Cancer treatments using ionizing radiation (IR) therapy are thought to act primarily through the induction of tumor cell damage at a molecular level. However, a new concept has recently emerged, suggesting that the immune system is required for effective IR therapy. Our work here has identified interferon gamma (IFN-γ) as an essential cytokine for the efficacy of IR therapy. Local IR (15 Gy) to mice bearing Colon38, a colon adenocarcinoma, decreases tumor burden in wild-type animals. Interestingly, IR therapy had no effect on tumor burden in IFNγKO mice. We further determined that intratumoral levels of IFN-γ increased 2 days following IR, which directly correlated with a decrease in tumor burden that was not a result of direct cytotoxic effects of IFN-γ on tumor cells. T cells from IR-treated tumors exhibited a far greater capacity to lyse tumor cells in a (51)Cr release assay, a process that was dependent on IFN-γ. CD8(+) T cells were the predominant producers of IFN-γ, as demonstrated by IFN-γ intracellular staining and studies in IFN-γ reporter mice. Elimination of CD8(+) T cells by antibody treatment reduced the intratumoral levels of IFN-γ by over 90%. More importantly, elimination of CD8(+) T cells completely abrogated the effects of radiation therapy. Our data suggest that IFN-γ plays a pivotal role in mediating the antitumor effects of IR therapy.

In species with endometrial decidualization and hemochorial placentation (humans, mice, and others), leukocytes localize to early implant sites and contribute to decidual angiogenesis, spiral arterial remodeling, and trophoblast invasion. Relationships between leukocytes, trophoblasts, and the decidual vasculature are not fully defined. Early C57BL/6J implant sites were analyzed by flow cytometry to define leukocyte subsets and by whole-mount immunohistochemistry to visualize relationships between leukocytes, decidual vessels, and trophoblasts. Ptprc(+) (CD45(+)) cells increased in decidua between Gestational Day (GD) 5.5 and GD 9.5. Uterine natural killer (uNK) cells that showed dynamic expression of Cd (CD) 69, an activating receptor, and Klrg1 (KLRG1), an inhibitory receptor, localized mesometrially and were the dominant CD45(+) cells between GD 5.5 and GD 7.5. At GD 8.5, immature monocytes that occurred throughout decidua exceeded uNK cells numerically and many leukocytes acquired irregular shapes, and leukocyte-leukocyte conjugates became frequent. Vessels were morphologically heterogeneous and regionally unique. Migrating trophoblasts were first observed at GD 6.5 and, at GD 9.5, breached endothelium, entered vascular lumens, and appeared to occlude some vessels, as described for human spiral arteries. No leukocyte-trophoblast conjugates were detected. Whole-mount staining gave unparalleled decidual vascular detail and cell-specific positional information. Its application across murine models of pregnancy disturbances should significantly advance our understanding of the maternal-fetal interface.

The omentum, an important peritoneal tissue, is studded with a high number of immune aggregates, or "milky spots," the number, function, and phenotype of which is largely unknown. We have analyzed the immune composition on the normal omentum and also have shown that both free immune cells and tumor cells in the peritoneal fluid bind preferentially to these immune aggregates. This binding may be mediated by the network of collagen I fibers, which overlay these areas. In addition, we have shown that not only do omental vessels express vascular endothelial growth factor receptor 3 (VEGFR3), a receptor that is only found on angiogenic blood vessels, but that tumor cells co-localize with these vessels, possibly increasing the ability of tumor to induce neovascularization and therefore thrive.

Summary The ability to alter the cytokine microenvironment has the potential to shape immune responses in many physiological settings, including the immunotherapy of tumours. We set out to develop a general approach in which cytokines could be functionally attenuated until activated. We report the development and initial characterization of fusion proteins in which human or mouse interleukin‐2 (IL‐2), a potent growth factor for immune cells, is joined to a specific IL‐2 inhibitory binding component separated by a protease site. The rationale is that upon cleavage by a protease the cytokine is free to dissociate from the inhibitory component and becomes biologically more available. We describe the successful development of two attenuation strategies using specific binding: the first uses the mouse IL‐2 receptor alpha chain as the inhibitory binding component whereas the second employs a human antibody fragment (scFv) reactive with human IL‐2. We demonstrated that the fusion proteins containing a prostate‐specific antigen or a matrix metalloproteinase (MMP) protease cleavage site are markedly attenuated in the intact fusion protein but had enhanced bioactivity of IL‐2 in vitro when cleaved. Further, we showed that a fusion protein composed of the IL‐2/IL‐2 receptor alpha chain with an MMP cleavage site reduced tumour growth in vivo in a peritoneal mouse tumour model. This general strategy should be applicable to other proteases and immune modulators allowing site‐specific activation of immunomodulators while reducing unwanted side‐effects.

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