Tolerance to Self and the Processing and Presentation of Self Antigens

Antigen processing and presentation is critical to the generation and maintenance of self tolerance. The hemoglobin system has provided important data on self antigen processing and presentation in vivo. Hemoglobin/la complexes were detectable in the thymus before the time of positive and negative selection. In addition, thymic epithelial cells were shown to lack the costimulatory factors necessary to trigger T cell clone proliferation. We have extended these findings to the Renal proximal tubule. This class II MHC-expressing epithelial cell was demonstrated to process and present foreign as well as self antigens to T cell hybridomas. Current studies are examining whether this epithelial cell possesses the costimulatory factors Required to fully stimulate T cell clones, or whether the proximal tubule may play an important Role in the maintenance of self tolerance. In addition we describe the exciting model of murine autoimmune myocarditis. We have demonstrated that this is a T cell mediated disease and believe that cardiac antigen presenting cells constitutively process and present the inciting self antigen, myosin. These studies may provide important insights into autoimmunity and self tolerance.     

Inducing long-term survival with lasting anti-tumor immunity in treating B cell lymphoma by a combined dendritic cell-based and hydrodynamic plasmid-encoding IL-12 gene therapy

In a previous study we showed that immunization with dendritic cells (DC) pulsed with idiotype (Id) fused with CD40 ligand (CD40L) could break the tolerance to Id which is expressed on B lymphoma cells and restored the responsiveness of T(h) cells, and, subsequently, induced IgG antibody response. However, this treatment had no therapeutic effect. In the present study, we found that using a hydrodynamic transfection-based technique, a high level of IL-12 production was noticed as early as 7 h after administering plasmid encoding IL-12 (pIL-12) and persisted at a detectable level for at least 9 days. In evaluating the efficacy of DC-based and/or IL-12 gene-based therapy in the treatment of 38C13 B cell lymphoma, it was found that either treatment alone was ineffective. However, a combined treatment induced 100% long-term survival. Furthermore, a long-lasting anti-tumor immunity was induced in these mice which resisted further tumor challenge at 58 days after initial inoculation. The surviving mice showed a strong IFN-gamma-producing T(h) cell response and humoral antibody response, but there were no detectable cytotoxic T lymphocytes. The antibody from the immune sera mediated a complement-dependent lysis of tumor cells that was tumor specific. Furthermore, immunization of mice with DC-based vaccine and pIL-12 treatment elicited higher levels of anti-Id IgG titer and an enhanced IgG2a response which increased the efficacy in mediating 38C13 tumor lysis. On examining the mechanism for this isotype change, we found that IFN-gamma production by CD4(+) T cells is not the only determining factor for achieving a successful therapy. DC-based treatment alone could induce the increase of IFN-gamma production, but lacked any therapeutic effect. The deciding factor appears to be the abrogation of IL-4 production that was achieved by combing with IL-12 gene therapy. Our study provides a basis for exploring the combined use of cytokines or cytokine genes in DC-based treatment for achieving effective cancer immunotherapy.     

Oligoclonal T cells in human cancer

Many solid tumors are characterised by the infiltration of lymphocytes and their presence has been correlated with a more favourable prognosis. These tumor-infiltrating lymphocytes (TIL), have been shown to possess specific cytolytic reactivity towards autologous tumours, thus suggesting that tumour cells may express antigens capable of eliciting an immune response. Expression of such tumour-associated antigens (TAA) in combination with appropriate accessory signals would lead to thein vivo accumulation of T cells with anti-tumour specificity. Analysis of the composition of the specific T-cell receptor (TCR) of TIL could thus provide information on the nature of the antigen(s) recognised by TIL. In this review, different aspects of the presence of clonal T cells in patients with cancer are discussed.     

The separation of human serum IgG into subclass fractions by immunoaffinity chromatography and assessment of specific antibody activity

Murine monoclonal antibodies ( McAbs ) with specificity for subclass-specific or subclass-restricted determinants on human IgG have been coupled to Sepharose to generate affinity columns. The judicial use of positive and negative chromatography and the exploitation of the special properties of individual McAb affinity columns has allowed the preparation of individual IgG subclasses from polyclonal IgG containing less than 1% contamination by any other IgG subclass. The specificity of the antibodies present in each polyclonal IgG subclass preparation has been assayed against a bacterial toxoid (tetanus), 2 bacterial cell wall antigens (E. coli and pneumococcal) and coat antigen(s) of a DNA virus (CMV). Antibodies were predominantly IgG1 to tetanus toxoid, IgG2 to pneumovax and E. coli cell walls, and IgG1, 2 and 3 to CMV coat antigens.     

Anti‐idiotype and anti‐anti‐idiotype antibodies for aflatoxin from laying hens

Anti‐idiotype (anti‐id) antibodies (IgY2)for aflatoxin (AF) were obtained from the egg yolks of laying hens immunized with affinity‐purified rabbit polyclonal anti‐aflatoxin B₁ (AFB₁) carboxymethyloxime‐bovine serum albumin (BSA) antibodies (pAb1). The IgY2 were affinity purified and then subjected to various analyses. Inhibition of the binding of pAbl to the solid‐phase AFB₁‐BSA by IgY2 and the binding of pAb1 to the solid‐phase IgY2 by free AFB₁ were demonstrated in a biotin‐avidin amplified enzyme‐linked immunosorbent assay (ELISA) system. The concentration of IgY2 causing 50% inhibition (ID₅₀) of the binding of pAb1 to AFB₁‐BSA was found to be 2.45 μg/assay. The ID₅₀ concentration of the binding of pAb1 to IgY2 by free AFB₁ was found to be 0.30 μg/assay. Inhibition of the binding of AFB₁‐horseradish peroxidase (HRP) to the solid‐phase pAbl by IgY2 (ID50 = 9.65 μg/assay) was also demonstrated in the direct ELISA. Egg yolk anti‐anti‐id antibodies (IgY3) were obtained by immunizing laying hens with rabbit pAb2 against anti‐AFB₃‐hemisuccinate‐BSA monoclonal antibody. IgY3 was subjected to affinity chro‐matography purification with Sepharose gel armed with AFB₂‐carboxymethytoxime, and then subjected to various analyses. ELISA analysis revealed that IgY3 has characteristics similar to other anti‐AFB antibodies induced in various experimental animals. In the direct ELISA, the ID₅₀ of the binding of AFB₁‐HRP to solid‐phase lgY3 by AFB₁ was found to be 0.12 ng ml^‐1. In the indirect ELISA, the ID₅₀ of the binding of IgY3 to solid‐phase AFB₁‐BSA by AFB₁ was found to be 2.2 ng ml^‐1. The IgY3‐based ELISA analysis showed higher sensitivity than that of the egg yolk antibodies directly against AFB‐protein conjugates (IgY1). A good correlation was found for the data obtained from IgY3‐based and pAb1‐based ELISAs in the analysis of AFB in the fungal culture filtrates.     

Preparation of anti-idiotypic antibody against avian influenza virus subtype H9

To generate monoclonal anti-idiotypic antibodies(mAb2)against avian influenza virus subtype H9(H9 AⅣ),BALB/c mice were immunized with purified chicken anti-H9-AⅣ IgG and the splenocytes of immunized mice werefused with myeloma cells NS-1.Hybridoma cells were screened by indirect enzyme-linked immunosorbent assayswith both chicken and rabbit anti-H9-AⅣ IgG as coating antigens.One hybridoma cell clone secreting monoclonalantibody against idiotypes shared by both chicken and rabbit anti-H9-AⅣ IgG was established.Experimentsdemonstrated the mAb2 was able to inhibit the binding of hemagglutinin to anti-H9-AⅣ IgG and to inducechickens to generate hemagglutination inhibition antibodies,indicating this anti-species-sharing-idiotypic antibodybore the internal image of hemagglutinin on avian influenza virus.Cellular & Molecular Immunology.2005;2(2):155-157.     

Accelerated expression of anti-Sm and Y2 idiotype differ in dependence on T cell subsets in MRL/+ mice.

We investigated the role of MRl T cells in the induction of anti-Sm antibodies and Y2 idiotype. Four injections of Sm antigen in Freund's complete adjuvant were required to induce peak amounts of specific anti-Sm antibody in young BALB/c and MRL/+ mice. The Y2 idiotype was expressed in MRL/+ mice but not in BALB/c mice. Expression of both anti-Sm, predominantly IgG2a heavy chain, and Y2 idiotype was augmented in MRL/+ mice after two injections of Sm if, prior to immunization, mice received splenic T cells from naive MRL/lpr or immunized, but not naive MRL/+ mice. These results suggest that the lpr gene contributes to the ability of autoimmune T cells to augment the anti-Sm antibody response. Treatment of primed MRL/+ donor T cells with anti-CD4, but not anti-CD8, antibodies and complement removed the ability to augment anti-Sm antibody production. In contrast, augmentation of Y2 idiotype production was abrogated by pretreatment of donor T cells with either anti-CD4 or anti-CD8. These results suggest that, while MRL/+ CD4+ T cells play an important role in anti-Sm antibody production, additional interaction between CD4+ and CD8+ T cells augments Y2 expression.     

Interaction of heavy and light chains of myeloma immunoglobulin with pooled normal chains: quantitative analysis of association and restoration of idiotype

We have analyzed in vitro recombinants between the isolated heavy (H) or light (L) chains of mouse myeloma protein MOPC-21 and L or H chains of normal mouse serum immunoglobulin (Ig). In the first series of experiments using fixed H chain and solubilized L chain, we have found out that only about 30% of normal L chain pool interact efficiently with individual H chain. Moreover, fractions with different affinity to H-MOPC-21 appeared to exist among normal L chains. In the second set of experiments recombination of H and L chains in solution was used. Examination of recombinants between myeloma H chain and normal L chains revealed a set representing 6% of L chain repertoire capable of forming MOPC-21-like idiotypic structure.     

Modulation of immune response in vitro and in vivo by human granulocyte factors

The adherence of granulocytes induces secretion of specific granule contents. The secreted proteins were termed granulocyte factors (GF). The experiments in vivo provide evidence that GF play an essential role in the stimulation of PFC in BALB/c mice immunized with SRBC when applied before challenge three times (5 micrograms per mouse), but 50 micrograms per mouse given in the same way diminishes the response. To elucidate this discrepancy, the effect of GF on the generation of suppressor cells (SC) and helper cells (HC) in vitro has been investigated. Antigen specific nonadherent SC or HC were induced in vitro using CBA mice spleen cells incubated with 100 micrograms/ml or 0.1 mg/ml of TNP-KLH, respectively, for 4 days. GF in concentrations of 0.1 to 1 microgram/ml abolish antigen specific SC generation. SC and HC activity was tested in cooperative cultures. Antigen specific SC in delayed hypersensitivity (DTH) to BCG were induced in an in vitro system as above using normal BALB/c spleen cells and 100 micrograms/ml PPD. Nonadherent suppressor cells were transferred intravenously into cyclophosphamide (CY)-treated syngeneic recipients. The recipients were immunized to BCG immediately after the cell transfer. DTH was measured by foot-pad reaction. This reaction was positive to PPD in CY treated mice immunized to BCG, while it was suppressed by the transfer of in vitro induced SC. When the SC were induced in the presence of 1 microgram/ml GF, the suppression was abrogated. The higher GF concentrations stimulated SC activities when they were measured in response to a nonrelated antigen and in specific anti-PPD response, but the HC inhibition could not be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phosphorylcholine-lipopolysaccharide conjugates on the induction of anti-idiotype-mediated B-cell tolerance

Preincubation of BALB/c spleen cell cultures for 24 hr with phosphorylcholine (PC)-containing antigen together with antibody against the major idiotype (id) of anti-PC antibody renders them irreversibly unresponsive to subsequent stimulation with the antigen alone. In contrast, cultures preincubated for 24 hr with anti-id antibody, either alone or together with lipopolysaccharide (LPS), resulted in an anti-PC response comparable to that induced in control cultures incubated with mock anti-id antibody. After such a 24-hr preincubation with anti-id antibody and various PC-LPS conjugates possessing intact activity for polyclonal B-cell activation, the anti-PC response was inversely proportional to the epitope (PC) density on the LPS conjugates. In addition, similar preincubation of cultures with a non-mitogenic low dose of PC-LPS in the presence of anti-id antibody induced suppression of the anti-PC response as observed with a specific antigen. These results suggest that specific epitope delivers an additional tolerogenic signal during induction of B-cell suppression by anti-id antibody. This epitope effect cannot be replaced by, and is antagonistic to, the mitogenic signal of LPS in the course of B-cell inactivation.