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Functional CD8+ T cells in human tumors play a clear role in clinical prognosis and response to immunotherapeutic interventions. PD-1 expression in T cells involved in chronic infections and tumors such as melanoma often correlates with a state of T-cell exhaustion. Here we interrogate CD8+ tumor-infiltrating lymphocytes (TILs) from human breast and melanoma tumors to explore their functional state. Despite expression of exhaustion hallmarks, such as PD-1 expression, human breast tumor CD8+ TILs retain robust capacity for production of effector cytokines and degranulation capacity. In contrast, melanoma CD8+ TILs display dramatic reduction of cytokine production and degranulation capacity. We show that CD8+ TILs from human breast tumors can potently kill cancer cells via bi-specific antibodies. Our data demonstrate that CD8+ TILs in human breast tumors retain polyfunctionality, despite PD-1 expression, and suggest that they may be harnessed for effective immunotherapies.

Interferon regulatory factor-1 (IRF-1) is a nuclear transcription factor that mediates interferon and other cytokine effects and appears to have antitumor activity in vitro and in vivo in cancer cells. We have constructed a recombinant adenoviral vector (Ad-IRF-1) that infects mammary cells with high efficiency and results in high levels of functional IRF-1 protein in transfected cells. Overexpression of IRF-1 in two mouse breast cancer cell lines, C3-L5 and TS/A, resulted in apoptosis in these cell lines as assessed by Annexin V staining. The involvement of caspases was confirmed by significant inhibition of apoptosis by a caspase inhibitor, and by demonstration of caspase-3 activity, cleavage of caspase-3, and PARP cleavage. Interestingly, the growth of nonmalignant breast cell lines C127I and NMuMG did not appear to be inhibited by IRF-1 overexpression. Suppression of growth for breast cancer cell lines in vivo was demonstrated by both preinfection of breast cancer cells ex vivo and by intratumoral injection of Ad-IRF-1 into established tumors in their natural hosts. The mechanism of apoptosis may involve the transcriptional upregulation of bak, caspase-8, and caspase-7 expression. These data support the antitumor potential of IRF-1 and the use of agents that increase IRF-1 in breast cancer.

Interferon Regulatory Factor-1 (IRF-1) is a transcription factor which acts as a tumor suppressor and causes apoptosis in cancer cells. We evaluated IRF-1 induced apoptosis in gastric cancer cell lines. We established stable clones in AGS cells that have a tetracycline inducible IRF-1 expression system. We used these clones and recombinant adenovirus expressing IRF-1 to explore the mechanism of IRF-1 induced apoptosis in gastric cancer. Expression of IRF-1 causes apoptosis in gastric cancer cell lines as demonstrated by phosphatidylserine exposure and cleavage of caspase-8, caspase-3, and Bid with mitochondrial release of cytochrome c. However, inhibition of caspase-8 and Bid did not inhibit apoptosis and did not decrease cleaved caspase-9 or mitochondrial release of cytochrome c. We then demonstrate that IRF-1 up-regulates PUMA (p53 up-regulated modulator of apoptosis), that is known to activate apoptosis by the intrinsic pathway; this can be p53 independent. IRF-1 binds to distinct sites in the promoter of PUMA and activates PUMA transcription. Moreover, molecular markers of mitochondrial apoptosis are eliminated in PUMA knockout and knockdown cells and phospatidylserine exposure is decreased dramatically. Finally, we demonstrate that IFN-γ induces IRF-1 mediated up-regulation of PUMA in cancer cells. We conclude that IRF-1 can induce apoptosis by the intrinsic pathway independent of the extrinsic pathway by up-regulation of PUMA.

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Although some patients with Paget's disease might be well treated by breast conservation therapy, many patients have underlying multifocal carcinoma (including invasive cancer), which can be inapparent by examination and mammography. Selecting candidates with disease amenable to complete excision without mastectomy is problematic.