Abstract Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of blood-related cancers characterized by chronic inflammation and accumulation of malignant T cells in the skin. Among lymphomas, CTCL is uniquely characterized by the striking dermal tropism of transformed cells—where exposure to microbes might contribute to disease etiology. CTCL patients often succumb to bacterial infections, and a correlation between the presence of certain strains of Staphylococcus aureus and disease severity has been observed. Commensal and pathogenic bacteria can influence differentiation of naïve T lymphocytes, trigger proliferation and activation of T cells through production of superantigens (SAgs), and create a permissive microenvironment for tumor cells by influencing chemokine and cytokine expression. We hypothesize that, along with genetic predisposition of an individual and mutations acquired by T cells or T lymphocyte precursors, exposure to S. aureus and SAgs produced by some strains of this pathobiont promotes malignant transformation and clonal evolution in CTCL. Prior research by other groups and our preliminary studies implicate S. aureus specifically in CTCL pathogenesis. We will isolate, sequence and study S. aureus isolates from the skin of CTCL patients (from tumor sites and unaffected skin) and determine the repertoire of toxins and SAgs produced by these strains. We anticipate that CTCL-associated isolates will differ in both the ability to produce large numbers of SAg types per organism and the distribution of their SAg gene content. We will use in vitro methods to evaluate how SAgs and other toxins and exoproteins produced by skin microbiota of CTCL patients contribute to T cell activation, proliferation, and malignant transformation. To further understand the contribution of S. aureus to CTCL pathogenesis and to demonstrate a causal connection between S. aureus colonization and malignant disease, we will use our new animal model of CTCL and our germ-free facility to examine the impact of microbial exposure on CTCL initiation and progression. We will also take advantage of cutting-edge, single-cell, high-throughput technology that enables simultaneous analysis of surface epitopes, T cell receptor gene rearrangements, and transcriptomics to achieve unprecedented resolution of the tumor microenvironment in CTCL and to examine changes in the microenvironment upon S. aureus colonization. The studies outlined in our proposal will provide significant insight into CTCL pathogenesis and will inform development of future therapies that are more targeted and less toxic than current chemotherapeutic approaches.