Studies on immunity in hybridoma-bearing mice. A. Immune response to antigens. II. inhibition of production of anti-dinitrophenyl antibodies in mice which have rejected the B 53 anti-dinitrophenyl-producing IgE hybridoma

When BALB/c mice, which had rejected the anti-dinitrophenyl (DNP) IgE-producing hybridoma B 53, were immunized with DNP proteins, they produced much less anti-DNP antibodies than control (normal) mice. The anti-DNP plaque-forming cell (PFC) number was much less when spleen cells from mice immunized with DNP proteins were treated with sera of mice which had rejected the hybridoma B 53 than the PFC number from the same spleen cells not treated by the sera. The sera of mice which had rejected the hybridoma B 53 contained an inhibitor which was adsorbed and eluted from an anti-mouse immunoglobulin column and also a mouse anti-DNP IgG2a column. The inhibition of PFC was hapten-reversible. In Western blotting the eluates from the anti-DNP IgG2a column reacted as well with the blotted anti-DNP IgE B 53 as an anti-idiotypic antibody to anti-DNP IgE B 53. These criteria establish that the inhibitor in the sera of the mice which had rejected the B 53 tumor was an anti-idiotypic antibody of the type which mimics the epitope (DNP) of the immunizing antigen.     

Studies on immunity in hybridoma-bearing mice. B. Immunity against the hybridoma. I. Studies on the immune state of mice after rejection of the hybridoma

Anti-dinitrophenyl IgE secreting hybridoma B 53 cells may be rejected when injected subcutaneously in BALB/c mice. These mice are immune as they withstand without any ill effect the intraperitoneal injection of LD100 B 53 cells. Sera from mice which rejected the tumor have cytotoxic antibodies against the hybridoma, as shown by in vitro tests, but serum cannot transfer immunity to naive BALB/c mice against hybridoma B 53. Spleen cells from mice which have rejected the tumor might transfer immunity against B 53 hybridoma, and with Winn tests it has been shown that these spleen cells are very effective against the B 53 cells and also against the myeloma cells which were used for the fusion to construct the B 53 hybridoma. Subcutaneously injected B 53 cells not only produce anti-DNP IgE secreting tumors, but often also metastasize to spleen, and they are sometimes detected in the circulating blood. Mice with splenic metastasis or with detectable circulating B 53 cells generally die. However, we did observe one mouse with splenic metastasis which successfully rejected the tumor and became immune to B 53 cells.     

Immunogenicity of antigen complexed with antibody. I. Role of different isotypes.

IgM-, IgG1-, IgG2a-, IgG2b- and IgE-type anti-ovalbumin antibodies were isolated from rat immune sera and the complexes formed by antibodies of a defined isotype and the antigen were compared for antibody avidity and interaction with homologous complement. IgG2a-containing complexes consumed total complement with the greatest efficiency. IgG2a-, IgG2b- and IgM-containing complexes displayed similar activities in activation of the alternative pathway while IgG1 was less efficient. Reduction and alkylation of IgG2a antibodies abolished the capacity of the complex to activate the alternative pathway, but not their total complement consumption. The complement-dependent solubilization was greatest when complexes contained IgG1- or IgM-type antibodies and lowest with IgG2a-containing complexes. Inbred Long Evans rats immunized by complexes containing IgG1 or IgG2b antibodies displayed a markedly lower delayed-type hypersensitivity (DTH) compared with those immunized by antigen alone. Immunization with IgG2a- or IgE-containing complexes resulted in a slightly decreased DTH, while IgM-containing complexes induced DTH like ovalbumin alone. IgG2a-containing complexes elicited an antibody response markedly higher than that found in animals immunized by antigen alone. The same effect was found when complexes contained reduced-alkylated IgG2a. Immunization with complexes containing the other isotypes involved in the study induced an antibody response similar to the antigen in phosphate-buffered saline.     

Impaired antibody response against T-dependent antigens in rhino mice.

The antibody response in rhino mice, which carry a mutant gene hrrh, to thymus-dependent (TD) or thymus-independent (TI) antigens was compared with that of phenotypically normal littermates. The magnitude of antibody response to TD antigens in rhino mice decreased as they grew up, whereas the antibody response to TI antigens in rhino mice was indistinguishable from that of littermates. A transfer of thymus cells from littermates to rhino mice resulted in the partial restoration of the responsiveness to TD antigens. The experiments employing adoptive transfer of spleen cells from rhino mice to the irradiated normal mice suggested that the hyporesponsiveness of TD antigens of adult rhino mice was mainly due to the defect in the T helper cell activities rather than either the increase of the suppressor cells or defects in other cell types.     

Immune responses to hapten-lipopolysaccharide conjugates in mice. III. Genetics of the antibody response to polysaccharide antigens

Antibody responses to the polysaccharide antigens of bacterial lipopolysaccharides and their hapten derivatives are herein documented to be under multigenic control. Among the genes responsible for the antibody response to these polysaccharide antigens include those coded for by genes located on the X chromosome and those on chromosome 17 linked to genes in the H-2 and/or Qa loci. The use of B10 congenic mice revealed that two genes on chromosome 17 are involved in the regulation of the antibody response to bacterial polysaccharide antigens. The use of CXB recombinant inbred mice confirmed the multigenic control of the antibody response within their background array of genes. The data clearly demonstrate that immune responses to these polysaccharide antigens are under genetic control, the importance of which may be of significance with regard to humoral protection during gram-negative infection.     

Immune response to H-2 class I antigens on platelets. I. Immunogenicity of platelet class I antigens

Purified allogeneic murine platelet suspensions were found unable to induce primary anti-H-2 class I antibody or T cell proliferative responses. In contrast, the same platelet suspensions could elicit secondary anti-class I responses. The secondary responses were not due to contaminating leucocytes. Possible explanations, the lack of acolyte determinants (class II or non- H-2) on platelets or inappropriate layout and/or structure of their class I antigens, are discussed. These findings emphasize the importance of sufficient leucocyte depletion before platelet transfusion in the human.     

Immune response to glutaraldehyde-treated cells. I. Dissociation of immunological memory and antibody production.

Glutaraldehyde (GA)-treated sheep red blood cells (SRBC) or H-2-allogeneic spleen cells (SC), induced immunological memory with absent or markedly reduced primary antibody production. In contrast, a normal secondary response was obtained when GA-SRBC or GA-SC were given to mice primed with the corresponding untreated antigens. The secondary response of mice primed and boosted with GA-treated cells was relatively high with GA-SRBC, and negative or very low with GA-SC. Morphological studies of the fate of intraperitoneally injected cells showed that endocytosed GA-SRBC persisted much longer in peritoneal macrophages than untreated SRBC. Simultaneous challenge of mice with untreated and GA-treated SRBC revealed that phagocytosis and digestion of both types of cells in the same macrophage proceeded independently of each other. The primary response of mice receiving both SRBC and GA-SRBC was entirely similar to the response when SRBC alone was given.     

IgE antibody response to mite antigens in mite infested mice.

Mice infested at birth with the mouse mite Myocoptes musculinus developed positive skin tests to mite antigens at the age of 5 weeks. Serum IgE antibodies directed against mite antigens were first detected at 6 weeks of age and high levels of IgE were present as long as 1 year later. Similar kinetics of IgE formation were observed in mice infected as adults. Mast cell degranulation by mite extract was demonstrated in connective tissue obtained from the skin of mite infested mice.     

Immune response to phosphorylcholine. I. Characterization of the epitope-specific antibody

The antibodies to phosphorylcholine induced in BALB/c mice were isolated and studied with a variety of biochemical and immunochemical methods. Analysis of the idiotype and the hapten-binding specificities showed no differences to the phosphorylcholine-binding myeloma protein TEPC 15, indicating a high degree of homogeneity. However, disc electrophoresis, isoelectric focusing and amino acid composition data indicated large differences in the structure of anti-phosphorylcholine antibody and TEPC 15. By these criteria the anti-phosphorylcholine anti-bodies from BALB/c mice are of oligoclonal origin.     

Immune response to phosphorylcholine. I. Characterization of the epitope-specific antibody.

The antibodies to phosphorylcholine induced in BALB/c mice were isolated and studied with a variety of biochemical and immunochemical methods. Analysis of the idiotype and the hapten-binding specificities showed no differences to the phosphorylcholine-binding myeloma protein TEPC 15, indicating a high degree of homogeneity. However, disc electrophoresis, isoelectric focusing and amino acid composition data indicated large differences in the structure of anti-phosphorylcholine antibody and TEPC 15. By these criteria the anti-phosphorylcholine antibodies from BALB/c mice are of oligoclonal origin.     

Role of plasmid-encoded antigens of Yersinia enterocolitica in humoral immunity against secondary Y. enterocolitica infection in mice

In the present study the role of Yersinia enterocolitica antigens in humoral immunity against secondary Y. enterocolitica infection has been investigated. For this purpose, BALB/c mice, 4 weeks after immunization by primary infection with a sublethal dose of various Y. enterocolitica strains and mutant strains, were challenged with a lethal dose of highly virulent Y. enterocolitica strains of serotype O:8. As evident from the survival rate and protection against bacterial growth in the spleens of mice, only immunization with wild-type or attenuated strains harbouring an intact virulence plasmid mediated protection against a lethal challenge. Protection by immunization with plasmid-harbouring strains correlated with persistence of the bacteria in spleens for at least 7 days after immunization and high serum titres of Yersinia-specific antibodies directed against both chromosomal and plasmid-encoded determinants. Furthermore, the adoptive transfer of the purified IgG fraction of a rabbit serum specific for the adhesin YadA encoded by virulence plasmid pO8 from Y. enterocolitica O:8 mediated protection against a lethal challenge with the serotype O:8 strain harbouring the virulence plasmid pO8 but did not mediate protection when this strain harboured the virulence plasmid pO9 from serotype O:9. In summary, the results demonstrate that in our experimental mouse infection model: (i) the presence of the intact virulence plasmid in an immunizing Y. enterocolitica strain is essential for induction of protection against a lethal challenge with highly virulent Y. enterocolitica and (ii) that antibodies against plasmid-encoded determinants of Y. enterocolitica, especially YadA, are of major importance for achievement of protective immunity in mice.     

Comparative antibody response to Salmonella antigens in genetically resistant and susceptible mice.

The ELISA was used to titrate the antibody response in mice inoculated with salmonella antigens. The genetically resistant A/J and susceptible C57BL/6J mice were either infected with the virulent or the avirulent Salmonella typhimurium. Alternatively, they were inoculated either once or twice with the heat-killed salmonella vaccine. No appreciable difference could be detected in the relative ability of these two strains of mice to produce antibodies against the lipopolysaccharide antigens of this pathogen under these four conditions.     

Immune response deficiency of BSVS mice. II. Generalized deficiency to thymus-dependent antigens.

BSVS mice gave abnormally low IgG responses to 5 thymus-dependent antigens as well as a weak delayed-type hypersensitivity (DTH) response to sheep red blood cells. In contrast to IgG, the IgM antibody responses of these mice were normal to three T-independent antigens as well as to all five T-dependent antigens. The low immune responsiveness of BSVS mice was also reflected in the low levels of IgG(2)a, IgG(2)b and IgG(3) in their normal serum. The low T-dependent immune responses may result from BSVS mice having been selectively bred for susceptibility to infection with St. Louis encephalitis virus and Salmonella. C57BL/6J mice, which are also highly susceptible to Salmonella, gave low immune responses similar to, but genetically distinct from, those of BSVS mice. The levels of Ig-positive and theta-positive cells were normal in BSVS and C57BL/6J mice.     

Idiotypic modulation of the antibody response of mice to Echinococcus granulosus antigens.

In this work we analysed the modulation of the antibody response to Echinococcus granulosus antigens via anti-idiotype (Ab2) administration. In a first set of experiments, we determined the antibody response to hydatid cyst fluid antigen (HCFA) of mice immunized with Ab2. Results showed that Ab2 elicited anti-HCFA antibodies in mice that had never been exposed to HCFA before. Moreover, that response was characterized by booster effect, avidity maturation and an inverse correlation between avidity and Ab2 dose. We infected these mice and measured the titre and avidity of anti-HCFA antibodies during 250 days of infection. Lack of avidity maturation was the most important feature observed, suggesting that the parasite (either protoscolex or cyst) could modulate the antibody response of its host. In a second set of experiments, we investigated the presence of regulatory anti-idiotypes in the Ab2 preparation. In this context we treated neonates with different doses of Ab2, immunized them with HCFA 12 weeks later and determined their anti-HCFA titres. A specific and Ab2 dose-dependent suppression of the response to HCFA was observed, suggesting the existence of regulatory anti-idiotypes in the Ab2 preparation. Although this Ab2 could be involved in the regulation of the anti-HCFA response, other anti-idiotypes could also be involved. Our results show that it may be possible to improve the avidity of the anti-HCFA response via the administration of the anti-idiotype Ab. However, the live parasite could successfully revert this effect by mechanisms not yet characterized.     

Immune reactivity during aging. I. T-helper dependent and independent antibody responses to different antigens, in vivo and in vitro.

The immunological status during aging was assessed by measuring the antibody response of the long-lived (C3H/eb X C57Bl/6J)F1 mice to various antigens in vivo and in vitro. In vivo, a decrease in antibody production to DNP and NIP haptenic determinants coupled on to BGG, as well as the response to SRBC, was observed. The decline was more pronounced in the IgG as compared to IgM antibodies. The results were recorded when various parameters such as antigen dose and kinetics of the response, were considered. Reduction of the antibody response was also noted when PVP was employed as immunogen. Similar results were noted when the responses to SRBC and DNP--polylysine were induced and followed in spleen organ cultures. In all of these experimental systems, the peak response was observed in mice 6--12 months old. From then on a gradual decrease was manifested, mice 30--36 months old producing significantly low responses. The results demonstrate that decrease in antibody production is expressed in the isolated spleen tissue in the same manner as in the intact animal. Furthermore, they were interpreted as indicating that the lesion may be at the T helper and the B cell compartments.     

Immune responses incongenitally thymus-less mice. I. Absence of response to oxazolone

Cell proliferation in regional lymph nodes of mice sensitized with oxazolone was measured by uptake of ¹²⁵IUdR. Experimental factors affecting incorporation of IUdR into lymph nodes are described. The delayed hypersensitivity response was assessed by measuring the increase in ear thickness after subsequent challenge with oxazolone. Unlike normal mice, thymus-less `nude' mice showed no detectable response to oxazolone. Implantation of an allogeneic neonatal thymus, either subcutaneously or under the kidney capsule, adoptively conferred on `nude' mice the ability to mount a lymphoproliferative response.     

Immune response to H-2 class I antigens on platelets. II. Specific decrease of H-2 class I-specific antibody response induced by treatment with allogeneic platelets

Mice pretreated with injections of allogeneic platelets were found to mount a decreased antibody response upon challenge by lymphocytes of the same donor strain. This decrease was mediated by platelets themselves, and not by leucocytes and red cells contaminating the platelet suspension. It affected specifically antibodies reactive with H-2 class I antigens present on donor platelets. This phenomenon may be related to the lack of class II or some non-H-2 antigens on platelets, and/or to properties of their class I antigens (soluble molecules adsorbed from the plasma). These findings emphasize the potential usefulness of purified platelet transfusions preceding organ transplantation in man.     

Immunofluorescent antibody response to lactic dehydrogenase virus in different strains of mice.

The development of antibody against lactic dehydrogenase virus in five strains of mice (NZB x NZWF1, BALB/c, C.B-17, ICR and C.B-17 scid or SCID mice) was examined by indirect immunofluorescence (IIF) of infected liver sections. IIF antibody appeared 1 to 3 weeks and rose progressively 2 to 4 weeks after infection in four strains of mice (NZB x NZWF1, BALB/c, C.B-17 and ICR mice). SCID mice did not develop antibody. These results suggest that IIF may be applicable for detecting LDV infection in many other ordinary strains of mice.     

CD59a deficient mice display reduced B cell activity and antibody production in response to T-dependent antigens

CD59a is the primary regulator of membrane attack complex in mice. Recently, we have shown that CD59a-deficient (Cd59a-/-) mice exhibit enhanced CD4+ T cell responses. Here, we explored the effects of CD59a on B cell function and antibody production. Contrary to our expectations, Cd59a-/- mice showed a decreased humoral immune response to a T cell dependent antigen, sheep red blood cells. We found that the decreased humoral immune response was associated with a reduction in plasma cell number in vivo and reduced ability to respond to stimuli during in vitro culture experiments. Using MLR studies in which purified wild type or Cd59a-/- CD4+ T cells were mixed with purified B cells from each source, we found that the reduced B cell activation was largely due to the absence of CD59a on CD4+ T cells. Furthermore, a CD59a fusion protein bound specifically to mouse B cells, and enhanced B cell proliferation in a MLR, demonstrating that B cells express an as yet unidentified ligand for CD59a that aids in B cell activation.     

Immune response to tumor antigens in a patient with colorectal cancer after immunization with anti-idiotype antibody.

An active vaccination protocol was performed on one patient with colon carcinoma as a pilot to a prospective randomized double-blind clinical trial with the vaccine SDZ SCV 106. This vaccine is an anti-idiotype goat antibody to the monoclonal antibody 17-1A, which is directed against the tumor antigen 17-1A. To study the effect of the therapy on the immune reactivity, several tests were performed to detect anti-tumor antibodies in the serum as well as in eluates of metastatic tissue. Furthermore metastases removed from the lung were examined by immunohistochemistry. The results suggest that the humoral and cellular immune reactivity against the tumor are enhanced.