Abstract Background: Breast cancer (BC) has historically been considered immunologically silent; however, several observations indicate that potentiation of immune functions can benefit BC patients. Intratumor T-cell infiltration has prognostic significance in patients with BC across different molecular and histological categories. In addition, PD-L1 can be overexpressed in BC, in particular in the highly aggressive triple negative BC (TNBC) subtype. This is associated with poor prognosis specifically in patients with luminal B and basal-like phenotypes, thus making these subtypes rational targets of PD-1/PD-L1 axis blockade treatments. However, initial results from early-phase clinical trials show a modest activity of immune checkpoint blockade monotherapy against BC, with 19% TNBC and 3-12% hormone receptor-positive patients achieving a clinical response to anti-PD-1/PD-L1 therapies. Lack of inflammation and T-cell infiltration at the tumor site are characteristic features of tumors that do not respond to checkpoint blockade. Since BC is typically poorly infiltrated by T cells, having strategies that reverse this immune exclusion as well as non-invasive modalities to predict this effect are crucial to improve the clinical efficacy of immune checkpoint blockade. Experimental Design: Our working model is that tumor glycolysis (lactate) and T-cell infiltration are mechanistically interdependent, since we reasoned that a highly glycolytic tumor microenvironment (due to lactate production) could hamper survival, expansion and effector functions of T cells in BC lesions, thus explaining T-cell exclusion. Therefore, we tested whether genetic and/or pharmacologic inhibition of lactate production/consumption could restore intratumor T-cell infiltration and immune function in BC models, thus favoring tumor responsiveness to checkpoint blockade. Results: By interrogating a compendium of 4 BC patient gene expression datasets, we found that patients harboring tumors with high expression of lactate dehydrogenase A (LDH-A) or the lactate transporters MCT-1/4 have a significantly higher risk to develop metastases (p<10−16), whereas those having tumors with increased expression of CD3 and CD8 transcripts experience a better prognosis (p<10−16). Gene expression data from MSKCC's cBio Portal showed an inverse correlation between glycolysis- and immune-related gene expression signatures in BC patients, which was in agreement with our hypothesis. To mechanistically demonstrate the impact of lactate metabolism on intratumor T-cell infiltration in BC, we generated an LDH-A-knocked-down variant (LDH-A KD) of the metastatic TNBC murine model 4T1, and studied its growth and immune infiltrate in vivo in comparison with the control 4T1 tumor. Animals implanted with LDH-A KD 4T1 showed a 5-fold increase in tumor-infiltrating CD3+ T cells and a 4-fold reduction in tumor-associated macrophages, and experienced a significantly prolonged survival. LDH-A KD tumors could be completely eradicated in immunocompetent but not in immunodeficient mice, further supporting the immunologic basis of the antitumor effects of LDH-A inhibition. In vitro analyses showed that proliferation, activation and pro-inflammatory cytokine release of activated CD8 T cells were significantly hampered when they were co-cultured with 4T1 cells. Of note, blocking lactate transport with two new MCT-1/4 small-molecule inhibitors promoted immune functions of CD8 T cells cultured with 4T1, without showing significant toxicity in cultures of T cells alone. Citation Format: Roberta Zappasodi, Arnab Ghosh, Inna Serganova, Ivan Cohen, Yasin Senbabaoglu, Masahiro Shindo, Mayuresh M. Mane, Avigdor Leftin, Ellen Ackerstaff, Jason A. Koutcher, Jedd D. Wolchok, Ronald G. Blasberg, Taha Merghoub. Overcoming intratumor T-cell exclusion by modulation of lactate metabolism to improve immune checkpoint therapies in aggressive breast cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR06.