Genetic control of the immune response to poly(Glu52Lys33Tyr15) in neonatally thymectomized high and low responder rats.

The immune response to poly (Glu52Lys33Tyr15) is under polygenic control and linked to the major histocompatibility complex of the rat. Aggregation of this antigen with methylated bovine serum albumin (MeBSA) eliminates the expression of genetic control by increasing the response of low responders and decreasing that of high responders. Humoral and cellular aspects of the immune response to both unaggregated and aggregated poly (Glu52Lys33Tyr15) were investigated in neonatally thymectomized high-responder ACI and low-responder F344 rats. T cells are necessary for responses to unaggregated poly (Glu52Lys33Tyr15) since thymectomy significantly decreased numbers of antibody-forming cells and serum antibody levels, and delayed hypersensitivity responses and antigen-induced in vitro proliferation. However, thymectomy had no significant effect on these parameters of immune responsiveness in either ACI or F344 rats immunized with poly (Glu52Lys33Tyr15)/MeBSA. Aggregation also increased IgG production and delayed hypersensitivity and antibody affinity in low responders.     

Fine specificity and idiotypic expression of monoclonal antibodies directed against poly(Tyr,Glu)-poly(DLAla)--poly(Lys) and its ordered analogue (Tyr-Tyr-Glu-Glu)-poly(DLAla)--poly(Lys).

In order to study the repertoire of poly(Tyr,Glu)-poly(DLAla)--poly(Lys) [(T,G)-A--L] specific antibodies, monoclonal antibodies were prepared by fusing myeloma cells with spleen cells from C3H.SW mice immunized with (T,G)-A--L and boosted with (Tyr-Tyr-Glu-Glu)-poly(DLAla)--poly(Lys)](T-T-G-G)-A--L]. Eleven clones which secreted homogeneous antibodies were obtained. In general, two families of monoclonal antibodies were detected: those which bind exclusively (T-T-G-G)-A--L and those which bind both (T-T-G-G)-A--L and (T,G)-A--L. Analysis for idiotypic expression revealed that only two antibodies (clones no. 103 and 160), which were found to be similar in their fine specificity, cross-reacted with antibodies against the major idiotypes of (T,G)A--L specific antibodies. Guinea-pig antibodies against clone no. 160 reacted with the polyclonal (T,G)-A--L specific antibodies, whereas antibodies against 103 monoclonal antibodies did not react with C3H.SW anti-(T,G)-A--L antibodies, but did cross-react with four other monoclonal antibodies. It appears that the idiotypic determinants expressed on polyclonal (T,G)-A--L specific antibodies are heterogeneous, and consist of at least two serologically different idiotypes detected by clones no. 103 and 160.     

Genetic regulation of delayed-type hypersensitivity responses to poly (Tyr,Glu)-poly(DLAla)--poly(Lys): expression of the genetic defect in the induction and manifestation phases in H-2s and H-2f mice.

The genetic defect of H-2s and H-2s non-responder mouse strains in both the induction and manifestation phases of delayed-type hypersensitivity (DTH) responses to poly(LTyr,LGlu)-poly(DLAla)--poly(LLys)[(T,G)-A--L] was analysed. Utilizing an in vitro system to activate DTH effector T cells, we observed that non-adherent T cells of (H-2f X H-2b) F1 or (H-2s X H-2b)F1 responder mice, could not be activated on antigen bearing adherent cells of H-2f or H-2s haplotypes. On the other hand, these T cells were effectively sensitized on adherent cells derived from either F1 or parental (H-2b) responder mice. These results indicate that in these mouse strains the genetic defect, in the induction phase of DTH, is expressed at the level of the antigen presenting cell. In subsequent experiments, we were able to "correct' the non-responsiveness of H-2s recipients by transfer of educated and irradiated (H-2s X H-2b)F1 T cells together with normal F1 adherent cells. Normal non-adherent and nylon wool enriched T cells failed to restore these responses. Similarly, antigen-pulsed F1 irradiated peritoneal exudate cells could stimulate DTH responses in SJL recipients of (SJL X C57BL/6)F1 (T,G)-A--L educated cells. The genetic defect of H-2s mice in the manifestation phase of the DTH reaction is thus also expressed on the antigen presenting cell.     

NH2-terminal amino-acid sequences of poly (Glu60, Ala30, Tyr10) (GAT) and poly (Glu60, Ala40) (GA) monoclonal antibody heavy chains

The heavy-chain NH₂-terminal sequences of five IgM, kappa monoclonal anti-GAT4 antibodies, and five IgG, kappa anti-GA antibodies (all derived from immune DBA/2 mice), have been determined. These monoclonal hybridoma antibodies were previously characterized with respect to their idiotypic properties. Extensive identity, covering possibly up to residue 33, was observed between the five IgM, kappa anti-GAT antibodies, whether they expressed the cross-reactive idiotypic specificities (CGAT) or not. One anti-GAT γ₁-chain derived from (B6 × D2)F1 mice was blocked. Among the five CGAT-negative, monoclonal anti-GA antibodies from DBA/2 mice, strong homologies were observed between the four heavy-chain amino-acid sequences of the antibodies expressing the cross-reactive GA-1 idiotypic specificities. These NH₂-terminal V_H sequences differed from each other and from those of the anti-GAT monoclonal heavy chains from the DBA/2 strain by only one amino acid, on average. Conversely, of the three GA-1-negative anti-GA antibodies for which sequencing was attempted, two were blocked, and one differed sharply from the others.     

Elicitation and detection of in vitro H-2-linked Ir gene regulated antibody responses to poly(LTyr, Glu)-poly(DLAla)--poly(LLys)

A microculture system is described in which secreted antibody responses to the synthetic polypeptide (T,G)-A--L were obtained in vitro. Responses were highly reproducible, antigen-dependent, antigen-specific, and under H-2-linked Ir gene control. Critical elements in the system include the schedule of in vivo antigen-priming, removal of the stimulating antigen after 3 days of culture, and a sensitive detection system (double-antibody ELISA). This system should be useful in the analysis of the mechanism of action of Ir genes as well as the mechanisms by which anti-idiotype antibodies modulate immune responses.     

Antibody response against poly (Glu60Ala30Tyr10) terpolymer and bacterial levan in ϰ-deficient mice

In murine species, the ?⁺-bearing immunoglobulins-dominate the antibody (Ab) repertoire with a ?/λ ratio of 95 : 5. The aim of the present study is to investigate the characteristics of the antibody response in ?-deficient (K ?/?) mice immunized with a T-dependent synthetic antigen, poly (Glu⁶⁰Ala³⁰Tyr¹⁰) (GAT) and a T-independent antigen, bacterial levan (BL). K ?/? mice were obtained by targeted deletion of the J?C? gene segments. In response to GAT, K ?/? mice respond by producing increasing amounts of anti-GAT Igλ1 and Igλ2 in the primary as well as secondary response, although anti-GAT specific monoclonal antibodies (mAb) raised in K ?/? mice are mostly of IgM isotype. The GAT public idiotype, GATIdX, present on all GAT-specific Ab bearing x light chain, is not detected in the sera of K ?/? mice or on any of the anti-GATλ1 mAb. In response to BL, the amount of Ig?1⁺ Ab in K ?/? mice is comparable to the amount of Ig?⁺ Ab in normal mice. However, λ2⁺ Ab are detected neither in wild-type nor in K ?/? mice. Like ?⁺ Ab, the majority of λ1⁺ mAb are specific for β2–6 fructosan present in BL and rye levan and, to some extent, express the BL-specific idiotype, A48ld. Our results show that important compensatory mechanisms occur in ?-deficient mice, restoring their ability to mount immune responses against a variety of T-dependent and T-independent antigens by the alternative usage of the clonally restricted λ repertoire.     

Ir gene control of T and B cell responses to determinants in (Glu Lys Ala) terpolymers

All mice responding to the terpolymer GLA40 make GL, GA and GLA specific antibodies irrespective of their response to GL or GA alone. The mice displayed positive T cell proliferative responses against the homologous terpolymer, but no T cell responses were obtained with GL, which is non-immunogenic in mice. T cells from GLA immune mice, which are also responders to GA, such as mice of H-2 haplotypes a, b, d, k and r, could be stimulated by GA. T cells from GLA immune mice of H-2 haplotypes p and q which are non-responders to GA could not be stimulated by GA. On the other hand, T cells from H-2s mice immune to GLA and which are also responders to GA alone could not be stimulated by GA. Thus mice of H-2 haplotypes p, q and s recognize the terpolymer via 'GLA' determinants alone, whereas mice of H-2 haplotypes a, b, d, k and r may recognize both GA and GLA determinants in GLA terpolymer.     

Genetic control of resistance to Salmonella typhimurium infection in high and low antibody responder mice.

Mice selected by Biozzi for high and low responses to sheep erythrocytes were investigated for resistance to subcutaneous Salmonella typhimurium infection. The resistance was measured by LD50 values, viable bacterial counts in liver and spleen at 10 days, and the kinetics of infection over 4 weeks. High responder mice were susceptible to S. typhimurium injected subcutaneously (LD50 less than 10) and low line resistant (LD50 3 x 10(6)). Control of natural resistance to S. typhimurium in inbred mice is primarily by a single gene. Ity, on chromosome 1. Results with hybrid generations of Biozzi mice with either BALB/c (sensitive) or CBA (resistant) inbred mice indicated additional genetic control of resistance in Biozzi mice. Analysis of resistance data of backcrosses of (high x low)F1 with either parental strain showed this genetic control to be at least one other gene in the Biozzi mice, not linked to Ity. The antibody responses in the hybrid generations and inbred and Biozzi parental strains were tested by haemagglutination assays and ELISA. After specific stimulation of the mice there was an inverse relationship between resistance to S. typhimurium and antibody levels.     

Public and individual idiotopes in the anti-poly(Glu60, Ala30, Tyr10) response: analysis by monoclonal antibodies

From BALB/c mice immunized with anti-GAT monoclonal antibody (mAb) G5, we have obtained anti-idiotypic mAb against individual (or private) idiotopes, expressed by G5 as well as anti-GAT mAb, that are heteroclitic because they recognize poly-(Glu50, Tyr50) (GT) better than poly(Glu60, Ala30, Tyr10) (GAT). From BALB/c mice immunized with BALB/c polyclonal anti-GAT antibodies, anti-idiotypic mAb directed against public idiotopes expressed following GAT immunization in all individuals of all mouse strains tested have been obtained. Nine anti-idiotypic mAb were studied in detail. One of these mAb recognizes only polyclonal anti-GAT antibodies; the other eight recognize polyclonal anti-GAT antibodies and anti-GAT mAb. The distribution of the structures recognized by the different anti-idiotypic mAb on a battery of 20 anti-GAT mAb allows definition of two families of public idiotopes.     

Induction by monoclonal anti-idiotypic antibodies of an anti-poly(Glu60 Ala30 Tyr10) (GAT) immune response in GAT-responder and GAT-nonresponder mice

Two different monoclonal anti-idiotypic (Id) antibodies, HP-Id20 and HP-Id22, recognizing two discrete idiotopes characteristic of the anti-poly(Glu60 Ala30 Tyr10) (GAT) response were used to immunize BALB/c (GAT-responder) and DBA/1 (GAT-nonresponder) mice. The monoclonals were injected either copolymerized with keyhole limpet haemocyanin or polymerized with glutaraldehyde. The specific response was studied by two assays: (a) inhibition of binding of monoclonal anti-GAT antibody G5Bb2-2 to HP-Id20 and HP-Id22 and (b) GAT binding assays. In BALB/c GAT-responder mice, HP-Id20 and HP-Id22 immunization led to the preferential stimulation of immunoglobulin idiotypically related to anti-GAT antibodies (Ab1') and expressing anti-GAT activity. The results obtained with BALB/c nu/nu mice indicated that this response is T-cell-dependent. By means of the same experimental protocol GAT-nonresponder animals could be induced to produce anti-GAT antibodies after HP-Id immunization. This last result indicates that anti-Id immunization can bypass Ir gene control and does not preferentially stimulate the induction of GAT-specific T suppressor cells.     

Genetic studies in inbred rats. VI. Linkage relationships of mixed lymphocyte reactivity, serologically defined antigens (Ag-B, Ag-C) and the immune response to poly(Glu52Lys33Tyr15).

The Ag-B allotype, mixed lymphocyte reactivity (MLR) and the immune response to poly(Glu52Lys33Tyr15) were assayed in male rats from the F2 hybrid and two backcross generations of the F344 and DA strains in order to investigate the structure of the rat major histocompatibility complex. No disparity between Ag-B type and mixed lymphocyte reactivity was found in 263 animals. The immune response to poly(Glu52Lys33Tyr15) was closely linked to the Ag-B locus, and both antibody production and the delayed hypersensitivity response were under polygenic control. These results suggest that the genetic loci which determine these responses in the rat are closely linked and that recombinational events between the Ag-B and MLR loci are infrequent.     

Genetic control of the immune response to ferritin in F1 hybrid mice.

The immune response to the antigen horse spleen ferritin, has been investigated in ten inbred parental strains and seven different F1 hybrid strains of mice, using an antigen excess technique. The degree of dominance in an F1 hybrid system can be estimated by using the Fisher dominance index. The responses in F1 hybrid animals, obtained from crosses of high and low responder parents, varied from dominant to recessive but the overall mean dominance index for the ferritin immunogenetic system was found to be -0.0467 +/- 0.0083 (mean +/- s.e.), a value close to zero, which suggests a codominant mode of inheritance of IR-genes to ferritin and this is consistent with most published data in other F1 IR-gene systems.     

Immune response against poly(Glu60,Ala30,Tyr10) (GAT): immunization with monoclonal anti-idiotypic antibodies leads to the predominant stimulation of idiotypically similar immunoglobulins with anti-GAT activity

Two monoclonal anti-idiotypic antibodies (HP-Id20 and HP-Id22) recognizing two different public idiotopes expressed in the anti-poly(Glu60,Ala30,Tyr10) (GAT) response were used to immunize BALB/c and C57BL/6 mice. From these animals hybridomas were isolated. From BALB/c and C57BL/6 mice eight and seven monoclonal antibodies were characterized, respectively. The reagents were classified according to the expression of the public idiotypic specificity p.GAT (recognized by a rabbit antiserum). The anti-GAT activity and the expression of the various idiotopes characterized on anti-GAT polyclonal and monoclonal antibodies were also studied. Most of the reagents are Ab1'-type of antibody resembling anti-GAT antibodies. One anti-anti-idiotypic monoclonal antibody (Ab3) was also isolated from BALB/c mice. This suggests that in this experimental model the repertoire induced after HP-Id immunization and antigen stimulation is comparable. The idiotypic analysis of a large number of anti-GAT and of Ab1' monoclonal antibodies suggests that only two public idiotopes are involved in the anti-GAT response.     

Analysis of the role of different cell types in the genetic regulation of antibody production to the thymus-independent synthetic polypeptide poly (DTyr,DGlu)-poly(DPro)--poly (DLys)

The immune response potential of mice to the thymus-independent synthetic polypeptide poly (D Tyr, D Glu)-poly (D Pro)--poly(D Lys)[D (T, G)-Pro--L] is genetically regulated. The detect in the ability of low responder mice to mount an immune response to this antigen appears to be expressed in their B cell population since the presence of thymocytes, or addition of “educated T cells” or supernatant of T cells after stimulation with the antigen neither enhanced, nor suppressed the level of antibodies produced in both low and high responder mice. Low responsiveness could not be enhanced either by stimulation of macrophages or by injection of poly(A). poly(U) in contrast to the significant effect of these agents on low responses to the thymus-dependent poly(L Tyr, L Glu)-poly(L Pro)--poly(L Lys) [L (T, G)-Pro--L]. These results suggest that macrophages do not participate in the limiting step, or are not involved at all, in anti-body production towards the thymus-independent polypeptide. The antibodies produced in response to D (T, G)-Pro--L were found to be mainly of the 7 S class. T cells are not required for the production of mercaptoethanol resistant antibodies to this immunogen since they were found in intact mice as well as in T cell depleted animals.     

Analysis of the role of different cell types in the genetic regulation of antibody production to the thymus-independent synthetic polypeptide poly (DTyr, DGlu)-poly (DPro)--poly (DLys).

The immune response potential of mice to the thymus-independent synthetic polypeptide poly (DTyr, DGlu)-poly(DPro)--poly(DLys)[D(T,G)-Pro--L] is genetically regulated. The defect in the ability of low responder mice to mount an immune response to this antigen appears to be expressed in their B cell population since the presence of thymocytes, or addition of "educated T cells" or supernatant of T cells after stimulation with the antigen neither enhanced, nor suppressed the level of antibodies produced in both low and high responder mice. Low responsiveness could not be enhanced either by stimulation of macrophages or by injection of poly(A) - poly(U) in contrast to the significant effect of these agents on low responses to the thymus-dependent poly(LTyr, LGlu)-poly(LPro)--poly(LLys) [L(T,G)-Pro--L]. These results suggest that macrophages do not participate in the limiting step, or are not involved at all, in antibody production towards the thymus-independent polypeptide. The antibodies produced in response to D(T,G)-Pro--L were found to be mainly of the 7 S class. T cells are not required for the production of mercaptoethanol resistant antibodies to this immunogen since they were found in intact mice as well as in T cell depleted animals.     

The role of thymus cells in the immune response to poly(Tyr, Glu)-polyD L Ala--polyLys as a function of the genetic constitution of the mouse strain

Immune responsiveness to poly (Tyr, Glu)? polyD L Ala–polylys [(T, G)? A–L] was studied at the cellular level by comparing the limiting dilutions of immunocompetent cells from thymus and bone marrow of five mouse strains exhibiting either high or low responses to this immunogen. Cell cooperation between thymocytes and bone marrow cells was demonstrated for (T, G)? A–L. Limiting dilutions of thymus and bone marrow cells in the presence of an excess amount of the complementary cell type were performed with cells of C3H. SW, C57BL/6 (H? 2^b), C3H/HeJ, AKR/Cu (H? 2^k) and SJL/J (H? 2^s). A low precursor frequency was detected in the bone marrow population when the defect was at the determinant level only, as in the case of C3H/HeJ and AKR/Cu mice. These results were confirmed by allogeneic transfer experiments. Furthermore, results of a two-step experiment performed with C3H. SW and C3H/HeJ congenic strains indicated that there is no defect in the proliferative rate of the thymocytes of the low responder strain. In contrast, in the SJL low responder strain the genetic defect was reflected in both the thymus and bone marrow populations of immunocytes. This strain is a nonresponder to all determinats attached to A–L and, therefore, exhibits a carrier defect. The results presented here demonstrate the crucial role of the thymus-derived cells when the genetic control includes a defect at the carrier level.     

Serological analysis of antigen-specific helper factors specific for poly-L(Tyr, Glu)-poly-DLAla--poly-LLys [(T, G)-A--L] and L Glu60-LAla30-LTyr10 (GAT).

In vitro prepared antigen-specific helper factors reactive to the synthetic polypeptide antigens poly-L(Tyr, Glu)-poly-DLAla--poly-LLys [(T, G)-A--L] or LGlu60-LAla30-LTyr10 (GAT) and bearing Ia determinants were analyzed serologically to determine the nature of the Ia determinants they expressed. I subregion-specific mouse anti-Ia antisera were used, and showed that (T, G)-A--L-specific helper factor (HF) contains I-A subregion-controlled determinants, whereas GAT-specific HF carries I-J subregion-controlled antigens. This unexptected finding was confirmed in both the H-2k and H-2 b haplotypes, using a variety of anti-I-J antisera. Rabbit anti-Ia antisera also reacted with both HF which raised the possibility that the Ia determinants on HF may be carbohydrate in nature. The fact that HF has a low molecular weight and yet contains Ia determinants, antigen-binding capacity and idiotypic markers is compatible with this interpretation.     

V gene analysis of anti-cardiolipin antibodies from (NZW x BXSB) F1 mice.

In (NZW x BXSB) F1 (W/B F1) male mice, systemic lupus-like disease, thrombocytopenia and coronary vascular disease with myocardial infarction occur, due to the presence of platelet-associated antibodies, anti-platelet antibodies and anti-cardiolipin antibodies (aCL). We developed monoclonal aCL and analysed the specificity of aCL. In the W/B F1 mice, there are aCL with pathogenic properties, which have an IgG isotype and reveal a cofactor-dependent binding to CL, binding activity to platelets, and lupus anti-coagulant (LA) activity. Here, we analysed the usage of VH and V kappa genes of six aCL, including two pathogenic aCL, from W/B F1 mice, in an attempt to address the question of whether or not aCL with pathogenic properties use restricted Ig V genes. Sequence analysis of VH and V kappa genes of aCL showed that the pathogenic aCL had VHJ558 and V kappa 21 or V kappa 23 genes, whereas the other aCL without pathogenic features used mainly the 7183 VH family and the random V kappa gene group. However, two pathogenic aCL showed a 86.6% homology with the IgV region, each other, indicating that they were not closely related clones. Thus, these findings suggest the possibility that usage of Ig VH genes in pathogenic aCL is not random, but that there may exist a few epitopes of antigen recognized by the pathogenic aCL.     

Genetic control of immunity to Trichinella spiralis in mice: capacity of cells from slow responder mice to transfer immunity in syngeneic and F1 hybrid recipients.

Mice of the C57BL/10 (B10) strain are slow responders to infection with T. spiralis in terms of ability to expel worms from the intestine. Compared with rapid-responder NIH mice, infection stimulates a slower and reduced blast cell response in the draining mesenteric lymph node (MLN). Transfer of immune cells from the MLN (MLNC) does not accelerate worm expulsion from naive B10 recipient mice, even though MLNC from this strain effectively transfer immunity to (B10 X NIH) F1 recipients. In common with other B10 background mice C57BL/10 show an infection-dose related suppression of immunity to T. spiralis. Such suppression does not appear to determine the response to MLNC, as adoptive transfer into B10 recipients was not enhanced by reducing the level of challenge infection given, and transfer into F1 recipients was unaffected by simultaneous transfer of lymphocyte populations from donors infected at a level which would induce suppression. A hypothesis is proposed which relates slow response status to (i) the inherent capacity of the intestinal inflammatory component of worm expulsion, and (ii) the outcome of infection-dose related stimulatory and suppressive influences acting on the two interacting lymphocyte components of expulsion. The relevance of H-2-linked and non-H-2 genes to the control of the response is discussed.