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Despite unique genetic alterations within brain metastases (BrMs) and an immunologically distinct surrounding microenvironment, the composition and functional properties of tumor-infiltrating lymphocytes within BrM remain largely unexplored. In particular, the expression of coinhibitory receptors, such as programmed cell death 1 (PD-1), T cell immunoglobulin mucin receptor 3 (TIM-3), and lymphocyte activation gene 3 (LAG-3), within BrMs is unknown. Using multiplexed quantitative immunofluorescence (QIF), this study evaluates the localized expression of PD-L1, level and functional profile of major T cell subsets, and coinhibitory receptors within lung cancer-associated BrMs and primary lung tumors. Clinicopathologically annotated samples from 95 patients with lung cancer between 2002 and 2015 were represented in a tissue microarray format. Spatially resolved and multiplexed QIF was used to evaluate PD-L1 protein, phenotype markers for major T cell subsets (CD3, CD4, CD8, and FOXP3), cell-localized activation and proliferation markers (granzyme B and Ki67), and coinhibitory receptors (PD-1, LAG-3, and TIM-3). The signal for each marker was measured in marker-selected tissue compartments, and associations between marker levels, tumor location, and major clinicopathological variables were studied. In total, 41 primary lung tumors and 65 BrMs were analyzed, including paired samples from 11 patients. Levels of tumor PD-L1 expression were comparable between BrMs and primary lung tumors. BrMs had significantly lower levels of all T cell subsets relative to primary lung tumors, and T cells in BrMs displayed lower levels of granzyme B than primary lesions. PD-1, TIM-3, and LAG-3 levels in CD3 + T-cells were also significantly lower in BrMs. Marker expression in patients with paired samples from BrMs and primary lung tumors showed comparable results. High CD3 + T-cells, as well as high levels of TIM-3 and LAG-3 in CD3 + T-cells, were associated with longer overall survival in BrMs but not primary lung tumors. Lung cancer-associated BrMs display lower T cell infiltration, markers of cytolytic function, and immune regulatory signals than primary lung tumors. Despite these differences, high TIM-3 and high LAG-3 expressions in CD3 + T-cells were associated with longer survival. These features are accompanied by comparable levels of PD-L1 protein expression compared with primary lung tumors. These results highlight unique aspects of the tumor immune microenvironment within the brain and provide further support for intracranially focused therapies., Competing Interests: Competing interests: BYL, RG, AA, HW, MR, VLC, JNC, AJA, and LBJ have nothing to disclose. HMK reports grants and personal fees from Merck and Bristol-Myers Squibb, grants from Apexigen, personal fees from Iovance, Immunocore, Celldex, Array BioPharma, Elevate Bio, Instil Bio, Clinigen, Shionogi, and Chemocentryx, outside the submitted work. KAS reports research funding from Navigate Biopharma, Tesaro/GlaxoSmithKline, Moderna Inc, Takeda, Surface Oncology, Pierre-Fabre Research Institute, Merck-Sharp & Dohme, Bristol-Myers Squibb, AstraZeneca, Ribon Therapeutics, and Eli Lilly. He has received honoraria for consultant/advisory roles from Moderna Therapeutics, Shattuck Labs, Pierre-Fabre, AstraZeneca, EMD Serono, Ono Pharmaceuticals, Clinica Alemana de Santiago, Dynamo Therapeutics, PeerView, Abbvie, Fluidigm, Takeda/Millenium Pharmaceuticals, Merck-Sharp & Dohme, Bristol Myers-Squibb, Agenus and Torque Therapeutics. SBG reports grants and personal fees from AstraZeneca and Boehringer Ingelheim, and personal fees from Bristol-Myers Squibb, Amgen, Spectrum, Blueprint Medicine, Sanofi Genzyme, Daiichi-Sankyo, Regeneron, Janssen, and Takeda., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Little is known about tumor-associated vasogenic edema in brain metastasis, yet it causes significant morbidity and mortality. Our purpose was to characterize edema in patients treated with anti-PD-1 and to study potential causes of vessel leakage in humans and in pre-clinical models., Methods: We analyzed tumor and edema volume in 18 non-small cell lung (NSCLC) and 18 melanoma patients with untreated brain metastases treated with pembrolizumab on a phase II clinical trial. Melanoma brain metastases were stained with anti-CD34 to assess vessel density and its association with edema. We employed an in vitro model of the blood-brain barrier using short-term cultures from melanoma brain and extracranial metastases to determine tight junction resistance as a measure of vessel leakiness., Results: Edema volumes are similar in NSCLC and melanoma brain metastases. While larger tumors tended to have more edema, the correlation was weak (R 2  = 0.30). Patients responding to pembrolizumab had concurrent shrinkage of edema volume and vice versa (R 2  = 0.81). Vessel density was independent of the degree of edema (R 2  = 0.037). Melanoma brain metastasis cells in culture caused loss of tight junction resistance in an in vitro blood-brain barrier model system in some cases, whereas extracerebral cell cultures did not., Conclusions: Edema itself should not preclude using anti-PD-1 with caution, as sensitive tumors have resultant decreases in edema, and anti-PD-1 itself does not exacerbate edema in sensitive tumors. Additional factors aside from tumor mass effect and vessel density cause perilesional edema. Melanoma cells themselves can cause decline in tight junction resistance in a system void of immune cells, suggesting they secrete factors that cause leakiness, which might be harnessed for pharmacologic targeting in patients with significant perilesional edema.