Riboswitch-controlled IL-12 gene therapy reduces hepatocellular cancer in mice.

Hepatocellular carcinoma (HCC) and solid cancers with liver metastases are indications with high unmet medical need. Interleukin-12 (IL-12) is a proinflammatory cytokine with substantial anti-tumor properties, but its therapeutic potential has not been realized due to severe toxicity. Here, we show that orthotopic liver tumors in mice can be treated by targeting hepatocytes via systemic delivery of adeno-associated virus (AAV) vectors carrying the murine IL-12 gene. Controlled cytokine production was achieved in vivo by using the tetracycline-inducible K19 riboswitch. AAV-mediated expression of IL-12 led to STAT4 phosphorylation, interferon-γ (IFNγ) production, infiltration of T cells and, ultimately, tumor regression. By detailed analyses of efficacy and tolerability in healthy and tumor-bearing animals, we could define a safe and efficacious vector dose. As a potential clinical candidate, we characterized vectors carrying the human IL-12 (huIL-12) gene. In mice, bioactive human IL-12 was expressed in a vector dose-dependent manner and could be induced by tetracycline, suggesting tissue-specific AAV vectors with riboswitch-controlled expression of highly potent proinflammatory cytokines as an attractive approach for vector-based cancer immunotherapy.

Modulation of the skin microbiome in cutaneous T-cell lymphoma delays tumour growth and increases survival in the murine EL4 model.

Cutaneous T-cell lymphomas (CTCL) are a group of lymphoproliferative disorders of skin-homing T cells causing chronic inflammation. These disorders cause impairment of the immune environment, which leads to severe infections and/or sepsis due to dysbiosis. In this study, we elucidated the host-microbial interaction in CTCL that occurs during the phototherapeutic treatment regime and determined whether modulation of the skin microbiota could beneficially affect the course of CTCL. EL4 T-cell lymphoma cells were intradermally grafted on the back of C57BL/6 mice. Animals were treated with conventional therapeutics such as psoralen + UVA (PUVA) or UVB in the presence or absence of topical antibiotic treatment (neomycin, bacitracin, and polymyxin B sulphate) as an adjuvant. Microbial colonisation of the skin was assessed to correlate with disease severity and tumour growth. Triple antibiotic treatment significantly delayed tumour occurrence (p = 0.026), which prolonged the survival of the mice (p = 0.033). Allocation to phototherapeutic agents PUVA, UVB, or none of these, along with antibiotic intervention, reduced the tumour growth significantly (p = 0.0327, p ≤ 0.0001, p ≤ 0.0001 respectively). The beta diversity indices calculated using the Bray-Curtis model showed that the microbial population significantly differed after antibiotic treatment (p = 0.001). Upon modulating the skin microbiome by antibiotic treatment, we saw an increase in commensal Clostridium species, e.g., Lachnospiraceae sp. (p = 0.0008), Ruminococcaceae sp. (p = 0.0001)., Blautia sp. (p = 0.007) and a significant reduction in facultative pathogens Corynebacterium sp. (p = 0.0009), Pelomonas sp. (p = 0.0306), Streptococcus sp. (p ≥ 0.0001), Pseudomonas sp. (p = 0.0358), and Cutibacterium sp. (p = 0.0237). Intriguingly, we observed a significant decrease in Staphylococcus aureus frequency (p = 0.0001) but an increase in the overall detection frequency of the Staphylococcus genus, indicating that antibiotic treatment helped regain the microbial balance and increased the number of non-pathogenic Staphylococcus populations. These study findings show that modulating microbiota by topical antibiotic treatment helps to restore microbial balance by diminishing the numbers of pathogenic microbes, which, in turn, reduces chronic inflammation, delays tumour growth, and increases survival rates in our CTCL model. These findings support the rationale to modulate the microbial milieu during the disease course of CTCL and indicate its therapeutic potential.

Phototherapy Restores Deficient Type I IFN Production and Enhances Antitumor Responses in Mycosis Fungoides.

Transcriptional profiling demonstrated markedly reduced type I IFN gene expression in untreated mycosis fungoides (MF) skin lesions compared with that in healthy skin. Type I IFN expression in MF correlated with antigen-presenting cell-associated IRF5 before psoralen plus UVA therapy and epithelial ULBP2 after therapy, suggesting an enhancement of epithelial type I IFN. Immunostains confirmed reduced baseline type I IFN production in MF and increased levels after psoralen plus UVA treatment in responding patients. Effective tumor clearance was associated with increased type I IFN expression, enhanced recruitment of CD8+ T cells into skin lesions, and expression of genes associated with antigen-specific T-cell activation. IFNk, a keratinocyte-derived inducer of type I IFNs, was increased by psoralen plus UVA therapy and expression correlated with upregulation of other type I IFNs. In vitro, deletion of keratinocyte IFNk decreased baseline and UVA-induced expression of type I IFN and IFN response genes. In summary, we find a baseline deficit in type I IFN production in MF that is restored by psoralen plus UVA therapy and correlates with enhanced antitumor responses. This may explain why MF generally develops in sun-protected skin and suggests that drugs that increase epithelial type I IFNs, including topical MEK and EGFR inhibitors, may be effective therapies for MF.

Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma.

Leukemic cutaneous T-cell lymphomas (L-CTCL) are lymphoproliferative disorders of skin-homing mature T-cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L-CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L-CTCL, which correlated with the increased clonal T-cell count in the blood. Dual inhibition of STAT3/5 using small-molecule degraders and multi-kinase blockers abolished L-CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L-CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti-leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L-CTCL.

The molecular and phenotypic makeup of fetal human skin T lymphocytes.

The adult human skin contains a vast number of T cells that are essential for skin homeostasis and pathogen defense. T cells are first observed in the skin at the early stages of gestation; however, our understanding of their contribution to early immunity has been limited by their low abundance and lack of comprehensive methodologies for their assessment. Here, we describe a new workflow for isolating and expanding significant amounts of T cells from fetal human skin. Using multiparametric flow cytometry and in situ immunofluorescence, we found a large population with a naive phenotype and small populations with a memory and regulatory phenotype. Their molecular state was characterized using single-cell transcriptomics and TCR repertoire profiling. Importantly, culture of total fetal skin biopsies facilitated T cell expansion without a substantial impact on their phenotype, a major prerequisite for subsequent functional assays. Collectively, our experimental approaches and data advance the understanding of fetal skin immunity and potential use in future therapeutic interventions.

αßγδ T cells play a vital role in fetal human skin development and immunity.

T cells in human skin play an important role in the immune defense against pathogens and tumors. T cells are present already in fetal skin, where little is known about their cellular phenotype and biological function. Using single-cell analyses, we identified a naive T cell population expressing αß and γδ T cell receptors (TCRs) that was enriched in fetal skin and intestine but not detected in other fetal organs and peripheral blood. TCR sequencing data revealed that double-positive (DP) αßγδ T cells displayed little overlap of CDR3 sequences with single-positive αß T cells. Gene signatures, cytokine profiles and in silico receptor-ligand interaction studies indicate their contribution to early skin development. DP αßγδ T cells were phosphoantigen responsive, suggesting their participation in the protection of the fetus against pathogens in intrauterine infections. Together, our analyses unveil a unique cutaneous T cell type within the native skin microenvironment and point to fundamental differences in the immune surveillance between fetal and adult human skin.

A deep dive into UV-based phototherapy: Mechanisms of action and emerging molecular targets in inflammation and cancer.

UV-based phototherapy (including psoralen plus UVA (PUVA), UVB and UVA1) has a long, successful history in the management of numerous cutaneous disorders. Photoresponsive diseases are etiologically diverse, but most involve disturbances in local (and occasionally systemic) inflammatory cells and/or abnormalities in keratinocytes that trigger inflammation. UV-based phototherapy works by regulating the inflammatory component and inducing apoptosis of pathogenic cells. This results in a fascinating and complex network of simultaneous events-immediate transcriptional changes in keratinocytes, immune cells, and pigment cells; the emergence of apoptotic bodies; and the trafficking of antigen-presenting cells in skin-that quickly transform the microenvironment of UV-exposed skin. Molecular elements in this system of UV recognition and response include chromophores, metabolic byproducts, innate immune receptors, neurotransmitters and mediators such as chemokines and cytokines, antimicrobial peptides, and platelet activating factor (PAF) and PAF-like molecules that simultaneously shape the immunomodulatory effects of UV and their interplay with the microbiota of the skin and beyond. Phototherapy's key effects-proapoptotic, immunomodulatory, antipruritic, antifibrotic, propigmentary, and pro-prebiotic-promote clinical improvement in various skin diseases such as psoriasis, atopic dermatitis (AD), graft-versus-host disease (GvHD), vitiligo, scleroderma, and cutaneous T-cell lymphoma (CTCL) as well as prevention of polymorphic light eruption (PLE). As understanding of phototherapy improves, new therapies (UV- and non-UV-based) are being developed that will modify regulatory T-cells (Treg), interact with (resident) memory T-cells and /or utilize agonists and antagonists as well as antibodies targeting soluble molecules such as cytokines and chemokines, transcription factors, and a variety of membrane-associated receptors.

Long-term skin-resident memory T cells proliferate in situ and are involved in human graft-versus-host disease.

The skin contains a population of tissue-resident memory T cells (Trm) that is thought to contribute to local tissue homeostasis and protection against environmental injuries. Although information about the regulation, survival program, and pathophysiological roles of Trm has been obtained from murine studies, little is known about the biology of human cutaneous Trm Here, we showed that host-derived CD69+ αß memory T cell clones in the epidermis and dermis remain stable and functionally competent for at least 10 years in patients with allogeneic hematopoietic stem cell transplantation. Single-cell RNA sequencing revealed low expression of genes encoding tissue egress molecules by long-term persisting Trm in the skin, whereas tissue retention molecules and stem cell markers were displayed by Trm The transcription factor RUNX3 and the surface molecule galectin-3 were preferentially expressed by host T cells at the RNA and protein levels, suggesting two new markers for human skin Trm Furthermore, skin lesions from patients developing graft-versus-host disease (GVHD) showed a large number of cytokine-producing host-derived Trm, suggesting a contribution of these cells to the pathogenesis of GVHD. Together, our studies highlighted the relationship between the local human skin environment and long-term persisting Trm, which differs from murine skin. Our results also indicated that local tissue inflammation occurs through host-derived Trm after allogeneic hematopoietic stem cell transplantation.

Quality of Life, Anxiety, and Depression in Patients With Early-Stage Mycosis Fungoides and the Effect of Oral Psoralen Plus UV-A (PUVA) Photochemotherapy on it.

Background: Little is known about psychological discomfort and quality of life (QoL) in early stage mycosis fungoides (MF) and the effect of psoralen plus UV-A (PUVA) on it. Objective: To evaluate QoL, anxiety, and depression with validated instruments in early stage MF patients and whether PUVA treatment improves it. Methods: Patients with stage IA to IIA MF were treated with PUVA twice weekly for 12-24 weeks, followed by maintenance treatment or not, in a prospective randomized clinical trial. Patients completed a questionnaire on DLQI as well as the Hospital Anxiety and Depression Scale (HADS) prior to therapy, after their last PUVA exposure, and after the PUVA maintenance or observance phase. Results: For 24 patients with early stage MF, completed questionnaires were available and analyzed. Prior to treatment, 17% reported strong (DLQI > 10) and 29% moderate impairment (DLQI 6-10) in QoL; 33% of patients reported HADS scores indicating anxiety, and 21% reported scores indicating depression. PUVA significantly improved overall QoL by reducing mean DLQI scores by 58.6% (p = 0.003), HADS-A by 30% (p = 0.045), and HADS-D by 44% (p = 0.002). Improvements in QoL and psychological well-being seemed to be sustained, irrespective of maintenance treatment or not. Limitations: Small sample size. Conclusions: PUVA sustainably improves QoL and psychological well-being in patients with early stage MF. Clinical trial registration: ClinicalTrials.gov identifier: NCT01686594.

Extracorporeal Photopheresis: A Case of Immunotherapy Ahead of Its Time.

Extracorporeal photopheresis (ECP) is a cell-based immunotherapy that involves the reinfusion of autologous leukocytes after exposure to psoralen and UVA. The treatment has been used for over 30 years, at first on patients with cutaneous T-cell lymphoma (CTCL) and later for the management of patients with graft-versus-host disease (GvHD), sclerosing disorders, atopic dermatitis, and other diseases that may share the common driving factor of a pathogenic T-cell clone or clones in blood circulation. Patients with clinical improvement mount an antigen-specific immune response that may have tolerance traits in the case of GvHD or anticlonal cytotoxic characteristics in the case of CTCL. The exact mechanisms that dictate one response or the other are not fully understood, but the evidence accumulated so far indicates that multiple events occur simultaneously and consequentially contribute to the end result. These include contact of cells with the outside (plastics and tubing of the ECP apparatus), exposure to psoralen and UVA that activates platelets, monocytes, and other myeloid cells, the release of damage-associated molecular patterns, differentiation of monocytes into dendritic cells, and generation and successive presentation of numerous antigens after the phagocytosis of apoptotic cells. Once reintroduced, the ECP product increases the frequency and activity of regulatory T cells (Tregs), shifts the systemic cytokine balance, and promotes extravasation of immune cells that together shape the effects of this treatment. In this review, we summarize the seminal work and most recent literature of the therapeutic mechanisms and reflect on future avenues of improvements and applications of ECP.

Evaluation of Low-Dose, Low-Frequency Oral Psoralen-UV-A Treatment With or Without Maintenance on Early-Stage Mycosis Fungoides: A Randomized Clinical Trial.

Importance: Psoralen-UV-A (PUVA) photochemotherapy is standard first-line treatment for skin-limited, early-stage mycosis fungoides capable of producing high initial complete response (CR) rates. However, much remains unknown about PUVA's therapeutic mechanisms, optimal duration and frequency of treatment, dose escalation, or use as maintenance therapy. Objectives: To evaluate low-dose, low-frequency PUVA, and whether maintenance treatment extends disease-free remission in patients with mycosis fungoides. Design, Setting, and Participants: This prospective randomized clinical trial with defined PUVA dosing regimen was carried out in 5 centers (Graz, Vienna, Hietzing, Innsbruck, and Salzburg) across Austria. Patients with stage IA to IIA mycosis fungoides (n = 27) were enrolled in the study beginning March 13, 2013, with the last patient enrolled March 21, 2016. These patients were treated with oral 8-methoxypsoralen followed by UV-A exposure 2 times per week for 12 to 24 weeks until CR. Patients with CR were randomized to PUVA maintenance for 9 months (14 total exposures) or no maintenance. The study was conducted from April 27, 2012, to July 27, 2018. Data analysis of the primary end point was of the intention-to-treat population, and the secondary end point analysis was of the evaluable population. Main Outcomes and Measures: Efficacy of the PUVA regimen was determined by the rate of CR as defined by a modified severity-weighted assessment tool (mSWAT) score reduction to 0. Levels of proinflammatory molecules in serum and histologic features and percentage of clonal T cells in skin were assessed to search for biomarkers of clinical response. Results: In 27 patients with mycosis fungoides, 19 (70%) were male with mean (range) age 61 (30-80) years. At baseline, patients with CR had a mean (range) mSWAT score of 18.6 (1-66) compared with 16.8 (3-46) in patients with partial response. The 12- to 24-week PUVA induction regimen reduced the mSWAT score in all patients and led to CR in 19 (70%) of 27 patients and a low mean cumulative UV-A dose of 78.5 J/cm2. The subsequent standardized 9-month PUVA maintenance phase prolonged median (range) disease-free remission from 4 (1-20) months to 15 (1-54) months (P = .02). High density of histologic infiltrate and high percentage of clonal TCR sequences in skin biopsy specimens at baseline were inversely associated with therapeutic response. No severe adverse effects were seen during the PUVA induction or maintenance phase. Conclusions and Relevance: This proof-of-concept study identifies potential biomarkers for therapeutic response to PUVA in mycosis fungoides; it also demonstrates that low-dose, low-frequency PUVA appears to be highly effective, and maintenance treatment may extend disease-free remission. Trial Registration: ClinicalTrials.gov identifier: NCT01686594.

Benign T cells drive clinical skin inflammation in cutaneous T cell lymphoma.

Psoralen plus UVA (PUVA) is an effective therapy for mycosis fungoides (MF), the skin-limited variant of cutaneous T cell lymphoma (CTCL). In low-burden patients, PUVA reduced or eradicated malignant T cells and induced clonal expansion of CD8+ T cells associated with malignant T cell depletion. High-burden patients appeared to clinically improve but large numbers of malignant T cells persisted in skin. Clinical improvement was linked to turnover of benign T cell clones but not to malignant T cell reduction. Benign T cells were associated with the Th2-recruiting chemokine CCL18 before therapy and with the Th1-recruiting chemokines CXCL9, CXCL10, and CXCL11 after therapy, suggesting a switch from Th2 to Th1. Inflammation was correlated with OX40L and CD40L gene expression; immunostaining localized these receptors to CCL18-expressing c-Kit+ dendritic cells that clustered together with CD40+OX40+ benign and CD40+CD40L+ malignant T cells, creating a proinflammatory synapse in skin. Our data suggest that visible inflammation in CTCL results from the recruitment and activation of benign T cells by c-Kit+OX40L+CD40L+ dendritic cells and that this activation may provide tumorigenic signals. Targeting c-Kit, OX40, and CD40 signaling may be novel therapeutic avenues for the treatment of MF.

From Early Immunomodulatory Triggers to Immunosuppressive Outcome: Therapeutic Implications of the Complex Interplay Between the Wavebands of Sunlight and the Skin.

Phototherapy is an efficient treatment for many cutaneous diseases that involve the activation of inflammatory pathways or the overgrowth of cells with aberrant phenotype. In this review, we discuss recent advances in photoimmunology, focusing on the effects of UV-based therapies currently used in dermatology. We describe the molecular responses to the main forms of photo(chemo)therapy such as UVB, UVA-1, and PUVA that include the triggering of apoptotic or immunosuppressive pathways and help to clear diseased skin. The early molecular response to UV involves DNA photoproducts, the isomerization of urocanic acid, the secretion of biophospholipids such as platelet activating factor (PAF), the activation of aryl hydrocarbon receptor and inflammasome, and vitamin D synthesis. The simultaneous and complex interaction of these events regulates the activity of the immune system both locally and systemically, resulting in apoptosis of neoplastic and/or benign cells, reduction of cellular infiltrate, and regulation of cytokines and chemokines. Regulatory T-cells and Langerhans cells, among other skin-resident cellular populations, are deeply affected by UV exposure and are therefore important players in the mechanisms of immunomodulation and the therapeutic value of UV in all its forms. We weigh the contribution of these cells to the therapeutic application of UV and how they may participate in transferring the direct impact of UV on the skin into local and systemic immunomodulation. Moreover, we review the therapeutic mechanisms revealed by clinical and laboratory animal investigations in the most common cutaneous diseases treated with phototherapy such as psoriasis, atopic dermatitis, vitiligo, and cutaneous T-cell lymphoma. Better understanding of phototherapeutic mechanisms in these diseases will help advance treatment in general and make future therapeutic strategies more precise, targeted, personalized, safe, and efficient.

Cbl-b deficiency provides protection against UVB-induced skin damage by modulating inflammatory gene signature.

Exposure of skin to ultraviolet (UV) radiation induces DNA damage, inflammation, and immune suppression that ultimately lead to skin cancer. However, some of the pathways that regulate these events are poorly understood. We exposed mice to UVB to study its early effects in the absence of Cbl-b, a known suppressor of antitumor immune response in the skin. Cbl-b-/- mice were protected from UV-induced cell damage as shown by the lower number of cyclobutane pyrimidine dimers and sunburn cells in exposed skin compared to wild-type mice. Microarray data revealed that deficiency of Cbl-b resulted in differential expression of genes involved in apoptosis evasion, tumor suppression and cell survival in UV-exposed skin. After UVB, Cbl-b-/- mice upregulated gene expression pattern associated with regulation of epidermal cell proliferation linked to Wnt signaling mediators and enzymes that relate to cell removal and tissue remodeling like MMP12. Additionally, the skin of Cbl-b-/- mice was protected from chronic inflammatory responses and epidermal hyperplasia in a 4-weeks UVB treatment protocol. Overall, our results suggest a novel role for Cbl-b in regulating inflammation and physiologic clearance of damaged cells in response to UVB by modulating inflammatory gene signature.

STAT3/5-Dependent IL9 Overexpression Contributes to Neoplastic Cell Survival in Mycosis Fungoides.

PURPOSE: Sustained inflammation is a key feature of mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL). Resident IL9-producing T cells have been found in skin infections and certain inflammatory skin diseases, but their role in MF is currently unknown. EXPERIMENTAL DESIGN: We analyzed lesional skin from patients with MF for the expression of IL9 and its regulators. To determine which cells were producing IL9, high-throughput sequencing was used to identify malignant clones and Vb-specific antibodies were employed to visualize malignant cells in histologic preparations. To explore the mechanism of IL9 secretion, we knocked down STAT3/5 and IRF4 by siRNA transfection in CTCL cell lines receiving psoralen+UVA (PUVA) ± anti-IL9 antibody. To further examine the role of IL9 in tumor development, the EL-4 T-cell lymphoma model was used in C57BL/6 mice. RESULTS: Malignant and reactive T cells produce IL9 in lesional skin. Expression of the Th9 transcription factor IRF4 in malignant cells was heterogeneous, whereas reactive T cells expressed it uniformly. PUVA or UVB phototherapy diminished the frequencies of IL9- and IL9r-positive cells, as well as STAT3/5a and IRF4 expression in lesional skin. IL9 production was regulated by STAT3/5 and silencing of STAT5 or blockade of IL9 with neutralizing antibodies potentiated cell death after PUVA treatment in vitro IL9-depleted mice exhibited a reduction of tumor growth, higher frequencies of regulatory T cells, and activated CD4 and CD8 T lymphocytes. CONCLUSIONS: Our results suggest that IL9 and its regulators are promising new targets for therapy development in mycosis fungoides. Clin Cancer Res; 22(13); 3328-39. ©2016 AACR.