Hyperimmune Mouse Research Articles

Selective suppression of the generation of anti-tumor L3T4+ but not of Lyt-2+ T cell-mediated immunity in the tumor-bearing state.

C3H/He mice hyperimmune against syngeneic MH134 hepatoma were prepared by intradermal (id) inoculation of viable tumor cells followed by surgical resection of the tumor and by repeated id challenges with viable tumor cells. Winn assays performed utilizing spleen cells from these mice have revealed that both Lyt‐2+ and L3T4+ T cell subsets from MH134‐hyperimmune mice produced complete tumor protection. The in vivo tumor‐neutralizing activity was also found in spleen cells from tumor‐bearing mice at various times after id implantation of MH134 tumor cells. However, in contrast to comparable tumor‐neutralization by Lyt‐2+ and L3T4+ T subsets from hyperimmune mice, only the Lyt‐2+ T cell subset from tumor‐bearing mice was capable of mediating the in vivo protective immunity. L3T4+ T cell‐mediated immunity was not detectable in the tumor‐bearing state irrespective of the length of the sensitization period with a primary growing tumor, but emerged in the mice which resisted the first tumor challenge after the resection of the primary tumor. These results indicate that the emergence of L3T4+ T cell‐mediated anti‐tumor immunity is stage‐dependent and the Lyt‐2+ T cells represent the main functional subset in the tumor‐bearing state, although both subsets of T cells are potentially capable of effecting anti‐tumor in vivo immunity. The results are discussed in relation to the selective suppression of the L3T4+ but not of Lyt‐2+ T cell function in the tumor‐hearing state.

Anti-Ro (SS-A) antibodies from Ro (SS-A)-immunized mice.

Immunization of BALB/c mice with immunoaffinity-purified bovine or human Ro (SS-A) induces the production of antibodies reactive with Ro (SS-A). Fusion of spleen cells from the hyperimmunized mice to SP2/0 cells resulted in hybridoma cell lines that produced anti-Ro (SS-A) antibodies. Anti-Ro (SS-A) binding was established by solid-phase immunosorbent assay, immunoblotting, or RNA immunoprecipitation. Most of the anti-Ro (SS-A) antibodies bound to both human and bovine Ro (SS-A) in the solid phase, but only one of the monoclonal antibodies selectively bound to human Ro (SS-A); this suggests that there are species differences between the bovine and human Ro (SS-A) antigens. Indirect immunofluorescence studies demonstrated that most anti-Ro (SS-A) antibodies bound to cytoplasmic or nuclear HEp-2 cellular antigens, whereas others did not bind to fixed HEp-2 tissue culture cells. Nuclear staining of mouse substrates by one of the sera containing anti-Ro (SS-A) demonstrated that autoantibodies were induced by immunization with human Ro (SS-A).

Mediation of in vivo tumor-neutralizing activity by Lyt-2+ as well as L3T4+ T cell subsets.

The present study reexamines the cell surface nature of T cells mediating in vivo protective tumor immunity with the use of anti‐L3T4 and ‐Lyt‐2 antibodies. C3H/HeN mice hyperimmune against syngeneic MH134 hepatoma or MCH‐1‐A1 fibrosarcoma were prepared by intradermal (id) inoculation of viable tumor cells followed by surgical resection of the tumor and by repeated challenges with viable tumor cells. Spleen cells from these mice were fractionated into L3T4+ or Lyt‐2+ T cell subset by treatment with anti‐Lyt‐2 or ‐L3T4 antibody plus complement (C). Winn assays performed by utilizing such fractionated T cells have revealed that both L3T4+ and Lyt‐2+ T cell subsets from hyperimmune mice produced complete tumor protection. Flow microfluorometry study illustrated that the treatment with anti‐L3T4 or ‐Lyt‐2 antibody plus C resulted in the complete isolation of L3T4− Lyt‐2+ (Lyt‐2+) or L3T4+ Lyt‐2− (L3T4+) T cell subset, respectively. This contrasted with the failure of treatment with anti‐Lyt‐1 antibody plus C to isolate all T cells expressing Lyt‐2 marker. It was further demonstrated that each subset of T cells exerted its anti‐tumor effect in a tumor‐specific way and without a requirement for the other alternative subpopulation of unprimed T cells. These results indicate that Lyt‐2+ T cell subset can be successfully isolated by treatment with anti‐L3T4 but not with anti‐Lyt‐I antibody plus C, and that each single subset of Lyt‐2+ and L3T4+ T cells can function as in vivo effector T cells.

Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid.

Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125I-labeled proteins with a series of monoclonal antibodies (MAbs). Surface proteins p70, p65, p50, and p44 were shown to be integral membrane components by selective partitioning into the hydrophobic phase during Triton X-114 (TX-114)-phase fractionation, whereas p41 was concomitantly identified as a surface protein exclusively partitioning into the aqueous phase. Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [35S]methionine, 14C-amino acids, or [3H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Covalent lipid attachment was established by high-performance liquid chromatography identification of [3H]methyl palmitate after acid methanolysis of delipidated proteins. An additional, unidentified methanolysis product suggested conversion of palmitate to another form of lipid also attached to these proteins. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a larger p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11. These studies establish that a highly restricted set of distinct, lipid-modified hydrophobic membrane proteins are major surface antigens of M. hyopneumoniae and that structural variants of surface antigens occur within this species.

The development and analysis of species specific and cross reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the major histocompatibility complex for use in the study of the immune system in cattle and other species

We examined the potential of developing a set of species specific and cross reactive monoclonal antibodies (MoAbs) for use in the study of the phylogenetic and functional relation of class I and Class II antigens of the major histocompatibility complex (MHC) and leukocyte differentiation antigens in cattle and other species. Comparing immunization strategies demonstrated the number of hybrids producing cross reactive antibodies can be increased by hyperimmunization of mice with lymphoid cells from multiple species. Comparing various methods of assay (antibody-complement mediated cytotoxicity [CT], enzyme linked immunosorbent assay [ELISA] and flow microfluorimetry [FMF]), revealed FMF is the most useful technique for the primary assay of hybridomas producing MoAbs of potential interest. By using dual parameter and dual fluorescence analysis, we could determine whether a given MoAb reacted with mononuclear cells (lymphocytes and monocytes) and/or granulocytes, and also whether any two MoAbs of different isotype and specificity recognized antigens present on identical or separate populations of leukocytes. Comparing the patterns of MoAb reactivity with leukocytes obtained from cows, goats, sheep, pigs, horses and humans, as well as comparing the patterns of reactivity with a panel of lymphoid cell lines derived from cattle (with enzootic bovine leukemia) and humans (with various forms of leukemia), revealed sets of MoAbs reactive with unique antigenic determinants present on BoLA class I (15 MoAbs) and class II (9 MoAbs) antigens, and also MoAbs reactive with determinants present on leukocyte differentiation antigens (36 MoAbs). Dual fluorescence analysis demonstrated the antigens detected by some MoAbs are predominantly expressed on one lineage of leukocytes while others are expressed on two or more lineages of leukocytes. Dual and single fluorescence analysis also demonstrated the PNA receptor(s) is: 1) expressed on T cells, granulocytes and class II antigen monocytes and 2) absent or expressed in low amount on sIgM + B cells and a newly defined Non T/Non B population of cells. The strategies described for identifying and analyzing the specificity of MoAbs demonstrate the feasibility of developing a set of cross reactive MoAbs for identifying homologous molecules in multiple species and delineating their functional and phylogenetic relation.

Antigenic analysis of Strongyloides ratti infective larvae and adult worms.

The protein composition of Strongyloides ratti infective larvae and adult worms extracted sequentially in water, sodium deoxycholate and sodium dodecyl sulphate (SDS) and their excretory/secretory products were analysed by both one- and two-dimensional SDS-polyacrylamide gel electrophoresis. While many bands common to all preparations and both stages of the worm were seen, a number of bands unique to each stage and preparation were identified. Western blot analysis of these larval and adult preparations for reaction with IgG and IgM antibodies in hyperimmune mouse sera revealed a large number of antigens. These data provide a framework for analysis of protective and diagnostically useful antigens.

Expression of Japanese encephalitis virus antigens in Escherichia coli

The expression of Japanese encephalitis virus (JE) cDNA in Escherichia coli has been used to study the functional organization of the viral genome. JE protein coding sequences were expressed in E. coli by subcloning random fragments of cloned cDNA (P. C. McAda, P. W. Mason, C. S. Schmaljohn, J. M. Dalrymple, T. L. Mason, and M. J. Fournier, 1987, Virology 158, 348–360) into the bacteriophage λτ11 expression vector. Over 120 λτl1 recombinants expressing viral protein sequences as β-galactosidase fusion proteins were identified immunologically with monoclonal antibodies (MAbs) and polyclonal hyperimmune mouse ascites fluid (HMAF). This expression and immunological detection strategy has been used to (1) map viral protein coding sequences to the JE genome; (2) demonstrate that contiguous viral protein coding regions can be expressed as single polypeptides in E. coli, providing functional confirmation for a long viral open reading frame; (3) localize important antigenic domains within the envelope protein E; and (4) identify in JE-infected cells a form of the glycosylated nonstructural protein NS1 that contains a hydrophobic C-terminal extension encoded by portions of the “ns2a” region of the JE genome.

Production of a monoclonal antibody selective to human transitional cell carcinoma.

In order to detect the bladder tumor specific antigens, the monoclonal antibody (MoAb) No. 10 to human transitional cell carcinoma of the bladder (T.C.C.B.) was obtained. Hybridomas were prepared by cell fusion between the mouse myeloma cell line X 63 Ag 8.653 and the spleen cells of BALB/c mouse hyperimmune to the bladder cancer cells (grade 2) from a patient, and were cloned. Consequently 12 MoAb-producing clones were obtained for the panel screening by enzymeimmunoassay (EIA) and then 3 MoAbs (No. 10, 11 and 14) were selected for the testing of reactivity to bladder cancer cells from patients including normal epithelia. Finally No. 10 was selected as the most appropriate MoAb for this study and was determined IgM with kappa-light chains by EIA.

Development of a practicable method for isolation and identification of dengue viruses in developing countries.

We sought a practicable method for isolation and identification of dengue viruses in South-East Asia. We compared two mosquito cell lines, C6/36 and TRA-284-SFG, for virus isolation and two identification methods, immunofluorescent staining of infected cells with serotype-specific mouse monoclonal antibodies and a sandwich-type enzyme-linked immunosorbent assay with conventional mouse hyperimmune ascitic fluids. We found that the combination of TRA-284-SFG cells and ELISA is a useful and feasible method in developing countries.

Identification of antibody classes and Fc receptors responsible for phagocytosis of Trypanosoma musculi by mouse macrophages.

The phagocytosis of Trypanosoma musculi by macrophages in the presence of specific antibodies was investigated. In 14-day-infected mice, opsonic antibodies were detected in serum, and phagocytosis of parasites by peritoneal macrophages was observed. The mechanism of T. musculi phagocytosis was analyzed. The binding of trypanosomes to peritoneal macrophages and J774 cells was observed in the presence of serum from hyperimmune mice and from mice infected 14 or 28 days earlier, but not in the presence of control mouse serum or sera from 7-day-infected mice. Binding was partially inhibited by mouse monoclonal immunoglobulins G1 (IgG1) or IgG2a and almost completely inhibited by a mixture of both. Binding was also partially inhibited by the anti-Fc gamma 1/gamma 2b receptor monoclonal antibody 2.4G2. Binding of T. musculi was also induced by fractions of serum from 28-day-infected mice obtained by protein A-Sepharose chromatography. Only the IgG1-rich fraction eluted at pH 6.0 and the IgG2a-rich fraction eluted at pH 4.5 promoted binding which could be almost completely inhibited by monoclonal IgG1 and IgG2a. These data indicate that IgG1 and IgG2a anti-T. musculi antibodies are responsible for the phagocytosis of T. musculi by mouse macrophages and both Fc gamma 2a and Fc gamma 1/gamma 2b receptors are involved. Such a mechanism is likely to account for the elimination of parasites in T. musculi-infected mice.

Isolation and characterization of an antigen-specific suppressor inducer molecule from serum of hyperimmune mice by using a monoclonal antibody.

We have used a rat monoclonal antibody (mAb) (called 14-30) to affinity purify the antigen-binding chain of a suppressor inducer factor (TsiF-AB) from the serum of mice hyperimmune to heterologous erythrocytes. The TsiF-AB requires the addition of a second, antigen-nonspecific component for biologic activity as well as Lyt-2+ T cells in the assay culture. This mAb can be used to affinity purify suppressor inducer factor from a well-characterized TsiF but not suppressor effector factor (TseF) from culture supernatants. Binding of mAb 14-30 to TsiF is independent of the antigen specificity of the suppressor factor and of the strain of origin of the TsiF. The TsiF affinity purified from hyperimmune serum has an apparent m.w. of 68,000 by SDS-PAGE analysis. 2D gel analysis shows that the serum-derived TsiF has charge heterogeneity, all in the acid range.

Identification of an antigenic and genetic variant of dengue-4 virus from the Caribbean.

Twenty-one dengue (DEN) viruses isolated from the Caribbean (Dominica and Jamaica) during the 1981-1982 epidemic year were distinct serological and genetic variants of DEN-4 virus. These isolates were clearly identified as DEN-4 viruses using type-specific monoclonal antibodies in indirect immunofluorescence assays. However, they either were not neutralized, or were neutralized poorly using hyperimmune mouse ascitic fluids (HMAF) or rhesus monkey serum directed against the H-241 prototype strain of DEN-4 virus isolated in the Philippines in 1956. HMAF prepared against a representative Caribbean isolate, however, neutralized with similar effectiveness the homologous virus, the H-241 prototype strain, and virus strains isolated from the Pacific and Southeast Asian areas from 1973 to 1984. The Caribbean isolate exhibited no more than 30% and 16% oligonucleotide spot homology with the H-241 and Bangkok viruses, respectively, by RNA fingerprint analysis, while demonstrating 82% and 89% homology with the Gilbert and Niue Island isolates, respectively. The isolation of dengue viruses which are serologically and genetically distinct from the prototype virus emphasizes the need for continued dengue virus surveillance. The recognition of unique dengue isolates should allow the selection of reference strains and vaccine candidate strains which will induce antibodies that are equally effective in neutralizing viruses from all geographic areas.

The B10.A mouse B cell response to pigeon cytochrome c is directed against the same area of the protein that is recognized by B10.A T cells in association with the Ek beta:Ek alpha Ia molecule.

An analysis of the fine specificities of the primary and hyperimmune antibody responses of B10.A mice to pigeon cytochrome c showed that both were qualitatively very similar. Small amounts of antibody appeared to be directed against the regions of serine 15 and/or glutamic acid 44. The remaining antibodies (greater than 70%) bound to the same complex topographic determinant (including residues 3, 103, and 104) on the back surface of pigeon cytochrome c which had been found to dominate the rabbit antibody response to this protein, and to be involved in Ia-restricted T cell stimulation. The mouse antibodies reacted very poorly with fragmented forms of the immunogen or with tobacco hornworm moth cytochrome c, even though both of these antigens had been shown previously to strongly stimulate pigeon cytochrome c-primed T cells. The specificities of the primary IgG responses of seven other mouse strains were found to be very similar, but not identical, to that of B10.A mice. The cytochrome c-specific antibodies in the hyperimmune serum were shown to bind to determinants involving residues that vary between pigeon and mouse cytochromes c. Comparison of the binding of the antibodies to the immunogen and to the corresponding host protein enabled the calculation of the proportion of the overall binding energy contributed by the variant residues. This was as low as 19 to 35% for the primary response, rose to 25 to 46% for the hyperimmune mouse antibodies, and reached 40 to 63% for hyperimmune rabbit antibodies. The remaining energy of interaction (37 to 81%) was necessarily contributed by the surface of the protein surrounding the variant residues, which is the same for the immunogen and the host protein. These results illustrate the relatively subtle differences in binding affinities which can distinguish self from non-self recognition by antibody molecules.

Monoclonal Antibodies Against Bovine Tissue Factor, Which Block Interaction With Factor VIIa

Two monoclonal antibodies that recognize bovine tissue factor (coagulation factor III) have been obtained following the fusion of hyperimmune mouse spleen cells with NS-1 plasmacytoma cells. Both antibodies, TF1-E2 and TF1-F7, have gamma 1 heavy chains and lambda light chains. TF1-E2 and TF1-F7 have each been used to purify bovine tissue factor from a crude detergent extract of bovine brain by immunoaffinity chromatography. Both antibodies inhibit tissue factor procoagulant activity and block the association of factor VIIa with tissue factor. The association of TF1-F7 and tissue factor solubilized in Triton X-100 was measured under equilibrium conditions. The Kd for this antibody-antigen interaction was 2.1 +/- 0.2 nmol/L. TF1-E2 effectively competes with TF1-F7 for tissue factor binding, indicating that the monoclonal antibodies recognize overlapping sites on the protein. These antibodies will be useful reagents for large-scale purification and for structure-function studies of bovine tissue factor. In particular, since they appear to bind to the same region of the tissue factor molecule as factor VIIa, they will be useful as specific probes for studying the kinetics of tissue factor-initiated coagulation and for immunocytochemical localization of tissue factor in bovine cells.

Monoclonal antibodies against bovine tissue factor, which block interaction with factor VIIa

Two monoclonal antibodies that recognize bovine tissue factor (coagulation factor III) have been obtained following the fusion of hyperimmune mouse spleen cells with NS-1 plasmacytoma cells. Both antibodies, TF1-E2 and TF1-F7, have gamma 1 heavy chains and lambda light chains. TF1-E2 and TF1-F7 have each been used to purify bovine tissue factor from a crude detergent extract of bovine brain by immunoaffinity chromatography. Both antibodies inhibit tissue factor procoagulant activity and block the association of factor VIIa with tissue factor. The association of TF1-F7 and tissue factor solubilized in Triton X-100 was measured under equilibrium conditions. The Kd for this antibody-antigen interaction was 2.1 +/- 0.2 nmol/L. TF1-E2 effectively competes with TF1-F7 for tissue factor binding, indicating that the monoclonal antibodies recognize overlapping sites on the protein. These antibodies will be useful reagents for large-scale purification and for structure-function studies of bovine tissue factor. In particular, since they appear to bind to the same region of the tissue factor molecule as factor VIIa, they will be useful as specific probes for studying the kinetics of tissue factor-initiated coagulation and for immunocytochemical localization of tissue factor in bovine cells.

677 SPECIFIC IgA AND IgG INTERFERE WITH G. MURIS ADHERENCE TO INTESTINAL EPITHELIUM

Mucosal adherence may contribute to the pathogenicity of Giardia. Factors implicated as mediators of adherence of this pathogen to the mucosal surface include lectins and antibodies. We have developed a method to examine adherence of G. muris trophozoites(TR) to intestinal epithelium to evaluate the role of anti-Giardia milk and serum antibodies. Uninfected CF1 mice were anesthetized, and a 2 cm segment of small intestine isolated. The ends were ligated and resulting sac injected with TR (5 × 105) and 200 μl of normal(NM) or immune(IM) mouse milk (13% V/V), hyperimmune(IRS) or normal rabbit serum(NRS)(40% V/V) and their IgG fractions. Following one hour of incubation, the animals were sacrified by intracardiac infusion of Ringer's lactate 30mM EDTA. The sac was removed, inverted and agitated with liberation of epithelial sheets. Adherence was assessed microscopically by counting the number of TR adherent to 10 villi isolated from each animal studied. NM decreased the number of adherent TR from 21.8 ± 3.5/10 villi in media alone to 13.4 ± 3.2(p<.01). IM further decreased the number of adherent TR to 6.6 ± 2.5 (p<.001). Addtion of NRS had no effect on adherence. IRS decreased the number of adherent parasites to 8.2 ± 2.1(p<.001). A decrease was also observed in the presence of the IgG fractions of IRS 13.2 ± 7.9, compared to the IgG fraction of NRS 23.7 ± 2.5 (p<.05). These findings suggest that specific anti-Giardia antibodies interfere with parasite adherence to the mucosal surface. This may represent an important mechanism, resulting in clearance of Giardia infection by antiparasite antibodies.

A role for anaphylactic sensitization in tumor control.

In order to evaluate the possible preventive role of an anaphylactic condition on tumor development, C57BL/6J mice were immunized or hyperimmunized by the intraperitoneal route with egg albumin in alum before the chemical induction of fibrosarcoma. Preimmunization using a protocol which elicits an optimal IgE response produced: (1) a significant reduction of tumor incidence; (2) an increase of survival time, and (3) a decrease of tumor growth rate in animals with higher IgE titers (greater than 640). On the other hand, hyperimmunized mice, which were suppressed in their anti-egg albumin IgE response, showed no changes in tumor incidence and survival time when compared to controls. Our results suggest a suppressive effect on tumor development related to the ability of the animal to mount an IgE response to antigens unrelated to the tumor.

Immune response to ultraviolet-induced tumors. I. Transplantation immunity developing in syngeneic mice in response to progressor ultraviolet-induced tumors.

Ultraviolet-light-induced murine skin tumors were analyzed for the ability to induce transplantation immunity and cytotoxic lymphocytes in syngeneic mice. A correlation was found between tumor regression and the induction of cytotoxic T cells with specificity for a unique tumor-associated antigen. Processing tumors possessed tumor-associated transplantation antigens (TATA), which could be demonstrated by transplantation in hyperimmunized mice. Progression correlated with a lack of splenic cytotoxic T cell reactivity. High levels of in situ cytotoxic reactivity could be induced by presenting the tumor-specific antigen on nongrowing tumor cells. Tumor-bearer hosts were shown to be sensitized to TATA because cultured tumor-bearer T cells adoptively transferred protection against tumor outgrowth. Mechanisms of the in vivo suppression of antitumor immunity are discussed.

Serotyping of dengue viruses by an enzyme-linked immunosorbent assay.

We have developed a sandwich enzyme-linked immunosorbent assay for serotyping dengue viruses. In this assay, we used antibody from dengue hemorrhagic fever patients for detection of flavivirus common antigens to confirm virus isolation in C6/36 cells and that from hyperimmune mouse ascitic fluids for serotyping. The anti-dengue antibody was immobilized on microplate wells for capturing of dengue antigens, which were then sandwiched with the same biotinylated antibody. Then the biotin in the solid phase was detected with peroxidase-conjugated streptavidin. We found that all the dengue strains tested were unequivocally identified by this method.

Simultaneous development of humoral and cellular tumor-specific immunity against L1210 mouse leukemia

We have recently described that a variant of L1210 leukemia cell (L1210/LN-1) originally fused with Lesch-Nyhan fibroblast is highly immunogenic for inducing tumor-specific transplantation immunity in (BALB/cxDBA/2)F, mice. This finding has clearly been confirmed in the present study by in-vitro cell-mediated cytotoxicity assay. Direct cytotoxic tests and competitive inhibition tests using tumor cells such as P388, LS-1 and L5178Y as target or inhibitor cells showed that the cell-mediated immunity is specific to L1210 leukemia. In the process of this study, we found that the CD2F 1 mice hyperimmune to L1210 leukemia cells developed co-existing humoral anti-L1210 leukemia immunity. In an in vitro complement-dependent cytotoxicity test and absorption test, antisera from hyperimmune mice reacted specifically to L1210 leukemia cells, but not other tumor cells such as P388, L5178Y, LS-1, DB27C and BW5147 or normal cells from various tissues of DBA/2, BALB/c and AKR mice. In an in vitro cytotoxicity blocking test, the cytotoxic antisera specifically reactive to L1210 cells totally failed to inhibit lysis of L1210 cells by cytotoxic cells, suggesting that antigens recognized by cell-mediated cytotoxicity assays are not identical to serologically defined tumor-cell surface antigens.