Genome-wide transcriptome analysis of the STAT6-regulated genes in advanced-stage cutaneous T-cell lymphoma.

The signal transducer and activator of transcription 6 (STAT6) is a critical up-stream mediator of interleukin-13 (IL-13) and IL-4 signaling and is constitutively activated in malignant lymphocytes from Sezary syndrome (SS) and mycosis fungoides (MF), the most common subtypes of cutaneous T-cell lymphomas. By combining genome-wide expression profiling with pharmacological STAT6 inhibition, we have identified the genes regulated by STAT6 in MF/SS tumors. We found that STAT6 regulates several common pathways in MF/SS malignant lymphocytes that are associated with control of cell-cycle progression and genomic stability as well as production of Th2 cytokines. Using ex vivo skin explants from cutaneous MF tumors as well as Sezary cells derived from the blood of SS patients, we demonstrated that inhibition of STAT6 activation downregulates cytokine production and induces cell-cycle arrest in MF/SS malignant lymphocytes, inhibiting their proliferation but not their survival. Furthermore, we show that STAT6 promotes the protumoral M2-like phenotype of tumor-associated macrophages in the tumor microenvironment of advanced stage MF by upregulating the expression of genes associated with immunosuppression, chemotaxis, and tumor matrix remodeling. Thus, we show STAT6 to be a major factor in the pathogenesis and progression of MF/SS, promoting proliferation and invasion of the malignant lymphocytes while inducing a progressive depression of the antitumor immune response. Together, our results provide new insights into disease pathogenesis and offer new prospective targets for therapeutic intervention.

Single-cell transcriptome analysis identifies skin-specific T-cell responses in systemic sclerosis.

OBJECTIVES: Although T cells have been implicated in the pathogenesis of systemic sclerosis (SSc), a comprehensive study of T-cell-mediated immune responses in the affected skin of patients with progressive SSc is lacking. Droplet-based single-cell transcriptome analysis of SSc skin biopsies opens avenues for dissecting patient-specific T-cell heterogeneity, providing a basis for identifying novel gene expression related to functional pathways associated with severity of SSc skin disease. METHODS: Single-cell RNA sequencing was performed by droplet-based sequencing (10x Genomics), focusing on 3729 CD3+ lymphocytes (867 cells from normal and 2862 cells from SSc skin samples) from skin biopsies of 27 patients with active SSc and 10 healthy donors. Confocal immunofluorescence microscopy of progressive SSc skin samples validated transcriptional results and visualised spatial localisations of T-cell subsets. RESULTS: We identified several subsets of recirculating and tissue-resident T cells in healthy and SSc skin that were associated with distinct signalling pathways. While most clusters shared a common gene expression signature between patients and controls, we identified a unique cluster of recirculating CXCL13+ T cells in SSc skin which expressed a T helper follicular-like gene expression signature and that appears to be poised to promote B-cell responses within the inflamed skin of patients. CONCLUSIONS: Current available therapies to reverse or even slow progression of SSc lead to broad killing of immune cells and consequent toxicities, including death. Identifying the precise immune mechanism(s) driving SSc pathogenesis could lead to innovative therapies that selectively target the aberrant immune response, resulting in better efficacy and less toxicity.

The mycosis fungoides cutaneous microenvironment shapes dysfunctional cell trafficking, antitumor immunity, matrix interactions, and angiogenesis.

Malignant T lymphocyte proliferation in mycosis fungoides (MF) is largely restricted to the skin, implying that malignant cells are dependent on their specific cutaneous tumor microenvironment (TME), including interactions with non-malignant immune and stromal cells, cytokines, and other immunomodulatory factors. To explore these interactions, we performed a comprehensive transcriptome analysis of the TME in advanced-stage MF skin tumors by single-cell RNA sequencing. Our analysis identified cell-type compositions, cellular functions, and cell-to-cell interactions in the MF TME that were distinct from those from healthy skin and benign dermatoses. While patterns of gene expression were common among patient samples, high transcriptional diversity was also observed in immune and stromal cells, with dynamic interactions and crosstalk between these cells and malignant T lymphocytes. This heterogeneity mapped to processes such as cell trafficking, matrix interactions, angiogenesis, immune functions, and metabolism that affect cancer cell growth, migration, and invasion, as well as antitumor immunity. By comprehensively characterizing the transcriptomes of immune and stromal cells within the cutaneous microenvironment of individual MF tumors, we have identified patterns of dysfunction common to all tumors that represent a resource for identifying candidates with therapeutic potential as well as patient-specific heterogeneity that has important implications for personalized disease management.

Single-Cell RNA Sequencing Unveils the Clonal and Transcriptional Landscape of Cutaneous T-Cell Lymphomas.

PURPOSE: Clonal malignant T lymphocytes constitute only a fraction of T cells in mycosis fungoides skin tumors and in the leukemic blood of Sézary syndrome, the classic types of cutaneous T-cell lymphomas. However, lack of markers specific for malignant lymphocytes prevents distinguishing them from benign T cells, thus delaying diagnosis and the development of targeted treatments. Here we applied single-cell methods to assess the transcriptional profiles of both malignant T-cell clones and reactive T lymphocytes directly in mycosis fungoides/Sézary syndrome patient samples. EXPERIMENTAL DESIGN: Single-cell RNA sequencing was used to profile the T-cell immune repertoire simultaneously with gene expression in CD3+ lymphocytes from mycosis fungoides and healthy skin biopsies as well as from Sézary syndrome and control blood samples. Transcriptional data were validated in additional advanced-stage mycosis fungoides/Sézary syndrome skin and blood samples by immunofluorescence microscopy. RESULTS: Several nonoverlapping clonotypes are expanded in the skin and blood of individual advanced-stage mycosis fungoides/Sézary syndrome patient samples, including a dominant malignant clone as well as additional minor malignant and reactive clones. While we detected upregulation of patient-specific as well as mycosis fungoides- and Sézary syndrome-specific oncogenic pathways within individual malignant clones, we also detected upregulation of several common pathways that included genes associated with cancer cell metabolism, cell-cycle regulation, de novo nucleotide biosynthesis, and invasion. CONCLUSIONS: Our analysis unveils new insights into mycosis fungoides/Sézary syndrome pathogenesis by providing an unprecedented report of the transcriptional profile of malignant T-cell clones in the skin and blood of individual patients and offers novel prospective targets for personalized therapy.

Single-Cell Lymphocyte Heterogeneity in Advanced Cutaneous T-cell Lymphoma Skin Tumors.

PURPOSE: The heterogeneity of tumor cells presents a major challenge to cancer diagnosis and therapy. Cutaneous T-cell lymphomas (CTCL) are a group of T lymphocyte malignancies that primarily affect skin. Lack of highly specific markers for malignant lymphocytes prevents early diagnosis, while only limited treatment options are available for patients with advanced stage CTCL. Droplet-based single-cell transcriptome analysis of CTCL skin biopsies opens avenues for dissecting patient-specific T lymphocyte heterogeneity, providing a basis for identifying specific markers for diagnosis and cure of CTCL. EXPERIMENTAL DESIGN: Single-cell RNA-sequencing was performed by Droplet-based sequencing (10X Genomics), focusing on 14,056 CD3+ lymphocytes (448 cells from normal and 13,608 cells from CTCL skin samples) from skin biopsies of 5 patients with advanced-stage CTCL and 4 healthy donors. Protein expression of identified genes was validated in advanced stage CTCL skin tumors by immunohistochemistry and confocal immunofluorescence microscopy. RESULTS: Our analysis revealed a large inter- and intratumor gene expression heterogeneity in the T lymphocyte subset, as well as a common gene expression signature in highly proliferating lymphocytes that was validated in multiple advanced-stage skin tumors. In addition, we established the immunologic state of reactive lymphocytes and found heterogeneity in effector and exhaustion programs across patient samples. CONCLUSIONS: Single-cell analysis of CTCL skin tumor samples reveals patient-specific landscapes of malignant and reactive lymphocytes within the local microenvironment of each tumor, giving an unprecedented view of lymphocyte heterogeneity and identifying tumor-specific molecular signatures, with important implications for diagnosis and personalized disease treatment.