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Melanoma-derived brain metastases (MBM) represent an unmet clinical need because central nervous system progression is frequently an end stage of the disease. Immune checkpoint inhibitors (ICI) provide a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique elements of the MBM TME and correlates of MBM response to ICI, we collected 32 fresh MBM and performed single-cell RNA sequencing of the MBM TME and T-cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity in the MBM TME, most notably multiple distinct neutrophil states, including an IL8-expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. In addition, we observed significant relationships between intracranial T-cell phenotypes and the distribution of T-cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data identify unique features of the MBM TME that may represent potential targets to improve clinical outcomes for patients with MBM.

Supplementary Figure from Microenvironmental Landscape of Human Melanoma Brain Metastases in Response to Immune Checkpoint Inhibition

Supplementary Data from Microenvironmental Landscape of Human Melanoma Brain Metastases in Response to Immune Checkpoint Inhibition

Melanoma-derived brain metastases (MBM) represent an unmet clinical need because central nervous system progression is frequently an end stage of the disease. Immune checkpoint inhibitors (ICI) provide a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique elements of the MBM TME and correlates of MBM response to ICI, we collected 32 fresh MBM and performed single-cell RNA sequencing of the MBM TME and T-cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity in the MBM TME, most notably multiple distinct neutrophil states, including an IL8-expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. In addition, we observed significant relationships between intracranial T-cell phenotypes and the distribution of T-cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data identify unique features of the MBM TME that may represent potential targets to improve clinical outcomes for patients with MBM.

Melanoma-derived brain metastases (MBM) represent an unmet clinical need due to central nervous system (CNS) progression as a frequent, end-stage site of disease. Immune checkpoint inhibition (ICI) represents a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique MBM-TME elements and correlates of MBM-ICI response, we collected 32 fresh MBM and performed single cell RNA sequencing of the MBM-TME and T cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity, most notably multiple distinct neutrophil states including an IL-8 expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. Additionally, we observe significant relationships between intracranial T cell phenotypes and the distribution of T cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data demonstrate unique features of the MBM-TME, which may represent potential targets to improve clinical outcomes for patients with MBM.

Using single-cell RNA sequencing and TCR sequencing, Pauken et al. detect CD8+ T cell clones shared between blood and tumor in mice or melanoma patients, characterize matching clones in blood and tumor, and identify potential biomarkers for their isolation in blood., The ability to monitor anti-tumor CD8+ T cell responses in the blood has tremendous therapeutic potential. Here, we used paired single-cell RNA and TCR sequencing to detect and characterize “tumor-matching” (TM) CD8+ T cells in the blood of mice with MC38 tumors or melanoma patients using the TCR as a molecular barcode. TM cells showed increased activation compared with nonmatching T cells in blood and were less exhausted than matching cells in tumors. Importantly, PD-1, which has been used to identify putative circulating anti-tumor CD8+ T cells, showed poor sensitivity for identifying TM cells. By leveraging the transcriptome, we identified candidate cell surface markers for TM cells in mice and patients and validated NKG2D, CD39, and CX3CR1 in mice. These data show that the TCR can be used to identify tumor-relevant cells for characterization, reveal unique transcriptional properties of TM cells, and develop marker panels for tracking and analysis of these cells., Graphical Abstract

PD-1 restricts T cell effector functions, regulates T cell tolerance, and maintains immune homeostasis, thereby playing pivotal roles in cancer, autoimmune and infectious diseases. Our studies and others show that PD-1 can restrain Treg suppressive function in autoimmunity and cancer. Here, we investigate the role of Treg intrinsic PD-1 in regulating pathogenic and protective CD4+ T cell responses in experimental autoimmune encephalomyelitis (EAE) by inducing PD-1 deletion on FoxP3+ cells prior to disease development. This deletion of PD-1 only in Treg protects mice from severe disease, and both flow cytometry and transcriptional analysis of central nervous system (CNS) CD4+ T cells show enrichment of tissue effector Treg (eTreg) subsets at peak of disease. We find that PD-1 restrains the generation and suppressive function of these effector Treg. This tissue eTreg signature is also observed in human and mouse Treg subsets from the tumor microenvironment, and are potent suppressor populations. Further studies are underway to determine how PD-1 impacts TCR signaling and FoxP3 transcriptional co-activators in Treg cells. Our study provides deeper insights into how PD-1 regulates T cell tolerance, underscoring the important role of PD-1 in regulating Treg differentiation and suppressive function during autoimmune diseases, and provide mechanistic insights for PD-1 modulation in cancer and autoimmunity. Supported by NIH P01AI108545

The ability to monitor anti-tumor CD8+ T cell responses in the blood has tremendous therapeutic potential, but is currently challenging due to the limited number of reagents to track antigen-specific T cells. Here, we used paired single-cell RNA sequencing and T cell receptor (TCR) sequencing to detect and characterize “tumor matching” (TM) CD8+ T cells in the blood of mice with MC38 tumors or melanoma patients using the TCR as a molecular barcode. TM cells showed increased activation compared to non-matching T cells in blood, and appeared less exhausted than matching counterparts in tumor. Importantly, PD-1, which has been used to identify putative circulating anti-tumor CD8+ T cells, showed poor sensitivity for identifying TM cells in both mice and humans. By leveraging the transcriptome, we identified candidate cell surface marker panels for TM cells in mice and melanoma patients, and validated CX3CR1, NKG2D, and CD39 proteins in mice. Combinations of markers performed better than single markers in identifying TM cells from non-TM cells in the blood, providing a platform to potentially track TM cells based on surface markers instead of the TCR. These data demonstrate that the TCR can be used to identify tumor-relevant populations for comprehensive characterization, reveal unique transcriptional properties of TM cells, and develop marker panels for tracking and analysis of these cells.

The ability to monitor anti-tumor CD8+ T cell responses in the blood has tremendous therapeutic potential. Here, we used paired single-cell RNA sequencing and T cell receptor (TCR) sequencing to detect and characterize “tumor matching” (TM) CD8+ T cells in the blood of mice with MC38 tumors and melanoma patients using the TCR as a molecular barcode. TM cells showed increased activation compared to non-matching T cells in blood, and appeared less exhausted than matching counterparts in tumor. Importantly, PD-1, which has been used to identify putative circulating anti-tumor CD8+ T cells, showed poor sensitivity for identifying TM cells. By leveraging the transcriptome we identified candidate cell surface marker panels for TM cells in mice and melanoma patients, and validated markers in mice. Here, using combinations of markers provided better performance than single markers in identifying TM cells from non-TM cells in the blood. These data demonstrate that the TCR can be used to identify tumor-relevant populations for comprehensive characterization, reveal unique transcriptional properties of TM cells, and develop marker panels for tracking and analysis of these cells. Citation Format: Kristen E. Pauken, Osmaan Shahid, Kaitlyn A. Lagattuta, Kelly M. Mahuron, Jacob M. Luber, Margaret M. Lowe, Linglin Huang, Conor Delaney, Jaclyn M. Long, Megan E. Fung, Kathleen Newcomer, Katy K. Tsai, Melissa Chow, Samantha Guinn, Juhi R. Kuchroo, Kelly P. Burke, Jason M. Schenkel, Michael D. Rosenblum, Adil I. Daud, Arlene H. Sharpe, Meromit Singer. Single-cell analyses characterize circulating anti-tumor CD8 T cells and identify markers for their isolation [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO016.