Incidence and death rates from melanoma are rising more rapidly than those of any other cancer. Tumors persist and metastasize despite a robust and specific immune response, including infiltration of cytotoxic T cells into the tumor. T cell behavior is tightly controlled by both co-stimulatory and inhibitory receptors. Many human tumors evade the immune response by co-opting these receptors, thus dampening the T cell response. A wave of novel therapies aimed at boosting the T cell response to cancer, whether through agonizing co-stimulatory receptors or antagonizing inhibitory receptors have recently been FDA approved or entered clinical trials. Specifically, novel therapy blocking the interaction between PD-1 and its ligands was approved for use in patients with advanced melanoma. It is thought that binding of programmed death-1 (PD-1), expressed on T cells, to its ligand within the tumor microenvironment dampens T cell-mediated killing. While a-PD-1 induces a clinical response in approximately 25% of advanced melanoma patients, the majority will ultimately relapse and die of their disease. The field remains hampered by an understanding of the effects of inhibitory receptor blockade on the systemic T cell populations. Additionally, no biomarker exists to predict which patients will benefit from a-PD-1 therapy. To dissect immune response to a-PD-1 therapy, this project utilizes high dimensional, single-cell technology to observe multiplexed phenotype and function of many immune cell subsets in patients with melanoma. The goal of this project is to dissect the phenotype, function, and signaling biology of T cells obtained from patients before, during, and after treatment in melanoma patients treated with recently approved anti-PD-1 therapy. T cells are a complex and heterogeneous subset of immune cells. Their state and capabilities can be characterized by a wide range of measurements, including surface marker expression, intracellular biochemical signaling, and ability to release cytotoxic moieties. Additionally, rare subsets within the T cell compartment, lie T regulatory cells, have a large impact on tumor biology. For these reasons, high dimensional, single-cell biology techniques are crucial for dissecting T cell phenotype and behavior. This project will utilize mass cytometry (CyTOF), a cutting edge technique capable of measuring expression of 35+ parameters simultaneously at the single cell level. Mass cytometry will be used to characterize the phenotype, dissect functional capabilities, and uncover signaling networks in both primary human melanoma tumors and sequential peripheral blood samples from melanoma patients treated with a-PD-1. This project will advance cancer research and patient care by identifying mechanisms by which T cells clear melanoma tumors during successful a-PD-1 therapy. Results from the anti-PD-1 therapy can be used to study and assess the success of other immunotherapies, both approved and in clinical trials. Sequential patient samples over the course of therapy will identify biomarkers of response and novel targets by which to further boost the immune response.

Public Health Relevance

Incidence and mortality rates of melanoma have been rising for the past 30 years and patients with metastatic melanoma have an incredibly poor prognosis. This project aims to improve therapy by identifying novel ways in which to enhance the patient's immune system to clear melanoma tumors. The findings from this research can be widely applied to help treat other cancers and chronic viral infections.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Schmidt, Michael K
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Vanderbilt University Medical Center
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United States
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Doxie, Deon B; Greenplate, Allison R; Gandelman, Jocelyn S et al. (2018) BRAF and MEK inhibitor therapy eliminates Nestin-expressing melanoma cells in human tumors. Pigment Cell Melanoma Res 31:708-719
Greenplate, Allison; Wang, Kai; Tripathi, Rati M et al. (2018) Genomic Profiling of T-Cell Neoplasms Reveals Frequent JAK1 and JAK3 Mutations With Clonal Evasion From Targeted Therapies. JCO Precis Oncol 2018:
Diggins, Kirsten E; Greenplate, Allison R; Leelatian, Nalin et al. (2017) Characterizing cell subsets using marker enrichment modeling. Nat Methods 14:275-278
Wogsland, Cara Ellen; Greenplate, Allison Rae; Kolstad, Arne et al. (2017) Mass Cytometry of Follicular Lymphoma Tumors Reveals Intrinsic Heterogeneity in Proteins Including HLA-DR and a Deficit in Nonmalignant Plasmablast and Germinal Center B-Cell Populations. Cytometry B Clin Cytom 92:79-87
Roussel, Mikael; Ferrell Jr, P Brent; Greenplate, Allison R et al. (2017) Mass cytometry deep phenotyping of human mononuclear phagocytes and myeloid-derived suppressor cells from human blood and bone marrow. J Leukoc Biol 102:437-447
Leelatian, Nalin; Doxie, Deon B; Greenplate, Allison R et al. (2017) Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis. Curr Protoc Mol Biol 118:25C.1.1-25C.1.23
Leelatian, Nalin; Doxie, Deon B; Greenplate, Allison R et al. (2017) Single cell analysis of human tissues and solid tumors with mass cytometry. Cytometry B Clin Cytom 92:68-78
Nicholas, Katherine J; Greenplate, Allison R; Flaherty, David K et al. (2016) Multiparameter analysis of stimulated human peripheral blood mononuclear cells: A comparison of mass and fluorescence cytometry. Cytometry A 89:271-80
Greenplate, Allison R; Johnson, Douglas B; Roussel, Mikael et al. (2016) Myelodysplastic Syndrome Revealed by Systems Immunology in a Melanoma Patient Undergoing Anti-PD-1 Therapy. Cancer Immunol Res 4:474-480
Johnson, Douglas B; Estrada, Monica V; Salgado, Roberto et al. (2016) Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy. Nat Commun 7:10582

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