Antibodies to idiotypes of isologous immunoglobulins.

To determine if the immunoglobulins (Igs) capable of eliciting the formation of isologous anti-idiotypic antibodies are rare exceptions, BABL/c mice were immunized with five myeloma proteins of BALB/c origin. Anti-idiotypes were produced against all but one. The idiotype of the exception (T15) is remarkably abundant in BALB/c mice, whose unresponsiveness is probably due to tolerance. Nevertheless, prolonged immunization with T15 finally induced the formation of isologous antibodies that seemed largely to be specific for IgA proteins, especially those with k-light-chains; the reactions of a few of these isologous antisera with T15 were slightly inhibited by phosphorylcholine, suggesting that some anti-idiotypes were probably formed even to this unusually prevalent idiotype. It is likelythat under appropriate conditions almost any myeloma protein can elicit isologous anti-idiotypes.

Selective use of the VHQ52 family in functional VH to DJH rearrangements in a B precursor cell line.

AT11-2, an Abelson virus-transformed cell line has DJH complexes on both chromosomes and is able to form functional variable region genes by the joins of VH genes to the DJH complexes during culture. Therefore we examined which VH gene family was used in functional VH to DJH recombinations in AT11-2. Surprisingly, of 32 independent functional VH to DJH recombinational events in AT11-2, 31 events used the VH segments of the VHQ52 family, and the remaining one used the VH segment of the VH7183 family. Thus, we describe here the first B precursor cell line that almost selectively uses the VHQ52 family in functional VH to DJH rearrangements. The selective use of the VHQ52 family in this B precursor cell line strongly indicates nonrandom use of VH gene families, and the existence of a stage at which the VHQ52 family is preferentially used during the normal development of early pre-B cells and has important implications for understanding the ontogeny of VH repertoire development. Furthermore, this cell line should prove extremely valuable in further studies of this kind.

Direct identification of phosphotyrosine-containing proteins in some retrovirus-transformed cells by use of anti-phosphotyrosine antibody.

For direct identification of phosphotyrosine-containing proteins in lysates of various cells, phosphotyrosine (P-Tyr) was coupled to carrier proteins and anti-P-Tyr antibodies were raised in rabbits and mice. The antibodies were highly specific for P-Tyr and did not cross-react with phosphoserine or phosphothreonine. The mean association constant of rabbit anti-P-Tyr antibody to N-acetyl-P-Tyr was about four times that of rabbit anti-azobenzene phosphonate antibody. In addition, anti-P-Tyr antibody scarcely cross-reacted with the 5'-monophosphate of ribosyladenine or the 5'-monophosphate of ribosylinosine, whereas anti-azobenzene phosphonate antibody cross-reacted appreciably with these compounds. Anti-P-Tyr antibody immunoprecipitated three oncogenic gene products from cells transformed with Rous sarcoma virus, Fujinami sarcoma virus, and Abelson murine leukemia virus, respectively. The immunoprecipitates with anti-P-Tyr antibody from cells transformed with these three retroviruses all manifested tyrosine kinase activity including activity for phosphorylations of oncogene products. In addition to the proteins reported previously, the following new phosphotyrosine-containing proteins were immunoprecipitated from the respective retrovirus-transformed cells by anti-P-Tyr antibody: Mr 230,000, 74,000, and 24,000 proteins (Rous sarcoma virus); Mr 230,000, 69,000, and 24,000 proteins (Fujinami sarcoma virus); and Mr 230,000, 62,000, and 54,000 proteins (Abelson murine leukemia virus).

Isolation of chicken-bek and a related gene; identification of structural variation in the ligand-binding domains of the FGF-receptor family.

cDNA clones carrying the chicken-bek gene and a related gene were isolated. Deducing the amino acid sequence of chicken-bek allowed us to predict that it encodes for a receptor tyrosine kinase related to the fibroblast growth factor (FGF) receptor, and that the chicken-bek gene and Cek3 are closely related. However, a significant structural difference was identified between chicken-bek and Cek3 within the putative extracellular region, in such a manner that the structure of the immunoglobulin-like domain was conserved. A probe specific to the altered structure detected mRNA in the tissues as did a probe common to bek and Cek3, indicating heterogeneity in the FGF-receptor family in a novel manner. Furthermore, another bek-like gene was isolated and the expressions of its mRNA and protein product were analysed in tissues and cultured cells.

Crystallization of the complexes between M315 idiotope and its monoclonal anti-idiotopic antibody Fab fragment.

The Fab fragment of a monoclonal anti-idiotopic antibody against M315 has been isolated and its complexes with Fv and Fab' fragment of M315 have been crystallized by using poly(ethylene glycol) 6000 or ammonium sulfate. X-ray diffraction photographs showed that the crystal of the complex with Fv diffracts better than that with Fab'. The Fv-complexed crystal was shown to be tetragonal I4, with cell dimensions a = 152 A and c = 69 A, and to contain one complex molecule of about 75,000 molecular weight in the crystallographic asymmetric unit.

Maturation of the immune response to (4-hydroxy-3-nitrophenyl)-acetyl (NP) haptens in C57BL/6 mice.

Changes with time in specificity and affinity of anti-NP antibodies in C57BL/6 mice after immunization with NP22-chicken gamma-globulin (CGG) were studied by comparing the abilities of the antibodies to bind to NP3-bovine serum albumin (NP3-BSA) at pH 5 and 8. Early anti-NP antibodies (on day 14 after immunization) bound to NP3-BSA at pH 8, but not pH 5. This pH-dependence of binding was explained in terms of the low affinity of the antibody to the phenolic form of NP on the basis of results of fluorescence quenching titration of a monoclonal anti-NP antibody that showed similar specificity to that of the early anti-NP antibodies. Since NP on the CGG molecule ionized with an apparent pK of about 7.4, more than half the NP should be in the unionized (phenolic) form under the immunization conditions. However, early anti-NP antibodies bound preferentially to the ionized (phenolate) form of NP, which was a minor form at neutral pH, whereas later anti-NP antibodies showed ability to bind to both the phenolate and phenolic forms of NP. This change in specificity with time was observed on immunization with T cell-dependent (TD) antigens such as NP-CGG and NP keyhole limpet hemocyanin (KLH), but not with a T cell-independent (TI) antigen such as NP-Ficoll. The heavy (H) chains from the two monoclonal antibodies 3G6 and 3C6, which bound to the phenolate form and both the phenolate and phenolic forms, respectively, were recombined with lambda 1 chains (L3G6 and L3C6) from these antibodies as well as a lambda 1 chain (LHOPC-1) with the amino acid sequence of the germline. Ability to bind to the phenolate form of NP was recovered in all the reconstituted IgGs, while ability to bind to both the phenolate and phenolic form of NP was observed only with IgG reconstituted from H3C6 and L3C6. These results suggest that the specificity corresponding to early anti-NP antibodies were generated even by lambda 1 chains of a germline sequence, but that of late anti-NP antibodies was expressed only by an appropriate pair of H and L chains. The contribution of amino acid substitution by somatic point mutation to the change of specificity with time was discussed.

The His-probe method: effects of histidine residues introduced into the complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values.

We examined the effects of histidine residues that were artificially introduced into complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values. Using a monoclonal antibody specific for hen egg-white lysozyme and three mutant antibodies that contained a histidine residue, we measured binding constants for antibodies and lysozyme at different pH values (pH 5-8). No gross conformational changes were evident over this range of pH values, as determined by analysis of the spectra of circular dichroism. Since the charge on a histidine residue is the most likely factor that can vary over this range of pH values, differences on pH-dependent antigen-binding patterns observed between the wild-type and mutant antibodies should be due mainly to the effects of the charges on the histidine residues. The three mutant antibodies showed different and characteristic patterns of pH-dependent binding to lysozyme, which depended on the location of the artificially introduced histidine residues.

Regulation of delayed-type hypersensitivity (DTH) response to hen egg-white lysozyme (HEL).

Delayed-type hypersensitivity (DTH) can be demonstrated in A/J mice immunized with hen egg-white lysozyme (HEL) in complete Freund's adjuvant (CFA) by challenging primed animals in the ear with aqueous HEL. Normal A/J mice receiving soluble HEL derivative peptide, P-Ib, sequence 29-54 and 109-123 linked by a single S-S bond, 7 days prior to immunization with HEL showed much lower DTH response specific to the protein. The inhibition of DTH reactivity is due to active suppression and involves the generation of suppressor T-cells (Ts). Thus, the suppression induced with a single i.v. injection of P-Ib solution was able to be transferred into syngeneic recipients by the spleen cells from mice pretreated with P-Ib. These suppressor cells are T-cells, since their ability to suppress DTH is completely abrogated by treatment wit anti-Thy 1.2 and complement. Amongst HEL derivative peptides tested in the present study, only P-Ib could induce the tolerance.

A 1H NMR method for the analysis of antigen-antibody interactions: binding of a peptide fragment of lysozyme to anti-lysozyme monoclonal antibody.

A proton nuclear magnetic resonance (NMR) study is reported of the molecular structural basis of antigen-antibody interactions. An immunologically reactive proteolytic fragment corresponding to one of the antigenic regions on hen egg-white lysozyme (HEL) was used in combination with a monoclonal antibody that recognizes this site. Using spin diffusion, we prepared an antibody in which the magnetization of the antigen binding site was saturated by non-specific nuclear Overhauser effect. Under these conditions the effect of the saturation of the antibody was observed to spread over the peptide fragment through the antigen binding site. On the basis of the results obtained for the intermolecular nuclear Overhauser effect, we discuss how the peptide fragment interacts with the antibody. The side chains of aromatic residues, Trp, Tyr, and His, and of ionic residues, especially Arg, Lys, and Glu, are suggested to be important in the antigen-antibody interaction.

Specificity change of antibody to (4-hydroxy-3-nitrophenyl)acetyl haptens by somatic hypermutation.

Change in the specificity of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies (Abs) with time after immunization was studied. The early anti-NP Abs was specific to the ionized (phenolate) form of NP. The specificity changed with time and the late Abs became able to bind to the protonated (phenolic) form as well as the phenolate form of NP. The nucleotide sequences of mRNA coding for variable regions of heavy and light chains suggested that somatic hypermutation contributed to this change of the specificity.

Native and non-native conformation-specific antibodies directed to the loop region of hen egg-white lysozyme.

Two types of antibodies were differentiated in conventional guinea pig anti-hen egg-white lysozyme (HEL) antisera. The specificities of both antibodies were directed to the loop I region (mainly directed to Cys64--Cys80 loop) but the antibodies were distinct in respect of reactivities with native HEL. One type of antibody reacted with HEL and loop-peptides of HEL but not with the completely reduced and carboxymethylated form of loop-peptides (native conformation specific antibody: NC-Ab). On the other hand, the other type of antibody did not react with HEL but reacted with loop-peptides and also with the completely reduced and carboxymethylated form of loop-peptides (non-native conformation specific antibody: NNC-Ab). The percentage of NNC-Ab in loop I reactive antibody fraction from pooled guinea pig anti-HEL antisera obtained by two different immunization methods was about 25%. Since the affinities of the NNC-Ab to loop-related peptides were higher by one order of magnitude than those of the NC-Ab to the same peptides, care is necessary in evaluating antigenic determinants in native protein. The immunization of guinea pigs with Ploop I . II [sequence 57-107 (Cys64-Cys80, Cys76-Cys94)] evoked an antibody population having specificity similar to but not identical with that of the NNC-Ab type anti-loop I antibody in conventional anti-HEL antisera.

Inducibility of protein-reactive antibodies by peptide immunization: comparison of three epitope peptides of hen egg-white lysozyme.

Three epitope peptides of hen egg-white lysozyme (HEL) were tested for ability to induce antibodies reactive with native HEL. Each peptide was coupled to bovine gamma-globulin (B gamma G) and 4 rabbits were immunized with each peptide-B gamma G conjugate in complete Freund's adjuvant. The mean association constants (K0s) of HEL-reactive antibodies (HEL-R-Abs) from each immunizing group to [3H]acetyl HEL or to [3H]acetyl-peptide were measured in solution by a double antibody method. Only peptide loop I.II (sequences 57-107 containing Cys64-Cys80 and Cys76-Cys94) induced high-affinity antibodies to HEL (K0 = 2.5 x 10(6)-2.3 x 10(7) M-1) among the three epitope peptides tested. The association constants of antipeptide loop I.II to [3H]acetyl peptide loop I.II were always one to two orders of magnitude higher than those to HEL. In addition, 50 to 80% of the anti-peptide loop I.II antibodies were reactive with native HEL. The specificity of anti-peptide loop I.II was directed to a conformational feature of the peptide rather than to native HEL and reactivity of the antibody to HEL was interpreted as a kind of cross-reaction. The HEL-R-Abs from anti-Ploop I.II antisera also manifested neutralizing activities against the enzymic activity of HEL when Micrococcus luteus was used as the substrate.

Characteristics of the epitope of protein-reactive anti-peptide antibodies.

The specificity of hen egg-white lysozyme (HEL)-reactive rabbit antibodies induced by the peptide loop I.II (sequences 57-107 containing Cys64-Cys80 and Cys76-Cys94) of HEL was clarified by analyzing their cross-reactions with various avian lysozymes and their reaction with synthetic peptides (sequences 59-82) in which alanine was substituted for the amino acid at certain positions. The Arg-68 residue of HEL plays a dominant role in the binding, while Gly-71, Ser-72, Arg-73, and Pro-79 also contribute to the binding of two anti-Ploop I.II antibodies (rabbit number 125 and 126). These residues, although remote in sequence, are grouped together in the crystal structure of HEL and may form an area of contact with the antibody. Contributions by Trp-63, Ile-78, and Asn-77 to the binding of the two antibodies to HEL were excluded. These results support the idea that the anti-Ploop I.II antibodies recognize a conformational type of epitope which is similar to that of native HEL. The immunogenicity of the reduced and alkylated form of Ploop I.II was also tested, but it failed to induce an HEL-reactive antibody.

Random expression of human immunodeficiency virus-1 (HIV-1) p17 (epitopes) on the surface of the HIV-1-infected cell.

Twenty monoclonal antibodies (MAbs) were obtained by immunizing Balb/c mice with recombinant p17 (rp17) of HIV-1. Epitope specificity of each MAb was determined using six peptides that cover the entire region of p17. We found that each MAb reacts with only one of the peptides, residues 12-29, 30-52, 53-87, and 87-115 (P12-29, P30-52, P53-87, P87-115) of p17 with the exception of one MAb. Three kinds of MAbs that recognize P30-52, P87-115, and a conformational epitope, suppressed the infectivity of HIV-1 (JMH-1) when they added in the culture of MT-4 cells infected by HIV-1 within 24 h of the infection.

Suppression of the delayed-type hypersensitivity response to hen egg white lysozyme (HEL) by HEL peptides in a genetically high-responder mouse strain: evidence for requirement of the loop structure for induction of suppressor T cells.

The delayed-type hypersensitivity (DTH) response of C3H/HeN mice to hen egg white lysozyme (HEL) can be blocked by a single iv injection of a solution of HEL in buffered saline 7 days before sensitization of animals with HEL in complete Freund's adjuvant (CFA). The minimal structure of HEL required for the suppression was examined by determining the abilities of various HEL-derivative peptides to inhibit HEL-DTH. Treatment of normal mice with Ploop I X II, sequence 57-107 (Cys64-Cys80, Cys76-Cys94), or P17 (sequences 1-27 and 123-129 linked by Cys6-Cys127) 7 days before immunization with HEL resulted in marked suppression of the DTH response. This inhibition of DTH involved generation of suppressor T cells (Ts). The results suggested that two suppressor pathways are involved. These data, together with another recent finding (1) that an entirely different portion of HEL is a suppressor determinant (SD) in A/J mice, indicate that different epitopes act as SDs in different strains of mice. Of the loop region peptides tested, Plc (intact loop I joined to a linear peptide, residues 84-97) was found to be the minimum structure capable of suppressing the HEL-DTH response; loop I or II alone did not cause suppression. Activation of Ts cells by the loop peptide depended on its conformational structure; completely reduced and carboxymethylated Ploop I X II did not cause suppression.

Characterization of the combining site of mouse myeloma protein M315.

The interaction of M315 with 2,4-dinitrophenyl haptens was studied. 2,4-Dinitroaniline (DNP-NH2) showed maximum affinity to M315 at about pH 4. The pH dependence of the association constant of DNP-NH2 to M315 showed three transitions at pH 4.7, at pH 7.2, and below pH 9, respectively. Since the DNP-NH2 molecule has no charged group in this pH range, the transitions were explained in terms of amino acid residues with ionizable side chains in M315. Judging from the pK values and the effect of succinylation, these transitions were concluded to be related to ionizations of carboxyl, imidazole, and phenol groups, respectively. Measurement of the fluorescence of affinity-labeled M315 suggested that the transition at pH 4.7 reflected an equilibrium between two forms of M315 with different conformations of the combining site. The contribution of the amino acid sequence on the light (L) chain to the interaction with haptens was studied by use of antibodies (Abs) reconstituted from the heavy chain of M315 (H315) and either a homologous or a heterologous L chain. The reconstituted heterologous Ab (H315L952) showed similar pH dependence of binding to DNP-NH2 to that of the homologous Ab (H315L315). Moreover, the two Abs showed no appreciable difference in binding to DNP-haptens of different sizes. These results suggested that the difference in the amino acid sequences of L315 and L952, which originated by a somatic hypermutation, has little effect on the ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatic mutations in the variable region of the lambda 2 chain of M315 suppression of antibodies to the idiotype of M315.

BALB/c mice immunized with purified BALB/c myeloma protein M315 (alpha, lambda 2) produce anti-idiotypic antibody directed predominantly to a combinational (VH-315 + VL-315) determinant(s) of the M315 paratope (Sirisinha and Eisen, 1971; Tungkanak and Sirisinha, 1976). We examined whether the unique B cell response is influenced by pretreatment of mice with fragments or chains derived from M315 before immunization with M315. Intravenous (i.v.) injection of the Fv-315 fragment (VH-315 + VL-315) into normal BALB/c mice seven days before immunization with M315 resulted in marked suppression of anti-M315 idiotype antibodies. Studies on the structural requirement for suppression indicated that VL-315, but not VH-315, is involved. Structural comparison with a defined lambda 2 light (L) chain suggested that three contiguous amino acid residues in the third hypervariable loop of the variable (V) domain of the L chain of M315 are important for down-regulation of production of antibodies to the M315 idiotype.