While significant advances in radiotherapy have increased its effectiveness in many

While significant advances in radiotherapy have increased its effectiveness in many cancer settings general strategies to widen the therapeutic window between normal tissue toxicity and malignant tumor destruction would still offer great value. establishing that CD47 expression in the microenvironment was sufficient to limit tumor radiosensitivity. Mechanistic investigations revealed increased tumor infiltration by cytotoxic CD8+ T cells in a CD47-deficient microenvironment with an associated increase in T cell-dependent intratumoral expression of granzyme Polygalasaponin F B. Correspondingly an inverse correlation between CD8+ T cell infiltration and CD47 expression was observed in Polygalasaponin F human melanomas. Our findings establish that blocking CD47 in the context of radiotherapy enhances antitumor immunity by directly stimulating CD8+ cytotoxic T cells with the potential to increase curative responses. Introduction CD47 is a widely expressed counter-receptor for the inhibitory phagocyte receptor SIRPα. Blocking this interaction enhances macrophage-mediated clearance of tumor cells (1-3). Correspondingly elevated CD47 expression on cancer cells is proposed to suppress anti-tumor innate immunity (4 5 However CD47 also Polygalasaponin F functions as a signaling receptor that determines cell fate through the regulation of several death/survival pathways mainly through its interactions with the matricellular protein thrombospondin-1 (TSP1). Binding of the C-terminal signature domain of TSP1 to CD47 causes a profound inhibition of the nitric oxide/cGMP signaling Polygalasaponin F in vascular cells and T cells (6-8). In the immune system binding of TSP1 to CD47 inhibits T cell activation (9-11) in part by inhibiting the autocrine activating function of hydrogen sulfide signaling in T cells (12). TSP1 is the relevant CD47 ligand in T cells because these cells do not express detectable levels of SIRPα (13 14 Signaling through CD47 also regulates T cell differentiation and adhesion as well as NK and dendritic cell functions that regulate adaptive immunity (15-22). Thus we propose that treatment of tumor-bearing animals with CD47 blocking antibodies which are known to inhibit both SIRPα and TSP1 binding to CD47 could directly modulate adaptive as well as innate anti-tumor immunity. Indeed cytotoxic T cells were recently implicated in the anti-tumor effects of a CD47-blocking antibody but this outcome was attributed to an indirect effect of inhibiting SIRPα engagement on macrophages (23). We previously demonstrated that blockade of CD47 enhances the radiation-induced delay in tumor growth in two syngeneic mouse models (24). The Mouse monoclonal antibody to PA28 gamma. The 26S proteasome is a multicatalytic proteinase complex with a highly ordered structurecomposed of 2 complexes, a 20S core and a 19S regulator. The 20S core is composed of 4rings of 28 non-identical subunits; 2 rings are composed of 7 alpha subunits and 2 rings arecomposed of 7 beta subunits. The 19S regulator is composed of a base, which contains 6ATPase subunits and 2 non-ATPase subunits, and a lid, which contains up to 10 non-ATPasesubunits. Proteasomes are distributed throughout eukaryotic cells at a high concentration andcleave peptides in an ATP/ubiquitin-dependent process in a non-lysosomal pathway. Anessential function of a modified proteasome, the immunoproteasome, is the processing of class IMHC peptides. The immunoproteasome contains an alternate regulator, referred to as the 11Sregulator or PA28, that replaces the 19S regulator. Three subunits (alpha, beta and gamma) ofthe 11S regulator have been identified. This gene encodes the gamma subunit of the 11Sregulator. Six gamma subunits combine to form a homohexameric ring. Two transcript variantsencoding different isoforms have been identified. [provided by RefSeq, Jul 2008] reduction of tumor burden when CD47 blockade was combined with ionizing radiation (IR) was associated with radioprotection of the cells in the tumor microenvironment increased oxygenation of the tumor by increasing blood flow and enhanced migration of cytotoxic lymphocytes. More recently we have demonstrated that blocking CD47 signaling provides radioprotection in T cells and endothelial cells through an up-regulation of pro-survival autophagy (25). Thus the increased survival of these cells in the irradiated tumor stroma could enhance anti-tumor immunity. IR activates the immune system and its role in the abscopal effect of radiation therapy is primarily attributed to activation of T-cell anti-tumor immunity (26-28). These results suggested that CD47 expression by stromal cells may play a significant role in modulating T cell anti-tumor immunity activated as a consequence of damage to tumor cells caused by IR. To date the ablation of tumor growth by CD47 blockade has been attributed to restoration of macrophage-mediated immune surveillance by reducing the ability of CD47 on tumor cells to engage SIRPα on tumor-associated macrophages. In contrast here we show that the reduction in tumor growth by CD47 blockade is dependent on an intact adaptive immune system specifically CD8+ cytotoxic T cells. Moreover blockade or loss of CD47 signaling in effector T cells is sufficient to directly increase CD8+ T cell killing of irradiated cancer cells and to reduce tumor burden in vivo. Materials and Methods Model of T-Cell Adoptive Transfer Athymic nu/nu mice in a BALB/c background (NCI-Frederick) were injected in the hind limbs with 1×106 15-12RM fibrosarcoma cells expressing HIV gp160 (29). Treatment was Polygalasaponin F initiated once tumors reached an average 100 mm3 volume. Tumor Polygalasaponin F irradiation was accomplished by securing each animal in a Lucite jig fitted with lead shielding that protected the.