Suppression of reaginic antibodies with modified allergens. II. Abrogation of reaginic antibodies with allergens conjugated to polyethylene glycol

The two immunogenic preparations, ovalbumin (OA) and the nondialysable constituents of the aqueous extract of ragweed pollen (RAG), were conjugated with polyethylene glycols of molecular weights of 6,000 and 20,000 (PEG6 and PEG20) with the aid of cyanuric chloride. The i.v. administration of OA-PEG6 or OA-PEG20 into normal mice or into mice sensitized to a state of immediate hypersensitivity to dinitrophenylated OA (DNP3-OA) resulted, respectively, in the suppression of the primary or secondary IgE responses to DNP and OA. Similarly, the administration of RAG-PEG6 abrogated the primary as well as the ongoing anti-RAG reaginic responses in mice sensitized to RAG. The unresponsiveness of spleen cells from animals which had received a tolerogenic dose of OA-PEG6 was also maintained after transfer into X-irradiated (550 rad) mice. The suppressive effects of the tolerogenic PEG conjugates of OA and RAG were shown to be immunologically specific. It is, therefore, suggested that PEG-modified allergens may prove useful for the abrogation of the IgE response to a variety of allergens responsible for conditions of common hypersensitivity in man.     

A canine model for the study of hapten-specific suppression of IgE-mediated bronchoconstriction and anaphylaxis

Newborn mongrel dogs were sensitized with conjugates of ovalbumin (OA) and 2,4-dinitrophenol (OA-DNP3) in the presence of Al(OH)3 to produce high levels of anti-OA and anti-DNP IgE antibody. At 4-6 months of age, when anti-DNP and anti-OA antibody levels reached titers of 64 by passive cutaneous anaphylaxis, the dogs underwent separate inhalation and intravenous challenges with conjugates of DNP and bovine gamma globulin (DNP15-BGG) and OA. Inhalation challenge with DNP15-BGG and OA resulted in 5- and 10-fold increases in airflow resistance, respectively. Intravenous challenge with either DNP15-BGG or OA produced profound anaphylaxis with 60-80% decreases in blood pressure, cardiac output and regional blood flows in the carotid, superior mesenteric and renal arteries, and the distal aorta. Treatment of sensitized dogs with 5 doses of 20 mg of conjugates of DNP and polyvinyl alcohol (DNP2-PVA) on alternate days resulted in suppression of anti-DNP IgE antibody production; abrogation of established airway and vascular anaphylactic sensitivities; no change in regional blood flows, and no effect on sensitivities to challenge with OA.     

The effect of hapten-specific suppression of IgE on antigen-induced histamine release from mouse peritoneal mast cells.

Subcutaneous injections of a mixture of dinitrophenylated ovalbumin (DNP3-OA) and dextran sulfate into Swiss-Webster mice elicited short-lived primary and long-lasting secondary IgE antibody responses to both DNP and OA. Histamine was released on in vitro challenge with antigen (OA or DNP22-BSA) of washed peritoneal mast cells (PMC) obtained from mice during a primary or a secondary IgE response. Administration of an intravenous injection of a tolerogenic conjugate of DNP8-mouse gamma-globulin, either prior to immunizationor during an ongoing IgE response, resulted in almost complete disappearance of circulating anti-DNP IgE antibody and in a very marked decrease in histamine release from PMC on challenge with DNP22-BSA. However, the IgE response to OA of these mice and the histamine release from their PMC on challenge with OA were not affected. Moreover, the PMC of mice, which had been tolerized to DNP, could be passively sensitized with serum containing DNP-specific IgE antibody for the release of histamine on DNP22-BSA challenge. The most significant finding of this study is the observation that the time course for the loss of reactivity of PMC to DNP22-BSA, after administration of the tolerogen during an ongoing secondary response, paralleled the decrease in circulating anti-GNP IgE antibody.     

Suppression of IgE antibody response by the fatty acid-modified antigen

Ovalbumin (OA) of hens was chemically coupled with fatty acids (lauric acid, myristic acid, palmitic acid and stearic acid). These hydrophobically modified antigens were unable to react with mouse antiserum against native OA and were incapable of eliciting primary and secondary anti-OA antibody responses in BALB/c mice. Preadministration of these modified antigens, especially of palmitoyl OA (OA-pal), suppressed both primary and secondary anti-OA IgE antibody responses without affecting IgG antibody production. Administration of OA-pal after the primary immunization resulted in a rapid decrease of the ongoing anti-OA IgE antibody production and inhibited the anamnestic anti-OA IgE antibody response upon subsequent immunization with OA. The passive transfer of spleen cells from OA-pal-treated animals with OA-primed spleen cells suppressed the adoptive secondary anti-OA IgE antibody response in irradiated recipients. The suppressive effect was abrogated by treatment with an anti-T-cell antiserum indicating that suppressor T cells were primed by administration of hydrophobically modified antigens.     

Down-regulation of secondary in vitro antibody responses by suppressor T cells of mice treated with a tolerogenic conjugate of ovalbumin and monomethoxypolyethylene glycol, OVA(mPEG)13

In previous studies from this laboratory it was shown that OVA(mPEG)n conjugates induced: (i) tolerance in mice with respect to IgG and IgE antibody responses to dinitrophenylated OVA (DNP-OVA); and (ii) OVA-specific suppressor T (Ts) cells which could down-regulate a primary immune response in vivo. For the present study, we have developed an in vitro culture system for assessing the activity of Ts cells of mice tolerized by an OVA(mPEG)13 conjugate. Spleen cells from mice which had been primed with DNP4-OVA in Al(OH)3 gel were cultured with DNP4-OVA to induce a secondary antibody response in vitro. After 6 days, cells secreting anti-DNP antibodies of the IgG1 class were enumerated by an immunoenzymatic plaque-forming cell assay. Addition to the culture of T cells from mice treated with 3 i.p. injections of 500 micrograms of OVA(mPEG)13 resulted in a 29-61% reduction in the number of IgG1 anti-DNP antibody-forming cells, in comparison with the effect of T cells from mice treated with PBS. It was concluded that this tolerogenic conjugate induced splenic Ts cells which were capable of suppressing secondary in vitro anti-DNP responses.     

IgE-isotype-specific suppressor cells in the mouse: characterization using tetraparental chimera mice of high (DBA/2) and low (SJL) IgE responder embryos

Tetraparental chimera mice were developed by aggregation of IgE high responder (DBA/2) and IgE low responder (SJL) embryos. Anti-dinitrophenyl (DNP) IgE antibody response in such mice (SJL----DBA/2) upon challenge with DNP-keyhole-limpet hemocyanin (KLH) in alum was clearly suppressed, while anti-DNP IgG antibody response was not. High-titer anti-DNP IgE and IgG antibody response developed in F1 hybrid mice of SJL and DBA/2 (SDF1) mice. The experimental results suggest that high IgE antibody production is the dominant trait, and the IgE-specific suppressor gene in SJL mice is autosomal recessive. IgE-specific suppressor T cells in SJL mice actively suppressed IgE antibody formation by DBA/2 immuno-competent cells across the histocompatibility barrier. Hapten-specific B cells and carrier-specific T cells were prepared in SJL----DBA/2 and SDF1 mice by immunization with DNP-KLH or ovalbumin (OA) in alum and transferred to irradiated SDF1 mice followed by challenge with DNP-OA. Hapten-specific B cells and carrier-specific helper T cells clearly developed in SDF1 mice. Recipient mice transferred with DNP-KLH-primed SDF1 spleen cells and OA-primed SDF1 spleen cells showed high-titer anti-DNP IgE and IgG antibody responses. OA-primed SJL----DBA/2 spleen cells cotransferred with DNP-KLH-primed SDF1 spleen cells and OA-primed SDF1 spleen cells completely abolished secondary anti-DNP IgE antibody response. The data suggest that carrier-specific helper T cells for IgE and IgG antibody responses are distinct. The regulatory role of IgE-isotype-specific suppressor cells were considered to be the interference of cooperative cellular interaction between IgE B cells and carrier-specific, IgE-specific helper T cells.     

The use of polyvinyl alcohols as tolerogenic carriers for suppression of anti-hapten IgE antibody responses

Conjugates of haptens [i.e. the dinitrophenyl (DNP) or benzylpenicilloyl (BPO) groups] with polyvinyl alcohols (PVA), with molecular weights in the range of 10–14 × 10³ daltons, were shown to suppress not only the primary anti-hapten IgE and hemagglutinating antibody responses of B₆D₂F₁ mice but, more importantly, also the ongoing antibody responses of presensitized mice. For this study, mice were immunized with the corresponding haptenated-ovalbumin (OA) conjugates (i.e. DNP₃-OA and BPO₄-OA) in presence of Al(OH)₃. In addition to the fact that the tolerogenic PVA-hapten conjugates did suppress exclusively hapten-specific responses, i.e. without affecting the anti-OA IgE antibody levels, their remarkable feature was that they were immunosuppressive even at the low average epitope density of about one. The immunosuppressive effectiveness of these tolerogenic conjugates was dose dependent, e.g. complete suppression of ongoing anti-hapten IgE responses was achieved with a single dose of 1 mg of the appropriate hapten-PVA conjugate.     

Suppression of reaginic antibodies with modified allergens. I. Reduction in allergenicity of protein allergens by conjugation to polyethylene glycol.

The conjugates of ovalbumin (OA) and of the nondialysable constitutents of the aqueous extract of ragweed pollen (RAG) with polyethylene glycols of molecular weights of 6,000 or 20,000 (PEG6 or PEG20) were shown to be nonantigenic, nonallergenic and nonimmunogenic. Thus, the i.v. administration of OA-PEG and RAG-PEG conjugates into mice did not elicit antibodies to OA and RAG, respectively, and these conjugates were shown to suppress in an immunologically specific manner the capacity of these animals to mount primary as well as secondary IgE responses to sensitizing doses of dinitrophenylated OA or of RAG. Moreover, the Peg-modified antigens did not combine either in vitro or in vivo with IgE antibodies directed against the natural antigens. Hence, OA-PEG and RAG-PEG conjugates were incapable of triggering allergic reactions in animals possessing IgE antibodies to the unmodified antigens. These PEG-modified antigens were also shown to be tolerogenic.     

Suppression of IgE antibody production in sensitized mice and rats by tolerogenic conjugates of synthetic hydrophilic polymers with antigen or hapten: effect on antigen-induced histamine release from peritoneal mast cells

IgE antibodies were produced in mice and rats by immunization with ragweed pollen extract (RAG) or dinitrophenylated ovalbumin (DNP3-OA). Treatment of these animals with tolerogenic conjugates of (i) the antigen (RAG or OA) with monomethoxypolyethylene glycol (mPEG), or (ii) DNP with polyvinyl alcohol (DNP-PVA) resulted, within 7-14 days, in a fall in circulating IgE antibodies and in mast cell sensitivity, as assessed by the radioallergosorbent test (RAST) and in the in vitro antigen-induced histamine release (HR) test, respectively. The reduction in responsiveness was more marked in mice than rats; 10 days after a booster immunization, the IgE antibody titres in the RAG-mPEG-treated group of mice were approximately 10-fold lower than in the saline-treated group, with a 100-fold difference in cell sensitivity. DNP-PVA treatment of mice produced a more than 10-fold reduction in IgE anti-DNP titres with a substantial reduction in histamine release.     

Effects of tolerogenic conjugates in a canine model for reaginic hypersensitivity. I. suppression of hapten-specific IgE antibody response

Intraperitoneal administration to dogs of conjugates consisting of the 2,4-dinitrophenyl (DNP) groups coupled to nonimmunogenic macromolecules such as the copolymer of D-glutamic acid and D-lysine (DNP16-DGL) prior to sensitization with DNP2-ovalbumin led to the development of hapten-specific tolerance with respect to the IgE antibody response. Administration of these same conjugates to sensitized dogs resulted in complete abrogation of the ongoing anti-DNP IgE antibody production. A similar hapten-specific suppression of the ongoing anti-DNP response was also observed using the conjugates of DNP9-canine gamma globulins, and the tolerogenic effect was dose-dependent. The state of hapten-specific immunosuppression induced by these two types of tolerogenic conjugates was maintained despite repeated booster injections of the sensitizing antigens at biweekly inervals.     

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. A. Immune response to antigens. I. Role of subcutaneously injected IgE-producing hybridoma on antibody production of different isotypes against immunizing antigens

When immunized with ovalbumin (OVA), mice bearing subcutaneously injected anti-dinitrophenyl (DNP) IgE-secreting hybridoma cells do not produce anti-OVA IgE antibodies as long as anti-DNP IgE is present in abundance in their sera. As soon as the titer of anti-DNP IgE antibody falls, anti-OVA IgE antibody appears. This inhibition of production of anti-OVA IgE is attributed to Fc epsilon-bearing T cells which have a suppressive action of limited duration. Other isotypes of anti-OVA antibodies are produced in similar amounts as in controls which were immunized with OVA but not injected with hybridoma cells.     

Antigen- and IgE class-specific suppression mediated by T suppressor cells of mice treated with glutaraldehyde-polymerized ovalbumin

Various size polymers are obtained following glutaraldehyde treatment of native ovalbumin (OA). OA-POL, approximately 35 X 10(6) daltons, was prepared at the isoelectric point of OA. Treatment of CBA mice with microgram amounts of OA-POL led to efficient antigen-specific suppression of IgE responses. IgG anti-OA antibodies were not suppressed. Transfer of cells from OA-POL-treated donors into normal, unprimed recipients interfered with the ability of these animals to mount a primary or secondary IgE response. In addition, cotransfer of spleen cells from OA-POL-treated mice along with OA (in alum)-primed cells, into irradiated syngeneic recipients resulted in IgE class-specific suppression that was abrogated by treatment of OA-POL donor cells with monoclonal anti-Thy 1.2 + complement. The presence or absence of T cells in the OA-POL population had no effect on IgG levels in the recipients. Analysis of the antigenic properties of OA-POL revealed 5-15% cross-reactivity with native OA as perceived by IgG or IgE antibodies. In contrast, OA-POL was highly cross-reactive at the T cell level as shown functionally by its potent induction of OA-specific, IgE-selective suppressor T cells. The results suggest that the beneficial effects of glutaraldehyde-modified allergens, recently introduced in the immunotherapy of atopic individuals may be due to the preferential exposure on the polymerized protein, of antigenic determinants generating T suppressor cells and to the selective loss of B cell-reactive determinants.     

Regulation of the IgE antibody response in mice. II. Radioresistance of established IgE antibody production.

The protracted IgE anti-ovalbumin (OA) response given by BDF1 mice was studied using an adoptive transfer model. Spleen cells taken from immunized BDF1 mice can produce IgE antibody in irradiated recipients without further overt antigenic challenge. Depletion of macrophages in active spleen cell suspensions did not diminish the capacity of the remaining cells to give an adoptive response. Evidently the cells subserving the adoptive response are not fully developed in donor mice until 4 weeks after immunization, since spleen cells removed at shorter intervals after immunization gave either no or weak adoptive responses. The production of IgE antibody in irradiated recipient mice is prevented if transferred B or T lymphocytes are treated in vitro with either gamma irradiation or mitomycin C, suggesting proliferation of both B and T lymphocytes is essential for the adoptive response to develop. However, the requirement for proliferation is only transient, since one IgE antibody production reached a steady state in the adoptive recipients, it manifested extreme resistance to high dose irradiation. Whole body irradiation of 800 and 1000 rad was without effect on sustained IgE production. This latter observation was valid for both intact mice which were irradiated 8 weeks after immunization and also for irradiated adoptively immunized mice. It is suggested that the IgE anti-OA antibody measured in serum of BDF1 mice several months after immunization with 1 microgram OA and 1 mg Al(OH)3 is the product of long-lived antibody secreting cells.     

Antigen-specific suppressor cells in hapten-specific carrier-determined tolerance.

Tolerance to the hapten 2,4-dinitrophenyl (DNP) induced by the injection of DNP coupled to isologous IgG (carrier-determined tolerance) is associated with a receptor blockade of antigen-binding lymphocytes. In the present study, hapten-specific suppressor cells were detected in the spleens of mice made tolerant by intravenous injection of 20 microgram DNP-IgG. When spleen cells from mice rendered tolerant to DNP were co-cultured with normal spleen cells in Marbrook cultures, the response to DNP-Ficoll was suppressed, while the response to sheep red blood cells was not altered. Depletion of T cells from these spleens restored the normal anti-DNP response. The suppressor cells were not detectable in the spleen lymphocyte population of mice in the early stages of tolerance but were present on day 7 after injection of tolerogen, and disappeared by day 14. Mice injected with larger doses of 1 mg or four weekly doses of 200 microgram DNP-IgG did not have detectable suppressor cells. Thus, it appears that a short-lived suppressor T cell is generated in carrier-determined tolerance. This cell most likely plays a minor role in the mechanism of carrier-determined tolerance and may be associated with the receptor blockade which is seen early in tolerance.     

Induction of immunity and tolerance in vitro by hapten protein conjugates. II. Carrier independence of the response to dinitrophenylated polymerized flagellin

The response to dinitrophenylated polymeric flagellin (DNP POL) in vitro, unlike that to DNP monomeric flagellin (DNP MON) or erythrocyte antigens does not require the presence of T cells. It was thus proposed that antigens with repeating determinants, like DNP POL, may immunize B cells without the cooperation of the usual helper cells. Since carrier specificity in secondary anti-hapten responses is based upon the cooperation of carrier-reactive T cells and hapten-reactive cells, the requirement for carrier-reactive cells in the response to “thymus-independent” DNP POL and “thymus-dependent” DNP MON was investigated. Using flagellin-primed spleen, the anti-DNP response to DNP MON was enhanced, but not that caused by DNP POL. Furthermore, there was no carrier specificity in the anti-DNP responses with DNP POL, which elicited similar responses in spleen cells primed with various DNP proteins, whereas DNP MON only immunized spleen cells which were primed with that carrier. Carrier-hapten cooperation has also been demonstrated by the suppressive effect of unconjugated carrier on the anti-hapten response to carrier hapten conjugates. Neither the induction of tolerance to flagellin, the carrier, nor the presence of free flagellin in vitro suppressed the anti-DNP response to DNP POL. In contrast, both these manoeuvres suppressed the anti-flagellin response to DNP POL, and both the anti-DNP and anti-flagellin responses elicited by DNP MON. Thus, by several criteria, there was no cooperation between carrier-reactive and hapten-sensitive cells in the genesis of the response to DNP POL. Spleen cell suspensions deprived of their content of phagocytes responded normally to DNP POL. Thus, since T cells, carrier-reactive cells and phagocytes are not needed for the induction of a response to DNP POL, this antigen immunizes B cells directly. The use of this simple system should facilitate our understanding of the mechanism of binding of antigen molecules to the surface of B cells in the process of immunization.     

Antigenic competition in IgE antibody production. III. Suppressive effect on Th and B cells

An adoptive cell transfer system was utilized to evaluate the site of action of the suppressive mechanism involved in antigenic competition in IgE antibody production. Carrier-primed (OA) and hapten-primed (DNP-KLH) spleen cells were transferred to syngeneic irradiated recipients that were challenged with a heterologous conjugate (DNP-OA). To study the effect of antigenic competition on T and B cells, donor mice of one or the other cell type received in addition the competitor antigen (Asc) at immunization. The adoptive secondary IgE anti-DNP antibody response was suppressed in both situations. This effect could not be attributed to transfer of Asc-primed cells. Irradiation of donor mice before immunization with the two antigens abrogated the suppressive effect. These results indicate that both Th and B cells primed to the test antigen were affected by antigenic competition.     

Demonstration of long-lived memory T suppressor cells in the IgE response

Administration to mice of repeated small doses of glutaraldehyde-polymerized ovalbumin (OA-POL) results in drastic suppression of the IgE antibody response. The high anamnestic IgE antibody responses usually obtained after 2 injections of native OA in alum were completely abrogated when OA-POL was administered between the 2 immunizations. Treatment with OA-POL prior to a primary immunization with 2 micrograms DNP-OA in alum induced a profound suppression of both anti-DNP and anticarrier IgE responses. The suppression, which was detectable for at least 7 months, was antigen-specific, selective for the IgE class and was sensitive to treatment of spleen cells with anti-Thy-1.2 antibody and complement. Adoptive transfer of spleen cells from donors treated with OA-POL 200 days, 16 days or 200 and 16 days prior to transfer, resulted in different kinetics of suppression in the recipient. The results are consistent with the presence of long-lived IgE-selective T suppressor cell memory. The possibility that separate Ts and Ts memory cell populations are generated is discussed.     

Role of interferon-gamma in the modulation of the IgE response by 2,4-dinitrophenyl-Bordetella pertussis vaccine in the mouse

Immunization of mice with 2,4-dinitrophenyl-Bordetella pertussis (DNP-BP) failed to induce anti-DNP IgE responses. Administration of DNP-BP induced, however, the formation of anti-DNP IgE B memory cells, as demonstrated by adoptive transfer. Furthermore, mice pretreated with DNP-BP and primed with 2 micrograms DNP-ovalbumin (OA) in alum 2 weeks later produced high day-7 anti-DNP IgE levels. These subsided to near undetectable levels by day 12-14. The transient expression of serum IgE levels was accompanied by normal levels of anti-DNP IgG. The anti-OA response induced as a result of priming with DNP-OA in alum was not affected by pretreatment with DNP-BP. IgG subclass analysis revealed that mice pretreated with DNP-BP had elevated levels of IgG2a and reduced levels of IgG1 as compared to control (TNP-keyhole limpet hemocyanin-pretreated) mice. Treatment of mice with an anti-interferon-gamma monoclonal antibody, shortly after immunization with DNP-BP, not only reduced anti-DNP IgG2a levels, but prevented the sharp anti-DNP IgE decline that occurred after priming with DNP-OA in alum. These results suggest that DNP-BP-induced interferon-gamma production modulates Ig isotype expression in vivo in an anti-gen-specific manner.     

Suppression of IgE antibodies to the (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) group and induction of NIP-specific suppressor cells with NIP-poly-N-vinylpyrrolidone conjugates

The capacity of (C57BL/6 × DBA/2)F₁ mice to produce anti-(4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) IgE antibodies, as a result of immunization with 1 μg of NIP₂-ovalbumin (OA) in the presence of Al(OH)₃, was specifically suppressed by treatment of mice, either before or after immunization, with tolerogenic conjugates consisting of the hapten coupled to poly-N-vinylpyrrolidone (PVP) with an average mol. wt. of 10000. The suppression was hapten-specific, since it did not affect the immune response of the host to OA. The unresponsive state of the spleen cells of mice which had been tolerized with respect to NIP was maintained even after cell transfer into X-irradiated, syngeneic recipients and was shown to be due to hapten-specific suppressor cells. However, the splenic B cells of these mice did not possess any suppressive activity. The generation of an effective number of suppressor cells required between 1 and 2 days following the administration of the tolerogen. The suppressive effects observed on adoptive transfer could not be attributed to the carryover of the tolerogen, which might have been associated with the spleen cells of the donor mice, since the transfer of B cells of tolerized mice or of mice which had received 2 mg of the tolerogen 2-24 h before cell transfer did not abrogate the capacity of spleen cells of immune mice to mount an anti-NIP response. Hence, it may be concluded that NIP₂-PVP induced, in addition to a probable receptor blockade of B cells, a central inhibitory mechanism which led to the development of an effective number of suppressor cells within 1 to 2 days after administration of NIP₂-PVP.