Abstract While T cells contribute to the eradication of several cancers, including melanoma, the role of T cells present in healthy-appearing, tumor-adjacent tissue is less well described. Memory T cells contain distinct populations of effector memory and central memory, distinguished by their surface phenotype and functions. Effector T cells are thought to be terminally differentiated. We hypothesize that T cells present in healthy-appearing breast tissue contribute to anti-tumor immunity and immune surveillance. Here, we studied breast cancer patients, comparing tumor-infiltrating T cells in breast cancer to T cells from healthy-appearing, tumor-adjacent breast tissue. Since the amount of tissue available for study from excised primary tumors is limited, we utilized a previously established three-dimensional explant method to expand resident T cells from fresh breast tissue. T cell differentiation phenotypes were defined for ten breast cancer specimens and nine tumor-adjacent breast specimens. Six of the tumor/tumor-adjacent tissue specimens were paired. T cell phenotype and function were studied using multiparameter flow cytometry. Tumor-infiltrating CD4 effector T cells (TEFF CD45RO-, CD62L-) were more highly enriched compared to T cells derived from tumor-adjacent breast tissue (mean 21% vs. 6%, p=0.05). In contrast, effector memory CD4 T cells (CD45RO+, CD62L-) were enriched in the healthy adjacent lymphocytes (mean 71% vs. 54%, p<0.05) whereas central memory CD4 T cell (CD45RO+, CD62L+) frequency was equivalent (23% vs. 24%, p=0.83). Functionally, T cells (CD4 and CD8) derived from tumor tissue spontaneously released granules at a higher level than tumor-adjacent T cells as indicated by CD107a expression (1.8% vs 0.8%, p<0.001). In contrast, we detected a higher frequency of mitogen-induced CCL4-producing CD4 T cells from tumor-adjacent tissue vs. tumor (62% vs. 51%, p<0.05), as well as a higher frequency of IFN-γ-producing CD8 T cells (80% vs. 65%, p<0.05). These studies demonstrate the feasibility of using this three-dimensional explant approach to investigate the role of T cells in immune surveillance against breast cancer. Our findings indicate that T cell phenotypes differ in breast tissue; tumor-adjacent T cells are enriched in effector memory cells whereas TILs are characterized by terminally differentiated effector T cells. The data further suggest that breast TILs, through their comparatively reduced frequency of CCL4- and IFN-γ-producing cells have a diminished functional capacity to restrict tumor growth and metastasis. Citation Format: Yi Guo, Jaime Shamonki, Michelle Kinnaird, Maggie DiNome, Maureen Chung, Peter Sieling, Delphine J. Lee. Comparison of tumor-infiltrating lymphocytes from breast cancer to T cells from adjacent tumor tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-267. doi:10.1158/1538-7445.AM2014-LB-267