ovalbumin and Fibrosarcoma

Effective antitumor immunotherapy requires the identification of suitable target Ags. Interestingly, many of the tumor Ags used in clinical trials are present in preparations of secreted tumor vesicles (exosomes). In this study, we compared T cell responses elicited by murine MCA101 fibrosarcoma tumors expressing a model Ag at different localizations within the tumor cell in association with secreted vesicles (exosomes), as a nonsecreted cell-associated protein, or as secreted soluble protein. Remarkably, we demonstrated that only the tumor-secreting vesicle-bound Ag elicited a strong Ag-specific CD8(+) T cell response, CD4(+) T cell help, Ag-specific Abs, and a decrease in the percentage of immunosuppressive regulatory T cells in the tumor. Moreover, in a therapeutic tumor model of cryoablation, only in tumors secreting vesicle-bound Ag could Ag-specific CD8(+) T cells still be detected up to 16 d after therapy. We concluded that the localization of an Ag within the tumor codetermines whether a robust immunostimulatory response is elicited. In vivo, vesicle-bound Ag clearly skews toward a more immunogenic phenotype, whereas soluble or cell-associated Ag expression cannot prevent or even delay outgrowth and results in tumor tolerance. This may explain why particular immunotherapies based on these vesicle-bound tumor Ags are potentially successful. Therefore, we conclude that this study may have significant implications in the discovery of new tumor Ags suitable for immunotherapy and that their location should be taken into account to ensure a strong antitumor immune response.

Topics: Animals; Antigens, Neoplasm; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Epitopes, T-Lymphocyte; Exosomes; Fibrosarcoma; Immunoglobulin G; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Receptors, Fc; Transfection

Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

Topics: Animals; Antigens, Neoplasm; Antigens, Surface; Cancer Vaccines; CD8-Positive T-Lymphocytes; Chick Embryo; Fibrosarcoma; Mice; Mice, Inbred C57BL; Milk Proteins; Ovalbumin; Secretory Vesicles; Vaccines, DNA

Tumor-draining lymph node (TDLN) cells develop substantial antitumor activity after activation on immobilized alphaCD3 and culture in low-dose IL-2. This study found that the minor subset of TDLN T cells expressing binding sites for the adhesion receptor P-selectin (Plig(high) T cells) produced T lymphoblasts with the most tumor-specific IFN-gamma synthesis in vitro and antitumor activity following adoptive transfer in vivo. The Plig(high) T cells constituted <25% of the cells with the phenotype of recently activated cells including high levels of CD69, CD44, or CD25, and low levels of CD62L. The cultured Plig(high) TDLN were 10- to 20-fold more active against established pulmonary micrometastases than cultured unfractionated TDLN, and >30-fold more active than cultured TDLN cells depleted of the Plig(high) fraction before expansion (Plig(low) cells). Tumor-specific IFN-gamma synthesis in vitro paralleled the antitumor activities of the cultured fractions in vivo, implying that increased Tc1 and Th1 effector functions contributed to the tumor suppression. Neither nonspecific interaction with the P-selectin chimera used for sorting nor endogenous costimulatory activity in the Plig(high) fraction accounted for the marked increase in antitumor activities after culture. The cultured Plig(high) fraction contained a variety of potential effector cells; however, the CD8 and CD4 subsets of alphabeta T cells accounted for 95-97% of its antitumor activity. The authors propose that P-selectin sorting increased antitumor activities by concentrating Tc1 and Th1 pre-effector/effector cells before culture.

Topics: Adoptive Transfer; Animals; Antigens, CD; Cell Differentiation; Cells, Cultured; E-Selectin; Female; Fibrosarcoma; Immunization; Immunoglobulin M; Immunomagnetic Separation; Immunophenotyping; Inflammation; Interferon-gamma; Lung Neoplasms; Lymph Nodes; Lymphocyte Activation; Lymphokines; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; P-Selectin; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Fibroblast Growth Factor; Recombinant Fusion Proteins; Sialoglycoproteins; T-Lymphocyte Subsets; Tumor Cells, Cultured

Cells present in the polyoma virus-induced murine ascites tumor SEYF-a showed the capacity to fix soluble immune complexes of ovalbumin anti-ovalbumin. The fixation could be inhibited by preincubating the cells with antisera directed against H-2 antigens and with syngeneic anti-tumor antibodies. The latter did not react with normal cells. Depletion of phagocytes from the ascites cell population, thus enriching for tumor cells, increased the inhibition of complex fixation by the syngeneic anti-tumor antiserum. The inhibition of complex fixation by the anti-tumor antibodies was not mediated by "third-party" complexes. Pepsin-treated globulin derived from the syngeneic anti-tumor antiserum could still inhibit complex fixation by SEYF-a cells. These results raise the possibility that tumor cells, per se, expressed receptors for immune complexes. Macrophages, apparently of host origin, residing in the SEYF-a tumor, also expressed such receptors.

Topics: Animals; Antibodies; Antigen-Antibody Complex; Antigens; Binding Sites; Female; Fibrosarcoma; Histocompatibility Antigens; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred Strains; Neoplasms, Experimental; Ovalbumin; Phagocytes; Polyomavirus

Surface receptor sites for the Fc portion of antigen-antibody complexes were demonstrated on cells derived from three methylcholanthrene-induced fibrosarcomas, one of strain C3H and two of strain BALB/c origin, two spontaneously occurring malignant melanomas (B16 in strain C57BL/6 and Harding-Passey in strain BALB/c mice), a Moloney sarcoma virus-induced tumor of strain BALB/c origin and the Walker 256 carcinosarcoma of Holtzman rats. Primary cell cultures derived from these tumors adsorbed technetium-99m labeled, antibody-sensitized sheep erythrocytes (99mTc EA) as determined either by visual scoring of adherence or radioisotopic quantitation. Depending upon the tumor tested, from 20% to greater than 95% of the target cells absorbed 99mTc EA. All cells lost their reactivity after 1 or 2 passages in vitro, but this was regained after a single passage in vivo. Indicator erythrocytes coated with F(ab')2 fragments of the sensitizing sheep erythrocytes (SRBC) antiserum did not adhere thereby demonstrating that the hemadsorption required an intact Fc portion of the antibody molecule. Adherence of 99mTc EA was blocked by soluble immune complexes prepared with ovalbumin and rabbit antibody directed against it and Escherichia coli 055:B5 lipopolysaccharide and mouse antibody directed against it. Normal rabbit or mouse serum, immune serum, or antigen alone did not block adherence of 99mTc EA thereby demonstrating that the receptors had greater affinity for immune complexes than for either antigen or antibody alone. The existence of membrane receptors on tumor-derived cells which react with the Fc portion of antigen-antibody complexes may provide an explanation for the mechanism by which immune complexes are capable of blocking cell-mediated tumor cell destruction irrespective of whether the receptors are on the tumor cells themselves or on admixed lymphocytes and macrophages.

Topics: Animals; Antigen-Antibody Complex; Binding Sites, Antibody; Binding, Competitive; Culture Techniques; Erythrocytes; Escherichia coli; Fibrosarcoma; Hemadsorption; Immune Sera; Immunization; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Melanoma; Mice; Neoplasms, Experimental; Ovalbumin; Rabbits; Sarcoma, Experimental; Sheep; Technetium

Topics: Animals; Cattle; Cell Adhesion; Cell Division; Cells, Cultured; Cricetinae; Embryo, Mammalian; Enzyme Repression; Fibroblasts; Fibrosarcoma; Glycine max; Ovalbumin; p-Dimethylaminoazobenzene; Pancreas; Sarcoma, Experimental; Tosyl Compounds; Trypsin Inhibitors

Topics: Animals; Anti-Bacterial Agents; Antibody Formation; Cattle; Chemotherapy, Cancer, Regional Perfusion; Cyclophosphamide; Diphtheria Toxoid; Fibrosarcoma; Geriatrics; Immunity; Immunosuppressive Agents; Mechlorethamine; Melanoma; Neoplasm Metastasis; Neoplasms; Ovalbumin; Pharmacology; Rabbits; Rats; Research; Sarcoma, Yoshida; Serum Albumin; Serum Albumin, Bovine; Toxicology