Soji Bitoh

Inhibition of the Effector Phase of IgE-Mediated Allergies by Tolerogenic Conjugates of Allergens and Monomethoxypolyethylene Glycol

In this study we established that the conjugate of ovalbumin (OVA), which was used as a model allergen, with monomethoxypolyethylene glycol (mPEG), i.e., OVA(mPEG)11, was not only tolerogenic and essentially non-allergenic, but also capable of inactivating mast cells sensitized with anti-OVA IgE antibodies (Abs). Moreover, mast cells sensitized with a mixture of anti-OVA and anti-DNP IgE Abs were also desensitized by OVA(mPEG)11 with respect to both sensitivities. Most importantly, treatment of OVA-sensitive mice by OVA(mPEG)11 protected them from systemic anaphylaxis on challenge with OVA. The possibility of inactivating IgE-sensitized mast cells with mPEG conjugates of single epitopes of a given allergen is being investigated.

Specific immunosuppression of human anti-murine antibody responses in hu-PBL-SCID mice

Severe combined immunodeficient (SCID) mice were reconstituted with normal human peripheral blood leukocytes (PBLs) and were shown to produce a human anti-mouse immunoglobulin antibody response on immunization with heat-treated murine monoclonal IgG1 antibody to ovalbumin, referred to as ha-Mab-2. The human anti-mouse antibody response was proportional tot the number of B cells and mononuclear cells transferred from a given batch of PBL. However, pretreatment of hu-PBL-SCID mice with a tolerogenic covalent conjugate of monomethoxypolyethylene glycol (mPEG) and Mab-2 suppressed this response on subsequent injections of ha-Mab-2, and this suppression was shown to be antigen-specific, i.e., it did not suppress the antibody response to ovalbumin and did not affect the level of production of human immunoglobulin of hu-PBL-SCID mice. The suppression was due to the generation of human suppressor CD8+ T (Ts) cells, which down regulated CD4+ helper T cells in an antigen and HLA class I specific manner, i.e., these findings were in accord with the previously shown immunosuppressive effect of tolerogenic mPEG conjugates in normal mice.

Potential Therapeutic Efficacy of Allergen-Monomethoxypolyethylene Glycol Conjugates for in vivo Inactivation of Sensitized Mast Cells Responsible for Common Allergies and Asthma

Skin sites of rats, which had been systemically sensitized to ovalbumin (OVA) were injected intradermally with murine anti-DNP IgE mAbs or with murine polyspecific IgE to recombinant Bet v 1. Injection of OVA(mPEG)10-11 conjugates into these skin sites inhibited passive cutaneous anaphylaxis (PCA) on subsequent intravenous challenge with DNP44-BSA and rBet v 1; by contrast, neither OVA nor an unrelated mPEG conjugate affected the PCA reactions. In dogs sensitized to both OVA and ragweed pollen extract (RAG), inhalation of either allergen (AL) caused a dramatic increase in airway resistance (Rrs). By contrast, administration of an aerosol of OVA(mPEG) caused no change in Rrs. Moreover, thereafter, (1) in spite of repeated challenges with aerosolized OVA over many months, the increase in Rrs on inhalation of OVA was blocked and (2) insufflation of RAG resulted in increase in Rrs of only about 50% in relation to that prior to inhalation of the conjugate; this dogs anti-RAG hyperreactivity remained blunted over many months. It is concluded that AL-mPEG conjugates of optimal composition inactivate sensitized mast cells and basophils, as manifested by a significant decrease of cutaneous or airway responses on subsequent challenge with the respective AL(s).

Downregulation of helper T cells by an antigen-specific monoclonal Ts factor

The findings of previous studies in this laboratory demonstrating that conjugates of human monoclonal (myeloma) IgG (HIgG) and monomethoxypolyethylene glycol (mPEG) were able to induce in mice antigen-specific tolerance and CD8+ suppressor T (Ts) cells were confirmed in the present study. An extract (TsF) of a nonhybridized clone of Ts cells (viz., clone 23.32), which had been derived from spleen cells of mice tolerized with HIgG(mPEG)26, was shown to possess antigen-specific suppressive activity. This monoclonal TsF was able to specifically suppress in vitro antibody formation only if it was present from the beginning of the culture. From the results of the cellular dissection of the system used it was concluded that (i) the TsF had no effect on fully differentiated primed B cells or plasma cells, and (ii) the TsF inactivated carrier-primed Th cells when the culture contained concomitantly naive CD8+ T cells, accessory cells, and antigen. These data support the view that the monoclonal TsF exerted its downregulating effect on Th cells only if it could first interact with a CD8+ T cell, in the presence of accessory cells and antigen.

Suppression of human anti-mouse idiotypic antibody responses in hu-PBL-SCID mice

Severe combined immunodeficient (SCID) mice were engrafted with appropriate numbers of T cells and B plus mononuclear cells, which had been fractionated from normal human peripheral blood leukocytes (hu-PBLs). Treatment of these hu-PBL-SCID mice with a tolerogenic covalent conjugate of monomethoxypolyethylene glycol (mPEG) and an anti-ovalbumin, IgG1 murine monoclonal antibody, Mab-2, suppressed the human anti-mouse antibody responses to both the common (gamma 1,kappa) and the idiotypic determinants of Mab-2. Moreover, the Mab-2(mPEG)36 conjugate suppressed the immune responses of hu-PBL-SCID mice to the common and idiotypic determinants of murine monoclonal antibodies to the 2,4-dinitrophenyl residue and to human CD4, consisting also of gamma 1 and kappa chains. It is concluded that a tolerogenic mPEG conjugate of a murine monoclonal antibody induces pan-suppression of the human lymphoid system with respect to other murine monoclonal antibodies that share the isotypic determinants of the original one (here, Mab-2) incorporated in the conjugate. Hence, it may be anticipated that human anti-mouse antibody responses to any murine IgG monoclonal antibody would be suppressed by one of eight mPEG conjugates, each incorporating one of the four subclasses of IgG and one of the two light chains.

Regulation of immune responses via genetically restricted cellular interactions: II. I-Region-restricted cooperation between idiotypic and anti-idiotypic B lymphocytes☆

Immunization of BALB/c mice with MOPC-104E myeloma protein induced idiotype-specific enhancing cells which acted on anti-dextran antibody-producing cells. The enhancing cells have surface phenotypes of B cells. Using BALB/c H-2 congenic strains, it was found that the cooperation between anti-idiotypic-enhancing B lymphocytes and dextran-primed B lymphocytes was controlled by major histocompatibility gene complex. Here we have described the loci which restrict the successful cooperation between B lymphocytes, wherein it was revealed that the interaction was restricted to the I-A and I-E subregions in H-2k haplotype and the I-A subregion in H-2b haplotype. Utilizing several monoclonal antibodies specific for Ia antigens, it was revealed that the enhancing B lymphocyte activity was completely inhibited by the pretreatment of antibody-producing B cells with anti-Ia.7 in H-2d haplotype as well as H-2k, and with anti-I-A antibody in H-2b haplotype. The results suggest that the anti-idiotypic B-lymphocyte response to the self idiotype is under control of H-linked immune response (Ir) gene.

Long-term-cultured mouse B-lymphocyte line: II. Modulation of Ia-antigen expression on B-lymphocyte line☆

Mouse B-cell line, established by culturing anti-Thy-1 and complement-treated splenic B cells with concanavalin A-stimulated conditioned medium, expressed immunoglobulins and Ia antigens on its surface. The long-term-cultured B-cell line was split in two and maintained with or without 3300 R X-irradiated T-cell-depleted syngeneic splenic adherent cells (SAC). Interestingly, the B-cell line cultured without SAC lost its Ia antigen but not its Ig expression, whereas the cell line with SAC maintained both Ia and Ig expression. The ability to express Ia antigens was restored by culturing them only in the presence of Ia-positive feeder cells. Neither recombinant interferon-gamma or lectin-stimulated conditioned medium nor cell-free culture supernatant SAC had the ability to restore Ia antigen expression on the B-cell line. Incubation of Ia-negative B-cell line with phorbol esters restored the Ia expression. It is suggested that the expression of Ia antigen on B lymphocytes was controlled differently from that on macrophage lineage. The B-cell line expressing Ia antigens acts as stimulator cells for alloantigen-activated T lymphocytes and as antigen-presenting cells on the KLH-specific Ia-restricted proliferative T-cell clone in the presence of a specific antigen.

Long-term-cultured mouse B-lymphocyte line: I. Establishment and characterization of mouse B-lymphocyte line

Mouse B-cell line was established by culturing anti-Thy-1 antibody and complement-treated splenic B cells with the conditioned medium of concanavalin A-stimulated spleen cell-culture supernatant. At the eighth week of culture, it was revealed that 100% of the long-term-cultured cells had both cytoplasmic and surface immunoglobulin. These cells were then maintained in the conditioned medium together with T-cell-depleted splenic and then splenic adherent feeder cells. Flow cytometric studies of the B-cell line showed that they had surface mu, delta, and kappa but no gamma, lambda, Lyt-1, or Lyt-2. The growth of the B-cell line was dependent on the factor(s) derived from concanavalin A-stimulated conditioned medium. It was found that IL-2 was the major factor supporting the B-cell growth. The B-cell line did not secrete immunoglobulin spontaneously, but it could differentiate into antibody-forming cells through the stimulation of bacterial lipopolysaccharides. The technique for obtaining mouse B-cell lines are reproducible in our laboratory and one of those lines has been propagated and maintained for 16 months to the present.

Dual Effects of Allergen-Mpeg Conjugates

Since the early 1970s the research in this laboratory has been directed toward the development of methods for the specific suppression of IgE antibodies (1,2). Among the more recent strategies used, the administration of tolerogenic conjugates—consisting of different allergens/antigens coupled to optimal numbers of molecules of monomethoxy-polyethylene glycol (mPEG)—proved to be the most effective method for the specific long-term suppression of the induction of antibody responses in mice and rats to the corresponding immunogenic compounds (3,4). This method involves two steps: Step I. Injection of tolerogenic conjugates of the appropriate immunogenic allergen (AL), i.e., AL(mPEG)n, Step II. Administration of the unmodified AL 7 days after injection of the immunosuppresive AL(mPEG)n.

Molecular Analysis of Immunoglobulin Heavy Chain Genes Coding for Idiotypic and Anti-Idiotypic Antibodies Involved in B-B Cellular Interaction

We recently reported that a unique B cell clone (B19‐1d), specific for a cross‐reactive idiotype (CRI) on MOPC104E myeloma protein (M104E), enhances Igh‐restricted CRI+ antibody production. In this paper, we report the nucleotide sequences of immunoglobulin heavy chain variable regions (VH) of both M104E and B19‐1d‐derived hybridoma (HB19) antibodies. The sequence data revealed that both belong to the J558 germ line VH gene subfamily. Strikingly, not only the VH region, but also the leader sequences of M104E and HB19 are very similar to each other at 88% (VH) and 91 % (leader) homology, but they use different D and J segments. The VH region sequence similarity is highest among the germ line VH gene sequences of the BALB/c J558 subfamily so far screened. Southern hybridization data, using 5′‐noncoding regions of either M104E or HB19 genomic VH gene clones as probes, revealed that both VH genes are conserved in the M104E CRI producer strains of mice. Moreover, these probes show the restriction length polymorphism pattern of mouse VH genes in various strains. That the HB19 VH gene locates to the 5′ upper arm of the M104E VH gene on the chromosome was suggested by Southern blot hybridization. Immunoglobulin VH gene restriction of idiotypic and antiidiotypic B‐B cellular interaction is discussed from a molecular point of view.