T-cell activation and bacterial infection in skin wounds of recessive dystrophic epidermolysis bullosa patients.

Recessive dystrophic epidermolysis bullosa (RDEB) patients develop poorly healing skin wounds that are frequently colonized with microbiota. Because T cells play an important role in clearing such pathogens, we aimed to define the status of adaptive T cell-mediated immunity in RDEB wounds. Using a non-invasive approach for sampling of wound-associated constituents, we evaluated microbial contaminants in cellular fraction and exudates obtained from RDED wounds. Infectivity and intracellular trafficking of inactivated Staphylococcus aureus was accessed in RDEB keratinocytes. S. aureus and microbial antigen-specific activation of RDEB wound-derived T cells were investigated by fluorescence-activated cell sorting-based immune-phenotyping and T-cell functional assays. We found that RDEB wounds and epithelial cells are most frequently infected with Staphylococcus sp. and Pseudomonas sp. and that S. aureus essentially infects more RDEB keratinocytes and RDEB-derived squamous cell carcinoma cells than keratinocytes from healthy donors. The RDEB wound-associated T cells contain populations of CD4+ and CD8+ peripheral memory T cells that respond to soluble microbial antigens by proliferating and secreting interferon gamma (IFNγ). Moreover, CD8+ cytotoxic T lymphocytes recognize S. aureus-infected RDEB keratinocytes and respond by producing interleukin-2 (IL-2) and IFNγ and degranulating and cytotoxically killing infected cells. Prolonged exposure of RDEB-derived T cells to microbial antigens in vitro does not trigger PD-1-mediated T-cell exhaustion but induces differentiation of the CD4high population into CD4high CD25+ FoxP3+ regulatory T cells. Our data demonstrated that adaptive T cell-mediated immunity could clear infected cells from wound sites, but these effects might be inhibited by PD-1/Treg-mediated immuno-suppression in RDEB.

Immunological mechanisms underlying progression of chronic wounds in recessive dystrophic epidermolysis bullosa.

Hereditary epidermolysis bullosa (EB) is a mechanobullous skin fragility disorder characterized by defective epithelial adhesion, leading to mechanical stress-induced skin blistering. Based on the level of tissue separation within the dermal-epidermal junction, EB is categorized into simplex (EBS), junctional (JEB), dystrophic (DEB) and Kindler syndrome. There is no cure for EB, and painful chronic cutaneous wounds are one of the major complications in recessive (RDEB) patients. Although RDEB is considered a cutaneous disease, recent data support the underlying systemic immunological defects. Furthermore, chronic wounds are often colonized with pathogenic microbiota, leading to excessive inflammation and altered wound healing. Consequently, patients with RDEB suffer from a painful sensation of chronic, cutaneous itching/burning and an endless battle with bacterial infections. To improve their quality of life and life expectancy, it is important to prevent cutaneous infections, dampen chronic inflammation and stimulate wound healing. A clear scientific understanding of the immunological events underlying the maintenance of chronic poorly healing wounds in RDEB patients is necessary to improve disease management and better understand other wound healing disorders. In this review, we summarize current knowledge of the role of professional phagocytes, such as neutrophils, macrophages and dendritic cells, the role of T-cell-mediated immunity in lymphoid organs, and the association of microbiota with poor wound healing in RDEB. We conclude that RDEB patients have an underlying immunity defect that seems to affect antibacterial immunity.

Intracellular escape strategies of Staphylococcus aureus in persistent cutaneous infections.

Pathogenic invasion of Staphylococcus aureus is a major concern in patients with chronic skin diseases like atopic dermatitis (AD), epidermolysis bullosa (EB), or chronic diabetic foot and venous leg ulcers, and can result in persistent and life-threatening chronic non-healing wounds. Staphylococcus aureus is generally recognized as extracellular pathogens. However, S. aureus can also invade, hide and persist in skin cells to contribute to wound chronicity. The intracellular life cycle of S. aureus is currently incompletely understood, although published studies indicate that its intracellular escape strategies play an important role in persistent cutaneous infections. This review provides current scientific knowledge about the intracellular life cycle of S. aureus in skin cells, which can be classified into professional and non-professional antigen-presenting cells, and its strategies to escape adaptive defense mechanisms. First, we discuss phenotypic switch of S. aureus, which affects intracellular routing and degradation. This review also evaluates potential intracellular escape mechanism of S. aureus to avoid intracellular degradation and antigen presentation, preventing an immune response. Furthermore, we discuss potential drug targets that can interfere with the intracellular life cycle of S. aureus. Taken together, this review aimed to increase scientific understanding about the intracellular life cycle of S. aureus into skin cells and its strategies to evade the host immune response, information that is crucial to reduce pathogenic invasion and life-threatening persistence of S. aureus in chronic cutaneous infections.

Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa.

Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin ß3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB-associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin ß3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin ß3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin ß3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6ß4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.

Prime-Boost Immunization Eliminates Metastatic Colorectal Cancer by Producing High-Avidity Effector CD8+ T Cells.

Heterologous prime-boost immunization with plasmid DNA and viral vector vaccines is an emerging approach to elicit CD8+ T cell-mediated immunity targeting pathogens and tumor Ags that is superior to either monotherapy. Yet, the mechanisms underlying the synergy of prime-boost strategies remain incompletely defined. In this study, we examine a DNA and adenovirus (Ad5) combination regimen targeting guanylyl cyclase C (GUCY2C), a receptor expressed by intestinal mucosa and universally expressed by metastatic colorectal cancer. DNA immunization efficacy was optimized by i.m. delivery via electroporation, yet it remained modest compared with Ad5. Sequential immunization with DNA and Ad5 produced superior antitumor efficacy associated with increased TCR avidity, whereas targeted disruption of TCR avidity enhancement eliminated GUCY2C-specific antitumor efficacy, without affecting responding T cell number or cytokine profile. Indeed, functional TCR avidity of responding GUCY2C-specific CD8+ T cells induced by various prime or prime-boost regimens correlated with antitumor efficacy, whereas T cell number and cytokine profile were not. Importantly, although sequential immunization with DNA and Ad5 maximized antitumor efficacy through TCR avidity enhancement, it produced no autoimmunity, reflecting sequestration of GUCY2C to intestinal apical membranes and segregation of mucosal and systemic immunity. Together, TCR avidity enhancement may be leveraged by prime-boost immunization to improve GUCY2C-targeted colorectal cancer immunotherapeutic efficacy and patient outcomes without concomitant autoimmune toxicity.

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities.

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.

Analysis of chemotactic molecules in bone marrow-derived mesenchymal stem cells and the skin: Ccl27-Ccr10 axis as a basis for targeting to cutaneous tissues.

BACKGROUND AIMS: Adult stem cells produce a plethora of extracellular matrix molecules and have a high potential as cell-based therapeutics for connective tissue disorders of the skin. However, the primary challenge of the stem cell-based approach is associated with the inefficient homing of systemically infused stem cells to the skin. METHODS: We examined chemotactic mechanisms that govern directional migration of mesenchymal stem cells (MSCs) into the skin by conducting a comprehensive expression analysis of chemotactic molecules in MSCs and defined cutaneous tissues from normal and hereditary epidermolysis bullosa (EB)-affected skin. RESULTS: Analysis of chemokine receptors in short-term and long-term MSC cultures showed tissue culture-dependent expression of several receptors. Assessment of epidermis-derived and dermis-derived chemokines showed that most chemotactic signals that originate from the skin preferentially recruit different sets of leukocytes rather than MSCs. Analysis of the chemotactic molecules derived from EB-affected non-blistered skin showed only minor changes in expression of selected chemokines and receptors. Nevertheless, the data allowed us to define the Ccl27-Ccr10 chemotactic axis as the most potent for the recruitment of MSCs to the skin. Our in vivo analysis demonstrated that uniform expression of Ccr10 on MSCs and alteration of Ccl27 level in the skin enhance extravasation of stem cells from circulation and facilitate their migration within cutaneous tissue. CONCLUSIONS: Collectively, our study provides a comprehensive analysis of chemotactic signals in normal and EB-affected skin and proof-of-concept data demonstrating that alteration of the chemotactic pathways can enhance skin homing of the therapeutic stem cells.

Systemic CXCL10 is a predictive biomarker of vitiligo lesional skin infiltration, PUVA, NB-UVB and corticosteroid treatment response and outcome.

Vitiligo is an acquired pigmentary skin disorder that currently lacks standardized treatment and validated biomarkers to objectively evaluate disease state or therapeutic response. Although prior studies have linked vitiligo autoimmunity with CXCL10/CXCL9-mediated recruitment of leukocytes to the skin, only limited clinical data are available regarding CXCL10 as vitiligo biomarker. To evaluate the utility of systemic CXCL10 as a predictor of disease progression and treatment response on a large cohort of vitiligo patients. CXCL10 levels in lesional, perilesional, and unaffected skin of vitiligo patient (n = 30) and in the serum (n = 51) were measured by quantitative ELISA. CXCL10 expression, recruitment of leukocytes, and inflammatory infiltrates were evaluated by histochemical (n = 32) and immunofluorescence (n = 10) staining. Rigorous cross-sectional and longitudinal biostatistical analysis were employed to correlate CXCL10 levels with disease variables, treatment response, and outcome. We demonstrated that elevated CXCL10 level (2 pg/mm2 and higher) in lesional skin correlates with increased leukocytic infiltrate, disease duration (< 2 year), and its higher level in the serum (50 pg/ml and higher). Changes in CXCL10 serum levels in patients treated with psoralen plus UVA (PUVA) phototherapy, narrowband UVB (NB-UVB) phototherapy, and systemic steroids (SS) correlated with changes in the intralesional CXCL10 levels in repigmented skin. NB-UVB and SS regimens provided most consistent CXCL10 mean change, suggesting that these regimens are most effective in harnessing CXCR3-mediated inflammatory response. Serum CXCL10 is a useful vitiligo biomarker, which predicts lesional skin leukocytic infiltration, and vitiligo treatment response and outcome.

Gene expression signatures of mouse bone marrow-derived mesenchymal stem cells in the cutaneous environment and therapeutic implications for blistering skin disorder.

BACKGROUND AIMS: Multiple studies have demonstrated that mesenchymal stromal cells (MSC) can be utilized therapeutically for various congenital and acquired disorders. The involvement of MSC in the maintenance of skin homeostasis and their curative application for the treatment of skin wounds have also been documented. However, it is not known whether MSC can commit to cutaneous lineages, produce structural proteins essential for the skin integrity or be used for hereditary skin disorders. METHODS: To address these questions, we conducted a comparative expression analysis between MSC and potentially adjacent cutaneous cells, fibroblasts and keratinocytes, with specific emphasis on extracellular matrix encoding and related genes. RESULTS: Our data demonstrated that MSC share many features with cutaneous fibroblasts. We also observed that under direct influence of cutaneous fibroblasts in vitro and fibroblast-derived matrix in vivo, MSC acquired a fibroblastic phenotype, suggesting that specific cell-cell interactions play a key regulatory role in the differentiation of MSC. Additionally, the observed fibroblastic transition of MSC was underlined by a significant up-regulation of several cutaneous-specific genes encoding lumican, decorin, type VII collagen, laminin and other structural proteins. As many of the identified genes have considerable therapeutic value for dermatologic afflictions, particularly type VII collagen, we evaluated further the therapeutic potential of congenic MSC in the skin of Col7a1-null mice recapitulating human recessive dystrophic epidermolysis bullosa (RDEB). Remarkably, MSC-derived type VII collagen was sufficient for restoration of the damaged dermal-epidermal junction and partial reversal of the RDEB phenotype. CONCLUSIONS: Collectively, our results suggest that MSC may offer promising therapeutics for the treatment of RDEB and potentially other genodermatoses.

Orthotopic Human Metastatic Uveal Melanoma Xenograft Mouse Models: Applications for Understanding the Pathophysiology and Therapeutic Management of Metastatic Uveal Melanoma.

The propensity of uveal melanoma to metastasize to the liver hinders the accrual of micro-metastatic and end-stage disease tissue samples and restricts the investigation of metastatic uveal melanoma (MUM). Pre-clinical experimental animal models of MUM can help elucidate the pathophysiology of metastatic lesions and provide a tool for designing new therapeutic approaches for MUM. Here, we present an advanced model of hepatic metastases that enables quantitatively visualizing the development of individual hepatic tumor clones and estimating their growth kinetics and colonization efficiency. Similar to clinically observed liver metastases, these models enable the assessment of growth kinetics of the liver micro-metastases and the testing of therapeutic approaches for the treatment of MUM. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Experimental patient-derived xenograft mouse model of metastatic uveal melanoma Basic Protocol 2: Experimental liver micro-metastatic mouse model using splenic injection of metastatic uveal melanoma cells.

Congenital muscular dystrophy-associated inflammatory chemokines provide axes for effective recruitment of therapeutic adult stem cell into muscles.

BACKGROUND: Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness. The two most prevalent forms of CMD, collagen VI-related myopathies (COL6RM) and laminin α2 deficient CMD type 1A (MDC1A), are both caused by deficiency or dysfunction of extracellular matrix proteins. Previously, we showed that an intramuscular transplantation of human adipose-derived stem cells (ADSC) into the muscle of the Col6a1-/- mice results in efficient stem cell engraftment, migration, long-term survival, and continuous production of the collagen VI protein, suggesting the feasibility of the systemic cellular therapy for COL6RM. In order for this therapeutic approach to work however, stem cells must be efficiently targeted to the entire body musculature. Thus, the main goal of this study is to test whether muscle homing of systemically transplanted ADSC can be enhanced by employing muscle-specific chemotactic signals originating from CMD-affected muscle tissue. METHODS: Proteomic screens of chemotactic molecules were conducted in the skeletal muscles of COL6RM- and MDC1A-affected patients and CMD mouse models to define the inflammatory and immune activities, thus, providing potential markers of disease activity or treatment effect. Also using a pre-clinical animal model, recapitulating mild Ullrich congenital muscular dystrophy (UCMD), the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations. RESULTS: Comprehensive proteomic screens evaluating relevant human and mouse skeletal muscle biopsies offered chemotactic axes to enhance directional migration of systemically transplanted cells into CMD-affected muscles, including CCL5-CCR1/3/5, CCL2-CCR2, CXCL1/2-CXCR1,2, and CXCL7-CXCR2. Also, the specific populations of ADSC selected with an affinity for the chemokines being released by damaged muscle showed efficient migration to injured site and presented their therapeutic effect. CONCLUSIONS: Collectively, identified molecules provided insight into the mechanisms governing directional migration and intramuscular trafficking of systemically infused stem cells, thus, permitting broad and effective application of the therapeutic adult stem cells for CMD treatment.

Aberrant recruitment of leukocytes defines poor wound healing in patients with recessive dystrophic epidermolysis bullosa.

BACKGROUND: Poorly healing wounds are one of the major complications in patients suffering from recessive dystrophic epidermolysis bullosa (RDEB). At present, there are no effective means to analyze changes in cellular and molecular networks occurring during RDEB wound progression to predict wound outcome and design betted wound management approaches. OBJECTIVES: To better define mechanisms influencing RDEB wound progression by evaluating changes in molecular and cellular networks. METHODS: We developed a non-invasive approach for sampling and analysis of wound-associated constituents using wound-covering bandages. Cellular and molecular components from seventy-six samples collected from early, established and chronic RDEB wounds were evaluated by FACS-based immuno-phenotyping and ELISA. RESULTS: Our cross-sectional analysis determined that progression of RDEB wounds to chronic state is associated with the accumulation (up to 90 %) of CD16+CD66b+ mature neutrophils, loss of CD11b+CD68+ macrophages, and a significant increase (up to 50 %) in a number of CD11c+CD80+CD86+ activated professional antigen presenting cells (APC). It was also marked by changes in activated T cells populations including a reduction of CD45RO+ peripheral memory T cells from 80 % to 30 % and an increase (up to 70 %) in CD45RA+ effector T cells. Significantly higher levels of MMP9, VEGF-A and cathepsin G were also associated with advancing of wounds to poorly healing state. CONCLUSIONS: Our data demonstrated that wound-covering bandages are useful for a non-invasive sampling and analysis of wound-associated constituents and that transition to poorly healing wounds in RDEB patients as associated with distinct changes in leukocytic infiltrates, matrix-remodeling enzymes and pro-angiogenic factors at wound sites.

Targeted manipulation of mammalian genomes using designed zinc finger nucleases.

Targeted introduction of a double-stranded break (DSB) using designer zinc finger nucleases (ZFNs) in mammalian cells greatly enhances gene targeting - homologous recombination (HR) at a chosen endogenous target gene, which otherwise is limited by low spontaneous rate of HR. Here, we report that efficient ZFN-mediated gene correction occurs at a transduced, transcriptionally active, mutant GFP locus by homology-directed repair, and that efficient mutagenesis by non-homologous end joining (NHEJ) occurs at the endogenous, transcriptionally silent, CCR5 locus in HEK293 Flp-In cells, using designed 3- and 4-finger ZFNs. No mutagenesis by NHEJ was observed at the CCR2 locus, which has ZFN sites that are distantly related to the targeted CCR5 sites. We also observed efficient ZFN-mediated correction of a point mutation at the endogenous mutant tyrosinase chromosomal locus in albino mouse melanocytes, using designed 3-finger ZFNs. Furthermore, re-engineered obligate heterodimer FokI nuclease domain variants appear to completely eliminate or greatly reduce the toxicity of ZFNs to mammalian cells, including human cells.

Human interleukin 10 receptor 1/IgG1-Fc fusion proteins: immunoadhesins for human IL-10 with therapeutic potential.

Interleukin 10 (IL-10) is produced by various types of human cancer, including malignant melanoma, and plays an important role in negative regulation of cell-mediated immune responses against tumors. We have developed chimeric molecules (immunoadhesins), combining the extracellular domain of human interleukin 10 receptor 1 (IL-10R1) with the Fc regions of human IgG1 heavy chain and investigated their capability of blocking the biological activities of human IL-10. Monomeric and dimeric immunoadhesins (IL-10R1/IgG1) constructs were tested for capturing human IL-10 and blocking its biological activities. Plasmid vectors that contained the IL-10 immunoadhesin constructs were directly transfected into human melanoma cell lines. Transfection of plasmid vectors into melanoma cell lines resulted in capturing of exogenously added as well as endogeneously produced IL-10. The supernatants obtained from an IL-10 non-producing melanoma cell line transfected with monomeric IL-10 immunoadhesin plasmids most efficiently captured exogenously added IL-10, compared to those obtained with the dimeric IL-10R1/IgG1 plasmid vector. Transfection of IL-10-producing melanoma cells with the monomeric IL-10 immunoadhesin plasmids totally captured endogenously produced IL-10 and enhanced T cell responses against allogeneic melanoma cells. Furthermore, purified monomeric IL-10 immunoadhesin protein showed IL-10 capturing efficacy compatible with that of IL-10-specific monoclonal antibodies. Collectively, these studies indicate that IL-10 immunoadhesins, especially in monomeric form, are potent inhibitors of biological activities of IL-10 and suggest that these molecules, alone or in conjunctions with other immunotherapeutic approaches, can be utilized for the immuno-targeting of IL-10 producing tumors.

Retroviral delivery of ECM genes.

The use of recombinant DNA has become a powerful tool in the analysis of functional and structural properties of the extracellular matrix proteins. During last decade, various procedures of plasmid DNA delivery using liposome-based or electroporation-based transfection have been developed. However, in many instances, these procedures were shown to be not effective in DNA transfer or toxic for the mammalian cells. On contrary, retrovirus-mediated infection represents a superior mode of gene delivery with a success rate and viability of the cells approaching 100% in in vitro conditions. The use of the retroviral system also allows permanent insertion of the gene of interest into the chromosome of the infected cell, resulting in efficient gene transfer in which most recipient cells will incorporate and express the transduced gene. In this chapter, we will describe several retrovirus-based systems and provide step-by-step protocols applicable for the production of the recombinant virus and efficient delivery of the ECM genes.

Characterization of the CCL21-mediated melanoma-specific immune responses and in situ melanoma eradication.

Previous studies have shown that secondary lymphoid chemokine, CCL21, can be used for modulation of tumor-specific immune responses. Here, using B16F0 melanoma cells stably expressing CCL21 under the control of cytomegalovirus and ubiquitin promoters, we showed that CCL21-activated immune responses depend on the amount of melanoma-derived chemokine, which, in turn, depends on the strength of the promoter. We showed that ubiquitin promoter-driven expression of CCL21 enabled massive infiltration of tumors with CD4(+)CD25(-), CD8(+) T lymphocytes, and CD11c(+) dendritic cells, and consequent activation of cellular and humoral immune responses sufficient for complete rejection of CCL21-positive melanomas within 3 weeks in all tumor-inoculated mice. Mice that rejected CCL21-positive tumors acquired protective immunity against melanoma, which was transferable to naive mice via splenocytes and central memory T cells. Moreover, melanoma-derived CCL21 facilitated immune-mediated remission of preestablished, distant wild-type melanomas. Overall, these results suggest that elevated levels of tumor-derived CCL21 are required for the activation of strong melanoma-specific immune responses and generation of protective immunologic memory. They also open new perspectives for the development of novel vaccination strategies against melanoma, which use intratumoral delivery of the optimized CCL21-encoding vectors in conjunction with DNA-based vaccines.

STAT5A/B Blockade Sensitizes Prostate Cancer to Radiation through Inhibition of RAD51 and DNA Repair.

Purpose: The standard treatment for organ-confined prostate cancer is surgery or radiation, and locally advanced prostate cancer is typically treated with radiotherapy alone or in combination with androgen deprivation therapy. Here, we investigated whether Stat5a/b participates in regulation of double-strand DNA break repair in prostate cancer, and whether Stat5 inhibition may provide a novel strategy to sensitize prostate cancer to radiotherapy.Experimental Design: Stat5a/b regulation of DNA repair in prostate cancer was evaluated by comet and clonogenic survival assays, followed by assays specific to homologous recombination (HR) DNA repair and nonhomologous end joining (NHEJ) DNA repair. For HR DNA repair, Stat5a/b regulation of Rad51 and the mechanisms underlying the regulation were investigated in prostate cancer cells, xenograft tumors, and patient-derived prostate cancers ex vivo in 3D explant cultures. Stat5a/b induction of Rad51 and HR DNA repair and responsiveness to radiation were evaluated in vivo in mice bearing prostate cancer xenograft tumors.Results: Stat5a/b is critical for Rad51 expression in prostate cancer via Jak2-dependent mechanisms by inducing Rad51 mRNA levels. Consistent with this, genetic knockdown of Stat5a/b suppressed HR DNA repair while not affecting NHEJ DNA repair. Pharmacologic Stat5a/b inhibition potently sensitized prostate cancer cell lines and prostate cancer tumors to radiation, while not inducing radiation sensitivity in the neighboring tissues.Conclusions: This work introduces a novel concept of a pivotal role of Jak2-Stat5a/b signaling for Rad51 expression and HR DNA repair in prostate cancer. Inhibition of Jak2-Stat5a/b signaling sensitizes prostate cancer to radiation and, therefore, may provide an adjuvant therapy for radiation to reduce radiation-induced damage to the neighboring tissues. Clin Cancer Res; 24(8); 1917-31. ©2018 AACR.

Pro-Inflammatory Chemokines and Cytokines Dominate the Blister Fluid Molecular Signature in Patients with Epidermolysis Bullosa and Affect Leukocyte and Stem Cell Migration.

Hereditary epidermolysis bullosa (EB) is associated with skin blistering and the development of chronic nonhealing wounds. Although clinical studies have shown that cell-based therapies improve wound healing, the recruitment of therapeutic cells to blistering skin and to more advanced skin lesions remains a challenge. Here, we analyzed cytokines and chemokines in blister fluids of patients affected by dystrophic, junctional, and simplex EB. Our analysis revealed high levels of CXCR1, CXCR2, CCR2, and CCR4 ligands, particularly dominant in dystrophic and junctional EB. In vitro migration assays demonstrated the preferential recruitment of CCR4+ lymphocytes and CXCR1+, CXCR2+, and CCR2+ myeloid cells toward EB-derived blister fluids. Immunophenotyping of skin-infiltrating leukocytes confirmed substantial infiltration of EB-affected skin with resting (CD45RA+) and activated (CD45RO+) T cells and CXCR2+ CD11b+ cells, many of which were identified as CD16b+ neutrophils. Our studies also showed that abundance of CXCR2 ligand in blister fluids also creates a favorable milieu for the recruitment of the CXCR2+ stem cells, as validated by in vitro and in-matrix migration assays. Collectively, this study identified several chemotactic pathways that control the recruitment of leukocytes to the EB-associated skin lesions. These chemotactic axes could be explored for the refinement of the cutaneous homing of the therapeutic stem cells.

Misbalanced CXCL12 and CCL5 Chemotactic Signals in Vitiligo Onset and Progression.

Generalized nonsegmental vitiligo is often associated with the activation of melanocyte-specific autoimmunity. Because chemokines play an important role in the maintenance of immune responses, we examined chemotactic signatures in cultured vitiligo melanocytes and skin samples of early (≤2 months) and advanced (≥6 months) vitiligo. Analysis showed that melanocytes in early lesions have altered expression of several chemotaxis-associated molecules, including elevated secretion of CXCL12 and CCL5. Higher levels of these chemokines coincided with prominent infiltration of the skin with antigen presenting cells (APCs) and T cells. Most of the intralesional APCs expressed the CD86 maturation marker and co-localized with T cells, particularly in early vitiligo lesions. These observations were confirmed by in vivo animal studies showing preferential recruitment of APCs and T cells to CXCL12- and CCL5-expressing transplanted melanocytes, immunotargeting of the chemokine-positive cells, continuous loss of the pigment-producing cells from the epidermis, and development of vitiligo-like lesions. Taken together, our studies show that melanocyte-derived CXCL12 and CCL5 support APC and T-cell recruitment, antigen acquisition, and T-cell activation in early vitiligo and reinforce the role of melanocyte-derived CXCL12 and CCL5 in activation of melanocyte-specific immunity and suggest inhibition of these chemotactic axes as a strategy for vitiligo stabilization.