Abstract Combination of immune checkpoint blockade (ICB) with chemotherapy is becoming more commonly used for the treatment of various cancer types, including advanced non-small cell lung cancer, and triple negative breast cancer (TNBC) in patients who have PD-L1 positive tumors, among others. Although chemotherapy-ICB modalities have been shown to improve clinical responses over those observed with ICB alone, long-term disease control is achieved only in a small subset of patients. To provide a rational basis for combination therapy, and to potentially develop more effective approaches, in this study we investigated the impact of chemotherapy on the tumor microenvironment (TME) in the context of PD-1/PD-L1 axis blockade. ICB refractory murine models were utilized, including the TNBC 4T1 model and the Lewis Lung Carcinoma (LLC) model. Following treatments with anti-PD-L1, docetaxel, or the combination anti-PD-L1 plus docetaxel, tumors were collected 4 days after the last treatment to evaluate early changes in the TME. Analyses included the characterization of tumor immune infiltrates via flow cytometry, RNA expression profiling via RT-PCR, RNA in situ hybridization, and immunohistochemistry for detection of immune infiltrates, cancer-associated fibroblasts, and phospho-Smad2 signaling. RNA analysis of 4T1 and LLC tumors revealed a significant upregulation of several transforming growth factor-beta (TGF-beta) downstream target genes in the anti-PD-L1 monotherapy group. This increase was associated with enhanced expression of TGF-beta in the TME, as determined by RNA in situ hybridization, together with enhanced TGF-beta signaling denoted by increased expression of phospho-Smad2. Tumors treated with anti-PD-L1 monotherapy also exhibited increased numbers of alpha-smooth muscle actin positive fibroblasts in the TME. The effects observed with anti-PD-L1 as a monotherapy were not observed in tumors treated with docetaxel monotherapy or in those treated with the combination docetaxel plus anti-PD-L1 therapy, suggesting that upregulation of immunosuppressive TGF-beta signaling in the TME could be prevented by the addition of chemotherapy. Ongoing and future studies are aimed at understanding the mechanisms involved in the upregulation of TGF-beta signaling in the TME in response to anti-PD-L1 therapy in non-responsive tumors, the effect of various chemotherapies in this phenomenon, and the study of additional tumor models for optimization of chemotherapy-ICB modalities. Citation Format: Masafumi Iida, Lucas A Horn, Haiyan Qin, Kristen Fousek, Jeffrey Schlom, Claudia Palena. Chemotherapy reduces TGF-beta signaling triggered by blockade of the PD-1/PD-L1 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6100.