Abstract Background: Increased CD8+ tumor-infiltrating lymphocytes (TIL) is associated with improved prognosis in triple-negative breast cancer (TNBC) suggesting that T-cell responses at the tumor site can be harnessed for autologous T-cell therapy using TIL expanded ex vivo. Although TIL therapy has been developed for solid tumors such as melanoma, cervical, and ovarian cancer. Moreover, methods facilitating CD8+ TIL expansion from TNBC are desirable given their cytotoxic potential against tumor cells. One approach to address this need is to provide agonist signals through the 4-1BB/CD137 pathway during TIL expansion selectively costimulating CD8+ T-cell activation. In this study, we established a method of expanding TIL from surgical specimens and core biopsies from primary TNBC patients and compared the phenotype and function of these TIL to lymphocytes from peripheral blood. Patients and methods: Eight primary human TNBC tumor samples were obtained by surgical resection or core biopsy after neo-adjuvant chemotherapy. Small (4-6 mm2) tumor fragments were cultured for 28 days in 24-well plates in medium containing 3000 IU/ml IL-2 alone or in combination with 10 µg/ml agonistic anti-4-1BB IgG4 (BMS663513) added at the start of culture. Viable cell numbers and the expression of CD3, CD8, CD4, CD27, CD28, CD56, CD16, Granzyme B, and Perforin were determined by flow cytometry on day 28 after culture. Cytotoxic function of the TIL was evaluated by measuring Caspase 3 cleavage in target cells. Blood samples collected at surgery were immunophenotyped for subsets of T cells and NK cells, and assessed for ability of T cells to synthesize Th1/Th2 cytokines following activation through the T-cell receptor (TCR), and potential of NK cells to kill K562 targets. Results: TIL were successfully expanded from tumor fragments in 6/8 of the cases, with addition of anti-4-1BB greatly increasing the percentage and yield of CD8+CD3+ T cells. CD8+ TIL isolated from cultures receiving 4-1BB costimulation however had decreased CD27 and CD28 expression together with increased cytotoxic T-cell activity. Gene expression analysis also found that TIL from these 4-1BB costimulated cultures had a more differentiated CD8+ T-cell gene profile. Peripheral blood CD3+, CD8+, and CD4+ T cells were lower than those of healthy controls, but TCR-activated cytokine synthesis was not significantly different. Peripheral blood NK cells expressed normal Granzyme A/B and Perforin levels and exhibited normal IFN-? secretion and CD107a-release following exposure to IL-12/IL-18 or with K562 targets. Conclusions: TIL can be reproducibly expanded from TNBC tumors with IL-2 after neo-adjuvant therapy, with CD8+ TIL outgrowth and effector activity increased by provision of 4-1BB/CD137 costimulatory signals during culture initiation. These CD8+ TIL however had a more differentiated phenotype with higher cytotoxic activity. Peripheral blood T-cell and NK cell function was comparable to those of healthy donors. Our results support further development of an autologous TIL expansion protocol after neo-adjuvant therapy for use in an adoptive cell therapy approach to treat TNBC recurrence or metastasis. Citation Format: Michiko Harao, Hui Gao, Jie Qing Chen, Elizabeth A Mittendorf, Gildy V Babiera, Sarah M DeSnyder, Korrene F Rockwood, Savitri Krishnamurthy, Huiming Sun, Jie S Willey, Naoto T Ueno, James M Reuben, Laszlo G Radvanyi. Co-stimulation through 4-1BB/CD137 improves expansion and function of tumor-infiltrating T lymphocytes from primary and metastatic triple-negative breast cancer and inflammatory breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-05.