Abstract Checkpoint inhibitors (CIs) have shown an unprecedented clinical activity in a large variety of cancer types, but only in a fraction of patients. To increase the number of responders, preclinical models are studied to define effective combinatorial regimens and the best window of therapeutic opportunities. We recently described a synergy between anti-PD-1 and -PD-L1 CIs and several types and dosages of chemotherapy in immunocompetent models of triple negative breast cancer (TNBC), likely due to unique effects of chemotherapeutics over circulating and tumor-infiltrating, suppressive, antigen-presenting and effector immune cell populations (Orecchioni et al, Br J Cancer 2018). To find a high-order drug combination, we have further studied by multiparametric flow cytometry and single-cell transcriptomic a panel of different chemotherapy drugs (including microtubular poisons, alkylating agents, topoisomerase inhibitors and antimetabolites) used in vivo at different dosages and schedule. Our aim was to define a combination, dose and schedule able to promote a) dendritic cell maturation, b) release and c) uptake of cancer-associated antigens from targeted neoplastic cells, d) cross-priming between antigen-presenting cells (APCs) and T cells, e) NK cell activation, and f) inhibition of suppressor immune cell populations. Vinca alkaloids vinblastine and vinorelbine (V), at low-medium dosages, were the most effective in a), b), c) and d). After V administration, Tregs were reduced, and circulating and tumor-infiltrating APCs showed a “maturation to activation” program with increased expression of several antigens including CD40, CD80, and CD86. The alkylating agent cyclophosphamide (CTX) targeted Tregs, mobilized APC progenitors, activated and increased the number of circulating and intratumoral NK cells. These effects were significantly increased when CTX was administered in an intermittent fashion (every 6 days) at low-medium doses. As the APC-NK cell axis has been recently found to be pivotal in CI-mediated, anti-tumor cell immunity, our data suggested a preclinical trial in immunocompetent orthotopic models of metastatic (post-mastectomy) BALB mice injected with 4T1 or EMT-6 TNBC cells. Among a large panel of different combinations and dosages, the sequential administration of V, intermittent CTX and anti-PD-L1 was the only able to completely abrogate TNBC local and metastatic growth, and this effect was not observed in T and NK cell-depleted mice. Taken together with recent data from randomized, combinatorial studies in TNBC patients (Schmid et al, NEJM 2018), our findings suggest that CIs might be included in future combinatorial regimens aimed at improving the percentage of patients receiving a clinical benefit and prolonging the duration of this benefit. Citation Format: Stefania Orecchioni, Loredana Vecchi, Paolo Falvo, Lucilla Luzi, Patrizia Mancuso, Chiara Camisaschi, Francesco Bertolini. The sequential administration of vinca alkaloids and intermittent cyclophosphamide primes antigen-presenting and NK cells and significantly improves in vivo efficacy of anti-PD-L1 in triple-negative breast cancer models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3948.