Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to γ-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy.

The V protein of the paramyxovirus simian virus 5 (SV5) is responsible for targeted degradation of STAT1 and the block in alpha/beta interferon (IFN-␣/␤) signaling that occurs after SV5 infection of human cells. We have analyzed the growth properties of a recombinant SV5 that was engineered to be defective in targeting STAT1 degradation. A recombinant SV5 (rSV5-P/V-CPI؊) was engineered to contain six naturally occurring P/V protein mutations, three of which have been shown in previous transfection experiments to disrupt the V-mediated block in IFN-␣/␤ signaling. In contrast to wild-type (WT) SV5, human cells infected with rSV5-P/V-CPI؊ had STAT1 levels similar to those in mock-infected cells. Cells infected with rSV5-P/V-CPI؊ were found to express higher-than-WT levels of viral proteins and mRNA, suggesting that the P/V mutations had disrupted the regulation of viral RNA synthesis. Despite the inability to target STAT1 for degradation, single-step growth assays showed that the rSV5-P/V-CPI؊ mutant virus grew better than WT SV5 in all cell lines tested. Unexpectedly, cells infected with rSV5-P/V-CPI؊ but not WT SV5 showed an activation of a reporter gene that was under control of the IFN-␤ promoter. The secretion of IFN from cells infected with rSV5-P/V-CPI؊ but not WT SV5 was confirmed by a bioassay for IFN. The rSV5-P/V-CPI؊ mutant grew to higher titers than did WT rSV5 at early times in multistep growth assays. However, rSV5-P/V-CPI؊ growth quickly reached a final plateau while WT rSV5 continued to grow and produced a final titer higher than that of rSV5-P/V-CPI؊ by late times postinfection. In contrast to WT rSV5, infection of a variety of cell lines with rSV5-P/V-CPI؊ induced cell death pathways with characteristics of apoptosis. Our results confirm a role for the SV5 V protein in blocking IFN signaling but also suggest new roles for the P/V gene products in controlling viral gene expression, the induction of IFN-␣/␤ synthesis, and virus-induced apoptosis.Alpha and beta interferons (IFN-␣ and IFN-␤) are important cytokines that are produced during virus infection (reviewed in references 5 and 46). IFN-␣/␤ signaling is initiated when secreted IFN binds to its receptor on the cell surface, resulting in the phosphorylation of a family of latent transcription factors called STAT1 and STAT2 (signal transducers and activators of transcription). IFN-activated STAT1 and STAT2 heterodimerize and associate with p48 (also known as IRF-9) to form the transcription factor ISGF3. This transcription factor binds to interferon-sensitive response elements (ISRE) located in the promoter region of IFN-inducible genes (reviewed in reference 24). Many of the identified IFN-induced gene products have potent antiviral activity and can contribute to the inhibition of host and viral protein synthesis, induction of apoptosis, and clearance of viral infections. As such, viruses have evolved mechanisms that counteract the induction of IFN, the activation of the IFN signaling pathways, or both of these processes (reviewed in references ...

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to -irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our fi ndings indicate that directed radiotherapy can improve the effi cacy of tumor immunotherapy.

Purpose: Saccharomyces cerevisiae, a nonpathogenic yeast, has been used previously as a vehicle to elicit immune responses to foreign antigens, and tumor-associated antigens, and has been shown to reduce tumor burden in mice. Studies were designed to determine if vaccination of human carcinoembryonic antigen (CEA)-transgenic (CEA-Tg) mice (where CEA is a selfantigen) with a recombinant S. cerevisiae construct expressing human CEA (yeast-CEA) elicits CEA-specificT-cell responses and antitumor activity. Experimental Design: CEA-Tg mice were vaccinated with yeast-CEA, and CD4 + and CD8 + T-cell responses were assessed after one and multiple administrations or vaccinations at multiple sites per administration. Antitumor activity was determined by tumor growth and overall survival in both pulmonary metastasis and s.c. pancreatic tumor models.Results: These studies demonstrate that recombinant yeast can break tolerance and that (a) yeast-CEA constructs elicit both CEA-specific CD4 + and CD8 + T-cell responses; (b) repeated yeast-CEA administration causes increased antigen-specificT-cell responses after each vaccination; (c) vaccination with yeast-CEA at multiple sites induces a greater T-cell response than the same dose given at a single site; and (d) tumor-bearing mice vaccinated with yeast-CEA show a reduction in tumor burden and increased overall survival compared to mock-treated or control yeast-vaccinated mice in both pulmonary metastasis and s.c. pancreatic tumor models. Conclusions: Vaccination with a heat-killed recombinant yeast expressing the tumor-associated antigen CEA induces CEA-specific immune responses, reduces tumor burden, and extends overall survival in CEA-Tg mice. These studies thus form the rationale for the incorporation of recombinant yeast-CEA and other recombinant yeast constructs in cancer immunotherapy protocols.