Abstract Background: Triple-negative breast cancer (TNBC) have had significantly worse survival outcome compared to other BC subtypes even though treatment strategies of TNBC have been advanced. The advent of immune checkpoint inhibitors (ICIs), especially pembrolizumab, a PD-1 inhibitor, has started to revolutionize TNBC treatment, showing encouraging effects in improving survival outcomes regardless of treatment settings. In neoadjuvant setting, pembrolizumab with cytotoxic chemotherapy achieved 13.6 % of pathologic complete response rate improvement and 36% of three-year event reduction. Therefore, pembrolizumab has highlighted new dimensions of TNBC's immunogenic characteristics. However, no immunogenic characteristics to predict the response of pembrolizumab have not been revealed. Methods: This study was designed as prospective, translational research for stage II or III TNBC patients scheduled for neoadjuvant chemotherapy with or without pembrolizumab followed by curative surgery. Tissue and blood samples were collected at baseline, 1 or 3 weeks after the first treatment initiation and curative surgery. We employed various single-cell and bulk RNA techniques for our multiomic analysis, including peripheral blood mononuclear cell (PBMC) single-cell RNA sequencing, T cell receptor sequencing (scTCR), high-resolution single-cell spatial transcriptomics (Xenium) targeting 380 genes, and bulk PBMC and tissue TCR analysis Results: From March 2023 to October 2023, 37 patients were enrolled in this study. Of 37 patients, we collected 36 blood and BC tissue samples at baseline, as well as 35 blood and 14 BC tissues at follow-up. These samples were analyzed and also compared with sex and age-matched healthy controls (n = 65). Of 37 patients, two patients were treated with only cytotoxic chemotherapy without pembrolizumab. We observed substantial increases in both systemic and localized immune responses, primarily marked by an upsurge in activated T cells and a decrease in regulatory T cells. A notable rise in TCR diversity post one week of treatment was followed by clonal expansion of activated T cells after three weeks in both blood and tissue. In addition, we observed a significant enrichment of pathways related to inflammatory response in classical monocytes following treatment. Conclusion: The findings from this study significantly enhance our understanding of the dynamics of circulating and local immune cells in TNBC, analyzed at a single-cell level with spatial information. These insights reveal how pembrolizumab alters the immune landscape and correlates these changes with treatment responses. Importantly, the data provide valuable indicators for predicting which TNBC patients are more likely to respond to neoadjuvant pembrolizumab therapy. Citation Format: Eun Seop Seo, Hae Hyun Jung, Kibyung Kim, Kyunghee Park, Eun Young Ko, Byung Joo Chae, Jeong Eon Lee, Eun Yoon Cho, Ji-Yeon Kim, Woong-Yang Park. Identification of circulating and spatial dynamics of immune cells response to to immune checkpoint inhibitors in triple-negative breast cancer at single-cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1176.