The presence of a common idiotype in anti-H-2 immune sera as detected by anti-idiotype to a monoclonal anti-H-2 antibody

To study the idiotypes (Id) of the humoral response to H-2L^d antigens, xenogeneic antisera were produced to three independent monoclonal antibodies, recognizing L^d determinants H-2.64 or 65. The reactivity of each of these anti-Id was specific for the immunizing antibody, no cross-reactions among the three anti-L hybridomas being detectable. Antisera produced in BALB/c H-2^dm2 mice hyperimmunized with BALB/c tissue were examined for the presence of cross-reactive Id (Id^CR). When the ability of these anti-Id to inhibit the binding of alloantibodies to L^d splenic antigens was tested, a broadly shared Id was detected by one anti-Id (anti-23-10-1S). The shared Id represented between one-fourth and one-half of the total anti-L^d humoral response and was found in every BALB/c H-2^dm2 anti-BALB/c serum tested. The Id^CR was limited to those alloantibodies reacting with the determinant H-2.65 which corresponds with the serologic reactivity of monoclonal antibody 23-10-1S. The other two anti-Id did not detect components of the anti-L^d humoral response, even though one of these was made against monoclonal antibody 30-5-7S which also detects serologic specificity H-2.65. The detection of a Id^CR in anti-L^d responses stands in contrast to our previous failure to detect Id^CR in anti-H-2K^k responses. Among the possible reasons for this contrast are (a) the fact that 23-10-1S is an IgM antibody, whereas the anti-H-2K^k antibodies studies were IgG; (b) the chance occurrence of anti-H-2 hybridomas with dominant Id and (c) the possibility that the anti-L^d antibody repertoire may be less heterogeneous than that directed to K^k antigens.     

Inhibition of killer-target cell interaction by monoclonal anti-H-2 antibodies.

The sites on target cells with which cytotoxic T lymphocytes interact were characterized using three different monoclonal BALB/c anti-CBA antibodies derived from plasma cell hybrids. The antibodies all reacted with H-2K^k and appeared to recognize public specificities H-2.5,11 and 25, respectively. Allogeneic killing directed at products of H-2K^k was inhibited by all three antibodies, irrespective of the H-2 haplotype of the responder; cytotoxicity directed at products of another allele, H-2K^d, or of the H-2 D region on the same target cell was not affected. The antibodies did not inhibit killer cells carrying H-2K^k. Cytotoxic reactions against minor histocompatibility antigens, including the male-specific antigen H-Y, were also blocked by all three monoclonal antibodies when restricted by H-2K^k, but not when restricted by H-2D on the same target cell. Cytotoxic T lymphocytes thus appear to interact with their target via the same, serologically defined H-2K^k molecule which carries public specificities, whether they recognize it as an alloantigen or as self. This argues against the existence of separate H-2 K-encoded molecules recognized by killer cells only and against H-2 specific modifications of minor histocompatibility antigens as the basis of H-2 restriction. One of the antibodies, 27 R9, which reacted with H-2K^k and H-2′ and was thought to recognize specificity H-2.25, showed a weak cytotoxic reaction but bound with a high titer to H-2 D^k, a reaction that has not previously been described. This antibody selectively and with a very high titer inhibited male-specific cytotoxicity restricted by H-2D^k, but did not significantly interfere with allogeneic killing against products of the H-2 D^k region nor apparently with H-2D^k- restricted cytotoxicity specific for other minor antigens. The results suggest the existence of at least two different restriction elements controlled by the H-2D^k region.     

Restricted blocking of cytotoxic T-cell function by anti-H-2K/D antibodies

Antibodies specific for H-2K and H-2D, the murine major histocompatibility complex (MHC)-encoded class I antigens, can block cytotoxic T (Tc)-cell function. Antibodies specific for the Tc cell and not the target cell have been used to map inhibition to the effector cell, suggesting a role for class I antigens in Tc-cell function. These antibody effects have been demonstrated for both alloreactive and MHC-restricted Tc cells, but inhibition has only been revealed by measuring cytotoxicity in a short-term assay. Using the neutral red assay for cytotoxicity, blocking effects evident after a 1.5-hr assay were lost by 2.5 hr. For some Tc-cell responses, only anti-H-2K antibodies have been found to be inhibitory, despite evidence of the expression of both H-2K and H-2D molecules on these cells. Some Tc-cell populations can be blocked by antibodies specific for both the H-2K and H-2D molecules. B10.A(4R) anti-Sendai Tc cells can be inhibited by anti-H-2Kk antibodies, but five different anti-H-2Db antibodies have been ineffective inhibitors. In contrast, B10.A(4R) anti-ectromelia Tc cells can be inhibited very effectively by each of these anti-H-2Db antibodies, as well as by anti-H-2Kk antibodies. Anti-H-2 antibodies also inhibit the function of cloned alloreactive Tc-cell lines such that the inhibitory capacity of antibodies specific for K versus D determinants appears to be consistent and specific for each Tc-cell line. A long-term Tc-cell clone, AR1, has been inhibited specifically by anti-H-2Kb and not anti-H-2Db antibodies, suggesting a clonally 'restricted' phenomenon.     

Production of anti-H-2 antibodies in thymectomized mice

Neonatally and adult thymectomized (Tx) mice of strains C57BL/10Sn and A. BY (H-2^b) were immunized at the age of 5 – 8 weeks with lethally irradiated spleen and lymph node cells from adult B 10. A and A (H-2^a) donors, respectively. The titer of H-2 hemagglutinins and cytotoxins was determined at different intervals after immunization. Both untreated antibodies and antibodies reduced with 2-mercaptoethanol (2-Me) were tested. The non-Tx control mice responded with production of both 2-Me-sensitive and 2-Me-resistant H-2 antibodies. In the Tx mice, only 2-Me-sensitive H-2 antibodies were detected. Thus, the anti-H-2 IgM response appears to be thymus-independent and the anti-H-2 IgG response thymus-dependent.     

Comparative study of idiotypes on monoclonal antibodies derived from patients with lupus and leprosy and from normal individuals

A collaborative study was performed to compare the expression of a series of idiotypes defined on human anti-DNA and other autoantibodies. Three panels of human monoclonal antibodies were tested: eight derived from patients with systemic lupus erythematosus (SLE); 13 from an individual with lepromatous leprosy; and 38 from normal subjects. The following rabbit anti-idiotype sera were used: one (RId16/6) raised against the lupus-derived monoclonal anti-DNA antibody 16/6, four (RId8E7, RId4G7, RId4D5 and RIdTH9) against leprosy-derived monoclonal antibodies of various specificities, and one (anti-4.6.3) against a normal-derived anti-DNA monoclonal (KIM 4.6). In addition, two other anti-idiotypes were used--one a murine monoclonal (3I), the other a rabbit polyclonal (RIdD)--which had been raised against polyclonal anti-DNA antibodies from lupus serum. Further experiments were performed with immunoabsorbed fractions of RId8E7. Direct-binding and competition assays were used. All of the anti-idiotypes produced different patterns of positivity among the three panels of human monoclonal antibodies, with the exception of RId8E7 and RId4G7, which showed considerable concordance. There was a tendency towards anti-idiotypes being disease- or group-specific: thus anti-4.6.3 failed to bind to any of the lupus or leprosy-derived monoclonals, while RId16/6 and RId8E7 bound most strongly to the lupus- and leprosy-derived antibodies respectively. KIM 4.6 itself was bound only weakly by RId16/6, while 16/6 was not recognized by anti-4.6.3; 16/6 was, however, bound by 3I, while KIM 4.6 was not. 3I bound to several other monoclonals but RIdD, which has been shown to be specific for the anti-DNA fraction of lupus serum, did not bind to any of them. These results indicate that the majority of these anti-idiotype preparations recognize largely separate sets of determinants. The monoclonal antibodies which bind to DNA may be only partly representative of anti-DNA antibodies in the serum of lupus patients.     

Studies of Monoclonal Antibodies Specific for Major Histocompatibility Complex Products of the Rat

Polyclonal syngeneic, allogeneic, and xenogeneic and monoclonal syngeneic anti-anti-idiotypic antibodies have been produced against previously described monoclonal anti-idiotypic antibodies with specificity for monoclonal RT1 alloantigen-specific antibodies. The anti-anti-idiotypes could again be shown to be highly specific for the monoclonal anti-idiotype used for the induction of the anti-anti-idiotypic antibodies and to carry the same, or a very similar, idiotype as the original monoclonal idiotypic antibody used to induce the monoclonal anti-idiotypic. Among the 30 syngeneic and allogeneic and the five xenogeneic polyclonal anti-anti-idiotypic antisera and the three monoclonal anti-anti-idiotypes, only one polyclonal antiserum showed binding capacity to the corresponding RT1-encoded antigenic determinants on spleen cells. All the other antibodies were idiotypic but not antigen binding.     

Idiotypic analysis of monoclonal anti-fluorescyl antibodies: Localization and characterization of idiotypic determinants

Nine monoclonal IgG anti-fluorescyl antibodies, which exhibit diverse affinities for fluorescein (Fl) (K_a values ranging from 5 × 10⁶ to 10¹⁰ M⁻) were analyzed idiotypically. Each of the BALB/c hybridoma proteins (γ, κ) exhibited unique idiotypic determinants although two clones (6-10-6 and 20-19-1) were partially (15–20%) cross-reactive. Of two other clones (4-6-9 and 4-6-10) derived from the same cell line, 4-6-9 contained γl heavy (H) chains and 4-6-10 contained both γl and γ2b H-chains. In addition, 4-6-9 shared idiotypic determinants with 4-6-10 although the latter also displayed unique idiotypic specificities. Collectively, the nine clones demonstrated structural diversity analogous to previous studies which denned binding mechanism diversity. The location of determinants recognized by antiidiotype reagents directed against each of the monoclonal antibodies was examined by binding inhibition with free Fl and fluorescein-BSA (Fl-BSA). All clones contained determinants both within the active site (Fl-inhibitable) and in close proximity to it (Fl-BSA-inhibitable), although the relative proportions of these determinants varied among the clones. Inhibitor concns required for 50% inhibition varied independently of ligand binding affinity, and therefore were more likely influenced by the heterogeneous nature and affinity of the anti-idiotype reagents toward the individual determinants. Idiotypic analysis of H- and light (L) chains derived from five monoclonal antibodies of diverse affinities was performed. Fl binding and expression of idiotypic determinants by all clones required both H- and L-chains. Restoration of the idiotye by reassociated H- and L-chains was found to be highly restricted to homologous H- and L-chain pairs, as heterologous combinations did not result in the expression of either parental idiotype. The latter was true whether the heterologous pairs were derived from clones of the same isotype or the heterologous combination associated to form an intact molecule with greater affinity than the parental H- and L-chain combination. Heterologous recombinants from the two clones (6-10-6 and 20-19-1) exhibiting partial idiotypic cross-reactivity were able to restore a fraction (˜25%) of their idiotypic determinants. Results demonstrated the extensive conformational requirements of ligand binding and idiotype expression and indicated that a high degree of specificity in the V_H- and V_L-chain interaction must exist for the expression of these idiotypes.     

Identification of molecules detected by three different anti-H-2Kk monoclonal antibodies.

Three different monoclonal antibodies specific for murine Class I H-2Kk-encoded determinants have been found to bind to molecules other than the classical 45 Kd Class I glycoprotein. These results were obtained after electrophoresis of a detergent lysate of spleen cells, and extraction of molecules present in multiple gel fractions. The antigenic activity in each gel fraction was assessed after removal of detergent by capacity to inhibit the binding of each of these antibodies to Ig-capped spleen cells in a rosetting assay. Only the H100-27R9 antibody was inhibited by an extract representing 45-50 Kd proteins resembling Class I molecules. This antibody also bound material extracted from a higher 65-70 Kd fraction of the gel. The binding of the 11-4.1 and H100-30R3 antibodies was inhibited by unique molecules in the molecular weight range of less than 20 Kd. The inhibitory material was not sensitive to pronase but was sensitive to glycosidases. This material could be absorbed by each of the H100-30R3 and 11-4.1 antibodies but not by H100-27R9. All data are consistent with unique, low molecular weight carriers of carbohydrate-defined epitopes which can bind monoclonal antibodies having specificity for H-2K-encoded gene products. It is predicted that these are glycolipids, but it is not yet known whether they are cell-surface or cytoplasmic molecules.     

Separation of anti-Ia (I-region associated antigens) from anti-H-2 antibodies in complex sera, by absorption on blood platelets. description of three new Ia specificities.

H-2 antigens are expressed in substantial amounts of murine blood platelets (for H-2 antigenic content 1 lymphocyte approximately 50 platelets) whereas Ia antigens are probably not expressed at all (minimal Ia antigenic content more than 35 times lower than for H-2). This property of blood platelets makes them very useful for the selective absorption of anti-H-2 antibodies from complex sera and for the preparation of specific anti-Ia antibodies from such sera. In 20 sera produced against the complete H-2 complex, 12 sera contained anti-Ia antibodies beside the expected anti-H-2 antibodies. In two sera, separation of the anti-Ia antibodies was easily obtained by absorption of the anti-H--2 antibodies on platelets. The analysis of one serum (C3H.Q X B10.D2) anti-C3H [(q X d) anti-k] showed that, in addition to be expected anti-H--2.23 and anti-Ia.2 antibodies, it contained at least three other Ia antibodies, separable by absorption on lymphocytes, which recognized three antigens--Ia.17, determined by the haplotypes k, f, s, r, j; Ia.18, determined by the haplotypes k, f, s; and Ia.19 determined by the haplotypes k and r. The genes are located in the I--A and/or I--B subregions of the H--2 complex.     

Induction of an immune response to the porin of Haemophilus influenzae type b by monoclonal anti-idiotypic antibodies.

Monoclonal anti-idiotypes were generated against monoclonal antibody (mAb) Hb-2 which recognized a highly conserved epitope on the outer membrane porin protein from Haemophilus influenzae type b (Hib). Four hybridomas reacting with F(ab') 2 fragments of Hb-2 were selected and characterized. Inhibition studies using syngeneic anti-anti-idiotypic antisera suggested that at least three different antigenic determinants on Hb-2 were recognized by these monoclonal anti-idiotypes. The binding of each anti-idiotype to Hb-2 was inhibited by Hb-2 whereas the reaction was not affected by any other anti-Hib mAb. Complete inhibition of the binding of anti-idiotype to the idiotype could be achieved with 10 micrograms of total outer membrane protein (OMP) from Hib suggesting that the anti-idiotypes might be directed against paratope-associated idiotypes. Outer membrane antigens not recognized by mAb Hb-2 did not inhibit the reaction. Furthermore, the pre-incubation of Hb-2 with each anti-idiotype specifically prevented the reaction of Hb-2 with its antigen. Antibodies with specificity for the porin were generated in guinea pigs immunized with anti-idiotypes AHb-22 and AHb-23. This study indicates that these particular monoclonal anti-idiotypes may be used as an antigen substitute for the porin of Hib in a xenogeneic species.     

Aberrant H-2-like allospecificities on K36.16 thymoma. Studies by radiobinding and immunoprecipitation with anti-H-2 monoclonal antibodies

The K36.16 thymoma expresses an H-2Dd-like allospecificity as detected by the 34-5-8 monoclonal antibody directed against H-2Dd using immunoprecipitation techniques. This monoclonal antibody gives a negative result by radio-immune assay but other anti-Dd antibodies react positively with this tumour. These results confirm and extend previous observations and are attributed to probable gene-conversion events possibly involving a donor gene from the Qa/Tla region, the products of which cross-react with Dd.     

Idiotypic relationship of anti-Ia.2 antibodies

Alloantiserum and hybridoma-derived anti-Ia antibodies were investigated for their idiotypic similarity using 4 monoclonal anti-idiotypes raised against monoclonal anti-Ia. With one anti-idiotype, idiotypic cross-reactivity could be found between three independently derived monoclonal BALB/c anti-Ia antibodies, all of which were directed against the same serological determinant, Ia.2. The respective idiotype, tentatively designated la.2 idiotope, could also be demonstrated on anti-Ia.2 antibodies in BALB/c alloantisera, but not on anti-Ia against other specificities. The expression of the Ia.2 idiotope is linked to the Igh allotype; the use of Ig-recombinant mice allowed us to map the Igh-Ia.2 locus to the left of the T1S marker in the Igha V region.     

Fine specificity of antibodies produced in rhesus monkeys following in vivo treatment with anti-T cell murine monoclonal antibodies

The immune response of 23 rhesus monkeys against different murine monoclonal antibodies (mAb) administered in vivo as immunosuppressive agents has been analyzed. Seven mAb specific for either helper-inducer (CD4 molecules) or cytotoxic/suppressor (CD8 molecules) T cells, that cross-react with monkey lymphocytes, were administered i.v. for 10 consecutive days in rhesus monkeys. Nineteen of the animals were recipients of a skin or renal allotransplant. Nineteen out of the 23 monkeys developed a significant immune response against the injected monoclonal. This response was restricted in its specificity since unrelated murine monoclonals were not recognized by the monkeys' anti-monoclonal immunoglobulins. Fine analysis of the monkeys' sera revealed that the antibodies produced against the xenogeneic proteins selectively exhibited two major specificities i.e., anti-isotypic and anti-idiotypic. On a practical basis, these results suggest that an animal already immunized against a given mAb should still be sensitive to the therapeutic effect of another monoclonal sharing the same specificity but different idiotype.     

Monoclonal antibody detection of two classes of H-2Kk molecules

Studies described in this paper indicate that two anti-H-2K^k monoclonal antibodies, namely 27R9 (H-2.25) and 30R3 (H-2.5) recognize different H-2K^k molecules on the surface of lymphocytes. Initial experiments in support of this conclusion were cocapping experiments which showed mutual exclusiveness between H-2K^k molecules which bind either of the two monoclonal antibodies 27R9 (H-2.25) or 30R3 (H-2.5) whereas conventional anti-H-2K^k (H-2.23) alloantiserum binds to both types of H-2 molecules. This result was confirmed by experiments using solubilized H-2 antigen preparations to inhibit antibody binding to spleen cells. Preabsorption of the preparation with one monoclonal antibody did not remove its inhibitory activity for the other monoclonal antibody, and only partially removed its inhibitory activity for the conventional anti-H-2K^k serum. These results suggest that at least two antigenically distinct H-2K^k molecules arc controlled by the H-2K region. Subsequent blocking studies have indicated that the two different molecules are associated, to some extent, in the cell membrane. Furthermore, in ¹²⁵I-protein A radioimmunoassay.each monoclonal antibody was found to bind to only half of the estimated total number of H-2K^k molecules recognized by conventional anti-H-2K^k sera. Several interpretations for the existence of the two classes of H-2K^k molecules are discussed.     

Induction of H-2-specific antibodies by injections of syngeneic Sendai virus-coated cells

The capacity of the B cell immunoglobulin receptor to recognize complexes of Sendai viral and H-2b antigens was investigated by studying the antibody response to injections of syngeneic Sendai virus-coated (SV+) spleen cells in C57BL/6 (B6) mice. Almost all mice produced alloreactive anti-H2 lymphocytotoxic antibodies. In contrast, such antibodies were found very exceptionally in mice injected with normal (SV-) cells or with Sendai virus (SV) only. The reaction pattern of the cytotoxic antibodies induced was variable and ranged from almost anti-private to widely cross-reactive serotypes. The results of reactions on H-2-congenic, -recombinant and -mutant mouse strains, and of capping and immunoprecipitation experiments showed that the cytotoxic antibodies were directed against H-2 class I molecules. The anti-H-2 antibodies exhibited enhanced binding for SV+ target cells, but absorption experiments showed that this was not the result of cross-reactions with cell surface Sendai viral determinants or with a molecular complex of H-2 plus SV. This conclusion was supported by the observation that syngeneic SV+ cells were not the predominant targets for the induced lymphocytotoxic antibodies. Our results do not support the existence of MHC-restricted antiviral antibodies, but show the induction of anti-class I H-2 alloantibodies by injections with syngeneic SV-coated cells. We present a model for regular induction of anti-H-2 antibodies without intentional alloimmunization.     

Public H-2 specificities are target determinants for alloreactive cytotoxic T lymphocytes

The specificity of the cross-killing exerted by cytotoxic T lymphocytes (CTL's) generated against H-2 region products was investigated in a 51Cr release assay on a panel of target cells from a number of different H-2 haplotypes. Their pattern of reaction shows that: (1) The target cells, expressing public specificities (H-2.28, H-2.1, H-2.3 or H-2.8) which should, theoretically, be recognized by the CTL's were killed, while those expressing no public specificities, recognized according to the H-2 chart by the CTL's were not killed. (2) The CTL's generated against the H-2.28 specificity expressed on the D region products cross react with target cells expressing this specificity in their K region products and vice versa. The same phenomenon was observed with the H-2.1 specificity. These results provide evidence that public specificities are targets for CTL's. Antiserum reacting against the public specificity recognized by the CTL's was found to block the cross-killing, however, to the same extent as antisera directed against any specificity (private or public) expressed on the same molecule as the target determinant. Finally both the inhibition studies using anti-H-2 antisera and the direct cytotoxic assays showed that the public specificities of the H-2.28 family carried by the H-2.D and H-2.L molecules were recognized by different subpopulations of CTL's.     

Private specificity of H-2Ldx molecule detected serologically by a surface antigen redistribution method (capping)

The presence of H-2·1 positive-H-2·63 negative molecules, H-2L^dx, in the products of the ^dx region was originally detected in H-2^dx inbred strains GRS/A and LIS/A (Snoek et al. 1979).
This study confirms the existence of H-2L^dx molecules in 2 congeneic strains with a H-2^dx haplotype on C3H (C3H·LG strain) and B10 (B10·GR strain) background. Besides anti-H-2·1-like antibodies present in anti-D^dx allo-antiserum, monoclonal antibody d anti- k (H100-5/28, H-2·m3) was shown to react with H-2L^dx molecule. Furthermore, evidence is shown that H-2L^dx molecule has a unique specificity, which is detected serologically in a capping experiment.     

Anti-idiotypic antibody with potential use as an Eimeria tenella sporozoite antigen surrogate for vaccination of chickens against coccidiosis.

Anti-idiotypic antibodies were raised in rabbits against four monoclonal antibodies with specificity for the surface antigenic determinants of Eimeria tenella sporozoites, the infective stage of the coccidial parasite. Two of the monoclonal antibodies (1073 and 15-1) transferred passive protection in chickens against E. tenella infection. The polyclonal anti-idiotype antibody preparations against protective monoclonal antibodies contained specificities for the paratope-associated idiotypes of these monoclonal antibodies, as assessed by the competitive inhibition of binding of the homologous idiotype-anti-idiotype by the sporozoite antigen. Competitive inhibition of binding of homologous idiotype-anti-idiotype by the parasite antigen was not observed when the anti-idiotype antibody preparations against monoclonal antibodies 1546 and 1096 were tested. The anti-idiotype 1073 and 15-1 antibodies functioned as surrogate antigens in vivo when used for vaccination of young chickens, as evidenced by the induction of partial protective immunity against subsequent challenge infection with virulent parasites and induction of antisporozoite antibodies. These data clearly support the view that anti-idiotypic antibodies raised against the paratope-associated idiotypes can mimic pathogen antigens and therefore can provide a possible alternative approach for the vaccination of chickens against coccidiosis.     

Intrastrain recurrent idiotypes among anti-DNA antibodies of (NZB X NZW)F1 hybrid mice

Immunization of NZB and A/J mice against an anti-DNA hybridoma antibody (F227) derived from (NZB x NZW)F₁ (B/W) mice allowed the preparation of two anti-idiotype antisera. These two reagents were shown to recognize different idiotopes of the F227 monoclonal antibody. NZB anti-idiotypic antibodies recognized non-ligand-modifiable idiotypic determinants. These idiotopes were private or present at undetectable level in BW mouse sera since it was found that only two of the 24 B/W mouse sera tested were recognized by these antibodies. Conversely, A/J anti-idiotypic antibodies recognized partially ligand-modifiable idiotopes which were found in all B/W mouse sera tested. These results demonstrate that anti-DNA antibodies share similar idiotypic specificities and suggest that these autoantibodies occur as families of structurally related proteins.