Keratinocytes Present Staphylococcus aureus Enterotoxins and Promote Malignant and Nonmalignant T Cell Proliferation in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma is characterized by malignant T cells proliferating in a unique tumor microenvironment dominated by keratinocytes (KCs). Skin colonization and infection by Staphylococcus aureus are a common cause of morbidity and are suspected of fueling disease activity. In this study, we show that expression of HLA-DRs, high-affinity receptors for staphylococcal enterotoxins (SEs), by KCs correlates with IFN-γ expression in the tumor microenvironment. Importantly, IFN-γ induces HLA-DR, SE binding, and SE presentation by KCs to malignant T cells from patients with Sézary syndrome and malignant and nonmalignant T-cell lines derived from patients with Sézary syndrome and mycosis fungoides. Likewise, preincubation of KCs with supernatant from patient-derived SE-producing S aureus triggers proliferation in malignant T cells and cytokine release (including IL-2), when cultured with nonmalignant T cells. This is inhibited by pretreatment with engineered bacteriophage S aureus-specific endolysins. Furthermore, alteration in the HLA-DR-binding sites of SE type A and small interfering RNA-mediated knockdown of Jak3 and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that upon exposure to patient-derived S aureus and SE, KCs stimulate IL-2Rγ/Jak3-dependent proliferation of malignant and nonmalignant T cells in an environment with nonmalignant T cells. These findings suggest that KCs in the tumor microenvironment play a key role in S aureus-mediated disease activity in cutaneous T-cell lymphoma.

Stage-related increase in PIM2 expression in mycosis fungoides.

The oncogene PIM2 is upregulated in several malignancies but has never been investigated in mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma (CTCL). PIM2 is a well-known oncogene and is regulated by cell signaling pathways like the JAK/STAT- and NF-kB-pathway, key regulators in the pathogenesis of CTCL. The aim of this study was to examine the role of PIM2 in MF. PIM2 gene expression was measured in 81 formalin-fixed paraffin-embedded skin biopsies from patients with MF and 46 control biopsies from healthy skin (HS) and benign inflammatory skin disease (BID). Validation of PIM2 protein expression was performed on selected biopsies with immunohistochemical staining. We found a significant difference in gene expression levels between both early stage MF and HS (p < 0.0001), and BID (p < 0.0001). In addition, the PIM2 gene expression was higher in advanced-stage MF compared to early stage disease (p = 0.0001). No significant difference in gene expression levels was found between patients with and without disease progression. In conclusion, we found PIM2 expression is significantly increased in MF compared to controls, and in advanced-stage MF compared to early stage MF. These findings could potentially have diagnostic value in discriminating early stage MF from BID.

Lifestyle and demographic associations with 47 inflammatory and vascular stress biomarkers in 9876 blood donors.

BACKGROUND: The emerging use of biomarkers in research and tailored care introduces a need for information about the association between biomarkers and basic demographics and lifestyle factors revealing expectable concentrations in healthy individuals while considering general demographic differences. METHODS: A selection of 47 biomarkers, including markers of inflammation and vascular stress, were measured in plasma samples from 9876 Danish Blood Donor Study participants. Using regression models, we examined the association between biomarkers and sex, age, Body Mass Index (BMI), and smoking. RESULTS: Here we show that concentrations of inflammation and vascular stress biomarkers generally increase with higher age, BMI, and smoking. Sex-specific effects are observed for multiple biomarkers. CONCLUSION: This study provides comprehensive information on concentrations of 47 plasma biomarkers in healthy individuals. The study emphasizes that knowledge about biomarker concentrations in healthy individuals is critical for improved understanding of disease pathology and for tailored care and decision support tools.

Blood-based biomarkers are circulating molecules that can help to indicate health or disease. Biomarker levels may vary depending on demographic and lifestyle factors such as age, sex, smoking status, and body mass index. Here, we examine the effects of these demographic and lifestyle factors on levels of biomarkers related to activation of the immune system and cardiovascular stress. Measurements of 47 different proteins were performed on blood samples from nearly 10,000 healthy Danish blood donors. Measurement data were linked with questionnaire data to assess effects of lifestyle. We found that immune activation and vascular stress generally increased with age, BMI, and smoking. As these measurements are from healthy blood donors they can serve as a reference for expectable effects and inflammation levels in healthy individuals. Knowledge about the healthy state is important for understanding disease progression and optimizing care.

Repeated immunization with ATRA-containing liposomal adjuvant transdifferentiates Th17 cells to a Tr1-like phenotype.

In many autoimmune diseases, autoantigen-specific Th17 cells play a pivotal role in disease pathogenesis. Th17 cells can transdifferentiate into other T cell subsets in inflammatory conditions, however, there have been no attempts to target Th17 cell plasticity using vaccines. We investigated if autoantigen-specific Th17 cells could be specifically targeted using a therapeutic vaccine approach, where antigen was formulated in all-trans retinoic acid (ATRA)-containing liposomes, permitting co-delivery of antigen and ATRA to the same target cell. Whilst ATRA was previously found to broadly reduce Th17 responses, we found that antigen formulated in ATRA-containing cationic liposomes only inhibited Th17 cells in an antigen-specific manner and not when combined with an irrelevant antigen. Furthermore, this approach shifted existing Th17 cells away from IL-17A expression and transcriptomic analysis of sorted Th17 lineage cells from IL-17 fate reporter mice revealed a shift of antigen-specific Th17 cells to exTh17 cells, expressing functional markers associated with T cell regulation and tolerance. In the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, vaccination with myelin-specific (MOG) antigen in ATRA-containing liposomes reduced Th17 responses and alleviated disease. This highlights the potential of therapeutic vaccination for changing the phenotype of existing Th17 cells in the context of immune mediated diseases.

Staphylococcus aureus induces drug resistance in cancer T cells in Sézary syndrome.

ABSTRACT: Patients with Sézary syndrome (SS), a leukemic variant of cutaneous T-cell lymphoma (CTCL), are prone to Staphylococcus aureus infections and have a poor prognosis due to treatment resistance. Here, we report that S aureus and staphylococcal enterotoxins (SE) induce drug resistance in malignant T cells against therapeutics commonly used in CTCL. Supernatant from patient-derived, SE-producing S aureus and recombinant SE significantly inhibit cell death induced by histone deacetylase (HDAC) inhibitor romidepsin in primary malignant T cells from patients with SS. Bacterial killing by engineered, bacteriophage-derived, S aureus-specific endolysin (XZ.700) abrogates the effect of S aureus supernatant. Similarly, mutations in major histocompatibility complex (MHC) class II binding sites of SE type A (SEA) and anti-SEA antibody block induction of resistance. Importantly, SE also triggers resistance to other HDAC inhibitors (vorinostat and resminostat) and chemotherapeutic drugs (doxorubicin and etoposide). Multimodal single-cell sequencing indicates T-cell receptor (TCR), NF-κB, and JAK/STAT signaling pathways (previously associated with drug resistance) as putative mediators of SE-induced drug resistance. In support, inhibition of TCR-signaling and Protein kinase C (upstream of NF-κB) counteracts SE-induced rescue from drug-induced cell death. Inversely, SE cannot rescue from cell death induced by the proteasome/NF-κB inhibitor bortezomib. Inhibition of JAK/STAT only blocks rescue in patients whose malignant T-cell survival is dependent on SE-induced cytokines, suggesting 2 distinct ways SE can induce drug resistance. In conclusion, we show that S aureus enterotoxins induce drug resistance in primary malignant T cells. These findings suggest that S aureus enterotoxins cause clinical treatment resistance in patients with SS, and antibacterial measures may improve the outcome of cancer-directed therapy in patients harboring S aureus.

Contact allergens in African countries: A review of published patch test studies.

Only few studies on contact allergy in African countries have been published. The aim of the present study was to provide an overview of the most common contact allergens identified by the use of patch tests in African countries based on a review of the existing literature. A total of twenty-four publications from eight African countries were initially identified by search in PubMed. The abstracts and method sections were screened, and 15 studies in which patch tests were actually used to identify the allergen causing the allergic contact dermatitis (ACD) were finally selected. Nickel, cobalt, chromium, fragrance mix and p-tert-butylphenol-formaldehyde resin were the dominating contact allergens responsible for 40%-90% of the positive patch test reactions. This study indicates that a targeted effort directed towards prevention, avoidance and regulation of reliably identified contact allergens could reduce the disease burden of ACD considerable in some African countries.

CD100 boosts the inflammatory response in the challenge phase of allergic contact dermatitis in mice.

BACKGROUND: Allergic contact dermatitis (ACD) is an inflammatory disease with a complex pathophysiology in which epidermal-resident memory CD8+ T (TRM ) cells play a key role. The mechanisms involved in the activation of CD8+ TRM cells during allergic flare-up responses are not understood. METHODS: The expression of CD100 and its ligand Plexin B2 on CD8+ TRM cells and keratinocytes before and after allergen exposure was determined by flow cytometry and RT-qPCR. The role of CD100 in the inflammatory response during the challenge phase of ACD was determined in a model of ACD in CD100 knockout and wild-type mice. RESULTS: We show that CD8+ TRM cells express CD100 during homeostatic conditions and up-regulate it following re-exposure of allergen-experienced skin to the experimental contact allergen 1-fluoro-2,4-dinitrobenzene (DNFB). Furthermore, Plexin B2 is up-regulated on keratinocytes following exposure to some contact allergens. We show that loss of CD100 results in a reduced inflammatory response to DNFB with impaired production of IFNγ, IL-17A, CXCL1, CXCL2, CXCL5, and IL-1ß and decreased recruitment of neutrophils to the epidermis. CONCLUSION: Our study demonstrates that CD100 is expressed on CD8+ TRM cells and is required for full activation of CD8+ TRM cells and the flare-up response of ACD.

The junctional adhesion molecule-like protein (JAML) is important for the inflammatory response during contact hypersensitivity.

BACKGROUND: The junctional adhesion molecule-like protein (JAML) plays important roles in wound healing and activation of epidermal γδ T cells in mice. Whether JAML plays a role in contact hypersensitivity (CHS), the animal model of allergic contact dermatitis (ACD), is not known. METHODS: To examine the role of JAML in CHS, we used various mouse models of CHS in JAML knockout (KO) and wild-type (WT) mice. Furthermore, the expression of the JAML ligand coxsackievirus and adenovirus receptor (CXADR) on keratinocytes was accessed in vitro and in vivo. RESULTS: JAML KO mice had a diminished inflammatory response during both the sensitization and elicitation phase of CHS and had reduced numbers of CD8+ and CD4+ T cells in the epidermis. Furthermore, interferon γ (IFNγ), interleukin 1ß (IL-1ß) and CXCL10 production were significantly reduced in JAML KO mice during the elicitation phase. We found that CD8+ T cells express JAML and that JAML is essential for rapid flare-up responses to contact allergens. Finally, we show that keratinocytes up-regulate the JAML ligand CXADR following exposure to contact allergens. CONCLUSION: Our study is the first to show a central role of JAML in CHS and reveals a potential new target for the treatment of ACD in humans.

In vitro differentiated human CD4+ T cells produce hepatocyte growth factor.

Differentiation of naive CD4+ T cells into effector T cells is a dynamic process in which the cells are polarized into T helper (Th) subsets. The subsets largely consist of four fundamental categories: Th1, Th2, Th17, and regulatory T cells. We show that human memory CD4+ T cells can produce hepatocyte growth factor (HGF), a pleiotropic cytokine which can affect several tissue types through signaling by its receptor, c-Met. In vitro differentiation of T cells into Th-like subsets revealed that HGF producing T cells increase under Th1 conditions. Enrichment of HGF producing cells was possible by targeting cells with surface CD30 expression, a marker discovered through single-cell RNA-sequencing. Furthermore, pharmacological inhibition of PI3K or mTOR was found to inhibit HGF mRNA and protein, while an Akt inhibitor was found to increase these levels. The findings suggest that HGF producing T cells could play a role in disease where Th1 are present.

Citrullination of myelin basic protein induces a Th17-cell response in healthy individuals and enhances the presentation of MBP85-99 in patients with multiple sclerosis.

The post-translational modification citrullination has been proposed to play a role in the pathogenesis of multiple sclerosis (MS). Myelin basic protein (MBP) is a candidate autoantigen which is citrullinated to a minor extent under physiological conditions and hypercitrullinated in MS. We examined immune cell responses elicited by hypercitrullinated MBP (citMBP) in cultures of mononuclear cells from 18 patients with MS and 42 healthy donors (HDs). The immunodominant peptide of MBP, MBP85-99, containing citrulline in position 99, outcompeted the binding of native MBP85-99 to HLA-DR15, which is strongly linked to MS. Moreover, using the monoclonal antibody MK16 as probe, we observed that B cells and monocytes from HLA-DR15+ patients with MS presented MBP85-99 more efficiently after challenge with citMBP than with native MBP. Both citMBP and native MBP induced proliferation of CD4+ T cells from patients with MS as well as TNF-α production by their B cells and CD4+ T cells, and citrullination of MBP tended to enhance TNF-α secretion by CD4+ T cells from HLA-DR15+ patients. Unlike native MBP, citMBP induced differentiation into Th17 cells in cultures from HDs, while neither form of MBP induced Th17-cell differentiation in cultures from patients with MS. These data suggest a role for citrullination in the breach of tolerance to MBP in healthy individuals and in maintenance of the autoimmune response to MBP in patients with MS.

CD4+ T cells inhibit the generation of CD8+ epidermal-resident memory T cells directed against clinically relevant contact allergens.

BACKGROUND: CD8+ epidermal-resident memory T (TRM ) cells play central roles in local flare-up responses to experimental contact allergens by inducing massive influx of neutrophils to the epidermis upon allergen challenge. Whether similar immunopathogenic mechanisms are involved in the responses to clinically relevant contact allergens is unknown. METHODS: The immune response to cinnamal, ρ-phenylenediamine (PPD) and methylisothiazolinone (MI) was studied in a well-established mouse model for allergic contact dermatitis that includes formation of TRM cells by ELISA, flow cytometry, fluorescence microscopy analyses and cell depletion protocols. RESULTS: We show that the formation of CD4+ and CD8+ epidermal TRM cells and the inflammatory response are highly allergen-dependent. However, the magnitude of the flare-up responses correlated with the number of epidermal CD8+ TRM cells, CXCL1/CXCL2 release and recruitment of neutrophils to the epidermis. Finally, depletion of CD4+ T cells strongly enhanced the number of epidermal CD8+ TRM cells, the flare-up response and the epidermal infiltration of neutrophils for all allergens. CONCLUSION: As the first, this study demonstrates that clinically relevant contact allergens have the ability to generate pathogenic, epidermal CD8+ TRM cells that recruit neutrophils following re-exposure to the allergen, but that this normally is counteracted by the simultaneous induction of anti-inflammatory CD4+ T cells.

Imbalanced IL-1B and IL-18 Expression in Sézary Syndrome.

Sézary syndrome (SS) is a rare and aggressive type of cutaneous T-cell lymphoma, with an abnormal inflammatory response in affected skin. The cytokines IL-1B and IL-18, as key signaling molecules in the immune system, are produced in an inactive form and cleave to the active form by inflammasomes. In this study, we assessed the skin, serum, peripheral mononuclear blood cell (PBMC) and lymph-node samples of SS patients and control groups (healthy donors (HDs) and idiopathic erythroderma (IE) nodes) to investigate the inflammatory markers IL-1B and IL-18 at the protein and transcript expression levels, as potential markers of inflammasome activation. Our findings showed increased IL-1B and decreased IL-18 protein expression in the epidermis of SS patients; however, in the dermis layer, we detected increased IL-18 protein expression. In the lymph nodes of SS patients at advanced stages of the disease (N2/N3), we also detected an enhancement of IL-18 and a downregulation of IL-1B at the protein level. Moreover, the transcriptomic analysis of the SS and IE nodes confirmed the decreased expression of IL1B and NLRP3, whereas the pathway analysis indicated a further downregulation of IL1B-associated genes. Overall, the present findings showed compartmentalized expressions of IL-1B and IL-18 and provided the first evidence of their imbalance in patients with Sézary syndrome.

Impact of U2AF1 mutations on circular RNA expression in myelodysplastic neoplasms.

Mutations in U2AF1 are relatively common in myelodysplastic neoplasms (MDS) and are associated with an inferior prognosis, but the molecular mechanisms underlying this are not fully elucidated. Circular RNAs (circRNAs) have been implicated in cancer, but it is unknown how mutations in splicing factors may impact on circRNA biogenesis. Here, we used RNA-sequencing to investigate the effects of U2AF1 mutations on circRNA expression in K562 cells with a doxycycline-inducible U2AF1S34 mutation, in a mouse model with a doxycycline-inducible U2AF1S34 mutation, and in FACS-sorted CD34+ bone marrow cells from MDS patients with either U2AF1S34 or U2AF1Q157 mutations. In all contexts, we found an increase in global circRNA levels in the U2AF1-mutated setting, which was independent of expression changes in the cognate linear host genes. In patients, the U2AF1S34 and U2AF1Q157 mutations were both associated with an overall increased expression of circRNAs. circRNAs generated by a non-Alu-mediated mechanism generally showed the largest increase in expression levels. Several well-described cancer-associated circRNAs, including circZNF609 and circCSNK1G3, were upregulated in MDS patients with U2AF1 mutations compared to U2AF1-wildtype MDS controls. In conclusion, high circRNA expression is observed in association with U2AF1 mutations in three biological systems, presenting an interesting possibility for biomarker and therapeutic investigation.

Endolysin Inhibits Skin Colonization by Patient-Derived Staphylococcus Aureus and Malignant T-Cell Activation in Cutaneous T-Cell Lymphoma.

Staphylococcus aureus is suspected to fuel disease activity in cutaneous T-cell lymphomas. In this study, we investigate the effect of a recombinant, antibacterial protein, endolysin (XZ.700), on S. aureus skin colonization and malignant T-cell activation. We show that endolysin strongly inhibits the proliferation of S. aureus isolated from cutaneous T-cell lymphoma skin and significantly decreases S. aureus bacterial cell counts in a dose-dependent manner. Likewise, ex vivo colonization of both healthy and lesional skin by S. aureus is profoundly inhibited by endolysin. Moreover, endolysin inhibits the patient-derived S. aureus induction of IFNγ and the IFNγ-inducible chemokine CXCL10 in healthy skin. Whereas patient-derived S. aureus stimulates activation and proliferation of malignant T cells in vitro through an indirect mechanism involving nonmalignant T cells, endolysin strongly inhibits the effects of S. aureus on activation (reduced CD25 and signal transducer and activator of transcription 5 phosphorylation) and proliferation (reduced Ki-67) of malignant T cells and cell lines in the presence of nonmalignant T cells. Taken together, we provide evidence that endolysin XZ.700 inhibits skin colonization, chemokine expression, and proliferation of pathogenic S. aureus and blocks their potential tumor-promoting effects on malignant T cells.

Phenotypic plasticity of malignant T cells in blood and skin of a Sézary syndrome patient revealed by single cell transcriptomics.

Background: Sézary Syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL). In SS patients, malignant T cells are circulating through the blood and cause erythroderma. Objective: To compare the transcriptome of single cells in blood and skin samples from a patient with advanced SS. Methods: We utilized combined single cell RNA and T-cell receptor (TCR) sequencing (scRNA-seq). Results: We scrutinized the malignant T cells in blood and skin in an unbiased manner without pre-sorting of cells. We observed different phenotypes of the same monoclonal malignant T-cell population, confirmed by TCR sequencing and inferred copy number variation analysis. Malignant T cells present in the circulating blood expressed genes resembling central memory T cells such as CCR7, IL7R and CD27. In the skin, we detected two major malignant T-cell populations: One subpopulation was closely related to the malignant T cells from the blood, while the other subpopulation expressed genes reminiscent of skin resident effector memory T cells including GZMB and NKG7. Pseudotime analysis indicated crucial transcriptomic changes in the transition of malignant T cells between blood and skin. These changes included the differential regulation of TXNIP, a putative tumor suppressor in CTCL, and the adaptation to the hypoxic conditions in the skin. Tumor cell proliferation in the skin was supported by stimulating interactions between myeloid cells and malignant T cells. Conclusions: Using scRNA-seq we detected a high degree of functional heterogeneity within the malignant T-cell population in SS and highlighted crucial differences between SS cells in blood and skin.

mRNA COVID-19 vaccine elicits potent adaptive immune response without the persistent inflammation seen in SARS-CoV-2 infection.

SARS-CoV-2 infection and vaccination elicit potent immune responses. Our study presents a comprehensive multimodal single-cell dataset of peripheral blood of patients with acute COVID-19 and of healthy volunteers before and after receiving the SARS-CoV-2 mRNA vaccine and booster. We compared host immune responses to the virus and vaccine using transcriptional profiling, coupled with B/T cell receptor repertoire reconstruction. COVID-19 patients displayed an enhanced interferon signature and cytotoxic gene upregulation, absent in vaccine recipients. These findings were validated in an independent dataset. Analysis of B and T cell repertoires revealed that, while the majority of clonal lymphocytes in COVID-19 patients were effector cells, clonal expansion was more evident among circulating memory cells in vaccine recipients. Furthermore, while clonal αß T cell responses were observed in both COVID-19 patients and vaccine recipients, dramatic expansion of clonal γδT cells was found only in infected individuals. Our dataset enables comparative analyses of immune responses to infection versus vaccination, including clonal B and T cell responses. Integrating our data with publicly available datasets allowed us to validate our findings in larger cohorts. To our knowledge, this is the first dataset to include comprehensive profiling of longitudinal samples from healthy volunteers pre/post SARS-CoV-2 vaccine and booster.

Immune cell composition in unipolar depression: a comprehensive systematic review and meta-analysis.

Depression has been associated with inflammatory pathophysiological mechanisms, including alterations in amount of circulating immune cells. However, no meta-analysis within the past 20 years have reevaluated the circulating immune cells in blood and cerebrospinal fluid (CSF) from patients with depression compared to healthy controls. The aim of this study was to systematically evaluate the circulating immune cells in blood and CSF from patients with unipolar depression compared to healthy controls. Databases were searched up until February 12, 2021. Data-extraction was performed by two independent reviewers. 104 studies were included in the meta-analysis using fixed and random-effects models. Patients with depression had a significantly higher overall leukocyte count (35 studies; SMD, 0.46; 95% CI: 0.31-0.60, I2 = 68%), higher neutrophil count (24 studies; SMD, 0.52; 95% CI: 0.33-0.71, I2 = 77%) and higher monocyte count (27 studies; SMD, 0.32; 95% CI: 0.11-0.53, I2 = 77%) compared to healthy controls. Leukocyte counts were higher in inpatients, indicating a relation to depression severity. Furthermore, there were significant alterations in several lymphocyte subsets, including higher natural killer cells and T cell subsets. Higher neutrophil/lymphocyte ratio (11 studies; SMD = 0.24; 95% CI: 0.06-0.42, I2 = 73%), CD4/CD8 cell-ratio (26 studies; SMD = 0.14; 95% CI: 0.01-0.28, I2 = 42%) and T helper 17/T regulatory ratio (2 studies; SMD = 1.05; 95% CI: 0.15-1.95, I2 = 86%) were found in patients compared to healthy controls. CSF white cell count was higher in patients compared to controls (3 studies; SMD = 0.20; 95% CI: 0.01-0.38, I2 = 0%). There were no data for CSF cell subsets. This study suggests that there are several blood immune cell alterations in patients with unipolar depression compared to healthy controls, both in major leukocyte subsets and more specialized immune cell subsets.