity remaining against anti-I-Jk (dot-blot 2, lane B) after absorbing anti-IgGl activity (dot-blot 2,lane A).

the activity of any residual anti-constant region antibodiesthat might be present. The serum of the rabbit immunizedwith anti-I-Jk also showed continued reactivity with anti-I-Jkdespite the presence of excess IgGi; therefore, this serum toohad anti-idiotypic activity. It was adsorbed on an IgG1column and then affinity-purified on an anti-I-Jk column. Allstudies were done with the IgGl-adsorbed, affinity-purifiedanti-I-Jk antibodies.

Inhibition of Suppressor T-Cell Activity by the RabbitAntiserum. To determine whether the anti-idiotypic antiserawere recognizing a marker involved in mediating T-cellsuppression, we tested whether the antisera could blocksuppressor T-cell activity. The antiserum to anti-I-Jd blockedthe suppression of (Glu50Tyr'0)n-specific plaque-forming cells(PFC) induced by a (Glu50Tyr50)n-specific TsF designatedTsF1 (Table 1). In this assay, spleen cells are incubated with(Glu50Tyr50)n conjugated to methylated BSA [(Glu50Tyr50)n-MeBSA] for 5 days under Mishell-Dutton conditions, and thenumber of cells making antibody to (Glu50Tyr50)n is deter-mined. When TsF1 was added, there was a marked reductionin (Glu50Tyr5)0l-specific PFC. When F(ab')2 fragments of theanti-idiotypic antiserum were added at initiation of theculture, the TsFi-induced suppression of PFC was reversed(Table 1, Exp. 1). Preimmune serum and anti-IgG2 antiserumhad no activity in this assay. The anti-idiotypic antiserum didnot itself cause polyclonal B-cell activation, as addition of theF(ab')2 fragments alone did not lead to an increase in(GluSGTyrW)L-specific PFC.The I-J TsF1 (WF11.3A11) induces (Glu50Tyr50)n-specific

suppression in both H-2d and H-2k haplotypes (4). Wecompared the blocking effect of anti-I-Jd anti-idiotypic anti-serum on TsF1 activity in BALB/c and in AKR spleen cellcultures (Table 1, Exp. 2). The anti-idiotype blocked I-J TsF1activity on BALB/c (H-2d, I-Jd) but not on AKR/NCr (H_2k,I-JJ spleen cells. Since this experiment uses the same I-JTsF1, the fact that AKR/NCr suppression was not blockedindicates that the anti-idiotype does not block TsF1, thus

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Table 1. Blocking of factor-induced suppression by F(ab')2fragments of anti-I-Jd anti-idiotype to BALB/c but not toAKR mice

(Glu50Tyr50)n-specificAnti- PFCt

(Glu1OTyr1°),,- (Glul°Tyr'°),,- idiotype~tTsFi* MeBSA Ag BALB/c AKR/Cum

Experiment 1- + - 1125 -

1:400 + <20 -1:400 + 1.75 840

- 1.75 <20Experiment 2

- + 970 7201:400 + 140 3501:400 + 1.75 1010 <20

+ 1.75 930 1310

*Culture supernatants containing monoclonal (Glu51Tyr1°)"-TsFj(WF11.3A1) were added at the indicated dilution at culture initia-tion.tRabbit anti-I-Jd (WF18.2B15) anti-idiotype F(ab')2 fragments wereadded at the indicated amounts at culture initiation.t(Glu51Tyr-'O),-specific PFC responses of Mishell-Dutton-type cul-tures 5 days after culture initiation.

eliminating the possibility that the anti-idiotype has anti-I-Jdactivity. The immune serum did not alter the normal T-cell-dependent PFC response, as addition of anti-idiotype tocultures did not change the (Glu50Tyr5)n-PFC response (Table1).Because the TsF1 used is active with cells of the H-2d and

H-2k haplotypes, the reciprocal experiment was possible totest the anti-idiotype to anti-IlJk with the same TsFj. Theaffinity-purified anti-anti-I-Jk inhibited the suppression ofH-2k cells by TsFj but not the suppression ofH-2d cells (Table2). These data show that anti-IlJk anti-idiotype also inhibitssuppressor T-cell activity and, like the anti-I-Jd anti-idiotypicantiserum, does so in a genetically restricted manner.

Effect on the Anti-I-Jd Antiserum on Activation of Iad-Restricted Helper T Cells. In order to test whether theantiserum nonspecifically interfered with the interaction ofH-2d T cells with accessory cells, we tested whether it wouldblock helper T-cell activation. T-cell hybridomas, one spe-cific for I-Ad and ovalbumin and one for I-Ed alone, secretedinterleukin 2 (IL-2) when incubated with Iad-bearing macro-phages and antigen (Table 3). There was no decrease in IL-2production when the rabbit anti-idiotypic antiserum wasadded to the cultures. The appropriate anti-Ia antibody didinhibit IL-2 production. Therefore, the anti-idiotypic antise-

Table 2. Blocking of factor-induced suppression by F(ab')2fragments of affinity-purified anti-I-Jk anti-idiotype toCBA/J but not to BALB/c mice

Anti- (Glu5OTyr51)-(Glu5°Tyr5°)"- (GIu50Tyr50),- idiotypet specific PFC

TsF,* MeBSA ,ug BALB/c CBA/J+ 530 890

1:400 + - 30 <201:400 + 0.25 <20 640

+ 0.25 450 790

*Culture supernatants containing monoclonal (Glu51Tyr1°),-TsFj(WF11.3A1) were added at the indicated dilution at culture initia-tion.tRabbit anti-I-Jk (WF8.C12.8) affinity-purified anti-idiotype F(ab')2fragments were added at the indicated amount at culture initiation.*(Glu-Tyr50)"-specific PFC responses of Mishell-Dutton-type cul-tures 5 days after culture initiation.

rum is not directed to conventional Ia molecules and does notinterfere with H-2d helper T-cell activation.

Reactivity of Primary Cells with the Anti-Idiotypic Antise-rum. We determined the cellular distribution of the mole-cule(s) seen by the anti-idiotypic antisera. First monocytes,B cells, and T cells from BALB/c mice were examined busing the anti-idiotypic antiserum generated against anti-I-JFig. 2 shows FACS data on splenic adherent cells (Left Top),splenic nonadherent cells (Left Middle), and on thymocytes(Left Bottom). The antiserum showed specific binding toapproximately 40% of splenic adherent cells and of splenicnonadherent cells. There was no detectable specific bindingto thymocytes, strongly suggesting that the nonadherent cellsrecognized by the immune serum were B cells. In theseexperiments preimmune serum and an antiserum to IgG2b,kwere used as controls to ensure that the binding reflects theactivity of anti-idiotypic antibodies and not preexisting anti-bodies to mouse cells or antibodies to Ig constant regiondeterminants. Cells from C57BL/6J (H-2b), C3H/HeJ (H-2k),and A.CA/J (H-2f) mice also were examined and showedsimilar FACS binding profiles, suggesting that, in addition tocontaining an internal image of I-Jd, the antiserum containssome antibodies to nonpolymorphic determinants on the I-Jreceptor (Fig. 2 Right Top and Middle).The affinity-purified antiserum to anti-IlJk was similarly

analyzed. It bound to about 30% of splenic adherent andnonadherent cells of the H-2k haplotype and showed noreactivity with H-2k thymocytes (data not shown). Like theother anti-idiotypic antiserum, its reactivity was not restrict-ed to cells of the H-2k haplotype.

Reactivity with Cell Lines. We wished to determine if thereare existing cell lines expressing the molecule(s) seen by therabbit antiserum to anti-I-Jd, as the existence of such a linewould facilitate biochemical analysis of the membrane mark-er(s). First we examined a number of murine macrophagelines. However, all of the macrophage lines tested showed ahigh degree of autofluorescence, making detection of specificsurface markers by immunofluorescent techniques difficult.Therefore, we analyzed a number of B-cell lines. Whereasmost of the myeloma and hybridoma lines did not react withthe anti-idiotypic antiserum, the MPC11 myeloma line wasfound to express the marker seen by the antiserum (Fig. 2Right Bottom). Preimmune serum showed no reactivity onthis line, and an antiserum directed against murine IgG2molecules showed little reactivity, as this line has littlemembrane immunoglobulin. The MPC11 cell line did notreact with anti-Ia antibodies (Fig. 2 Right Bottom). Again this

Table 3. IL-2 assay; anti-idiotypic antiserum does not blockhelper T-cell activation

System and treatment AcpmBAC1/DO11.10*/ovalbumin 16,010 ± 2793+ anti-IlJd anti-idiotypic antiserum 17,861 ± 5237+ anti-IgG2 antiserum 15,852 ± 4917+ anti-I-Ad 0 ± 1824

BAC1/3D062.1t 5,969 ± 2681+ anti-I-Jd anti-idiotypic antiserum 8,803 ± 3715+ anti-IgG2 antiserum 9,471 ± 4427+ anti-I-Ed 0 ±

682

When incubated with Iad-bearing macrophages (BAC1) and anti-gen (D011.10) or with BACI alone (3D062.1), T-cell hybridomassecrete IL-2. Numbers given indicate thymidine incorporation by theIL-2-dependent cell line CT6 and are averages of triplicate cultures.The Acpm values were obtained by subtracting the cpm values for Tcells alone (DO11.10, 17,816 ± 3,123 cpm; 3D062.1, 3,151 ± 959 cpm)from cpm values for experimental wells.*T-cell hybridoma specific for I-Ad and ovalbumin.tT-cell hybridoma specific for I-Ed alone.

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Adherent Cells H

,..anti-idiotype

ant-IgG2i /conjugate alone

'i , ,Icellis alones-

i-2 b,fk

Non-adherent SpleenCells H-2d 1 Non-adherent Cells H-2b'f'k

anti-idiotype t anti-idiotype

preimmune\ anti-IgG2 l anti-IgG2

~~conjugate alone

Thymocytes H-2d s'3

MPCII

anti-idiotype 'i

preimmmune ' nti-Iav1conjugate alone r! niiitp

cells alone \_anti-idiot,\zonil-thy , \ \ preimmune

,"' \\ X Ad ) conjugate alone

FIG. 2. Cellular reactivity as determined by FACS II analysisusing IJd anti-idiotype. (Left Top) Adherent spleen cells (H-2d) froma BALB/c mouse stain positively with an F(ab')2 preparation ofimmune serum. There is no significant staining with preimmune,anti-IgG2, conjugate alone, or cells alone. (Left Middle) Nonadher-ent spleen cells (H-2d) also show staining with the F(ab')2 preparationof immune serum and no other significant staining. (Left Bottom)Thymocytes (H-2d) isolated from a BALB/c mouse stain with rabbitanti-mouse Thy-1 antibody but do not stain with the anti-idiotypicF(ab')2 preparation. (Right Top and Middle) Staining of adherent andnonadherent cells, respectively, of the H-2bfk haplotypes is shown.(Right Bottom) Staining of the MPC11 line with the anti-idiotype andnot with anti-Ia antibodies is shown.

suggests that the antiserum is seeing a membrane moleculedifferent from Ia molecules.

Since our anti-idiotype is a polyvalent antiserum, it may haveseparate specificities recognizing B-cell lines and inhibiting(Glu50Tyr%)n suppression. Therefore, we adsorbed F(ab')2fragments of the antiserum on the MPC11 cell line until nofurther binding to MPC11 cells was detectable. The antiserumcontinued to display anti-IgG2b activity on dot-blot analysis butno longer showed anti-idiotypic activity against the anti-I-Jdantibody (data not shown). It also no longer reacted withprimary splenic B cells and monocytes. These data show thatthe anti-idiotypic antibodies bind to MPC11 cells and not tosome irrelevant antibodies with a fortuitous specificity. Theyfurther suggest that the molecule on MPC11 cells recognized bythe antiserum is similar to the molecule recognized on primarymonocytes and B cells and that biochemical analysis of thismolecule can be performed on the MPC11 cell line.

In order to show that the molecule(s) seen on the MPC11line and on primary cells is the molecule involved in medi-ating the inhibition of suppression, the MPC11-adsorbedF(ab')2 fragments were tested for their ability to inhibit TsF1activity in a (Glu50Tyr50)n-specific response. The adsorbedF(ab')2 fragments did not mediate inhibition of suppressionwhen used at the same concentration as the F(ab')2 fragmentsof anti-idiotypic antiserum prior to adsorption on the MPC11cell line (Table 4). Thus, adsorption with MPC11 cellsremoved those antibodies that block TsF1 activity.

DISCUSSIONIt has been suggested that antigen encountered by lymphocytesin the absence of an accessory cell can activate suppressor T

Table 4. Anti-idiotype that has been adsorbed on MPC11 cellsno longer has activity in suppression studies

(Glu5Tyr5),-(Glu0Tyry)n- (GluS°TyrS°)n_ specific

TsFi* MeBSA Anti-idiotypet PFCt- + 650

1:400 + Anti-I-Jd 6201:400 + Anti-I-Jd adsorbed <20

on MPC111:400 + Preimmune <20

*Culture supernatants containing monoclonal (Glu°0Tyr")n-TsFj(WF11.3A1) were added at the indicated dilution at culture initia-tion.tRabbit anti-IlJd (WF18.2B15) anti-idiotype F(ab')2 fragments orpreimmune F(ab')2 fragments were added at a 1:200 dilution atculture initiation.t(Glu"Tyr")n-specific PFC responses of Mishell-Dutton-type cul-tures 5 days after culture initiation.

cells (10, 11). However, recent evidence demonstrates thatthere are systems in which induction of suppressor T cells Ts1,TS2, and Ts3 requires an antigen-presenting cell (1, 2). Thephenotype of this antigen-presenting cell and the particularmembrane molecules on it that mediate the activation ofsuppressor T cells remain controversial.Suppressor T cells are characterized by the presence of an

I-J determinant on their membrane, and antibody to the I-Jdeterminant blocks suppressor T-cell activity (12). We haveattempted to identify the cells involved in interacting withsuppressor T cells by generating anti-idiotypic antisera toanti-I-J antibodies. If some of the antibodies in each serumreact with the binding site of the anti-I-J monoclonal anti-body, they should be structurally homologous to I-J andtherefore should bind to a cellular receptor for I-J.The anti-idiotypic antisera we made block the suppression

of antigen-specific PFC induced by TsF1. The inhibition ofTsFj mediated by our antisera appears to be geneticallyrestricted because the anti-idiotype made to anti-I-Jd blockssuppression in cultures of BALB/c cells but not in culturesof AKR cells, and the anti-idiotype made to anti-I-Jk blockssuppression in cultures of CBA/JNCr cells but not ofBALB/c cells. Because the TsFj used in both BALB/c andAKR/NCr or CBA/JNCr cultures is the same, the anti-idiotypes must bind directly to cells and not to TsF1 itself.The anti-I-J antibody used as an immunogen for one anti-idiotypic antiserum is specific to the d haplotype; therefore,this anti-idiotype must contain antibodies that are the internalimage of IJd and bind specifically to the site of the I-Jreceptor that binds I-Jd. Likewise, the anti-I-Jk antibody isspecific to the k haplotype, and the anti-idiotype generatedbinds to I-Jk receptor at the site where I-Jk binds.The antiserum to anti-I-J does not block activation of

helper T cells. Therefore, the membrane molecules on themacrophage responsible for suppressor T-cell induction aredifferent from the Ia molecules involved in activating helperT cells. If I-J is a set of determinants on suppressor T-cellreceptors, then the complementary molecule on cells inter-acting with suppressor T cells might be expected to sharedeterminants with Ia molecules. Our data do not showstructural dissimilarities between the molecule seen by ouranti-idiotypes to anti-I-J and classical Ia molecules butmerely that the molecule seen by our antisera is not identicalto classical Ia molecules. The molecule seen by our antiseramight in fact function in concert with classical Ia antigens.A subpopulation of both macrophages and B cells is

recognized by our anti-idiotypes. The antisera see a markersimilar to Ia molecules in that Ia molecules are also expressedon macrophages and B cells and are involved in mediating theinteraction of helper T cells with both macrophages and B

Adherent Spleen Cells H-2d

,I anti-idiotypeI preimmune\ anti-IgG2' conjugate alone

cells alone

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cells. We think that the marker we have identified may helpmediate the interactions between suppressor T cells and bothmacrophages and B cells. We speculate that the interactionbetween macrophages and suppressor T cells leads to theinduction of suppression. In fact, we have evidence that anantigen-presenting cell is required for presentation of TsF1(unpublished data). It is not clear what interaction occursbetween suppressor T cells and B cells. It may be thatI-J-positive suppressor T cells can act directly on B cells tosuppress antibody production. Evidence for this direct inter-action exists (13, 14). Alternatively, it may be that B cells,like adherent accessory cells, can induce I-J-positive sup-pressorT cells ifthey bear the I-J receptor, just as B cells maysometimes induce helper T cells. There is recent evidence forthis interaction also (refs. 15 and 16; unpublished data). Wedid not detect binding of the anti-idiotype to thymocytes.This does not mean that there are no mature T cells bearingthis marker but only that they do not exist in sufficientnumber in the thymus to be detected in our assay.While the inhibition of suppression mediated by the anti-

idiotypes is H-2-restricted, the binding of the anti-idiotype tomonocytes and B cells is not. The antisera we made shouldrepresent the internal image of I-Jd and I-Jk, respectively.That each binds to cells of several H-2 haplotypes suggeststhat the I-J receptor molecule may have conserved regionsexpressed on cells of every H-2 haplotype and polymorphicregions involved in recognition of I-J molecules of a specificH-2 haplotype. The anti-idiotypes would then recognizesome conserved sequences, but each would also recognizesome polymorphic sequences in the I-J receptor of either theH-2d haplotype or the H-2k haplotype. It is also possible thata nonpolymorphic I-J receptor has different binding sites fordifferent I-J molecules and that our anti-idiotypes recognizea region on the I-J receptor that interacts with I-Jd or I-Jk. Wefavor the first explanation, as it would make I-J and the I-Jreceptor analogous to the T-cell receptor on helper T cellsand Ia.The identification of the MPC11 mouse myeloma cell line,

which expresses the marker recognized by our anti-idiotypicantiserum to anti-IJd, should facilitate the biochemical anal-ysis of this membrane molecule. It is interesting that theMPC11 line does not express I-Ad or I-Ed determinants.Either the determinant seen by our antiserum need not beexpressed on Ia-positive cells or, when a cell undergoesmalignant transformation, expression of this determinant canbe dissociated from expression of Ia antigens.

Exploiting the approach used to make antibody to theinsulin receptor (3) and to the acetylcholine receptor (17), wehave identified a molecule predominantly on macrophagesand B cells that interacts with the I-J molecule on T cells andwhose function seems crucial to suppressor T-cell activity.

This technique can be used to develop anti-idiotypicantibodies, both polyvalent and monoclonal, that will char-acterize the receptor for I-J in different mouse strains anddetermine the degree of polymorphism, if any, and thestructural relationship to classical Ia antigens. Such antibod-ies can facilitate the isolation of the gene(s) encoding this

molecule. Although the receptor is not itself classical I-A orI-E, it may nevertheless be encoded by genes within themajor histocompatibility complex. If so, this would resolvethe apparent discrepancies between the gene mapping dataobtained by classical genetic studies and the studies showingT cell I-J does not map to the major histocompatibilitycomplex (18-20). There is, however, as yet no data regardingthe genetic locus for this receptor molecule, and it and T-cellI-J may be representative of a system of molecules governingcellular cooperation in the immune system that is indepen-dent of the major histocompatibility locus.

We thank Lou Boccumini, Catherine Rapelje, and JoanneTrojnacki for their expert technical assistance. We also thank DonnaJackson and Laurie Vitagliano for their help in preparing thismanuscript. This work was supported by National Institutes ofHealth Grants AI 16166 and Al 10702 and National Cancer InstituteGrants 3POCA1330 and CA 34109. B.D. is an Established Investi-gator of the American Heart Association.

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