ovalbumin and Lymphoma--B-Cell

Establishment of persistent infection in memory B cells by murid herpesvirus-4 (MuHV-4) depends on the proliferation of latently infected germinal center B cells, for which T cell help is essential. Whether the virus is capable of modulating B-T helper cell interaction for its own benefit is still unknown. Here, we investigate if the MuHV-4 latency associated M2 protein, which assembles multiprotein complexes with B cell signaling proteins, plays a role. We observed that M2 led to the upregulation of adhesion and co-stimulatory molecules in transduced B cell lines. In an MHC-II restricted OVA peptide-specific system, M2 polarized to the B-T helper contact zone. Furthermore, it promoted B cell polarization, as demonstrated by the increased proximity of the B cell microtubule organizing center to the interface. Consistent with these data, M2 promoted the formation of B-T helper cell conjugates. In an in vitro competition assay, this translated into a competitive advantage, as T cells preferentially conjugated with M2-expressing B cells. However, expression of M2 alone in B cells was not sufficient to lead to T cell activation, as it only occurred in the presence of specific peptide. Taken together, these findings support that M2 promotes the formation of B-T helper cell conjugates. In an in vivo context this may confer a competitive advantage to the infected B cell in acquisition of T cell help and initiation of a germinal center reaction, hence host colonization.

Topics: Animals; Cell Adhesion Molecules; Cell Line; Cell Line, Tumor; Cell Polarity; Cell Proliferation; Host-Pathogen Interactions; Immunologic Memory; Lymphocyte Activation; Lymphoma, B-Cell; Mice; Ovalbumin; Peptide Fragments; Rhadinovirus; T-Lymphocytes, Helper-Inducer; Viral Proteins; Virus Latency

The addition of an appropriate adjuvant that activates the innate immunity is essential to subsequent development of the adaptive immunity specific to the vaccine antigens. Thus, any innovation capable of improving the immune responses may lead to a more efficacious vaccine. We recently identified a novel immune modulator using a naturally occurring seed peptide called lunasin. Lunasin was originally isolated from soybeans, and it is a small peptide containing 43 amino acids. Our studies revealed stimulatory effects of lunasin on innate immune cells by regulating expression of a number of genes that are important for immune responses. The objective was to define the effectiveness of lunasin as an adjuvant that enhances immune responses. The immune modulating functions of lunasin were characterized in dendritic cells (DCs) from human peripheral blood mononuclear cells (PBMCs). Lunasin-treated conventional DCs (cDCs) not only expressed elevated levels of co-stimulatory molecules (CD86, CD40) but also exhibited up-regulation of cytokines (IL1B, IL6) and chemokines (CCL3, CCL4). Lunasin-treated cDCs induced higher proliferation of allogeneic CD4+ T cells when comparing with medium control treatment in the mixed leukocyte reaction (MLR). Immunization of mice with ovalbumin (OVA) and lunasin inhibited the growth of OVA-expressing A20 B-lymphomas, which was correlated with OVA-specific CD8+ T cells. In addition, lunasin was an effective adjuvant for immunization with OVA, which together improved animal survival against lethal challenge with influenza virus expressing the MHC class I OVA peptide SIINFEKL (PR8-OTI). These results suggest that lunasin may function as a vaccine adjuvant by promoting DC maturation, which in turn enhances the development of protective immune responses to the vaccine antigens.

Topics: Adjuvants, Immunologic; Animals; Antigens, CD; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Chemokines; Cytokines; Dendritic Cells; Female; Humans; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Lymphoma, B-Cell; Mice, Inbred BALB C; Orthomyxoviridae Infections; Ovalbumin; Soybean Proteins

To study mechanisms of T cell-mediated rejection of B cell lymphomas, we developed a murine lymphoma model wherein three potential rejection antigens, human c-MYC, chicken ovalbumin (OVA), and GFP are expressed. After transfer into wild-type mice 60-70% of systemically growing lymphomas expressing all three antigens were rejected; lymphomas expressing only human c-MYC protein were not rejected. OVA expressing lymphomas were infiltrated by T cells, showed MHC class I and II upregulation, and lost antigen expression, indicating immune escape. In contrast to wild-type recipients, 80-100% of STAT1-, IFN-γ-, or IFN-γ receptor-deficient recipients died of lymphoma, indicating that host IFN-γ signaling is critical for rejection. Lymphomas arising in IFN-γ- and IFN-γ-receptor-deficient mice had invariably lost antigen expression, suggesting that poor overall survival of these recipients was due to inefficient elimination of antigen-negative lymphoma variants. Antigen-dependent eradication of lymphoma cells in wild-type animals was dependent on cross-presentation of antigen by cells of the tumor stroma. These findings provide first evidence for an important role of the tumor stroma in T cell-mediated control of hematologic neoplasias and highlight the importance of incorporating stroma-targeting strategies into future immunotherapeutic approaches.

Topics: Animals; Antigens; B-Lymphocytes; Chickens; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Interferon-gamma; Lymphoma, B-Cell; Mice; Mice, Inbred C57BL; Ovalbumin; Proto-Oncogene Proteins c-myc; Signal Transduction

The mechanisms of immune evasion during haematological malignancies are poorly understood. As lymphomas grow in lymphoid organs, it would be expected that if these lymphomas express neo-antigens they should be readily detected by the immune system. To test this assumption, we generated a new non-Hodgkin B-cell lymphoma model expressing the model tumour neo-antigen Ovalbumin (OVA), and analysed the endogenous antigen-specific CD8(+) T-cell response that it elicited in recipient mice. The OVA+ lymphoma cells were eliminated by cytotoxic T lymphocytes (CTL) in mice that had been previously vaccinated against OVA. In contrast, the immune system of naïve mice ignored the malignant cells even though these continuously expressed and presented OVA on their MHC class I molecules. This state of ignorance could be overcome by therapeutic vaccination, which led to the expansion of endogenous anti-OVA-specific CD8(+) T cells. However, the cytotoxic and interferon-γ secretion capacity of these T cells were impaired. The tumour model that we describe thus reproduces several key aspects of human lymphoma; tumor ignorance can be broken by vaccination but the ensuing immune response remains ineffective. This model can be exploited to further understand the mechanisms of lymphoma immunoevasion and devise effective immunotherapy.

Topics: Animals; Antigens; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cytotoxicity, Immunologic; Disease Models, Animal; Epitopes; H-2 Antigens; Histocompatibility Antigens Class I; Immune Tolerance; Lymphoma, B-Cell; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; T-Lymphocytes, Cytotoxic; Vaccination

CD40, a member of the tumor necrosis factor receptor (TNFR) superfamily, is expressed on antigen-presenting cells (APCs) and is essential for immune activation. Although agonistic CD40 antibodies have been developed for immunotherapy, their clinical efficacy has been limited. We have found that coengagement of the Fc domain of agonistic CD40 monoclonal antibodies (mAbs) with the inhibitory Fcγ receptor FcγRIIB is required for immune activation. Direct comparison of mAbs to CD40 enhanced for activating FcγR binding, hence capable of cytotoxicity, or for inhibitory FcγRIIB binding, revealed that enhancing FcγRIIB binding conferred immunostimulatory activity and considerably greater anti-tumor responses. This unexpected requirement for FcγRIIB in enhancing CD40-mediated immune activation has direct implications for the design of agonistic antibodies to TNFR as therapeutics.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Antibody Affinity; Antibody-Dependent Cell Cytotoxicity; Antigen-Presenting Cells; CD40 Antigens; Cytotoxicity, Immunologic; Dendritic Cells; Humans; Immunoglobulin Fc Fragments; Lymphocyte Activation; Lymphoma, B-Cell; Mice; Mice, Inbred BALB C; Mutation; Ovalbumin; Receptors, IgG; T-Lymphocytes, Cytotoxic

Adoptive immunotherapy using antigen-specific T-helper type 1 (Th1) cells has been considered as a potential strategy for tumor immunotherapy. However, its application to tumor immunotherapy has been hampered by difficulties in expanding tumor-specific Th1 cells from tumor-bearing hosts. Here, we have developed an efficient protocol for preparing mouse antigen-specific Th1 cells from nonspecifically activated Th cells after retroviral transfer of T-cell receptor (TCR)-alpha and TCR-beta genes. We demonstrate that Th1 cells transduced with the TCR-alpha and -beta genes from the I-A(d)-restricted ovalbumin (OVA)(323-339)-specific T-cell clone DO11.10 produce IFN-gamma but not interleukin-4 in response to stimulation with OVA(323-339) peptides or A20 B lymphoma (A20-OVA) cells expressing OVA as a model tumor antigen. TCR-transduced Th1 cells also exhibited cytotoxicity against tumor cells in an antigen-specific manner. Moreover, adoptive transfer of TCR-transduced Th1 cells, but not mock-transduced Th1 cells, exhibited potent antitumor activity in vivo and, when combined with cyclophosphamide treatment, completely eradicated established tumor masses. Thus, TCR-transduced Th1 cells are a promising alternative for the development of effective adoptive immunotherapies.

Topics: Animals; Cell Line, Tumor; Cytotoxicity, Immunologic; Humans; Immunotherapy, Adoptive; Lymphoma, B-Cell; Mice; Mice, Inbred BALB C; Mice, Transgenic; Ovalbumin; Receptors, Antigen, T-Cell; T-Lymphocytes, Helper-Inducer; Th1 Cells

To investigate the precise role of antigen-specific Th1 and Th2 cells in tumor immunity, we developed a novel adoptive tumor-immunotherapy model using OVA-specific Th1 and Th2 cells and an OVA gene-transfected tumor. This therapeutic model demonstrated that both antigen-specific Th1 and Th2 cells had strong antitumor activity in vivo with distinct mechanisms. However, immunological memory suitable for the generation of tumor-specific cytotoxic T lymphocytes was induced only when tumor-bearing mice received Th1 cell therapy, but not Th2 cell therapy. Thus it was strongly suggested that Th1-dominant immunity is critically important for the induction of antitumor cellular immunity in vivo. We also proposed that several immunomodulating protocols using interleukin (IL)-12, IL-12 gene, the natural killer T cell ligand alpha-galactosylceramide, or Th1 cytokine-conditioned dendritic cells might be useful strategies for the induction of Th1-dominant immunity essential for the development of tumor-specific immunotherapy.

Topics: Animals; Cancer Vaccines; Cytokines; Dendritic Cells; Epitopes, T-Lymphocyte; Female; Galactosylceramides; Genetic Therapy; Immunity, Cellular; Immunologic Memory; Immunotherapy, Adoptive; Interleukin-12; Lymphoma, B-Cell; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Transplantation; Ovalbumin; Th1 Cells; Th2 Cells; Transfection

Cholera toxin (CT) can function as a potent adjuvant in the mucosal immune response. However, we have found that treatment of A20-HL murine B lymphoma cells with CT severely inhibits the presentation of ovalbumin (OVA) to cells of the T cell clone 42-6A specific for OVA(323-339)/I-Ad, whereas it does not affect the presentation of OVA(323-339) peptide. CT treatment did not affect the expression of B7-1, B7-2, ICAM-1, LFA-1 or MHC class II on, or the internalization of OVA into A20-HL cells. In CT-treated A20-HL cells, degradation of OVA was decreased, and intracellular pH was raised to a level approximately equivalent to that in CH3NH2-treated cells. Treatment with CH3NH2 is known to raise the pH in endocytic structures and thus inhibits antigen processing. Treatment of A20-HL cells with dibutyryl-cAMP similarly increased intracellular pH. The increase in intracellular pH following CT treatment was inhibited by a cAMP inhibitor, 2',3'-dideoxyadenosine. These results strongly suggest that CT treatment of A20-HL cells inhibits their antigen-presenting cell function by triggering the cAMP cascade, increasing intracellular pH, and reducing the degradation of OVA.

Topics: Animals; Antigen Presentation; Chickens; Cholera Toxin; Hydrogen-Ion Concentration; Intracellular Fluid; Lymphoma, B-Cell; Mice; Ovalbumin; Tumor Cells, Cultured

During the investigation of the role of protein synthesis in antigen-presenting cell (APC) function of A20-HL B lymphoma cells, we found that partial inhibition of protein synthesis enhanced their APC function. The treatment of A20-HL cells with 0.313-2.5 microM emetine, an irreversible inhibitor of protein synthesis, decreased protein synthesis by 60-70%, and enhanced their APC function to stimulate I-Ad/OVA323-339-specific T cells to produce interleukin-2 in response to ovalbumin (OVA). The emetine-treated and paraformaldehyde-fixed A20-HL cells required only 20 nM OVA323-339 peptide to stimulate the T cells, whereas those untreated and fixed required 200 nM peptide. This enhancement of APC function was mostly because of the induction of B7-1 expression on A20-HL cells by the emetine treatment, since B7-1 molecules were detected on the emetine-treated A20-HL cells, but only negligibly, if at all, on the untreated cells, and an anti-B7-1 monoclonal antibody, 1G10, inhibited the enhanced APC function of the emetine-treated A20-HL cells. The emetine-treatment also increased B7-1 mRNA expression in A20-HL cells, suggesting that the induction of B7-1 expression was due to the increase in the accumulation of mRNA and the translation with residual ability to synthesize protein. Thus, partial inhibition of protein synthesis in A20-HL cells increases B7-1 mRNA accumulation and its expression on the cell surface, which results in the enhancement of their APC function.

Topics: Antigen Presentation; Antigen-Presenting Cells; B7-1 Antigen; Emetine; Humans; Lymphoma, B-Cell; Neoplasm Proteins; Ovalbumin; RNA, Messenger; Tissue Fixation; Tumor Cells, Cultured

We have used the complex of antigen with class II major histocompatibility proteins (Ia) in membrane-bound form to target a phototoxic compound to antigen-specific T cell hybridomas in vitro. The iodoacetamidyl ester of phototoxic pyrene was bound covalently to antigen-presenting cells (APC), and protein antigens were added to the cells for processing, presentation and targeting of the drug to three different T hybridomas specific for myelin basic protein (MBP), ovalbumin (OVA) and keyhole limpet hemocyanin (KLH). The B hybridoma LS102.9 was used as APC to present MBP, KLH and either a tryptic digest of OVA or the synthetic peptide OVA323-339 to these T cells. A transformed B lymphoma, which expresses trinitrophenol (TNP)-specific surface IgM, A20-HL, was used to present TNP conjugates of KLH and OVA to T cells. Either the antigen-bearing intact APC or Ia+ membranes shed spontaneously from them were used as drug carriers to target pyrene to the T cells. In the dark, or in the absence of pyrene, both the intact APC or the shed membranes stimulated interleukin-2 (IL-2) production by the T cells in an antigen-specific way. After UVA (320-400 nm) irradiation, both forms of these drug carriers had an antigen-specific toxic effect on the T hybridoma cells with receptors for the antigen that they carried. Both spontaneous T cell proliferation and antigen-induced IL-2 production were inhibited. The shed membranes had a more antigen-specific toxic effect than the intact APC, which tend to settle out with the T cells in the microtiter plates, possibly causing nonspecific contact.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antigen-Presenting Cells; Cell Line; Cell Membrane; Drug Carriers; Hemocyanins; Histocompatibility Antigens Class II; Humans; Hybridomas; Interleukin-2; Lymphoma, B-Cell; Myelin Basic Protein; Ovalbumin; T-Lymphocytes; Tumor Cells, Cultured

Human-PBL-SCID animals were created by intraperitoneal (i.p.) transfer of human peripheral blood lymphocytes (PBL) or PBL depleted of CD8-expressing lymphocytes (CD8dL). Analysis of human immunoglobulin levels in these animals revealed that severe combined immunodeficiency (SCID) mice receiving CD8dL produced significantly higher levels of serum human immunoglobulin than those receiving PBL. In an attempt to induce antigen-specific human antibodies these human-PBL-SCID animals were vaccinated with soluble protein antigen [ovalbumin (OVA)] entrapped within liposomes as an immunological adjuvant. Vaccination produced antigen-specific human IgM and IgG in human-PBL-SCID mouse serum. The use of liposomes as adjuvant and the reconstitution of animals with CD8dL together enhanced the OVA-specific immune response as evidenced by the detection of significantly increased serum antibody titres. In the CD8dL reconstituted group, solid tumours of human B-cell origin became detectable in the peritoneal cavity of animals at 8-10 weeks post-reconstitution. These tumours were readily established in vitro and subsequent analysis of culture supernatants showed that these malignant cells continue to secrete human antibodies specific for the original immunizing antigen in vitro. We believe this vaccination of the human-PBL-SCID mouse to produce antigen-specific human antibodies, may find use in the future production of human monoclonal antibodies and in the testing and development of novel vaccine/adjuvant systems.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Antibody Specificity; CD8-Positive T-Lymphocytes; Epitopes; Female; Humans; Immunoglobulins; Liposomes; Lymphocyte Transfusion; Lymphoma, B-Cell; Mice; Mice, SCID; Ovalbumin; Tetanus Toxoid; Transplantation, Heterologous; Vaccination

Self antigens in the body fluids must be taken up, processed and presented by antigen-presenting cells (APC) in order to induce T cell tolerance. For self antigens like the fifth component of complement (C5) which is not picked up by APC via antigen-specific receptors, presentation has to rely on uptake by nonspecific means. C5 was used as a model soluble self antigen to study the capacity of different APC (B lymphoma cells, fibroblasts and macrophages) of taking up, processing and presenting low concentrations of soluble C5 to C5 specific T cell hybrids. Under conditions of limiting antigen amounts macrophages and fibroblasts exhibited similar presentation capacity for soluble C5 while B cells did not. C5 presentation by macrophages was enhanced in the presence of C5-specific antibody and augmented further if antigen was added in the form of particulate latex-antigen-antibody complexes indicating enhanced uptake via Fc receptor-mediated endocytosis or phagocytosis. B cells presented soluble C5 only in the presence of C5-specific antibody. Uptake of C5 under these conditions occurred via Fc receptors type II. This pathway of antigen uptake did not operate with other antigens which were presented efficiently after nonspecific endocytosis. In light of these findings it seems reasonable to propose that nonspecific endocytosis of serum proteins like C5 by B cells is normally limited in order to avoid interference with their critical role in antigen receptor-mediated uptake and presentation for the initiation of an antibody response. It seems likely that presentation of soluble self antigens present in the circulation may normally be the task of dendritic cells and macrophages depending on the physical shape of the antigen.

Topics: Animals; Antigen-Presenting Cells; B-Lymphocytes; Complement C5; Fibroblasts; Histocompatibility Antigens Class II; Lymphoma, B-Cell; Macrophages; Mice; Ovalbumin; Receptors, Fc; T-Lymphocytes

The kinetics of iodinated human alpha-fetoprotein (AFP) binding and uptake by 2 human neoplastic lymphoid cell lines (CEM and RAJI) have been studied. Three saturation plateaus were obtained by incubating CEM and RAJI cells at 4 degrees C with 125I-AFP at different concentrations. Scatchard analysis suggested the presence of 3 types of receptor site with different affinities and capacities on cells of both lines. AFP binding was inhibited by unlabelled human and bovine AFP, and to a lesser extent by human serum albumin (SAH); no significant competition was observed with human transferrin (Tf) or ovalbumin (Ova). Pulse-chase experiments showed that 125I-AFP was released practically undegraded from the cells. Covalent conjugates of AFP and Tf with horseradish peroxidase (HRP) were used to follow the endocytosis and intracellular pathway of these serum proteins by electron microscopy. Both proteins were observed in coated vesicles, endosomes and a tubular vesicular network localized in the Golgi-centrosphere region. SAH-HRP was internalized to a much lesser extent. Ova-HRP was poorly internalized and was observed in lysosome-like organelles.

Topics: alpha-Fetoproteins; Binding, Competitive; Endocytosis; Flow Cytometry; Humans; Immunoenzyme Techniques; In Vitro Techniques; Leukemia, T-Cell; Lymphoma, B-Cell; Microscopy, Electron; Ovalbumin; Receptors, Cell Surface; Receptors, Peptide; Serum Albumin; Temperature; Transferrin