Regulation of the murine IgE antibody response. I. Characterization of suppressor cells regulating both persistent and transient responses and their sensitivity to low doses of X-irradiation.

Anti-ovalbumin (OA) IgE antibody responses were measured in B6D2F1 mice as a function of time and antigen dose. One hundred to 200 microgram of OA in Al(OH)3 elicited transient responses, whereas 1 to 10 microgram of OA in Al(OH)3 elicited persistent anti-OA IgE responses of high titer. T cells isolated from the spleens of mice mounting either a persistent or a transient response strongly suppressed primary anti-DNP IgE responses in unirradiated recipient mice that were immunized with DNP-OA in Al(OH)3; it was, therefore, concluded that suppressor T cells (Ts cells) were activated during both the persistent and transient IgE responses. Nevertheless, in the present study it was not possible to completely rule out the contention that IgG antibodies may also have been suppressing the IgE response. With a modified adoptive transfer system, it was shown that these Ts cells were sensitive to low doses (250 R) of x-irradiation. The suppressive activity of long-term OA primed cells was also shown to be markedly enhanced when cultured for 24 hr with soluble OA; this finding was interpreted to indicate the presence of memory suppressor cells.     

Reaginic antibody formation in the mouse. X. Possible role of suppressor T cells in transient IgE antibody responses.

The kinetics of IgE antibody response to alum-absorbed dinitrophenyl derivatives of ovalbumin (DNP-OA) was dependent on the dose of immunogen. A persistent IgE antibody response was obtained when high responder BDF1 mice were immunized with a minimum (0.05 microgram) dose. An increase of the immunogen to 10 microgram depressed IgE antibody responses but enhanced IgG antibody responses of both hapten and carrier specificities. Determination of T helper cell activity and B memory cells after immunization with different doses of antigen indicated that minimum immunogen was favorable for developing helper activity, whereas 1 to 10 microgram immunogen were more favorable than a 0.05-microgram dose for developing both IgE and IgG B memory cells. Nevertheless, neither helper T cells nor B memory cells in the spleen explains a transient IgE antibody response to a high (10 microgram) dose of DNP-OA. Evidence was obtained that immunization with 10 microgram OA induced generation of antigen-specific suppressor T cells, which were not detectable after immunization with 0.05 microgram OA. Transfer of suppressor T cells to DNP-OA-primed mice depressed both anti-hapten and anti-carrier IgE antibody responses. The results suggested strongly that suppressor T cells are involved in a transient IgE antibody response to a high-dose immunogen.     

Reaginic antibody formation in the mouse. VI. Suppression of IgE and IgG antibody responses to ovalbumin following the administration of high dose urea-denatured antigen.

The major antigenic determinants in ovalbumin molecules (OA) were lost following denaturation in 8 M urea. The urea-denatured antigen (UD-OA) failed to combine with anti-OA antibody, but was capable of priming mouse T cells specific for OA. BDF₁ mice primed with alum-precipitated OA were given three intravenous injections of 100 μg UD-OA at 3, 5, and 7 days after the primary immunization. The treatment with UD-OA suppressed both IgE and IgG antibody responses to OA. The same treatment of OA-primed animals with intravenous injections of OA resulted in suppression of IgE antibody response but enhanced IgG antibody response. Intravenous injections of either OA or UD-OA suppressed both IgE and IgG anti-DNP antibody responses of DNP-OA-primed animals but failed to suppress anti-hapten antibody responses to DNP-keyhole limpet hemocyanin. The effect of the treatment on helper T cells and B memory cells in OA-primed animals was studied by adoptive transfer experiments. The results showed that the OA-treatment as well as UD-OA-treatment suppressed the development of both helper function of T cells and B memory cells in the spleen, but the UD-OA treatment was more effective than OA-treatment for the suppression of B cell development. Possible mechanisms for the suppression of the development of immunocompetent cells were discussed.     

Reaginic antibody formation in the mouse. VII. Depression of the ongoing IgE antibody formation by suppressor T cells.

The ongoing IgE antibody formation against ovalbumin (OA) in high responder mice was depressed by i.v. injections of either native or urea-denatured ovalbumin (UD-OA). Adoptive transfer experiments to determine the helper function of spleen cells from the treated animals showed that helper function for both IgE and IgG antibody responses diminished after treatment. Evidence was obtained that treatment suppressed the expansion of IgE-G memory cells. When the same treatment with OA or UD-OA was given to OA-primed mice before the appearance of IgE antibody in their serum, OA-specific splenic suppressor T cells were demonstrable. Thus, the transfer of splenic T cells from treated mice into normal mice suppressed the primary IgE and IgG antibody responses of the recipeints to DNP-OA. It was also found that the transfer of the splenic T cells from UD-OA-treated mice into OA-primed mice depressed ongoing IgE antibody formation in the recipients. The results suggested strongly that the decrease of helper function and the depression of ongoing IgE antibody formation by repeated injections of UD-OA was caused by generation of antigen (OA)-specific suppressor T cells.     

Stable Antigen Is Most Effective for Eliciting CD8+ T-Cell Responses after DNA Vaccination and Infection with Recombinant Vaccinia Virus In Vivo

The induction of strong CD8(+) T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8(+) T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VV in vitro was enhanced in the presence of short-lived antigens. In vivo, however, the highest induction of NP-specific CD8(+) T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responses in vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.     

Subcutaneous late phase responses are augmented during local inhalational tolerance in a murine asthma model

Acute exposure of sensitized mice to antigen elicits allergic airway disease (AAD) characterized by Th2 cytokine-dependent pulmonary eosinophilia, methacholine hyperresponsiveness and antigen-specific IgE elevation. However, chronic exposure induces a local inhalational tolerance (LIT), with resolution of the airway responses but persistent systemic IgE production. To further determine if systemic immunologic responses were maintained during LIT, we assessed subcutaneous late phase responses to ovalbumin in this model. Sensitized and AAD mice developed small subcutaneous responses to ovalbumin, with footpad thickness increasing to 113.7 and 113.6% of baseline, respectively. In comparison, LIT mice developed marked foot swelling (141.6%). Histologic examination confirmed increased inflammation in the chronic animals, with a significant contribution by eosinophils. Thus, the resolution of airway inflammatory responses with chronic antigen inhalation is a localized response, not associated with loss of systemic responses to antigen.     

Regulation of antibody response in different immunoglobulin classes. II. Induction of in vitro IgE antibody response in murine spleen cells and demonstration of a possible involvement of distinct T-helper cells in IgE and IgG antibody responses.

In vitro induction of anti-DNP IgE as well as IgG1, IgG2a antibody responses was shown in murine spleen cell culture. Spleen cells primed three times with 1 mug of DNP-OA or DNP-Asc produced significant amounts of anti-DNP IgE as well as IgG antibodies by the in vitro stimulation with DNP-OA or DNP-Asc, respectively. Collaboration between DNP-primed B cells and carrier-primed T cells was required for the induction of both IgE and IgG antibodies with DNP-coupled T-dependent antigen. Carrier-specific T cells induced with a low dose of Asc (0.01 mug) showed helper function only on IgE antibody response, whereas T cells primed with a higher dose of Asc (10 mug) cooperated only with IgG-B cells. T cells primed with Asc in CFA showed helper function mainly on IgG antibody response but not on IgE antibody response. The result indicated the presence of a distinct population of T helper cells for IgE and IgG antibody responses. T-independent antigen (DNP-Ficoll) induced both anti-DNP IgE and IgG antibody responses in DNP-primed spleen cell population without the requirement of the collaboration of helper T cells.     

Elicitation of antigen-induced primary and secondary murine IgE antibody responses in vitro

Described herein are methods for eliciting and quantitating primary and secondary murine IgE antibody responses in vitro, and the important role of antigen concentration in determining the level of IgE produced during an immune response. The methods for quantitating IgE antibody levels in culture supernatant fluids and in serum by ELISA are presented in detail. The specificity of such methods was confirmed in that (1) no other isotype of antibody registered in the IgE-ELISA, and (2) parallel determinations of IgE antibody concentrations could be obtained by independent analysis using Fc epsilon RI-dependent basophil degranulation. We examined various parameters of cell donor immunization and lymphoid cell culture which allow for optimal in vitro primary and secondary IgE responses. High relative antigen doses result in diminished IgE antibody responses in experimental animals, a finding confirmed herein. High antigen concentrations in vitro also result in relative suppression of IgE antibody synthesis. This was also true for in vitro production of IgG1 and IgA antibodies. Conversely, IgM and IgG2a responses were elicited at both low and high antigen concentrations; IgG2b and IgG3 were not produced under the conditions of priming and culture used herein. Finally, production of IgE in vitro depended on the presence of carrier-primed CD4+ T cells and hapten-specific B cells. Generation of maximal IgE antibody secretion, and hence elicitation of an allergic reaction, is thus dependent on the amount of antigen acting as stimulant for the immune response.     

Immunologic properties of mast cells from rats infected with Nippostrongylus brasiliensis.

The concentration of IgE in the serum of Sprague-Dawley rats increased after infection with Nippostrongylus brasiliensis (NB). The IgE concentration in normal rats was less than 1 mug/ml. After re-infection with NB, the concentration increased in 100 to 300 mug/ml. Mast cells were purified from peritoneal cells of both normal and NB-infected animals. Purified mast cells from the infected animals released histamine upon exposure to NB antigen. The antibody specific for IgE released histamine from purified mast cells of both normal and infected animals. Dose-reponse curves of histamine release suggested that mast cells from NB-infected animals bear more IgE molecules than normal mast cells. Binding of 125I-labeled rat E myeloma protein with normal mast cells was demonstrated by autoradiography. Under the same experimental conditions, mast cells of infected animals were not labeled with 125I-IgE. Mast cells from both normal and infected animals failed to combine 125I-labeled IgG. The number of IgE molecules bound per mast cell was determined by incubating 125I-labeled IgE with purified mast cells. When mast cells were incubated incubated in 0.6 to 2 mug/ml of IgE, the number of IgE molecules combined with the mast cells from infected animals was about 10% of that bound with normal mast cells. The results indicated that a large proportion of IgE receptors on mast cells of infected animals was occupied by their own IgE. No significant difference was observed between normal mast cells and those of infected animals with respect to histamine content and intracellular levels of cyclic nucleotides.     

Nippostrongylus brasiliensis: radioresistant IgE antibody-forming cells in infected rats

In Nippostrongylus brasiliensis-infected rats, anti-N. brasiliensis IgE antibody production was observed at 20 weeks postinfection, long after the worms, as a source of antigen, had been expelled. The persistent IgE production was not abrogated after whole body irradiation (800 R) administered at 12 or 20 weeks, suggesting the participation of radioresistant IgE-forming cells. Help of T cells and recruitment of B memory cells in the irradiated rats seems to be ruled out by the findings that the irradiation completely inhibited the initiation of anti-N. brasiliensis IgE production in rats shortly after the infection with N. brasiliensis or after primary and secondary immunization with N. brasiliensis-antigen. Moreover, clearance of anti-N. brasiliensis IgE antibody from circulation did not seem to be crucially affected by the irradiation. The radioresistant cells forming anti-N. brasiliensis IgE were most productive in mesenteric lymph nodes as compared to other lymph nodes. The recognition of antigens fractionated by chromatography on Sephadex G-200 was the same for IgE-forming cells from rats 12 weeks after infection as for those from 3 weeks after infection. Based on these results, one of the mechanisms of persistent elevation of IgE antibody in the host infected with helminth parasites might be explained by the participation of radioresistant IgE-forming cells.     

Relationships between cell-mediated immunity and the IgE antibody response: I. Lymphotoxin production to DNP-Ascaris conjugates

The purpose of this paper is to present evidence for the suitability of a lymphotoxin (LT) assay as an in vitro correlate of cell-mediated immunity (CMI) to a hapten-carrier conjugate known to stimulate a high IgE antibody response. This would be used in a study of the factors influencing the relationship(s) between CMI and IgE antibody responses to the same antigen. Antigen-induced LT was assayed on actinomycin-inhibited, chromium-51-labeled L929 fibroblasts, using supernatants obtained from spleen and lymph node cells of animals immunized with 2,4-dinitrophenyl-Ascaris conjugates. Although LT was present at all times tested, beginning at Day 10, its concentration varied with time after immunization, adjuvant used, and cell type assayed. The induction of LT was T-cell dependent and conjugate specific. LT was produced by nonadherent cells. Adherent cells from immunized animals produced L-cell cytotoxin in the absence of antigen stimulation when tested before Day 10.     

Immunogenic properties of modified antigen E. III. Effect of repeated injections of modified antigen on immunocompetent cells specific for native antigen.

It has been shown that ragweed antigen E loses its major antigenic determinants after denaturation in 8 M urea, but urea-denatured (UD) antigen and an alpha-polypeptide chain isolated from the denatured molecules are capable of priming mouse T cells specific for native antigen. Weekly injections of 10mug UD antigen or alpha-chain into antigen E-primed animals depressed the ongoing IgE antibody response, whereas injections of the same dose of antigen E failed to depress the antibody response. It was found by adoptive transfer experiments that helper activity of antigen E-primed splenic T cells was depressed by the treatment of the donors with either modified antigen or native antigen E. The same treatment of antigen E-primed animals depressed the DNA synthetic response of their splenic T cells to antigen E. The treatment of antigen E-primed animals with UD antigen resulted in a decrease of antigen E-specific IgE-B cells and IgG-B cells in their spleen, whereas the treatment with native antigen expanded the B cell populations. In view of the results obtained in the mouse, cellular basis for the immunologic effects of hyposensitization treatment is discussed.     

Effect of CpG oligonucleotides on vaccine-induced B cell memory

Adding synthetic oligodeoxynucleotides containing unmethylated CpG motifs to Anthrax vaccine adsorbed (AVA, the licensed human vaccine) increases the speed and magnitude of the resultant Ab response. Ab titers persist in the protective range for >1 year, significantly longer than in animals vaccinated with AVA alone. Unexpectedly, a majority of mice immunized with CpG-adjuvanted AVA maintained resistance to anthrax infection even after their Ab titers had declined into the subprotective range. The survival of these animals was mediated by the de novo production of protective Abs by high affinity memory B cells re-stimulated immediately after challenge. Thus, a previously unrecognized benefit of CpG oligodeoxynucleotides adjuvants is their ability to expand the long-lived memory B cell population. Current findings demonstrate that CpG-adjuvanted AVA mediates protection both by stimulating a strong/persistent serum Ab response and by generating a high-affinity long-lived pool of memory B cells.     

Antigen concentration determines helper T cell subset participation in IgE antibody responses.

A recently developed in vitro system for antigen-stimulated primary and secondary murine IgE antibody responses has been used to define (a) the relative participation of the Th1 and Th2 cell-derived lymphokines IFN-gamma and IL-4, respectively, in such responses, and (b) the role of antigen concentration in determining functional helper T cell activity. These studies confirm that IL-4 and IFN-gamma exert regulatory effects on IgE synthesis, but the nature and extent of their respective effects on primary and secondary IgE responses differ. Thus, primary IgE responses are considerably more sensitive to and dependent on IL-4 than are secondary IgE responses since (1) anti-IL-4 monoclonal antibody totally inhibited primary IgE responses, but only partially affected secondary responses; and (2) exogenously added IL-4 could stimulate primary IgE responses to optimal antigen concentrations, but had no effect on secondary IgE production. Likewise, antigen-stimulated primary IgE responses are about eightfold more sensitive than are secondary responses to the inhibitory effects of IFN-gamma. Studying the effect of antigen dose on the quantity of IgE antibody produced revealed that although IFN-gamma could be detected by ELISA in cultures exhibiting high-dose antigen-dependent diminution of IgE production, anti-IFN-gamma monoclonal antibody could not reverse this phenomenon. Thus, IFN-gamma is not solely responsible for decreased IgE synthesis associated with high-dose antigen exposure. IL-4 activity was detected in the fluid from cultures stimulated with low, but not high, levels of antigen. Moreover, addition of exogenous IL-4 restored IgE production to normal levels in cultures exposed to high antigen concentrations. Therefore, it appears that high levels of antigen result in selective stimulation of Th1 cells which produce IFN-gamma, and diminished activation of IL-4-producing Th2 cells. These results help explain observations regarding the influence of antigen dose on the generation of experimental and clinical IgE antibody responses in vivo.     

Interleukin-10 does not mediate inhalational tolerance in a chronic model of ovalbumin-induced allergic airway disease

OBJECTIVE: IL-10 is a potent anti-inflammatory cytokine, and IL-10-producing regulatory T cells are effective inhibitors of murine asthmatic responses. This study determined whether IL-10-dependent mechanisms mediated the local inhalational tolerance seen with chronic inhalational exposure to antigen. METHODS: Wildtype and IL-10(-/-) mice were sensitized with ovalbumin (OVA) and then challenged with daily OVA inhalations for 10 days or 6 weeks. RESULTS: The 10-day animals developed allergic airway disease, characterized by BAL eosinophilia, histologic airway inflammation and mucus secretion, methacholine hyperresponsiveness, and OVA-specific IgE production. These changes were more pronounced in IL-10(-/-) mice. The 6-week IL-10(-/-) and wildtype animals both developed inhalational tolerance, with resolution of airway inflammation but persistence of OVA-specific IgE production. CONCLUSION: IL-10 may have anti-inflammatory effects in the acute stage of murine allergic airways disease, but the cytokine does not mediate the development of local inhalational tolerance with chronic antigen exposure.     

IgE enhances parasite clearance and regulates mast cell responses in mice infected with Trichinella spiralis

Trichinella spiralis infection elicits a vigorous IgE response and pronounced intestinal and splenic mastocytosis in mice. Since IgE both activates mast cells (MC) and promotes their survival in culture, we examined its role in MC responses and parasite elimination in T. spiralis-infected mice. During primary infection, wild-type but not IgE-deficient (IgE(-/-)) BALB/c mice mounted a strong IgE response peaking 14 days into infection. The splenic mastocytosis observed in BALB/c mice following infection with T. spiralis was significantly diminished in IgE(-/-) mice while eosinophil responses were not diminished in either the blood or jejunum. Similar levels of peripheral blood eosinophilia and jejunal mastocytosis occurred in wild-type and IgE-deficient animals. Despite the normal MC response in the small intestine, serum levels of mouse MC protease-1 also were lower in parasite-infected IgE(-/-) animals and these animals were slower to eliminate the adult worms from the small intestine. The number of T. spiralis larvae present in the skeletal muscle of IgE(-/-) mice 28 days after primary infection was about twice that in BALB/c controls, and the fraction of larvae that was necrotic was reduced in the IgE-deficient animals. An intense deposition of IgE in and around the muscle larvae was observed in wild-type but not in IgE null mice. We conclude that IgE promotes parasite expulsion from the gut following T. spiralis infection and participates in the response to larval stages of the parasite. Furthermore, our observations support a role for IgE in the regulation of MC homeostasis in vivo.     

Genetic and biochemical evidence for a critical role of Janus kinase (JAK)-3 in mast cell-mediated type I hypersensitivity reactions

We investigated the role of JAK3 in IgE receptor/FcepsilonRI-mediated mast cell responses. IgE/antigen induced degranulation and mediator release were substantially reduced with Jak3-/- mast cells from JAK3-null mice that were generated by targeted disruption of Jak3 gene in embryonic stem cells. Further, treatment of mast cells with 3'bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154), a potent inhibitor of JAK3, inhibited degranulation and proinflammatory mediator release after IgE receptor/ FcepsilonRI crosslinking. Thus, JAK3 plays a pivotal role in IgE receptor/ FcepsilonRI-mediated mast cell responses and targeting JAK3 may provide the basis for new and effective treatment as well as prevention programs for mast cell-mediated allergic reactions.     

Fusion of the Mycobacterium tuberculosis antigen 85A to an oligomerization domain enhances its immunogenicity in both mice and non-human primates

To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4(+) and CD8(+) T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-γ responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability.     

Long-lived Th2 memory in experimental allergic asthma

Although life-long immunity against pathogens is beneficial, immunological memory responses directed against allergens are potentially harmful. Because there is a paucity of information about Th2 memory cells in allergic disease, we established a model of allergic asthma in BALB/c mice to explore the generation and maintenance of Th2 memory. We induced disease without the use of adjuvants, thus avoiding Ag depots, and found that unlike allergic asthma in mice immunized with adjuvant, immunizing with soluble and aerosol OVA resulted in pathological lung lesions resembling human disease. To test memory responses we allowed mice with acute disease to recover and then re-exposed them to aerosol OVA a second time. Over 400 days later these mice developed OVA-dependent eosinophilic lung inflammation, airway hyperresponsiveness, mucus hypersecretion, and IgE. Over 1 year after recuperating from acute disease, mice had persistent lymphocytic lung infiltrates, Ag-specific production of IL-4 and IL-5 from spleen and lung cells in vitro, and elevated IgG1. Moreover, when recuperated mice were briefly aerosol challenged, we detected early expression of Th2 cytokine RNA in lungs. Taken together, these data demonstrate the presence of long-lived Th2 memory cells in spleen and lungs involved in the generation of allergic asthma upon Ag re-exposure.     

Reaginic antibody formation in the mouse. VII. Induction of suppressor T cells for IgE and IgG antibody responses.

Intravenous injections of urea-denatured ovalbumin (UD-OA) into OA-primed high responder mice suppressed the antibody response not only to the priming antigen but also to subsequent immunization with dinitrophenyl derivatives of OA (DNP-OA). The transfer of normal spleen cells or OA-primed spleen cells into UD-OA-treated animals did not restore the capacity of responding to DNP-OA to form anti-DNP IgE and IgG antibodies. The transfer of splenic T cell fraction from the UD-OA-treated animals into normal syngeneic mice diminished both IgE and IgG antibody responses of the recipients to DNP-OA. The B cell-rich fraction from the same donors failed to affect the anti-hapten antibody response and enhanced anti-cancer (OA) IgG antibody response of the recipients. It was also found that the transfer of T cell-rich fraction of OA-primed spleen cells failed to suppress antibody response of the recipients to DNP-OA. The results indicated that spleen cells of UD-OA-treated mice contained suppressor T cells which are distinct from helper cells. Suppressive activity of T cells in the UD-OA treated animals was specific for OA. The transfer of the T cell-rich fraction failed to suppress anti-DNP antibody response of the recipients to DNP-KLH.