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.     

Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1-/-) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-beta 1-/- mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1-/- mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1-/- mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.     

Oral tolerance revisited: prior oral tolerization abrogates cholera toxin-induced mucosal IgA responses

Oral delivery of a large dose or prolonged feeding of protein Ags induce systemic unresponsiveness most often characterized as reduced IgG and IgE Ab- and Ag-specific CD4(+) T cell responses. It remains controversial whether oral tolerance extends to diminished mucosal IgA responses in the gastrointestinal tract. To address this issue, mice were given a high oral dose of OVA or PBS and then orally immunized with OVA and cholera toxin as mucosal adjuvant, and both systemic and mucosal immune responses were assessed. OVA-specific serum IgG and IgA and mucosal IgA Ab levels were markedly reduced in mice given OVA orally compared with mice fed PBS. Furthermore, when OVA-specific Ab-forming cells (AFCs) in both systemic and mucosa-associated tissues were examined, IgG AFCs in the spleen and IgA AFCs in the gastrointestinal tract lamina propria of mice given OVA orally were dramatically decreased. Furthermore, marked reductions in OVA-specific CD4(+) T cell proliferative and cytokine responses in spleen and Peyer's patches were seen in mice given oral OVA but were unaffected in PBS-fed mice. We conclude that high oral doses of protein induce both mucosal and systemic unresponsiveness and that use of mucosal adjuvants that induce both parenteral and mucosal immunity may be a better way to assess oral tolerance.     

Humoral immune responses to microbial infections in the genital tract

Human reproductive tracts represent components of the mucosal immune system with unique features. Although secretory IgA is present, IgG is more abundant, and typical mucosa-associated lymphoid tissue for generating common mucosal immune responses is absent. Antibody responses to genital infections or to locally applied vaccines are usually modest, but alternative strategies for eliciting genital tract antibodies are being developed.     

Adipose-Derived Mesenchymal Stem Cells Restore Impaired Mucosal Immune Responses in Aged Mice

It has been shown that adipose-derived mesenchymal stem cells (AMSCs) can differentiate into adipocytes, chondrocytes and osteoblasts. Several clinical trials have shown the ability of AMSCs to regenerate these differentiated cell types. Age-associated dysregulation of the gastrointestinal (GI) immune system has been well documented. Our previous studies showed that impaired mucosal immunity in the GI tract occurs earlier during agingthan is seen in the systemic compartment. In this study, we examined the potential of AMSCs to restore the GI mucosal immune system in aged mice. Aged (>18 mo old) mice were adoptively transferred with AMSCs. Two weeks later, mice were orally immunized with ovalbumin (OVA) plus cholera toxin (CT) three times at weekly intervals. Seven days after the final immunization, when fecal extract samples and plasma were subjected to OVA- and CT-B-specific ELISA, elevated levels of mucosal secretory IgA (SIgA) and plasma IgG antibody (Ab) responses were noted in aged mouse recipients. Similar results were also seen aged mice which received AMSCs at one year of age. When cytokine production was examined, OVA-stimulated Peyer’s patch CD4⁺ T cells produced increased levels of IL-4. Further, CD4⁺ T cells from the lamina propria revealed elevated levels of IL-4 and IFN-γ production. In contrast, aged mice without AMSC transfer showed essentially no OVA- or CT-B-specific mucosal SIgA or plasma IgG Ab or cytokine responses. Of importance, fecal extracts from AMSC transferred aged mice showed neutralization activity to CT intoxication. These results suggest that AMSCs can restore impaired mucosal immunity in the GI tract of aged mice.     

Thymic control of secretory antibody responses in the rat.

The effect of neonatal thymectomy on salivary and serum antibody responses was studied in rats. Local immunization of thymectomized rats with a T-dependent antigen (DNPBGG) elicited negligible amounts of IgA anti-DNP antibody in saliva. In contrast, both normal and sham-thymectomized animals demonstrated substantial levels of salivary IgA antibody. All thymectomized rats locally injected with a T-independent antigen (DNP-Lys-Ficoll) exhibited salivary IgA antibody production. Salivary IgG antibodies were somewhat decreased in thymectomized rats injected with either antigen; however, the final effect of T cell deprivation on IgG synthesis was not as pronounced as on IgA synthesis. Serum IgA antibody was induced in control rats injected with DNPBGG, whereas this Ig class of antibody was absent in thymectomized rats. The results suggest that thymus-derived cells exert a regulatory influence on both serum and secretory IgA responses to antigens.     

Dual mechanisms of potentiation of murine antigen-specific IgE production by cyclosporin A in vitro.

Concomitant administration of cyclosporin A (CsA) with Ag has been shown to augment the production of Ag-specific IgE in vivo. We demonstrate that addition of CsA also markedly potentiated Ag-specific IgE in vitro. Low doses of CsA (3 and 10 ng/ml) added at the time of culture initiation selectively enhanced Ag-specific IgE but not IgA or IgG1 production, whereas higher doses (30 ng/ml) suppressed production of all the isotypes. Augmented IgE production was found to correlate with enhanced production of IL-4 and diminished production of IFN-gamma. Delayed addition (after 2 days) of low doses of CsA to Ag-stimulated cultures did not potentiate IgE production, even though CsA differentially affected levels of IL-4 and IFN-gamma. CsA enhanced Ag-mediated cognate T/B interaction was not affected by neutralizing doses of anti-IL-4, suggesting Ag-mediated lymphocytic "synapses" may be inaccessible to anti-IL-4. The effect of CsA on Ag presentation was determined by pulsing peritoneal exudate cells, spleen cells, or primed B cells with Ag and low doses of CsA before incubation with primed splenocytes. Enhanced Ag-specific IgE responses were detected with no effect on IL-4 or IFN-gamma levels. Thus, our study indicates that CsA potentiation of Ag-specific IgE response is due to cumulative action of CsA on two independent pathways: first, CsA differentially modulates IL-4 and IFN-gamma levels during the early phase of cognate Th2/B cell interaction; and second, CsA directly affects APC and IgE isotype-specific amplifying cellular components without apparently affecting the secretory levels of IL-4 and IFN-gamma. Dual mechanisms of CsA-potentiated IgE production are consistent with the hypothesis of two-tiered T cell regulation of Ag-specific IgE responses.     

Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes

A murine model of IgA deficiency has been established by targeted deletion of the IgA switch and constant regions in embryonic stem cells. B cells from IgA-deficient mice were incapable of producing IgA in vitro in response to TGF-beta. IgA-deficient mice expressed higher levels of IgM and IgG in serum and gastrointestinal secretions and decreased levels of IgE in serum and pulmonary secretions. Expression of IgG subclasses was complex, with the most consistent finding being an increase in IgG2b and a decrease in IgG3 in serum and secretions. No detectable IgA Abs were observed following mucosal immunization against influenza; however, compared with those in wild-type mice, increased levels of IgM Abs were seen in both serum and secretions. Development of lymphoid tissues as well as T and B lymphocyte function appeared normal otherwise. Peyer's patches in IgA-deficient mice were well developed with prominent germinal centers despite the absence of IgA in these germinal centers or intestinal lamina propria. Lymphocytes from IgA-deficient mice responded to T and B cell mitogens comparable to those of wild-type mice, while T cells from IgA-deficient mice produced comparable levels of IFN-gamma and IL-4 mRNA and protein. In conclusion, mice with targeted deletion of the IgA switch and constant regions are completely deficient in IgA and exhibit altered expression of other Ig isotypes, notably IgM, IgG2b, IgG3, and IgE, but otherwise have normal lymphocyte development, proliferative responses, and cytokine production.     

IgE class switching is critically dependent upon the nature of the B cell activator, in addition to the presence of IL-4.

Cross-linkage of membrane IgD on resting murine B cells, by anti-IgD mAb conjugated to dextran (alpha delta-dex), induces high levels of proliferation, and in the presence of IL-2 or IL-5, Ig secretion in vitro. The structural and functional similarities between alpha delta-dex and TNP-Ficoll for B cell responses led us to propose that alpha delta-dex could provide a model system for studying B cell activation induced by T cell-independent, type II Ag. In this report, we study the effects of Ig class switch and differentiation factors on Ig isotype production by murine B cells activated by alpha delta-dex, and directly compare these to responses obtained after activation by LPS. We show that an IL-4-containing CD4+ T cell supernatant (Th2 SN) stimulates large increases in IgG1 and IgE production by LPS-activated B cells, but fails to stimulate detectable levels of IgE by alpha delta-dex-activated cells, despite inducing high levels of secreted IgM and IgG1. This is correlated with undetectable steady state levels of both germ-line and rearranged (productive) IgE-specific RNA in B cells stimulated with alpha delta-dex + Th2 SN. Alpha delta-dex is selective in its failure to costimulate IgE production in that IFN-gamma-containing T cell supernatant (Th1 SN) and transforming growth factor-beta-supplemented Th2 SN selectively stimulate a large IgG2a and IgA secretory response, respectively. Anti-IgD conjugated to Sepharose beads, in distinct contrast to dextran, costimulates a strong IgE response. These findings underscore the importance of the specific B cell activator, in addition to IL-4, in the regulation of IgE production.     

Evidence for differential induction of helper T cell subsets during Trichinella spiralis infection

The H-2-compatible mouse strains, AKR and B10.BR, exhibit disparate responses to infection with the parasitic nematode Trichinella spiralis. The resistant AKR mice expel intestinal adult worms faster than susceptible B10.BR mice. We tested antibody and lymphokine responses in these strains. With respect to antibody responses, the B10.BR mice had 3- to 10-fold more serum IgE and T. spiralis-specific IgG1 and IgA than AKR mice. The B10.BR mice also had greater numbers of IgG and IgA plaque-forming cells than AKR mice. In contrast, AKR mice produced T. spiralis-specific IgG2a, whereas the B10.BR mice did not. The antibody response kinetics of these strains were similar. We also analyzed lymphokine secretion after restimulating lymphocytes in vitro with T. spiralis Ag. The AKR mesenteric lymph node cells produced more IFN-gamma and less IL-4 than the B10.BR mesenteric lymph node cells. The B10.BR splenocytes produced more IL-4 than the AKR splenocytes, although splenocyte IFN-gamma production was not different. The kinetics of IL-4 production also differed between the two strains. In summary, resistant AKR mice produced more IFN-gamma and T. spiralis-specific IgG2a than susceptible B10.BR mice, which produced more IL-4, IgE, and T. spiralis-specific IgG1. Our results are consistent with differential activation of Th cell subsets in T. spiralis-infected AKR and B10.BR mice.     

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.     

Costimulation blockade inhibits allergic sensitization but does not affect established allergy in a murine model of grass pollen allergy

Type I allergy is characterized by the development of an initial Th2-dependent allergen-specific IgE response, which is boosted upon a subsequent allergen encounter. Although the immediate symptoms of allergy are mainly IgE-mediated, allergen-specific T cell responses contribute to the late phase as well as to the chronic manifestations of allergy. This study investigates the potential of costimulation blockade with CTLA4Ig and an anti-CD154 mAb for modifying the allergic immune response to the major timothy grass pollen allergen Phl p 5 in a mouse model. BALB/c mice were treated with the costimulation blockers at the time of primary sensitization to the Phl p 5 allergen or at the time of a secondary allergen challenge. Costimulation blockade (CTLA4Ig plus anti-CD154 or anti-CD154 alone) at the time of sensitization prevented the development of allergen-specific IgE, IgM, IgG, and IgA responses compared with untreated but sensitized mice. However, costimulation blockade had no influence on established IgE responses in sensitized mice. Immediate-type reactions as analyzed by a rat basophil leukemia cell mediator release assay were only suppressed by early treatment but not by a costimulation blockade after sensitization. CTLA4Ig given alone failed to suppress both the primary and the secondary allergen-specific Ab responses. Allergen-specific T cell activation was suppressed in mice by early as well as by a late costimulation blockade, suggesting that IgE responses in sensitized mice are independent of T cell help. Our results indicate that T cell suppression alone without active immune regulation or a shifting of the Th2/Th1 balance is not sufficient for the treatment of established IgE responses in an allergy.     

Fasciola hepatica: humoral and cytokine responses to a member of the saposin-like protein family following delivery as a DNA vaccine in mice

The humoral and cellular responses to DNA vaccination of BALB/c mice with a novel antigen from the Fasciola hepatica saposin-like protein family (FhSAP-2) have been investigated. Two constructs were produced containing the FhSAP-2 DNA sequence, one intended for extracellular secretion of FhSAP-2 protein, and one expressing FhSAP-2 in the cytoplasm of a transfected cell. The constructs were tested in HEK 293T cells, with the secretory construct producing less detectable FhSAP-2 relative to cytoplasmic construct when observed by fluorescence. The size of expressed protein was confirmed by Western blot of cell lysate, but FhSAP-2 was undetectable in cell supernatants. Both, secretory and cytoplasmic constructs as well as FhSAP-2 recombinant protein were tested in mice. The antibody response elicited in mice vaccinated with the rFhSAP-2 induced high levels of IgG(1), IgG(2), and IgE as well as high levels of IL-10 and IFNgamma indicating a mixed Th1/Th2 response. Vaccination of mice intramuscularly with the cytoplasmic FhSAP-2 construct resulted in a dominant IgG(2a) isotype antibody as well as a dominant IFNgamma cytokine, with significant IgE, IgG(1), and IL-10 responses also present, suggesting a mixed Th1/Th2 profile. Isotype and cytokine profiles elicited by the FhSAP-2 secretory construct were similar to those obtained with the cytoplasmic construct but at levels that were significantly lower. The results demonstrate that FhSAP-2 can be delivered as a DNA vaccine construct and induces a stronger Th1 response than the recombinant protein alone. This could result in an improvement in the immunoprophylactic potential of this candidate vaccine against F. hepatica.     

Blood concentrations of Th2-type immunoglobulins are selectively increased in weanling mice subjected to acute malnutrition

Male and female C57BL/6J mice, initially 19 days old, consumed a complete purified diet either ad libitum (age-matched control) or in restricted daily quantities (energy deficiency), or they consumed a purified isocaloric low-protein diet ad libitum (protein and energy deficit). In a 14-day experimental period, malnourished animals lost approximately 1.5% of their initial body weight daily. Zero-time controls, 19 days old, were also included in the study. Serum levels of Th2-type (IgG1 and IgE) and Th1-type (IgG2a and IgG3) immunoglobulins were quantified by enzyme-linked immunosorbent assay, and total IgG concentration was also assessed. Both malnourished groups exhibited high serum concentrations of IgG1 and IgE relative to the age-matched control group, whereas levels of the Th1-type immunoglobulins were unaffected. Total IgG concentration in the malnourished groups reflected the usual finding in humans (i.e., no effect or elevated). The results are consistent with the proposition of Th2-polarized immune competence in acute weanling deficiencies of energy, protein, or both.     

Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin

Cholera toxin (CT) has been found to be an extremely potent immunogen for mucosal IgA responses when administered via the intestine. This study has examined both mucosal and systemic immune responses after feeding CT and compared these responses with those obtained after feeding keyhole limpet hemocyanin (KLH), another protein that is strongly immunogenic in mice. Feeding CT to mice resulted not only in IgA antibody in intestinal secretions but also resulted in substantial plasma IgG and IgA antibody levels. Feeding KLH in much larger quantity resulted in little or no antibody response in intestinal secretions or plasma. Lymphoid cells from various tissues of mice fed CT were cultured in vitro for 10 days and the supernatant was tested for antibody to CT. Spontaneous antibody synthesis (no antigen added to cultures) was present in cultures of each cell type, but IgG anti-CT was found mainly in cultures of spleen and mesenteric lymph node cells and IgA anti-CT mainly in cultures of Peyer's patch and lamina propria cells. Peyer's patch cells cultured with CT as antigen synthesized both IgG and IgA anti-CT, suggesting that the antibody response to both isotypes originated in this site. Helper T cell activity for both IgA and IgG anti-CT was detected in spleens, mesenteric lymph nodes, and Peyer's patches. Lastly, when KLH and CT were fed to mice at the same time, an intestinal IgA anti-KLH and plasma IgG anti-KLH response was stimulated, a response pattern similar to that occurring to CT after CT was fed alone. We conclude that mucosal stimulation by CT generates both a systemic IgG and mucosal IgA response to this antigen, and that CT can cause a similar pattern of response to an unrelated protein antigen when both are administered into the intestine at the same time. The data favor the idea that both the IgG and IgA responses originate in GALT and then disseminate to other tissues. We propose that CT accomplishes these effects by altering the regulatory environment within GALT.     

IL-4 is required to generate and sustain in vivo IgE responses

Antibodies of the IgE isotype play a predominant role in immediate hypersensitivity reactions. IL-4, a T cell-derived lymphokine that stimulates increased Ia expression by resting B cells and increased IgG1 secretion by LPS-activated B cells in vitro, has also been shown to regulate in vitro and in vivo polyclonal IgE responses. We report that large quantities of a purified anti-IL-4 mAb inhibit primary in vivo polyclonal IgE responses by 99% in mice infected with Nippostrongylus brasiliensis or injected with anti-IgD antibodies, and totally inhibit secondary Ag-specific IgE responses to TNP-keyhole limpet hemocyanin without effect on either IgG1 or IgG2a responses to these stimuli. The lack of effect of anti-IL-4 antibody on IgG1 secretion cannot be explained simply by inadequate neutralization of IL-4, inasmuch as the doses of anti-IL-4 antibody used blocked an N. brasiliensis-induced increase in B cell Ia expression by more than 85%, whereas in vitro studies indicate that enhancement of B cell Ia expression requires less IL-4 than induction of IgG1 secretion. In addition to demonstrating that IL-4 plays a necessary role in the generation of an in vivo IgE response, we show that IL-4 has an important role in sustaining established IgE responses, because anti-IL-4 antibody, when given at the peak of an N. brasiliensis- or TNP-keyhole limpet hemocyanin-induced IgE response, accelerates the declines in total serum IgE and in IgE anti-TNP antibody levels, respectively. These observations suggest that the effects of IL-4 on in vivo immune responses may be more specific than might have been predicted from in vitro observations, and that regulation of IL-4 production or action may be useful for the prevention or therapy of immediate hypersensitivity disorders.     

Preferential role for NF-kappa B/Rel signaling in the type 1 but not type 2 T cell-dependent immune response in vivo.

T cell function is a critical determinant of immune responses as well as susceptibility to allergic diseases. Activated T cells can differentiate into effectors whose cytokine profile is limited to type 1 (IFN-gamma-dominant) or type 2 (IL-4-, IL-5-dominant) patterns. To investigate mechanisms that connect extracellular stimuli with the regulation of effector T cell function, we have measured immune responses of transgenic mice whose NF-kappa B/Rel signaling pathway is inhibited in T cells. Surprisingly, these mice developed type 2 T cell-dependent responses (IgE and eosinophil recruitment) in a model of allergic pulmonary inflammation. In contrast, type 1 T cell responses were severely impaired, as evidenced by markedly diminished delayed-type hypersensitivity responses, IFN-gamma production, and Ag-specific IgG2a levels. Taken together, these data indicate that inhibition of NF-kappa B can lead to preferential impairment of type 1 as compared with type 2 T cell-dependent responses.     

B cell stimulatory factor-1 enhances the IgE response of lipopolysaccharide-activated B cells

Supernatants from some mouse helper T cell (TH) lines contain an activity that can enhance IgE production by lipopolysaccharide (LPS)-stimulated B cells by at least two orders of magnitude. During purification, this activity could not be resolved from B cell stimulatory factor-1 (BSF-1). Highly purified BSF-1 from a different source, the T lymphoma cell line EL-4, enhanced IgE production to the same extent as TH supernatants, which suggests that BSF-1 is responsible for this increase in IgE production. Monoclonal antibody to BSF-1 totally inhibits the IgE-enhancing activity of a TH supernatant, lending further support to this conclusion. The effects of BSF-1 on LPS-stimulated B cells are specific for IgE and, as previously reported, IgG1 and IgG3, because the levels of IgM, IgG2a, IgG2b, and IgA in the cultures change relatively little when BSF-1 is added.     

TGF-beta receptor signaling is critical for mucosal IgA responses

TGF-beta receptor (TbetaR) signaling is important for systemic IgA production; however, its contribution to IgA secretion at mucosal sites remained uncertain. This important question was addressed using mice lacking the TbetaR in B cells (TbetaRII-B). Although reduced, IgA-secreting cells and IgA were still present in the systemic and mucosal compartments. The adaptive immune response was investigated after oral or nasal immunization using adjuvants acting on different molecular targets, namely, the cholera toxin B subunit and the macrophage-activating lipopeptide-2. Efficient Ag-specific cellular and humoral responses were triggered both in controls and TbetaRII-B mice. However, a significant reduction in Ag-specific IgG2b and increased levels of IgG3 were observed in sera from TbetaRII-B mice. Furthermore, Ag-specific IgA-secreting cells, serum IgA, and secretory IgA were undetectable in TbetaRII-B mice. These results demonstrate the critical role played by TbetaR in Ag-driven stimulation of secretory IgA responses in vivo.     

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.