hetIL-15 monotherapy increases intratumoral CD8+ T cytotoxic cells and reverses the metabolic dysfunction in murine breast tumors. Sevasti Karaliota(1), Dimitris Stellas(1), Vasiliki Stravokefalou(1,2), Bethany Nagy(1,3), Cristina Bergamaschi(3), Barbara K. Felber(3) and George N. Pavlakis(1). 1 Human Retrovirus Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick 2 Department of Pharmacology, Faculty of Medicine, University of Thessaly, Larissa, Greece 3 Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick Introduction CD8+ T cytotoxic cells have the capability to recognize and eliminate cancer cells, but their function is often impaired by the intratumoral immunosuppressive microenvironment. The cytokine IL-15 stimulates the generation, proliferation and cytotoxic function of tumor specific CD8+ T cells and NK cells. Several preclinical models have supported the anti-tumor activity of IL-15. We have produced the native heterodimeric IL-15 (hetIL-15) of human, macaque or mouse and also fusions to the Fc antibody fragments (hetIL-15FC), to further study its action. Experimental procedures: We studied the therapeutic efficacy of hetIL-15FC immunotherapy in the murine E0771 orthotopic breast cancer model in syngeneic C57BL/6 mice. The effects of hetIL-15FC on immune cells were analyzed in tumors by flow cytometry and immunohistochemistry (IHC). We also assessed the metabolic profile of splenic and tumor infiltrating T cells, with Seahorse analysis. Results and Conclusions: Locoregional administration of hetIL-15FC in the area of mammary pad resulted in complete tumor regression in 40% of the treated mice. hetIL-15FC monotherapy increased the overall survival and was accompanied with decreased or completely eradicated metastatic disease. hetIL-15 peritumoral enhanced the tumor infiltration of NK, CD8+ T cytotoxic and Dendritic Cells. Phenotypic analysis showed that these cells were activated and in an active proliferation state. Moreover, metabolic flux analysis of the tumor-infiltrating CD8+T cells from treated mice confirmed an increase in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity (a measure of mitochondrial reserve), which supports an activated/non exhausted phenotype. Consistent with the above finding, tumor infiltrating CD8+T cells from hetIL-15FC treated mice showed increased mitochondrial potential and/or mass, as evidenced by increased Mitotracker staining. Similar results were obtained from splenic CD8+T and NK cells of hetIL-15FC treated mice. In addition, tumor infiltrated CD8+T cells from hetIL-15FC treated mice presented elevated extracellural acidification rate (ECAR) and showed a pronounced shift in the OCR to ECAR ratio in comparison to control, confirming their increased proliferating status. We therefore conclude, that the locoregional administration of hetIL-15FC resulted in complete regression of EO771 primary breast cancer tumors, activating both cytotoxic arms of the immune system. hetIL-15FC supported also a favorable metabolic profile of intratumoral effector lymphocytes, important for their function. This supports our notion to further explore the use of hetIL-15 as a candidate immunotherapeutic agent for the treatment of breast cancer.