ovalbumin and Carcinoma--Lewis-Lung

β-Glucans are naturally occurring carbohydrates found in plants, fungi and some bacterial species, and currently are well-established and powerful immunomodulators with beneficial properties in cancer therapy. Recent studies suggested that some additional bioactive molecules have synergistic effects when combined with glucan. In the current study, we evaluated the anticancer properties of glucan, resveratrol, vitamin C combination. We found that compared to the individual components, the combination was the strongest activator of phagocytosis and antibody formation. Using two different models of cancer treatment, our results demonstrated that the combination strongly suppressed the growth of breast and lung tumors, most likely due to the stimulation of apoptosis.

Topics: Animals; Antibody Formation; Antioxidants; Ascorbic Acid; beta-Glucans; Carcinoma, Lewis Lung; Drug Therapy, Combination; Female; Granulocytes; Immunologic Factors; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Ovalbumin; Phagocytosis; Resveratrol; Stilbenes

Myeloid derived suppressor cells (MDSC) are important regulators of immune responses. We evaluated the mechanistic role of MDSC depletion on antigen presenting cell (APC), NK, T cell activities and therapeutic vaccination responses in murine models of lung cancer.. Individual antibody mediated depletion of MDSC (anti-Gr1 or anti-Ly6G) enhanced the antitumor activity against lung cancer. In comparison to controls, MDSC depletion enhanced the APC activity and increased the frequency and activity of the NK and T cell effectors in the tumor. Compared to controls, the anti-Gr1 or anti-Ly6G treatment led to increased: (i) CD8 T cells, (ii) NK cells, (iii) CD8 T or NK intracytoplasmic expression of IFNγ, perforin and granzyme (iv) CD3 T cells expressing the activation marker CD107a and CXCR3, (v) reduced CD8 T cell IL-10 production in the tumors (vi) reduced tumor angiogenic (VEGF, CXCL2, CXCL5, and Angiopoietin1&2) but enhanced anti-angiogenic (CXCL9 and CXCL10) expression and (vii) reduced tumor staining of endothelial marker Meca 32. Immunocytochemistry of tumor sections showed reduced Gr1 expressing cells with increased CD3 T cell infiltrates in the anti-Gr1 or anti-Ly6G groups. MDSC depletion led to a marked inhibition in tumor growth, enhanced tumor cell apoptosis and reduced migration of the tumors from the primary site to the lung compared to controls. Therapeutic vaccination responses were enhanced in vivo following MDSC depletion with 50% of treated mice completely eradicating established tumors. Treated mice that rejected their primary tumors acquired immunological memory against a secondary tumor challenge. The remaining 50% of mice in this group had 20 fold reductions in tumor burden compared to controls.. Our data demonstrate that targeting MDSC can improve antitumor immune responses suggesting a broad applicability of combined immune based approaches against cancer. This multifaceted approach may prove useful against tumors where MDSC play a role in tumor immune evasion.

Topics: Angiogenesis Inhibitors; Animals; Antigen-Presenting Cells; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Bone Marrow Cells; Carcinoma, Lewis Lung; Cell Adhesion; Cell Proliferation; Cytotoxicity, Immunologic; Disease Models, Animal; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Myeloid Cells; Neoplasm Metastasis; Ovalbumin; Spleen; T-Lymphocytes; Treatment Outcome; Tumor Burden; Vaccination

Staphylococcal enterotoxins A (SEA) and B (SEB) are classical models of superantigens (SAg), which induce potent T-cell-stimulating activity by forming complexes with MHC class II molecules on antigen-presenting cells. This large-scale activation of T-cells is accompanied by increased production of cytokines such as interferon-γ (IFN-γ). Additionally, as we previously reported, IFN-γ-producing CD8(+) T cells act as "helper cells," supporting the ability of dendritic cells to produce interleukin-12 (IL-12)p70. Here, we show that DC pulsed with SAg promote the enhancement of anti-tumor immunity. Murine bone marrow-derived dendritic cells (DC) were pulsed with OVA(257-264) (SIINFEKL), which is an H-2Kb target epitope of EG7 [ovalbumin (OVA)-expressing EL4] cell lines, in the presence of SEA and SEB and were subcutaneously injected into naïve C57BL/6 mice. SAg plus OVA(257-264)-pulsed DC vaccine strongly enhanced peptide-specific CD8(+) T cells exhibiting OVA(257-264)-specific cytotoxic activity and IFN-γ production, leading to the induction of protective immunity against EG7 tumors. Furthermore, cyclophosphamide (CY) added to SAg plus tumor-antigens (OVA(257-264), tumor lysate, or TRP-2) pulsed DC immunization markedly enhanced tumor-specific T-cell expansion and had a significant therapeutic effect against various tumors (EG7, 2LL, and B16). Superantigens are potential candidates for enhancing tumor immunity in DC vaccines.

Topics: Animals; Antigen-Presenting Cells; Antineoplastic Agents, Alkylating; Carcinoma, Lewis Lung; CD8-Positive T-Lymphocytes; Cyclophosphamide; Cytokines; Dendritic Cells; Flow Cytometry; Histocompatibility Antigens Class II; Interferon-gamma; Interleukin-12; Lung Neoplasms; Lymphocyte Activation; Lymphoma; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Ovalbumin; Receptors, G-Protein-Coupled; Superantigens; Survival Rate; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer; Tumor Cells, Cultured; Vaccines, Subunit

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4-5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

Topics: Adoptive Transfer; Animals; Antigen Presentation; Antigen-Presenting Cells; beta 2-Microglobulin; Carcinoma, Lewis Lung; CD11a Antigen; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Movement; Cell Proliferation; DNA-Binding Proteins; Fingolimod Hydrochloride; Granzymes; Hyaluronan Receptors; Immunosuppressive Agents; Integrin alpha4; Interferon-gamma; Lymph Nodes; Lymphocyte Activation; Lysosomal Membrane Proteins; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Monophenol Monooxygenase; Neoplasms; Ovalbumin; Peptide Fragments; Propylene Glycols; Receptors, Antigen, T-Cell; Sphingosine; T-Lymphocyte Subsets

Mice bearing established Lewis lung carcinoma (LLC) expressing model tumor antigen, ovalbumin (OVA) (LLC-OVA) marginally responded to local radiotherapy, but none of the mice was cured. In contrast, treatment of the tumor-bearing mice with intratumoral injection of tumor-specific T helper type 1 (Th1) cells and tumor antigen (OVA) after radiotherapy dramatically prolonged the survival days and induced complete cure of the mice at high frequency (80%). Radiation therapy combined with Th1 cells or OVA alone showed no significant therapeutic activity against LLC-OVA. Such a strong therapeutic activity was not induced by intratumoral injection of Th1 cells plus OVA. Compared with other treatment, radiation therapy combined with Th1 cells and OVA was superior to induce the generation of OVA/H-2(b) tetramer(+) tumor-specific cytotoxic T lymphocyte (CTL) with a strong cytotoxicity against LLC-OVA in draining lymph node (DLN). Moreover, the combined therapy is demonstrated to inhibit the growth of tumor mass, which grew at contralateral side. These results indicated that radiotherapy combined with Th1 cell/vaccine therapy induced a systemic antitumor immunity. These findings suggested that combination therapy with radiotherapy and Th1 cell/vaccine therapy may become a practical strategy for cancer treatment.

Topics: Animals; Antigens, Neoplasm; Carcinoma, Lewis Lung; Combined Modality Therapy; Immunotherapy, Adoptive; Mice; Mice, Inbred C57BL; Ovalbumin; T-Lymphocytes, Cytotoxic; Th1 Cells

Tumors contain many antigens that may be recognized by the immune system. It is not known whether these antigens, and the epitopes within these antigens, can all be recognized by the anti-tumor immune response or if such responses are restricted to a few dominant epitopes. Effector function of endogenous cytotoxic T lymphocytes (CTL) generated during tumor progression has previously been assessed by indirect, ex vivo assays, which often focused on a single antigen. Therefore, we evaluated the endogenous in vivo CTL response to multiple neo tumor antigens using murine Lewis lung carcinoma tumor cells transfected with ovalbumin or a polyepitope construct. Both express multiple MHC class I-restricted epitopes. Ovalbumin contains a known hierarchy of epitopes for given MHC molecules, whilst the polyepitope expresses a number of dominant epitopes. We show that as tumors progress, potent effector CTL are generated in vivo that are restricted to dominant epitopes; we did not see the responses to subdominant or cryptic epitopes. Our data show that the CTL recognizing tumor antigens vary in their lytic capacity, as the CTL responding to two of the four epitopes were particularly potent killers. The presence of these effector CTLs did not prevent tumor growth. However, intra-tumoral IL-2 treatment altered the potency, but not the hierarchy, of these CTL such that they mediated tumor regression. These results have implications for immunotherapy protocols.

Topics: Animals; Antigen Presentation; Antigens, Neoplasm; Carcinoma, Lewis Lung; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Epitopes, T-Lymphocyte; Female; Flow Cytometry; Immunodominant Epitopes; Immunotherapy; Interleukin-2; Mice; Mice, Inbred C57BL; Ovalbumin; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes, Cytotoxic; Transfection

Immunostimulating complex (ISCOM) vaccines are particulate antigen delivery vehicles composed of saponin, cholesterol, phospholipid and immunogen. Here we illustrate that ISCOM-based vaccines represent an attractive modality for the development of anti-cancer vaccines. Using murine models and a model cancer antigen, ISCOM vaccines were shown to induce potent CD8 T cell responses, to mediate protection in three different tumor models, to promote Th1-biased immunity, and to induce CD8 T cell responses in the absence of CD4+ T cell help. The former three activities were also found to be substantially improved when the vaccine antigen was associated with the ISCOM structure. Furthermore, the presence in vivo of pre-existing antibodies against the vaccine antigen did not inhibit CD8 T cell induction by the ISCOM vaccine. Although vaccination was effective against challenge with vaccine-antigen expressing tumors, no activity against neighboring vaccine-antigen negative tumor cells was observed, indicating that determinant spreading or bystander activity does not lead to significant anti-cancer activity.

Topics: Animals; Antibodies, Neoplasm; Antigens, Neoplasm; Cancer Vaccines; Carcinoma, Lewis Lung; CD8-Positive T-Lymphocytes; Epitopes; Female; Immunoglobulin G; Injections, Subcutaneous; ISCOMs; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Time Factors; Vaccination

To determine whether APC function or "arming" of CTL for lytic function are the points at which Ags from a nonimmunogenic tumor fail to induce an effective immune response, we established a murine tumor model that expressed intracellular OVA and selected a clone (cOVA-9) that remained susceptible to lysis by specific CD8(+) T cells throughout tumor growth. Viable cOVA-9 tumor cells grew in normal mice at a rate similar to the parental tumor, and vaccination with irradiated cOVA-9 cells did not induce protection against itself or the parental line, confirming its nonimmunogenic status. In vivo evaluation during tumor growth demonstrated persisting tumor Ag cross-presentation accompanied by the generation of potent, specific CTL which were detectable when tumors were barely palpable. Despite the presence of highly active CTL in the tumor-draining lymph nodes, there was no apparent lysis of tumor-associated APC. These data show that tumor-draining APC are not dysfunctional with regard to two crucial processes, in vivo tumor Ag cross-presentation and specific CTL arming, and that failure to prevent tumor growth is not in the induction phase, but in the effector phase and occurs within the tumor itself before the tumor matrix is established.

Topics: Animals; Antigen Presentation; Antigen-Presenting Cells; Antigens, Neoplasm; Carcinoma, Lewis Lung; Cell Division; Cell Movement; Cytoplasm; Cytotoxicity, Immunologic; Disease Progression; Egg Proteins; Epitopes, T-Lymphocyte; Female; Histocompatibility Antigens Class I; Lymph Nodes; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Transplantation; Ovalbumin; Peptide Fragments; Spleen; Stem Cells; T-Lymphocytes, Cytotoxic; Transfection; Tumor Cells, Cultured