Single-cell RNA sequencing profiling in a patient with discordant primary cutaneous B-cell and T-cell lymphoma reveals micromilieu-driven immune skewing.

BACKGROUND: Primary cutaneous lymphomas comprise a heterogeneous group of B-cell and T-cell malignancies which often show an indolent course, but can progress to aggressive disease in a subset of patients. Diagnosis is often delayed owing to clinical and histopathological similarities with benign inflammatory conditions. Especially during early disease, cancer cells are present at relatively low percentages compared with the inflammatory infiltrate, an interplay that is currently only insufficiently understood. OBJECTIVES: To improve diagnostics and perform molecular characterization of a complex type of primary cutaneous lymphoma. METHODS: Single-cell RNA sequencing (scRNA-seq) was performed and combined with T-cell and B-cell receptor sequencing. RESULTS: We were able to diagnose a patient with concurrent mycosis fungoides (MF) and primary cutaneous follicle centre lymphoma (PCFCL), appearing in mutually exclusive skin lesions. Profiling of tumour cells and the tissue microenvironment revealed a type-2 immune skewing in MF, most likely guided by the expanded clone that also harboured upregulation of numerous pro-oncogenic genes. By contrast, PCFCL lesions exhibited a more type-1 immune phenotype, consistent with its indolent behaviour. CONCLUSIONS: These data not only illustrate the diagnostic potential of scRNA-seq, but also allow the characterization of specific clonal populations that shape the unique tissue microenvironment in clinically distinct types of lymphoma skin lesions.

Analysis of anti-tumour necrosis factor-induced skin lesions reveals strong T helper 1 activation with some distinct immunological characteristics.

BACKGROUND: Psoriasiform and eczematous eruptions are the most common dermatological adverse reactions linked to anti-tumour necrosis factor (TNF)-α therapy. Yet, a detailed characterization of their immune phenotype is lacking. OBJECTIVES: To characterize anti-TNF-α-induced inflammatory skin lesions at a histopathological, cellular and molecular level, compared with psoriasis, eczema (atopic dermatitis) and healthy control skin. METHODS: Histopathological evaluation, gene expression (quantitative real-time polymerase chain reaction) and computer-assisted immunohistological studies (TissueFAXS) were performed on 19 skin biopsies from patients with inflammatory bowel disease (n = 17) and rheumatoid arthritis (n = 2) with new-onset inflammatory skin lesions during anti-TNF-α-therapy. RESULTS: Although most biopsies showed a psoriasiform and/or spongiotic (eczematous) histopathological architecture, these lesions were inconsistent with either psoriasis or eczema on a molecular level using an established chemokine (C-C motif) ligand 27/inducible nitric oxide synthase classifier. Despite some differences in immune skewing depending on the specific histopathological reaction pattern, all anti-TNF-α-induced lesions showed strong interferon (IFN)-γ activation, at higher levels than in psoriasis or eczema. IFN-γ was most likely produced by CD3/CD4/Tbet-positive T helper 1 lymphocytes. CONCLUSIONS: New-onset anti-TNF-α-induced eruptions previously classified as psoriasis or spongiotic dermatitis (eczema) exhibit a molecular profile that is different from either of these disorders.

Nonlesional atopic dermatitis skin shares similar T-cell clones with lesional tissues.

BACKGROUND: Atopic dermatitis (AD) is characterized by robust immune activation. Various T-cell subsets, including Th2/Th22 cells, are increased in lesional and nonlesional skin. However, there is conflicting literature on the diversity of the T-cell receptor (TCR) repertoire in lesional AD, and its relation to nonlesional skin remains unclear. METHODS: We performed high-throughput deep sequencing of the ß-TCR repertoire in 29 lesional and 19 nonlesional AD biopsies, compared to six healthy control and six cutaneous T-cell lymphoma (CTCL) samples from previously published cohorts. RESULTS: While greater T-cell infiltrates were observed in lesional vs nonlesional AD, TCR repertoire diversity was similar in lesional and nonlesional tissues, and absolute numbers of unique T-cell clones correlated with respective T-cell counts. Most (87%) top expanded lesional T-cell clones were shared with nonlesional tissues, and they were largely maintained after 16 weeks of successful treatment with topical triamcinolone. Nevertheless, both lesional and nonlesional AD showed a highly polyclonal TCR pattern, without evidence of oligoclonal expansion, or a preferred usage of certain V-ß genes in AD skin. Size of the overall T-cell infiltrate, but not the level of clonality, correlated with mRNA levels of key inflammatory mediators (e.g., IL-13, CCL17, IL23p19, CXCL10). CONCLUSION: While AD harbors a highly polyclonal T-cell receptor repertoire, and despite the lack of information on TCR antigen specificity, the sharing of top abundant clones between lesional and nonlesional skin, and their persistence after months of therapy, points to the continuous presence of potentially pathogenic skin resident memory T cells well beyond clinically inflamed lesions.

Proteome analysis identifies L1CAM/CD171 and DPP4/CD26 as novel markers of human skin mast cells.

BACKGROUND: The function of skin mast cells has been well documented in IgE-mediated allergic reactions, whereas other mast cell functions are poorly defined. This study aimed at identifying novel mast cell proteins by proteome analysis of primary human skin mast cells. METHODS: The proteome of skin mast cells was compared to other cell types and analyzed using bioinformatics. The expression and function of two proteins hitherto not described in skin mast cells was investigated in isolated mast cells as well as in mast cells in situ. RESULTS: Within the mast cell proteome, we identified 49 highly expressed proteins previously not described in mast cells; 21 of these proteins were found to be selectively expressed in mast cells. Two proteins, the neural cell adhesion molecule L1 and dipeptidyl peptidase 4, were further studied. L1 was found to be highly expressed in mast cells in normal, psoriasis, and mastocytosis skin. Dipeptidyl peptidase 4 was found to be expressed in mast cells in normal, psoriasis, and mastocytosis skin as well as in bone marrow mast cells in patients with systemic mastocytosis. In normal skin, mast cells were identified as a major source of dipeptidyl peptidase 4 and we also found that skin mast cells and fibroblasts secrete an active form of this enzyme. CONCLUSIONS: In a systematic proteomics approach we identified two novel mast cell proteins potentially relevant to skin homeostasis: neural cell adhesion molecule L1 and dipeptidyl peptidase 4.

Cold induces reactive oxygen species production and activation of the NF-kappa B response in endothelial cells and inflammation in vivo.

BACKGROUND: Organs intended for transplantation are generally stored in the cold for better preservation of their function. However, following transplantation and reperfusion, the microvasculature of transplanted organs often proves to be activated. Extensive leukocyte adhesion and microthrombus formation contribute to failure of the transplanted organ. OBJECTIVES: In this study we analyzed cold-induced changes to the activation status of cultured endothelial cells, possibly contributing to organ failure. METHODS: We exposed human umbilical vein endothelial cells (HUVECs) to temperatures below 37 °C (mostly to 8 °C) for 30 min and upon rewarming to 37 °C kept incubating them for up to 24 h. We also in vivo locally exposed mice to cold. RESULTS: The exposure to low temperatures induced, in HUVECs, expression of the prothrombotic factors plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) and of the inflammatory adhesion molecules, E-selectin, intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Furthermore, upon rewarming for 30 min, we detected activation of the inflammatory NF-κB pathway, as measured by transient NF-κB translocation to the nucleus and IκBα degradation. Using butylated hydroxytoluene (BHT), a scavenger of reactive oxygen species (ROS), we further demonstrated that cold-induced NF-κB activation depends on ROS production. Local exposure to cold also, in vivo, induced ROS production and ICAM-1 expression and resulted in leukocyte infiltration. CONCLUSIONS: Our results point to a causative link between ROS production and NF-κB activation, suppression of which had been shown to be beneficial during hypothermic storage and subsequent rewarming of organs for transplantation.

Clinical Challenges in the Management of Leishmania/HIV Coinfection in a Nonendemic Area: A Case Report.

We report on a 37-year-old male HIV-positive patient with generalized cutaneous leishmaniasis undiagnosed for several years. Upon presentation, visceral leishmaniasis was diagnosed in addition to cutaneous manifestation of the disease. Over a period of three years, several different treatment regimens including liposomal amphotericin B, liposomal amphotericin B with miltefosine, liposomal amphotericin B with interferon, and pentamidine combined fluconazole and allopurinol were applied until Leishmania PCR from blood turned negative. This case supports the necessity of multidrug combinational and sequential therapy over a very prolonged period of time in severely immunosuppressed patients infected with Leishmania and highlights the tremendous individual but also economic burden of this disease.