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Papers:
- Ostrand-Rosenberg, S., M. Grusby, and V. Clements, 2000. STAT6 knockout mice have enhanced tumor immunity to primary and metastatic mammary carcinoma. J. Immunol. 165: 6015-6019.
STAT4 and STAT6 are essential for the development of CD4+ Th1 and Th2 development, respectively. Tumor immunologists have hypothesized that Th1 cells are critical in tumor immunity because they facilitate differentiation of CD8+ T cells, which are potent anti-tumor effectors. We have used STAT4-/- and STAT6-/- mice to test this hypothesis. BALB/c and knockout mice were challenged in the mammary gland with the highly malignant and spontaneously metastatic BALB/c-derived 4T1 mammary carcinoma. Primary tumor growth and metastatic disease are reduced in STAT6-/- mice, relative to BALB/c and STAT4-/- mice. Antibody depletions demonstrate the effect is mediated by CD8+ T cells, and immunized STAT6-/- mice have higher levels of 4T1-specific CTL than BALB/c or STAT4-/- mice. Surprisingly, Th1 or Th2 cells are not involved, because CD4 depletion does not diminish the anti-tumor effect. Deletion of the STAT6 gene, therefore, facilitates development of potent anti-tumor immunity via a CD4+-independent pathway.
- Qi, L., J. Rojas, and S. Ostrand-Rosenberg, 2000. Tumor cells present MHC class II restricted nuclear and mitochondrial antigens and are the predominant antigen presenting cells in vivo. J. Immunol. 165: 5451-5461.
MHC class II-restricted tumor antigens presented by class II+ tumor cells identified to date are derived from proteins expressed in the cytoplasm or plasma membrane of tumor cells. It is unclear whether MHC class II+ tumor cells present class II-restricted epitopes derived from other intracellular compartments, such as nuclei and/or mitochondria, and if class II+ tumor cells directly present antigen in vivo. To address these questions, a model antigen, hen eggwhite lysozyme, was targeted to various sub-cellular compartments of mouse sarcoma cells and the resulting cells tested for presentation of three lysozyme epitopes in vitro and for presentation of nuclear antigen in vivo. In in vitro studies antigens localized to all tested compartments (nuclei, cytoplasm, mitochondria, and ER) are presented in the absence invariant chain (Ii) and H-2M (DM). Co-expression of Ii and DM inhibit presentation of some, but not all, of the epitopes. In vivo studies demonstrate that class II+ tumor cells, and not host-derived cells, are the predominant APC for class II-restricted nuclear antigens. Because class II+ tumor cells are effective APC in vivo and probably present novel tumor antigen epitopes not presented by host-derived APC, their inclusion in cancer vaccines may enhance activation of tumor-reactive CD4+ T cells.
- Pulaski, B., V. Clements, M. Pipeling, and S. Ostrand-Rosenberg, 2000. Immunotherapy with vaccines combining MHC Class II+/CD80+ tumor cells with IL-12 reduces established metastatic disease and stimulates immune effectors and monokine-induced by interferon-gamma. Canc. Immunol. Immunotherapy, 49: 34-45.
Because they are difficult to treat, animal models of wide-spread, established metastatic cancer are rarely used to test novel immunotherapies. Two such mouse models are used in this report to demonstrate the therapeutic efficacy and to probe the mechanisms of a novel combination immunotherapy consisting of the cytokine IL-12 combined with a previously described MHC class II, CD80 expressing cell-based vaccine. BALB/c mice with 3 week established primary 4T1 mammary carcinomas of up to 6 mm in diameter and with extensive, spontaneous lung metastases have a significant reduction in lung metastases following a 3 week course of immunotherapy consisting of weekly injections of the cell based vaccine plus 3 times/week injections of IL-12. C57BL/6 mice with 7 day established intravenous B16 melF10 lung metastases show a similar response following immunotherapy with IL-12 plus a B16-based MHC class II, CD80-expressing cell-based vaccine. In both systems the combination therapy of cells plus IL-12 is more effective than IL-12 or the cellular vaccine alone, although in the 4T1 system optimal activity does not require MHC class II and CD80 expression in the vaccine cells. The cell-based vaccines were originally designed to specifically activate tumor-specific CD4+ T lymphocytes and thereby provide helper activity to tumor-cytotoxic CD8+ T cells and IL-12 was added to the therapy to facilitate Th1 differentiation. In vivo depletion experiments for CD4+ and CD8+ T cells and NK cells and tumor challenge experiments in beige/nude/XID immunodeficient mice demonstrate that the therapy effect is not exclusively dependent on a single cell population, suggesting that T and NK cells are acting together to optimize the response. IL-12 may also be enhancing the immunotherapy via induction of the chemokine Mig, because reverse PCR experiments demonstrate that Mig is present in the lungs of therapy mice and is most likely synthesized by the tumor cells. These results demonstrate that the combination therapy of systemic IL-12 and a cell-based vaccine is an effective agent for the treatment of advanced, disseminated metastatic cancers in experimental mouse models and that multiple effector cell populations and anti-angiostatic factors are likely to mediate the effect.
- Pulaski, B., D. Terman, S. Khan, E. Muller, and S. Ostrand-Rosenberg, 2000. Cooperativity of SEB Superantigen , MHC Class II, and CD80 for Immunotherapy of Advanced Metastases in a Clinically Relevant Post-operative Mouse Breast Cancer Model. Canc. Res., 60: 2710-2715.
- Qi, L. and S. Ostrand-Rosenberg, 2000. Endogenously encoded MHC class II-restricted antigen traffics via the endocytic pathway and is unaffected by H-2M expression in cell-based cancer vaccines. Traffic, 1: 152-160.
We have developed cell-based cancer vaccines that activate anti-tumor immunity by directly presenting MHC class II-restricted, endogenously synthesized tumor antigens to CD4+ T helper lymphocytes. The vaccines are non-conventional antigen presenting cells because they express MHC class II, do not express invariant chain or H-2M, and preferentially present endogenous antigen. To further improve therapeutic efficacy we have studied the intracellular trafficking pathway of MHC class II molecules in the vaccines using ER-localized lysozyme as a model antigen. Experiments using endocytic and cytosolic pathway inhibitors (chloroquine, primaquine, and brefeldinA) and protease inhibitors (lactacystin, LLnL, E64, and leupeptin) indicate that trafficking is via the endocytic route, although antigen degradation is not mediated by endosomal or proteasomal proteases. Because of its ability to facilitate presentation of H-2M in exogenous antigen presentation via the endocytic route, vaccine cells were transfected with H-2M and tested for antigen presentation activity. In contrast to its role in presentation of exogenous antigen, H-2M has no effect on endogenous antigen presentation or vaccine efficacy. These results suggest that antigen/MHC class II complexes in the vaccines may follow a novel route for processing and presentation and may produce a repertoire of class II-restricted peptides different from those presented by professional APC. The therapeutic efficacy of the vaccines, therefore, may reside in their ability to present novel tumor peptides, thereby activating tumor-specific CD4+ T cells that would not otherwise be activated.
- Pulaski, B. and S. Ostrand-Rosenberg, 1998. Reduction of established spontaneous mammary carcinoma metastases following immunotherapy with MHC class II and B7.1 cell-based tumor vaccines. Cancer Res. 58: 1486- 493.
For many cancer patients, removal of primary tumor is curative, however, if metastatic lesions exist and are not responsive to treatment, survival is limited. Although immunotherapy is actively being tested in animal models against primary tumors and experimental metastases (i.v. induced), very few studies have examined immunotherapy of spontaneous, established metastatic disease. The shortage of such studies can be attributed to the paucity of adequate animal models and to the concern that multiple metastatic lesions may be more resistant to immunotherapy than a localized primary tumor. In this report we use the BALB/c-derived mouse mammary carcinoma, 4T1, and show that this tumor very closely models human breast cancer in its immunogenicity, metastatic properties, and growth characteristics. Additional studies demonstrate that treatment of mice with established primary and metastatic disease with MHC class II and B7.1 transfected tumor cells reduces or elliminates established, spontaneous metastases, but has no impact on primary tumor growth. These studies indicate that cell-based vaccines targeting the activation of CD4+ and CD8+ T cells may be effective agents for the treatment of malignancies, such as breast cancer, where the primary tumor is curable by conventional methods, but metastatic lesions remain refractile to current treatment modalities.
- Armstrong, T., V. Clements, and S. Ostrand-Rosenberg, 1998. MHC class II- transfected tumor cells directly present antigen to tumor-specific CD4+ T lymphocytes. J. Immunol. 160:661-666.
Review Articles:
- Ostrand-Rosenberg, S., B. Pulaski, V. Clements, L. Qi, S. Dissonayake, M. Gilbert, and C. Anderson, 2001. Immunological targets for the gene therapy of cancer. In: Gene Therapy of Cancer, E. Lattime and S. Gerson, eds., 2nd edition, in press.
During the past 10-15 years the field of immunotherapy of cancer has been inundated with novel experimental strategies for the treatment of malignancies. Many of these approaches use T lymphocyte-mediated immunity and are based on the explosion of knowledge in basic immunology combined with improving technical capabilities for the manipulation of genes. Studies in experimental animal systems using these approaches have shown impressive anti-tumor effects. To fully understand these strategies it is necessary to know how lymphocytes are activated in response to antigen and to appreciate the characteristics of tumor cells that make them potential targets for an immune response. In this chapter the process of CD4+ and CD8+ T lymphocyte activation is summarized to provide an overview of the critical requirements for the generation of tumor-specific T lymphocytes. Because induction of antitumor immunity in the immunotherapy setting will occur in cancer patients, the ability of tumor-bearing individuals to mount an immune response is discussed. Selection of tumor antigens to be targeted (tumor regression antigens) is an important consideration for immunotherapy strategies, and the repertoire of available antigens is evaluated. Numerous problems and questions must be considered when designing approaches using immunotherapy in the cancer patient, and some of these concerns are discussed in the context of the novel anti-cancer immunotherapeutic strategies currently being developed.
- Ostrand-Rosenberg, S., B. Pulaski, V. Clements, L. Qi, M. Pipeling, and L. Hanyok, 1999. Cell-based vaccines for the stimulation of immunity to metastatic cancers. Immunol. Rev. 170: 101-114.
We are developing vaccines for inducing immunity to metastatic cancers. Although primary tumors are frequently cured by surgery, chemotherapy, or radiation therapy, metastatic lesions often do not respond to these treatments or proliferate after conventional therapy is terminated. Vaccine therapy for established metastases as well as prophylactic vaccine treatment to prevent outgrowth of latent metastatic tumor cells would therefore be beneficial. Our goal is to activate CD4+ and CD8+ T lymphocytes, however, we have focused on activating tumor-specific CD4+ T helper lymphocytes because of their pivotal role as regulatory cells and in the generation of long-term immunological memory. The vaccines are based on the premise that tumor cells express potentially immunogenic antigens that could be targeted for T cell activation, and that if appropriately genetically modified, tumor cells could be antigen presenting cells for these antigens. To facilitate direct antigen presentation to CD4+ T cells, tumor cells have been transfected with syngeneic MHC class II, costimulatory molecule, and/or superantigen genes. In vivo studies in three mouse tumor models demonstrate that vaccination protects against future challenge with wild type tumor, cures some solid primary tumors, reduces established metastatic disease, and extends mean survival time. Antigen presentation studies demonstrate that in vivo vaccine efficacy is directly related to in vitro antigen presentation activity. The relevance of antigen presentation activity of the vaccines is further confirmed by in vivo studies demonstrating that during the immunization process, the vaccines directly present tumor encoded antigens to CD4+ T lymphocytes. Future clinical trials with these experimental vaccines are necessary to determine their efficacy for the treatment of human metastatic cancers.
- Armstrong, T., Pulaski, B. and S. Ostrand-Rosenberg, 1998. Tumor antigen presentation: Changing the rules. Canc. Immunol. Immunotherapy, 46:70-74.
Cell-based tumor vaccines have been developed based on the hypothesis that tumor cells can be genetically modified to directly present antigen to T lymphocytes. Contrary to expectations, cross-priming is the predominant pathway for activation of tumor-specific CD8+ T cells, while direct presentation of antigen dominates activation of tumor-specific CD4+ T cells. These results pose interesting paradoxes for the generation of immune responses, and have definite implications for the development of anti-cancer vaccines.
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