Proc. Nadl. Acad. Sci. USA Vol. 82, pp. 4788-4792, July 1985 Immunology Gene repertoire of the anti-poly(Glu60Ala30Tyr10) (GAT) immune response: Comparison of VH, V, and D regions used by anti-GAT antibodies and monoclonal antibodies produced after anti-idiotypic immunization (idlotope structure and expression/mRNA sequences) CLAUDE ROTH*, Jose ROCCA-SERRAt, GERARD SOMMO*, MICHEL FOUGEREAUt, AND JACQUES THPZE* *Unitd d'Immunogdn6tique Cellulaire, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France; and tCentre d'Immunologie de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France Communicated by Jacques Oudin, February 25, 1985 ABSTRACT Eight monoclonal antibodies were selected ported. A limited number of sequences for the heavy chain from BALB/c mice immunized with two different monoclonal variable region (VH) (12, 16) and K light chain variable region anti-idiotypic antibodies recognizing two discrete idiotopes (VK) (17-19) are used. At the idiotypic level the anti-GAT characteristic of the anti-poly(GluMAla"Tyrl) (GAT) anti- response is characterized by the expression of the public body response. These monoclonal antibodies were previously idiotypic specificity p.GAT defined by a xenogeneic antise- classified as AbN (anti-GAT-like) and Ab3 (anti-anti-idiotype) rum (20, 21) and expressed in all HP-GAT (15). More on the basis of expression of the public idiotypic specificity recently, two discrete idiotopes characteristic of the anti- (p.GAT) studied with a xenogeneic serum, anti-GAT activity, GAT responses have been identified (22-25). and expression of various public idiotopes. All the heavy chain In this paper we report the nucleotide sequences of two variable region (VH) sequences from Ab are nearly identical to sets of monoclonal antibodies obtained from BALB/c mice the VH sequences of Ab1 anti-GAT monoclonal antibodies. The immunized with two different monoclonal anti-idiotypic anti- same type of results has been found with the Ab' it light chain bodies (HP-Id) recognizing the two idiotopes previously variable region (VK) sequences. Confirming our classification, defined (25). The VH, VK, and diversity (D) sequences ofthese Ab3 VH and VK sequences were found to be completely different monoclonals are compared to the corresponding sequences from Ab1 VH and VK sequences. The Ab; diversity (D ) regions previously reported for HP-GAT. The possible contribution are different from one another and different from the D regions of VH, VK, and D segments to idiotope expression and found on monoclonal anti-GAT antibodies but function simi- antibody activity is discussed. larly. These D regions are not simply derived from already described D genes. Finally, our results suggest that in the MATERIALS AND METHODS anti-GAT response VH and VK sequences are mainly responsible Detection ofIdiotypic Specificities and ofAnti-GAT Activity. for idiotype expression. The detection of the p.GAT specificity and the percent maximal inhibition were calculated as described (20, 21). For Antigen immunization leads to the production of specific the detection of the two idiotopes identified in the GAT antibodies (Ab1), which can in turn be used as immunogens response by HP-Id20 and HP-Id22 a solid-phase radio- to produce anti-idiotypic reagents (1-3). Immunization with immunoassay (RIA) was used (25). The idiotope binding these reagents (Ab2) can also elicit an immune response (4, 5). inhibitory capacity (IBIC) ofeach AbN was determined. IBIC This response consists of immunoglobulins idiotypically was expressed as the percentage ofthe slope ofthe inhibition related to Ab1 with or without antibody activity (Ab;) and of curves, taking the slope obtained with HP-GAT G5 as anti-anti-idiotypic antibodies (Ab3). Structural analysis ofthe standard (IBIC = 100o). genes involved at the different steps of this "idiotypic The GAT-binding assay has been described (25). Results cascade" is a major tool in elucidating the origin ofthe B-cell are expressed as the reciprocal ofthe dilution of supernatant repertoire (6). More precisely, structural studies of antibod- giving 50% of maximal binding. Ig present in each fluid was ies obtained after anti-idiotypic (anti-Id) immunization measured and adjusted to 1 ,ug/ml before the assay. For the should unequivocally establish the genetic origin of Ab3 and GAT-binding inhibition assay, radiolabeled G5Bb2-2 (10 ng AbN and document the relationship between AbN and Ab1. per well) was used to measure binding to GAT and various Various systems have been analyzed at the Ab1 level, using dilutions of ascitic fluids were tested for their inhibitory either myelomas or hybridomas specific for well-defined capacity. Results are expressed as percent of maximal antigens. The immune response against arsonate (Ars) (7), inhibition obtained. nitrophenyl acetyl (NP) (8), phosphocholine (PC) (9), oxa- RNA Purification and Nucleotide Sequencing. The heavy- zolone (ox) (10), dextran (dex) (11), and poly(Glu6OAla30- and light-chain-encoding fractions of poly(A)+ RNAs were Tyr1') (GAT) (12) have been particularly studied. More prepared as described (12, 26). Nucleotide sequences were recently several reports dealing with the study ofAbN and Ab3 have been published (13, 14). Abbreviations: Abl, specific antibody; Ab2, anti-idiotypic antibody; In the immune response against GAT different monoclonal Ab3, anti-anti-idiotypic antibody; AbN, antibody-like immunoglob- ulin generated after anti-idiotypic immunization; Id, idiotype; GAT, anti-GAT antibodies (HP-GAT; HP, hybridoma product) random terpolymer poly(Glu6WAla3(TyrlO); VH, variable region of have been characterized (15). Their partial amino acid se- immunoglobuilin heavy chain; V,,, variable region ofinmmunoglobulin quences and complete nucleotide sequences have been re- K light chain; D, diversity region; J, joining region; HP, hybridoma product (monoclonal antibody); HP-Id, monoclonal anti-idiotypic antibody against public idiotopes; HP-GAT, monoclonal anti-GAT The publication costs of this article were defrayed in part by page charge antibody; IBIC, idiotope binding inhibitory capacity; G5 or G5Bb2.2, payment. This article must therefore be hereby marked "advertisement" HP-GAT used as reference;p.GAT, public idiotypic specificity in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. anti-GAT response; KLH, keyhole limpet hemocyanin. 4788 Downloaded by guest on September 26, 2021 Immunology: Roth et aL Proc. NatL Acad Sci USA 82 (1985) 4789 determined according to the modification of the original same characteristic idiotopes as all the BALB/c HP-GATs dideoxy method using the mRNA as template (27). Synthetic studied (22). As expected, Ab3 inhibited only the binding of oligonucleotides, d(CAGGGGCCAGTGG) specific for the HP-Id22 to G5 (25). constant region of the 4yl or y2a chain, d(GCTCTCGCAG- Primary Structure of VH Regions ofAb; and Ab3 Monoclonal GAGAC) specific for the constant region of the ,u chain, or Antibody. The nucleotide sequence of seven heavy chain d(TGGATGGTGGGAAGATG) specific for the constant re- regions, encompassing most of the VH, the entire D, and the gion of the K light chain, were synthesized according to Gait beginning of the joining (JH) segments was determined (Fig. and Sheppard (28) and used as primers. Each sequence was 1). Within the limits of the analysis (four residues were determined with 2 ,ug of enriched mRNA and 10 ng of undetermined), each of the five HPs has exactly the same VH oligonucleotide primer. The concentrations of nucleotides sequence as monoclonal anti-GAT G5. One (20.11) has only (unlabeled and labeled) and dideoxy analogs have already four substitutions, at position 56, 59, 67, and 68. These four been reported (12). To minimize possible ambiguities in changes do not lead to a net charge difference between the sequence determination on gels (27) labeling with each of the molecules (Gly -* Val, Lys Arg, Lys Arg, and Ala a-32P-labeled deoxynucleotides (dCTP, dATP, and dGTP) Gly). No silent substitution was identified. was performed in separate experiments and each was re- While Ab1 anti-GAT D regions usually code for five amino peated at least twice. acids, the D regions of AbN are very heterogeneous. None of these D segments is identical to D segments of monoclonal RESULTS anti-GAT antibodies, which were found to derive either from Characteristics of Monoclonal Antibodies Derived from DSP-2 or from DFL-16 (12). The D-J border is very hetero- HP-Id-Immunized BALB/c Mice. From BALB/c mice im- geneous, and two AbN D segments are very short and encode munized with two different HP-Ids coupled to keyhole limpet only three amino acids. The seven JH segments sequenced hemocyanin (KLH), two fusion experiments were performed are JH4, which is also used by most monoclonal anti-GATs and eight hybridomas were studied. Three hybridomas were (G5, G8Ca1 7, and G7Ab2-9). At the D-VH borders HP 20.33 isolated from animals immunized with HP-Id20-KLH and five has lost one triplet, while in all the other Ab's there is a silent hybridomas were from animals stimulated with HP-Id22- substitution. KLH. The characteristics ofthe corresponding HP have been HP 22.134 uses a completely different VH chain; this is determined (25) and are summarized in Table 1. consistent with the fact that it has been identified as an Ab3. The expression of the p.GAT idiotypic specificity charac- However, it belongs to the same VH subgroup as the AbN (29) teristic of the anti-GAT antibody response was measured on and also uses JH-4. these HPs by using the xenogeneic polyclonal antisera Primary Structure of Ab; and Ab3 V,, Regions. Three types previously defined (20). Seven of these HPs were found to of sequences were already defined in the VK involved in the inhibit the binding of radiolabeled monoclonal anti-GAT anti-GAT response. One was identified on HP-GAT G5 and antibody G5, which was taken as reference. One HP (22.134) on several other monoclonal anti-GAT antibodies derived was found not to inhibit in this assay.