Graziella Mazza

Destructive processing by asparagine endopeptidase limits presentation of a dominant T cell epitope in MBP.

Little is known about the processing of putative human autoantigens and why tolerance is established to some T cell epitopes but not others. Here we show that a principal human HLA-DR2–restricted epitope—amino acids 85–99 of myelin basic protein, MBP(85–99)—contains a processing site for the cysteine protease asparagine endopeptidase (AEP). Presentation of this epitope by human antigen-presenting cells is inversely proportional to the amount of cellular AEP activity: inhibition of AEP in living cells greatly enhances presentation of the MBP(85–99) epitope, whereas overexpression of AEP diminishes presentation. These results indicate that central tolerance to this encephalitogenic MBP epitope may not be established because destructive processing limits its display in the thymus. Consistent with this hypothesis, AEP is expressed abundantly in thymic antigen-presenting cells.

The separation and identification by monoclonal antibodies of dog IgG fractions

Four fractions of IgG from normal dog serum have been successfully isolated by gel filtration followed by protein A and protein G affinity chromatography using the fast protein liquid chromatography (FPLC) system. Protein A chromatography produced three peaks: peak 1 was fallthrough material consisting of components which did not bind to protein A, peak 2 consisted of bound material eluting at pH 6, and peak 3 contained bound material eluting at pH 3.5. The three peaks were then subjected individually to protein G affinity chromatography. Peak 1 from protein A chromatography produced a fallthrough peak followed by a weakly binding component which eluted at pH 8, and was called peak w. Peak 2 from protein A chromatography bound to protein G and eluted as a single peak at pH 3.8, and was called peak x. Peak 3 from protein A chromatography emerged as two separate peaks (y and z) off the protein G column; peak y bound and eluted at pH 4.1, and peak z bound weakly to protein G and emerged as a broad band at pH 8. Peaks w, x, y and z have been named gamma w, gamma x, gamma y and gamma z, respectively, and there purified IgG fractions were used to immunize mice for the preparation of monoclonal antibodies (McAbs). To date, two sets of McAbs have been produced: one which recognizes an epitope present in both gamma w and gamma z fractions and another set of McAbs which recognizes an epitope in the gamma x and gamma y fractions.

Natural and Induced Regulatory T Cells

Abstract: Mucosal antigen delivery can induce tolerance, as shown by suppression of subsequent responses to antigen. Our previous work showed that both intranasal and oral routes of antigen delivery were effective but indicated that the intranasal route might be more reliable. Intranasal peptide administration induced cells that could mediate bystander suppression of responses to associated antigenic epitopes. Here, we discuss further investigation into the nature of intranasal, peptide‐induced tolerance. Cells from mice treated with intranasal peptide became anergic and shut down secretion of cytokines such as IL‐2, but still secreted IL‐10. This latter cytokine was required for suppression of immune responses in vivo even though suppression of responses in vitro was IL‐10 independent. Intranasal peptide induced a subset of CD25−, CTLA‐4+ regulatory cells that suppressed naive cell function in vitro and in vivo. We provide evidence that these cells arise from CD25− precursors and differentiate independently from natural CD25+ regulatory cells. IL‐10‐secreting regulatory cells are also found in the peripheral blood of humans and can be induced by soluble peptide administration. This route of tolerance induction offers promise as a means of antigen‐specific immunotherapy of allergic and autoimmune conditions in humans.

DEVELOPMENT OF AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE DETECTION OF IGG SUBCLASSES IN THE SERUM OF NORMAL AND DISEASED DOGS

The present study describes the development of an enzyme-linked immunosorbent assay capable of quantifying serum antibody of all four canine IgG subclasses. A panel of subclass-restricted and subclass-specific monoclonal antibodies was used to measure IgG subclasses in the serum of healthy dogs, as well as in dogs with a range of clinical diseases. The subclasses have been redefined as IgG1, IgG2, IgG3 and IgG4 based on a comparison with the relative concentration and electrophoretic mobilities of human IgG subclasses. In serum samples from healthy dogs, the concentration of IgG1 (mean, 8.17 +/- 0.95 mg ml-1) and IgG2 (mean, 8.15 +/- 3.16 mg ml-1) were very similar and considerably higher than the levels of IgG3 (mean, 0.36 +/- 0.43 mg ml-1) and IgG4 (mean, 0.95 +/- 0.45 mg ml-1). There was no apparent difference in the level of subclasses between the different breeds comprising this normal population. Sera from dogs with a range of immune-mediated or inflammatory diseases all had markedly elevated levels of IgG2 (more than 13 mg ml-1), but IgG1 decreased (less than 4 mg ml-1) to levels below the normal range.

PREPARATION OF MONOCLONAL-ANTIBODIES SPECIFIC FOR THE SUBCLASSES OF CANINE IGG

Canine IgG is composed of four subclasses, which are defined as IgG1, IgG2, IgG3 and IgG4 on the basis of data from fast protein liquid chromatography, and their electrophoretic mobilities and relative concentrations in serum. This paper describes the preparation of mAbs specific for determinants on canine IgG2, IgG3 and IgG4. The mAb specific for IgG2 resulted from a conventional immunisation protocol. The mAb specific for IgG3 was a result of immunisation with IgG3 combined with the suppression of the immune response to IgG1 by passively administered anti-IgG1 antibody. The mAb specific for IgG4 resulted from immunisation with Fab or Fc fragments which were obtained by the cleavage of the IgG4 molecule with papain. The specificity of each mAb was established by using an enzyme-linked immunosorbent assay which showed that all three specific clones recognised a determinant in the Fd region of the canine immunoglobulin molecule.

T-helper 1 dominated responses to erythrocyte Band 3 in NZB mice.

Band 3, the red blood cell (RBC) anion channel protein, is the target autoantigen for the pathogenic RBC autoantibodies and T‐helper (Th) cells in New Zealand Black (NZB) mice with autoimmune haemolytic anaemia (AIHA). To determine the subpopulation of these Th cells, they were stimulated with Band 3 and the profile of the cytokines elaborated by the responding cells was measured. NZB T cells stimulated with Band 3 produced high levels of the Th1 cytokine, interferon‐γ (IFN‐γ), but little or no interleukin‐4 (IL‐4), IL‐5 or IL‐10. Similar patterns were produced by NZB T cells responding to a spectrin preparation from the RBC membrane skeleton, or to mycobacterial heat‐shock protein (hsp) 65 following immunization of mice with hsp  65 in incomplete adjuvant. By contrast, T cells from CBA mice similarly immunized with hsp  65 produced high levels of IL‐4 and IL‐5 in response to hsp  65. Examination of the isotype of the RBC‐bound immunoglobulins in NZB mice revealed that immunoglobulin G2a (IgG2a) autoantibodies were the first to be detected in most mice and that later in the disease, IgG3 autoantibodies were often prominent. It is concluded that, contrary to expectation, the development of RBC autoantibodies in NZB mice is associated with Th1 cytokine‐dominated responses.

Tissue immunoglobulin G subclasses observed in immune-mediated dermatopathy, deep pyoderma and hypersensitivity dermatitis in dogs

A panel of monoclonal antibodies has been used to define three of the four subclasses of canine immunoglobulin G (IgG2, IgG3 and IgG4) in formalin-fixed tissues. These reagents, together with a polyclonal antiserum specific for the Fc region of canine IgG, were used in an immunohistochemical study of biopsies of skin from five normal dogs and from the lesions of canine pemphigus foliaceus in seven dogs, discoid lupus erythematosus in eight dogs, bullous pemphigoid in one dog, cutaneous drug eruption in two dogs, deep pyoderma in 13 dogs and hypersensitivity dermatitis in eight dogs. IgG autoantibody was identified in the skin of all the dogs with immune-mediated dermatoses by using the polyclonal reagent, and antibody of the IgG2 and/or IgG4 subclass was identified in the epidermis of three of the dogs with pemphigus foliaceus, two of those with discoid lupus erythematosus and in the dog with bullous pemphigoid. In all the lesions, the infiltrate of dermal plasma cells consisted of similar numbers of IgG2 and IgG4 bearing cells, with relatively few IgG3 positive cells. The total number of these cells was generally approximately the same as or greater than the number of cells labelled with the polyclonal reagent. There was no significant difference between the IgG-bearing plasma cell infiltrate in German shepherd dogs with deep pyoderma and that in dogs of other breeds. The infiltration of IgG2 and IgG4 bearing plasma cells into the skin of dogs with a range of cutaneous disorders was related to the selectively enhanced serum levels of these subclasses in the diseased dogs.

Proliferative responses of peripheral blood mononuclear cells from normal dogs and dogs with autoimmune haemolytic anaemia to red blood cell antigens

Autoimmune haemolytic anaemia (AIHA), one of the most common autoimmune diseases of the dog, is characterised by binding of autoantibody to erythrocyte membrane antigens leading to a decreased red blood cell (RBC) life-span. Failure of self-tolerance with activation of autoreactive T-lymphocytes is thought to play a key role in the initiation of such autoimmune events. Peripheral blood mononuclear cells (PBMC) were obtained from 11 clinically normal dogs, six clinically normal relatives of two littermate dogs which died from AIHA, and four dogs which had recovered from primary AIHA. Cells were stimulated in vitro with a panel of canine RBC-derived antigens (RBC membranes, glycophorin, spectrin, five 15-mer glycophorin peptides), the non-recall antigen keyhole limpet haemocyanin (KLH), and the mitogen concanavalin A (Con A). The kinetics of the proliferative responses to specific antigens were assessed by serially sampling the cultures from days 4 to 10. PBMC from all dogs responded strongly to Con A (day 2) and to KLH (maximal response on days 7 to 10) under appropriate culture conditions. Two of 11 normal dogs responded weakly to RBC membranes (mean stimulation index = 4.25). In contrast, PBMC from all dogs recovered from AIHA responded to RBC membranes (mean SI = 9.2 +/- 2.5) and occasionally to other erythrocyte antigens. Similar responses were recorded with PBMC from dogs related to AIHA cases. It is considered that although normal individuals harbour erythrocyte-reactive lymphocytes, such cells are primed in dogs with AIHA or a genetic susceptibility to this disease.

BIOCHEMICAL CHARACTERIZATION OF CANINE BLOOD GROUP ANTIGENS: IMMUNOPRECIPITATION OF DEA 1.2, 4 AND 7 AND IDENTIFICATION OF A DOG ERYTHROCYTE MEMBRANE ANTIGEN HOMOLOGOUS TO HUMAN RHESUS

Despite the clinical significance of the canine blood group antigens, relatively little is known of the biochemistry of these molecules. In this study the canine blood group antigens DEA (dog erythrocyte antigen) 1.2, 4 and 7 were immunoprecipitated from red blood cells (RBC) bearing the corresponding blood group, and molecular weights of 85 kD (DEA 1.2), 32-40 kD (DEA 4) and 53-66 kD (DEA 7) assigned. DEA 1.2 and DEA 4 each appeared as a single band, whereas DEA 7 comprised three distinct bands (53, 58 and 66 kD). Polyclonal antisera specific for two peptides derived from the sequence of the human Rhesus blood group (Rh30A-C and Rh50A-C) were used in western blotting against canine and human erythrocyte membranes. The Rh30A-C antiserum identified a band of molecular weight 32 kD in both human and canine RBC, and the antiserum specific for Rh50A-C identified a band of 38-60 kD in human membranes and of 40-53 kD in canine RBC. This finding is consistent with conservation of areas of the Rhesus protein sequence between human and canine erythrocytes.

Quantitation of Erythrocyte-Bound IgG Subclass Autoantibodies in Murine Autoimmune Haemolytic Anaemia

A quantitative and sensitive cellular enzyme-linked immunosorbent assay was developed for determining the number of molecules of IgG of each subclass bound to the surface of murine red blood cells (RBC). To develop standard titration curves, RBC from normal mice were treated with tannic acid and coated with a known concentration of purified myeloma of each IgG subclass. The quantity of each subclass bound to the surface of erythrocytes was determined by calculating the protein concentration of the bound IgG, which was then converted into number of molecules of IgG/RBC. The assay was used to quantify the number of autoantibodies of all four IgG subclass bound to the erythrocytes of mice injected with rat RBC. Twenty one days after the first immunisation, a mean number of 84,000 molecules of IgG1/RBC were detected, which increased to 114,500 molecules/RBC on day 28. On days 56 and 96 the mean concentration of IgG1 remained high, however by day 110 the mean level of IgG1 had decreased slighty to 69,500 molecules/RBC. By contrast, the mean concentration of IgG2a autoantibodies was considerably lower throughout the experiment, starting at 40,200 molecules/RBC on day 21 and dropping to 2,500 molecules/RBC by day 110. The mean quantities of IgG2b and IgG3 autoantibodies were similar to each other, and intermediate between the levels of IgG1 and IgG2a autoantibodies.