Background: Despite the biological and clinical implications, the immune composition and functional characteristics of adaptive immune cells in brain metastases (BrM) are poorly understood. Using multiplexed quantitative immunofluorescence (QIF), this study evaluates the level and functional profile of major T-cell subsets in primary lung tumors, BrM, and extracranial metastases (ECM) from lung cancers. Methods: A tissue microarray was constructed from formalin-fixed, paraffin-embedded tumor samples of 94 lung cancer patients treated at Yale Cancer Center between 2002-2013. Multiplexed QIF was used to evaluate the cases with a panel containing phenotype markers for major T-cell subsets (CD3, CD4, CD8 and FOXP3), and cell-localized activation and proliferation (granzyme-B and Ki-67). Signal for each marker was measured in marker-selected tissue compartments using the Automated Quantitative Analysis (AQUA) platform. Associations between markers and major clinicopathologic variables were studied. Results: In total, 40 primary lung tumors, 63 BrM, and 15 ECM were analyzed, including paired samples from 22 patients. Lung cancer histology included adenocarcinoma 62.5%, squamous cell carcinoma 11.5%, small cell 9.4%, and other non-small cell 16.7%. BrM had both significantly lower levels of CD3+ T-cells (p< 0.0001) and T-cell granzyme B (p= 0.0188) compared with primary lung tumors. No significant differences were observed in T-cell Ki-67 levels across tissues. FOXP3 measured in CD4+ T-cells were significantly lower in BrM compared with primary malignancies (p= 0.0002) and ECM (p= 0.0404). Among patients with BrM, higher levels of CD3+ T-cells in BrM were associated with longer overall survival. Conclusions: Lung cancer-associated BrM have lower T-cell infiltration, cytolytic function, and regulatory T-cells than primary lesions. These results indicate lower adaptive anti-tumor responses in BrM and suggest the presence of a tolerogenic microenvironment in the brain. Overcoming this could be used to design optimal treatment strategies.