ovalbumin and Lymphoma--T-Cell

Therapeutic vaccines that arouse the cytotoxic T cell immune response to reject infected cells have been investigated extensively for treating disease. Due to the large amounts of resident antigen-presenting cells (APCs) and T cells in lymph nodes, great efforts have been made to explore the strategy of targeting lymph nodes directly with nanovaccines to activate T cells. However, these nanovaccines still have several problems, such as a low loading efficiency and compromised activity of antigens and adjuvants derived from traditional complicated preparation. There are also safety concerns about materials synthesized without FDA approval. Herein, we construct an assembled nanoparticle composed of an antigen (ovalbumin, OVA) and adjuvant (CpG) to ensure its safety and high loading efficiency. The activity of both components was well preserved due to the mild self-assembly process. The small size, narrow distribution, negative charge, and good stability of the nanoparticle endow these nanovaccines with superior capacity for lymph node targeting. Correspondingly, the accumulation at lymph nodes can be improved by 10-fold. Subsequently, due to the sufficient APC internalization and maturation in lymph nodes, ~60% of T cells are stimulated to proliferate and over 70% of target cells are specifically killed. Based on the effective and quick cellular immune response, the assembled nanoparticles exhibit great potential as therapeutic vaccines.

Topics: Adjuvants, Immunologic; Animals; Antigen-Presenting Cells; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Female; Lymph Nodes; Lymphocyte Activation; Lymphoma, T-Cell; Mice; Mice, Inbred C57BL; Nanoparticles; Oligodeoxyribonucleotides; Ovalbumin; T-Lymphocytes, Cytotoxic; Vaccines

T lymphocytes play a crucial role in the regulation of immune responses against the tumour cells. Tumour progression results in dysfunction and inhibition of T cells, which ultimately leads to impairment in the antitumour immune response. The impaired antitumour immune response in the host is represented by the decreased number of T cells and their incomplete and improper function. The immunosuppressive network in tumour-bearing host mediated by tumour cells also leads to the inequities of T cell subsets and imbalance of Th1/Th2 dichotomy. Therefore, in the present study, we sought to investigate the role of tumour progression in the development of T cell phenotype and the involvement of interleukin-13 thereof selecting Dalton's lymphoma (DL) as a tumour model. It was observed that a significant increase in the number of CD4(+) T cell population, whereas a significant decline in the CD8(+) T cells among lymphoid cell population of OVA-immunized DL-bearing BALB/c mice occurs. Similar observation was found following the administration of IL-13 to the normal healthy mice. It was further confirmed that expansion in Th2 type cells among CD4(+) T cell population occurs following the progression of tumour and administration of IL-13 to normal healthy mice by an yet to define mechanism. Therefore, it can be concluded that IL-13 has immense role in polarizing the immune responses by inducing the differentiation of Th2 type of cells.

Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Differentiation; Female; Flow Cytometry; Interleukin-13; Lymphoma, T-Cell; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Phenotype; Recombinant Proteins

Heat Shock protein-70 derived from tumor cells is highly immunogenic and induces specific anti-tumor immune response by directly activating cytotoxic CD8(+) T cells. Additionally, Hsp70 is known to be a strong activator of antigen presenting cells and therefore, up regulates the production of pro-inflammatory cytokines and chemokines. In this study, we have shown the effect of tumor-derived Hsp70 on the induction of delayed type hypersensitivity reaction in a T cell lymphoma bearing mice. The autologous Hsp70 augments contact hypersensitivity and delayed type hypersensitivity responses in mice challenged with allergen in vehicle and antigens respectively. The adoptive transfer of splenocytes derived from Hsp70 immunized mice is able to enhance delayed type hypersensitivity response in antigen challenged normal and DL-bearing host. Furthermore, adoptive transfer of macrophages incubated with autologous Hsp70 also enhances DTH reactivity in mice. The pro-inflammatory cytokines and C-C chemokines are found to be elevated in the DTH footpad extract of antigen challenged normal and DL-bearing mice. Increased production of IFN-gamma and MIP-1alpha+/- suggest that autologous Hsp70 augments the recruitment of antigen specific Th1 cells, which further secretes pro-inflammatory cytokines and C-C chemokines mediating the hypersensitivity reaction upon challenge with antigens.

Topics: Animals; Antigens; Cytokines; Female; HSP70 Heat-Shock Proteins; Hypersensitivity, Delayed; Lymphoma, T-Cell; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Th1 Cells

The TNF family member, a proliferation-inducing ligand (APRIL), has been suggested to act as a costimulatory molecule in T cell responses. However, studies addressing this role in vivo are largely lacking. Here, we evaluated the effects of APRIL on physiological T cell responses in vivo. Although receptors for APRIL are expressed on a subset of T cells, neither TCR transgenic (Tg) T cell responses nor endogenous TCR responses were affected by Tg APRIL expression in vivo. Moreover, APRIL did not significantly enhance the induction of T cell lymphomas upon Moloney murine leukemia virus (MLV) infection. This clearly contrasts current belief and indicates that APRIL does not serve a major role in T cell immunity or lymphomagenesis. However, we did observe a strong increase in erythroleukemia formation after MLV inoculation of APRIL Tg mice. Strikingly, this erythroleukemia-facilitating property of APRIL was confirmed using the erythroleukemogenic Friend-MLV. Erythroleukemia in APRIL Tg mice was characterized by low hematocrits and grossly enlarged spleens with an increased percentage of erythroid precursors. Altogether, these results unveil new proerythroleukemogenic properties of APRIL.

Topics: Animals; Autoimmunity; Flow Cytometry; Hematocrit; Heterozygote; Homozygote; Leukemia, Erythroblastic, Acute; Lymphoma, T-Cell; Mice; Mice, Inbred C57BL; Mice, Transgenic; Moloney murine leukemia virus; Ovalbumin; Receptors, Antigen, T-Cell; Stem Cells; T-Lymphocytes; Tumor Necrosis Factor Ligand Superfamily Member 13

Antigen-specific cytotoxic T lymphocytes (CTL) are essential for the immunotherapy against cancer or infection diseases although, conventionally, immunization with antigens in soluble form cannot induce CTL. In the present study, we have demonstrated for the first time that ovalbumin (OVA)-specific CTL can be induced without any adjuvants by immunization with soluble OVA with negative charges through scavenger-mediated delivery of antigens to antigen presenting cells (APC). Succinylated, maleylated and aconitylated derivatives were synthesized to allow the introduction of negative charges. All these derivatives can induce OVA-specific CTL and, especially, the CTL activity of mice immunized with maleylated derivatives was comparable with that with OVA emulsified with CFA, known to be the strongest adjuvant. Efficient antigen-specific T cell proliferation and IFN-gamma production were also observed for the OVA derivatives. The OVA derivatives also showed significant protective effects on the growth of OVA-expressing E.G7 tumor cells. In conclusion, the present study demonstrates that the introduction of negative charges to soluble antigens will be a useful strategy for the development of vaccines.

Topics: Animals; Antigen-Presenting Cells; Antigens; Cell Line, Tumor; Female; Immunization; Lymphocyte Activation; Lymphoma, T-Cell; Male; Mice; Mice, Inbred C57BL; Ovalbumin; Receptors, Scavenger; Solubility; T-Lymphocytes, Cytotoxic

Previous reports have shown that transcutaneous immunization (TCI) with proteins or peptides in combination with adjuvants efficiently induces specific cellular and humoral immune responses. However, depending on the kind of skin pretreatment, induction of cellular immune responses was restricted to generation of either specific cytotoxic T lymphocytes (CTLs) or T-helper (Th) cells. In this study, we induced antigen-specific CTL responses together with the appropriate Th responses by TCI of C57BL/6 mice. We applied ovalbumin protein or an ovalbumin-derived fusion peptide containing a CTL and Th epitope together with a combination of cholera toxin (CT) and CpG oligodeoxynucleotide (CpG) onto cold wax-depilated and hydrated bare skin. TCI with the ovalbumin fusion peptide induced more robust CTL and Th responses than that with ovalbumin protein. The fusion peptide in combination with the nontoxic CT derivative CTA1-D2D1 and CpG induced an antigen-specific CTL response, albeit less efficiently than in combination with complete CT. Further, we compared the potency of HPV-16 E7 oncoprotein-derived peptides containing single (CTL) or multiple (CTL + Th + B cell) epitopes to induce effective CTL responses. Strong E7-specific CTL responses were detected only after TCI with the E7 multiepitope peptide. This peptide was also shown to protect mice against tumor growth after challenge with HPV-16 E7-positive tumor cells. TCI with E7 protein and CT/CpG led to formation of an E7-specific humoral immune response.

Topics: Administration, Cutaneous; Animals; Antibody Formation; Epitopes; Female; Human papillomavirus 16; Humans; Immunization; Lymphoma, T-Cell; Mice; Mice, Inbred C57BL; Ovalbumin; Papillomavirus Infections; Peptides; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer; Thymoma; Transfection

To develop an efficient antitumor immunotherapy, we have examined if dendritic cells (DCs) loaded with soluble antigens by electroporation present more antigens via the MHC (major histocompatibility complex) class I pathway, which mediate a cytotoxic T-cell response. DCs loaded with ovalbumin (OVA) by electroporation presented more MHC class I-restricted determinants compared with DCs pulsed with OVA. When electroporated DCs were pulsed with OVA for additional times, both MHC class I- and II-restricted presentation of OVA were increased compared with each single procedure, including electroporation or simple pulse. Immunization with DCs loaded with OVA by electroporation induced higher cytotoxicity of splenocytes to E.G7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with DCs pulsed with OVA. In the animal study, immunization with DCs loaded with OVA or tumor cell lysates by electroporation induced an effective antitumor immunity against tumor of E.G7 cells or Lewis lung carcinoma cells, respectively. In addition, immunization with DCs loaded with antigen by combination of electroporation and pulse, completely protected mice from tumor formation, and prolonged survival, in both tumor models. These results demonstrated that electroporation would be a useful way to enhance MHC class I-mediated antitumor immunity without functional deterioration, and that the combination of electroporation and pulse could be a simple and efficient antigen-loading method and consequently lead to induction of strong antitumor immunity.

Topics: Animals; Antigen Presentation; Bone Marrow Cells; Cytotoxicity, Immunologic; Dendritic Cells; Electroporation; Female; Flow Cytometry; Histocompatibility Antigens Class I; Immunization; Immunotherapy, Adoptive; Lymphoma, T-Cell; Mice; Mice, Inbred C57BL; Ovalbumin; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Traditional methods for identifying T cell-recognized tumor antigens (Ags) are laborious and time-consuming. In an attempt to simplify the procedure, a novel strategy, SING (SIgnal transduction molecule-mediated, NFAT-controlled, GFP expression) was established as a direct approach for cloning T cell-recognized tumor Ags. In the SING system, a mouse T cell line (BW5147) was transduced with a chimeric H-2Kb construct containing T cell-signaling domains and a green fluorescent protein (GFP) reporter gene under the transcriptional control of nuclear factor of activated T cells (NFAT). The resultant BW5147 cells were named BS cells. This cell line could "sense" TCR stimulation through the T cell-signaling domains after coculture with Ag-specific T cells and then become fluorescent (expressing green fluorescence protein, GFP+) in the presence of Ag peptides. The interaction between BS cells and Ag-specific T cells could be enhanced by addition of costimulatory signals. Currently, BS cells have been optimized to "sense" TCR stimulation after being pulsed with the relevant peptides at concentrations as low as 10(-9) M. Endogenous Ag-expressing BS cells could also become fluorescent after coculture with Ag-specific T cells. Our results provide a proof of principle for using the SING system to directly isolate Ag-expressing BS cells from BS cell repertoires, which are retrovirally transduced with tumor-derived cDNA libraries. Once tumor Ag-marked BS cells are identified, the sequences encoding tumor Ags could be easily retrieved by PCR amplification of the genomic DNA using vector-specific primers.

Topics: 3T3 Cells; Animals; Antigen Presentation; Antigens, Neoplasm; Cell Line, Tumor; Cloning, Molecular; Coculture Techniques; DNA-Binding Proteins; Egg Proteins; Exocytosis; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; H-2 Antigens; Hybridomas; Ionomycin; Luminescent Proteins; Lymphoma, T-Cell; Macrolides; Mice; NFATC Transcription Factors; Nuclear Proteins; Ovalbumin; Peptide Fragments; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Signal Transduction; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Transcription Factors; Transcription, Genetic; Transduction, Genetic

Established EG7 tumors expressing OVA and growing at an intradermal site become rapidly reduced in size following adoptive therapy with in vitro-generated type I CD8 T cell (Tc1) effectors generated from naive CD8 T cells from transgenic TCR OVA-specific mice. Tc1 effectors kill EG7 target cells in vitro by a perforin-dependent mechanism. However, we show that there is no quantitative diminution of the initial phase of antitumor activity in vivo, whether the Tc1 effectors are derived from perforin-, Fas ligand-, or TNF-deficient transgenic TCR mice or whether the recipients are perforin deficient. Tumors are also equally well controlled whether the Tc1 effectors come from mice deficient in perforin plus Fas ligand or perforin plus TNF. Control of tumor growth is diminished when Tc1 effectors generated from IFN-gamma-deficient mice are used. We conclude that control of tumor growth is not in any way affected by loss of contact-mediated lytic mechanisms, and conclude that the CD8 effectors must act by recruiting host effector mechanisms to control tumor growth.

Topics: Animals; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cells, Cultured; Cytotoxicity, Immunologic; Egg Proteins; Fas Ligand Protein; Immunotherapy, Adoptive; Lymphoma, T-Cell; Lymphotoxin-alpha; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Mice, Transgenic; Neoplasm Transplantation; Ovalbumin; Peptide Fragments; Perforin; Pore Forming Cytotoxic Proteins; Transfection; Tumor Necrosis Factor-alpha

Although many tumors express tumor-specific antigens, most fail to stimulate effective immune responses. Tumors generally lack co-stimulatory molecules, which can lead to tolerance of tumor-specific T cells and progressive tumor growth. Here, we demonstrate that the ovalbumin (OVA) transfected EL4 tumor, E.G7-OVA, grows progressively in syngeneic mice even though the tumor can be rejected if the mice are immunized with OVA in adjuvant. E.G7-OVA grew more rapidly in RAG-1 deficient than sufficient mice suggesting that normal mice make an abortive immune response to this tumor. Depletion of gammadelta T cells or IL-10 augmented the ability of B6 mice to reject E.G7-OVA. Spleen cells from normal, but not IL-10 knockout, mice reconstituted rapid tumor growth in gammadelta T cell-deficient mice. Thus, gammadelta T cells play an important role in preventing immune elimination of this tumor by a mechanism that directly or indirectly involves IL-10.

Topics: Animals; Antibody Formation; CD8-Positive T-Lymphocytes; Crosses, Genetic; Enzyme-Linked Immunosorbent Assay; Female; Genes, RAG-1; Immune Tolerance; Interleukin-10; Lymphoma, T-Cell; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Phagocytosis; Receptors, Antigen, T-Cell, gamma-delta; Transfection; Tumor Cells, Cultured

Modification of tumor cells with one or more costimulatory adhesion molecules has been proposed as a means to develop therapeutic cancer vaccines for use in human immunotherapy. Expression of B7-1 (CD80) in tumors by gene transfer creates an immunogenic tumor cell that induces antitumor immunity and protects mice from further challenge with wild-type tumor cells. In this report, we demonstrate that protein transfer of glycosyl-phosphatidylinositol (GPI)-anchored costimulatory molecules into tumor cell membranes could be used as an alternative to gene transfer for tumor immunotherapy. Incubation of isolated tumor membranes with purified GPI-anchored B7-1 results in stable incorporation of B7-1 on tumor cell membranes within a few hours. Immunization of C57BL/6 mice with EG7 tumor membranes modified to express GPI-B7-1 by protein transfer induces tumor-specific T-cell proliferation and CTLs. In addition, immunization with these EG7 membranes protects mice from parental tumor challenge. The protein transfer approach used here does not require foreign vectors or live tumor cells and is completed within a matter of hours. Irradiated cells or membrane preparations from fresh or frozen tumor tissue can be used. Therefore, protein transfer of glycolipid-anchored molecules provides an efficient and novel approach to modify tumor membranes for human immunotherapy. This approach is not limited to costimulatory molecules because any cell surface protein can be converted to a GPI-anchored form by recombinant techniques.

Topics: Animals; B7-1 Antigen; Cell Membrane; Cytotoxicity, Immunologic; Epitopes; Female; Glycosylphosphatidylinositols; H-2 Antigens; Humans; Immunization; Immunotherapy; Lymphocyte Activation; Lymphoma, T-Cell; Membrane Proteins; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Ovalbumin; T-Lymphocytes, Cytotoxic

Delivery of antigen in a manner that induces effective, antigen-specific immunity is a critical challenge in vaccine design. Optimal antigen presentation is mediated by professional antigen-presenting cells (APCs) capable of taking up, processing and presenting antigen to T cells in the context of costimulatory signals required for T-cell activation. Developing immunization strategies to optimize antigen presentation by dendritic cells, the most potent APCs, is a rational approach to vaccine design. Here we show that cutaneous genetic immunization with naked DNA results in potent, antigen-specific, cytotoxic T lymphocyte-mediated protective tumor immunity. This method of immunization results in the transfection of skin-derived dendritic cells, which localize in the draining lymph nodes. These observations provide a basis for further development of DNA-based vaccines and demonstrate the feasibility of genetically engineering dendritic cells in vivo.

Topics: Amino Acid Sequence; Animals; Antigen Presentation; Biolistics; Cell Movement; Dendritic Cells; Epitopes; Feasibility Studies; Female; H-2 Antigens; Immunization; Lymph Nodes; Lymphoma, T-Cell; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasm Transplantation; Ovalbumin; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured; Vaccines, DNA

Antigens in extracellular fluids can be processed and presented with major histocompatibility complex (MHC) class I molecules by a subset of antigen presenting cells (APCs). Chicken egg ovalbumin (Ova) linked to beads was presented with MHC class I molecules by these cells up to 10(4)-fold more efficiently than soluble Ova. This enhanced presentation was observed with covalently or noncovalently linked Ova and with beads of different compositions. A key parameter in the activity of these conjugates was the size of the beads. The APC that is responsible for this form of presentation is a macrophage. These cells internalize the antigen constructs through phagocytosis, since cytochalasin B inhibited presentation. Processing of the antigen and association with MHC class I molecules appears to occur intracellularly as presentation was observed under conditions where there was no detectable release of peptides into the extracellular fluids. When injected in vivo in C57BL/6 mice, Ova-beads, but not soluble Ova, primed CD4- CD8+ cytotoxic T lymphocytes (CTLs). Similar results were obtained in BALB/c mice immunized with beta-galactosidase-beads. The implications of these findings for development of nonliving vaccines that stimulate CTL immunity are discussed.

Topics: Animals; Antigen-Presenting Cells; Azides; Cell Line; Cytochalasin B; Deoxyglucose; Female; Histocompatibility Antigens Class I; Kinetics; Lymphoma, T-Cell; Macrophages; Mast-Cell Sarcoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Phagocytosis; Spleen; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured