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Background Cancer immunotherapies have significantly improved treatment efficacy and patient survival by exploiting antigen-specific T cells to eliminate cancer cells. However, current approaches for identifying and isolating antigen-specific T cells typically require prior knowledge of target antigens, limiting discovery, and reducing the ability to consistently detect rare tumor-reactive T cells. We therefore sought to develop an unbiased platform for the identification and enrichment of antigen-specific T cells using naturally processed and presented tumor antigens.Methods We developed ATTACH (Assessment of T cells Tethered to Antigen Class I Histocompatibility), a microfluidic platform that applies controlled shear stress and leverages tumor cells as a natural source of endogenous major histocompatibility complex (MHC)-peptide complexes to isolate antigen-specific T cells based on MHC/peptide binding avidity. ATTACH was evaluated in both human and mouse systems for its ability to enrich rare tumor-reactive T-cell populations and deplete bystander virus-specific T cells.Results ATTACH resulted in up to a 10-fold enrichment of antigen-specific T cells across both human and mouse systems, enabling the isolation of clonotypes present at frequencies as low as 0.1%. In addition to enriching rare tumor-reactive T cells, ATTACH efficiently depleted virus-specific bystander T cells.Conclusions ATTACH provides a streamlined and unbiased approach for the rapid identification and isolation of antigen-specific T cells, and may facilitate the optimization of cellular therapies for the treatment of solid tumors.