Chemical and Pharmacological Prospection of the Ascidian Cystodytes dellechiajei.

Marine invertebrates are a traditional source of natural products with relevant biological properties. Tunicates are soft-bodied, solitary or colonial, sessile organisms that provide compounds unique in their structure and activity. The aim of this work was to investigate the chemical composition of the ascidian Cystodytes dellechiajei, selected on the basis of a positive result in biological screening for ligands of relevant receptors of the innate immune system, including TLR2, TLR4, dectin-1b, and TREM2. Bioassay-guided screening of this tunicate extract yielded two known pyridoacridine alkaloids, shermilamine B (1) and N-deacetylshermilamine B (2), and a family of methyl-branched cerebrosides (3). Compounds 2 and 3 showed selective binding to TREM2 in a dose-dependent manner. N-deacetylshermilamine B (2), together with its acetylated analogue, shermilamine B (1), was also strongly cytotoxic against multiple myeloma cell lines. TREM2 is involved in immunomodulatory processes and neurodegenerative diseases. N-deacetylshermilamine B (2) is the first example of a polycyclic alkaloid to show an affinity for this receptor.

Sulfavant A as the first synthetic TREM2 ligand discloses a homeostatic response of dendritic cells after receptor engagement.

OBJECTIVE: The immune response arises from a fine balance of mechanisms that provide for surveillance, tolerance, and elimination of dangers. Sulfavant A (SULF A) is a sulfolipid with a promising adjuvant activity. Here we studied the mechanism of action of SULF A and addressed the identification of its molecular target in human dendritic cells (hDCs). METHODS: Adjuvant effect and immunological response to SULF A were assessed on DCs derived from human donors. In addition to testing various reporter cells, target identification and downstream signalling was supported by a reverse pharmacology approach based on antibody blocking and gene silencing, crosstalk with TLR pathways, use of human allogeneic mixed lymphocyte reaction. RESULTS: SULF A binds to the Triggering Receptor Expressed on Myeloid cells-2 (TREM2) and initiates an unconventional maturation of hDCs leading to enhanced migration activity and up-regulation of MHC and co-stimulatory molecules without release of conventional cytokines. This response involves the SYK-NFAT axis and is compromised by blockade or gene silencing of TREM2. Activation by SULF A preserved the DC functions to excite the allogeneic T cell response, and increased interleukin-10 release after lipopolysaccharide stimulation. CONCLUSION: SULF A is the first synthetic small molecule that binds to TREM2. The receptor engagement drives differentiation of an unprecedented DC phenotype (homeDCs) that contributes to immune homeostasis without compromising lymphocyte activation and immunogenic response. This mechanism fully supports the adjuvant and immunoregulatory activity of SULF A. We also propose that the biological properties of SULF A can be of interest in various physiopathological mechanisms and therapies involving TREM2.

Effects of colchicine on tissue factor in oxLDL-activated T-lymphocytes.

Several studies have shown that T-cells might be involved in pathophysiology of acute coronary syndromes (ACS). Tissue factor (TF) plays a key role in ACS. Many evidences have indicated that some statins reduce TF expression in several cell types. However, literature about rosuvastatin and TF and about statins effects on T-cells is still scanty. Colchicine is an anti-inflammatory drug recently proven to have beneficial effects in ACS via unknown mechanisms. This study investigates the effects of colchicine and rosuvastatin on TF expression in oxLDL-activated T-cells. T-cells, isolated from buffy coats of healthy volunteers, were stimulated with oxLDL (50 µg/dL). T-cells were pre-incubated with colchicine (10 µM) or rosuvastatin (5 µM) for 1 h and then stimulated with oxLDL (50 µg/mL). TF gene (RT-PCR), protein (western blot), surface expression (FACS) and procoagulant activity (FXa generation assay) were measured. NF-κB/IκB axis was examined by western blot analysis and translocation assay. Colchicine and rosuvastatin significantly reduced TF gene, and protein expression and procoagulant activity in oxLDL stimulated T-cells. This effect was associated with a significant reduction in TF surface expression as well as its procoagulant activity. These phenomena appear modulated by drug effects on the transcription factor NF-kB. Rosuvastatin and colchicine prevent TF expression in oxLDL-stimulated T-cells by modulating the NF-κB/IκB axis. Thus, we speculate that this might be another mechanism by which these drugs exert benefic cardiovascular effects.

Human T cells interacting with HNSCC-derived mesenchymal stromal cells acquire tissue-resident memory like properties.

Tissue-resident memory (Trm) cells are specialized components of both CD4+ and CD8+ T cell subsets that persist in peripheral nonlymphoid tissues following infections and provide fast response in case of a secondary invasion by the same pathogen. Trm cells express the surface markers CD69, CD103, and the immune checkpoint molecule PD-1. Trm cells develop not only in the context of infections but also in tumors, where they can provide a line of defense as suggested by the positive correlation between the frequency of tumor-infiltrating Trm cells and patients' survival. Trm cells persistence in peripheral tissues depends on their adaptation to the local microenvironment and the presence of survival factors, mainly IL-7, IL-15, and Notch ligands. However, the cell sources of these factors are largely unknown, especially in the context of tumors. Here, we show that head-neck squamous cell carcinoma (HNSCC) is enriched in CD4+ and CD8+ T cells with a Trm phenotype. Moreover, we show that mesenchymal stromal cells that accumulate in HNSCC are a source of survival factors and allow proper expression of Trm-typical markers in a VCAM1-dependent manner.

Probing the Therapeutic Potential of Marine Phyla by SPE Extraction.

The marine environment is potentially a prolific source of small molecules with significant biological activities. In recent years, the development of new chromatographic phases and the progress in cell and molecular techniques have facilitated the search for marine natural products (MNPs) as novel pharmacophores and enhanced the success rate in the selection of new potential drug candidates. However, most of this exploration has so far been driven by anticancer research and has been limited to a reduced number of taxonomic groups. In this article, we report a test study on the screening potential of an in-house library of natural small molecules composed of 285 samples derived from 57 marine organisms that were chosen from among the major eukaryotic phyla so far represented in studies on bioactive MNPs. Both the extracts and SPE fractions of these organisms were simultaneously submitted to three different bioassays-two phenotypic and one enzymatic-for cytotoxic, antidiabetic, and antibacterial activity. On the whole, the screening of the MNP library selected 11 potential hits, but the distribution of the biological results showed that SPE fractionation increased the positive score regardless of the taxonomic group. In many cases, activity could be detected only in the enriched fractions after the elimination of the bulky effect due to salts. On a statistical basis, sponges and molluscs were confirmed to be the most significant source of cytotoxic and antimicrobial products, but other phyla were found to be effective with the other therapeutic targets.

A New Bioassay Platform Design for the Discovery of Small Molecules with Anticancer Immunotherapeutic Activity.

Immunotherapy takes advantage of the immune system to prevent, control, and eliminate neoplastic cells. The research in the field has already led to major breakthroughs to treat cancer. In this work, we describe a platform that integrates in vitro bioassays to test the immune response and direct antitumor effects for the preclinical discovery of anticancer candidates. The platform relies on the use of dendritic cells that are professional antigen-presenting cells (APC) able to activate T cells and trigger a primary adaptive immune response. The experimental procedure is based on two phenotypic assays for the selection of chemical leads by both a panel of nine tumor cell lines and growth factor-dependent immature mouse dendritic cells (D1). The positive hits are then validated by a secondary test on human monocyte-derived dendritic cells (MoDCs). The aim of this approach is the selection of potential immunotherapeutic small molecules from natural extracts or chemical libraries.

Prognostic Factors and Biomarkers of Responses to Immune Checkpoint Inhibitors in Lung Cancer.

Manipulation of the immune response is a game changer in lung cancer treatment, revolutionizing management. PD1 and CTLA4 are dynamically expressed on different T cell subsets that can either disrupt or sustain tumor growth. Monoclonal antibodies (MoAbs) against PD1/PDL1 and CTLA4 have shown that inhibitory signals can be impaired, blocking T cell activation and function. MoAbs, used as both single-agents or in combination with standard therapy for the treatment of advanced non-small cell lung cancer (NSCLC), have exhibited advantages in terms of overall survival and response rate; nivolumab, pembrolizumab, atezolizumab and more recently, durvalumab, have already been approved for lung cancer treatment and more compounds are in the pipeline. A better understanding of signaling elicited by these antibodies on T cell subsets, as well as identification of biological determinants of sensitivity, resistance and correlates of efficacy, will help to define the mechanisms of antitumor responses. In addition, the relevance of T regulatory cells (Treg) involved in immune responses in cancer is attracting increasing interest. A major challenge for future research is to understand why a durable response to immune checkpoint inhibitors (ICIs) occurs only in subsets of patients and the mechanisms of resistance after an initial response. This review will explore current understanding and future direction of research on ICI treatment in lung cancer and the impact of tumor immune microenvironment n influencing clinical responses.

Musculin inhibits human T-helper 17 cell response to interleukin 2 by controlling STAT5B activity.

We recently demonstrated that human T-helper (Th) 17 cells, unlike Th1 cells, do not proliferate in response to T-cell receptor stimulation, mainly because of their reduced capacity to produce and respond to IL-2. In this study, we show that their lower responsiveness to IL-2 is due to the selective expression of Musculin (MSC), a member of the basic helix-loop-helix transcription factors. We show that MSC expression in human Th17 cells is retinoic acid orphan receptor (ROR)γt-dependent, and allows the upregulation of PPP2R2B, a regulatory member of the protein phosphatase 2A (PP2A) enzyme. High PPP2R2B levels in human Th17 cells were responsible for the reduced STAT5B Ser-193 phosphorylation upon IL-2 signalling and, therefore, impaired STAT5B DNA binding and transcriptional activity on IL-2 target genes. PP2A, observed in Th17 cells, controls also STAT3, dephosphorylating Ser727, thus increasing its activity that plays a crucial role in Th17 development and/or maintenance. Thus, our findings identify an additional mechanism responsible for the limited expansion of human Th17 cells, and could provide a further explanation for the rarity of these cells in inflamed tissues.

Pregnancy-associated plasma protein-A promotes TF procoagulant activity in human endothelial cells by Akt-NF-κB axis.

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase with a controversial role in pathophysiology of cardiovascular disease. It seems involved in progression of atherosclerosis and is widely represented in atherosclerotic plaque. PAPP-A plasma levels are elevated in patients with acute coronary syndromes (ACS), thus it has been suggested that it might be a prognostic marker for developing major cardiovascular events. However, the pathophysiological link(s) between PAPP-A and ACS are still unknown. Several studies have indicated that tissue factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates whether PAPP-A, at concentrations measurable in ACS patients, might induce TF expression in human endothelial cells in culture (HUVEC). In HUVEC, PAPP-A induced TF-mRNA transcription as demonstrated by real time PCR and expression of functionally active TF as demonstrated by FACS analysis and pro-coagulant activity assay. PAPP-A induced TF expression through the activation of Akt/NF-κB axis, as demonstrated by luciferase assay and by suppression of TF-mRNA transcription as well as of TF expression/activity by Akt and NF-κB inhibitors. These data indicate that PAPP-A promotes TF expression in human endothelial cells and support the hypothesis that this proteinase, besides being involved in progression of atherosclerosis, does not represent an independent risk factor for adverse cardiovascular events, but it rather might play an "active" role in the pathophysiology of ACS as an effector molecule able to induce a pro-thrombotic phenotype in endothelial cells.

Chitinase 3-like-1 is produced by human Th17 cells and correlates with the level of inflammation in juvenile idiopathic arthritis patients.

BACKGROUND: CHI3L1 is a chitinase-like protein without enzymatic activity, produced by activated macrophages, chondrocytes, neutrophils. Recent studies on arthritis, asthma, and inflammatory bowel diseases suggest that chitinases are important in inflammatory processes and tissue remodeling, but their production by human T cells, has never been reported. METHODS: A microarray analysis of gene expression profile was performed on Th17 and classic Th1 cell clones and CHI3L1 was found among the up-regulated genes on Th17 cells. Different types of helper T cell clones (TCCs) were then evaluated by Real Time PCR (RT-PCR) for CHI3L1 mRNA expression; protein expression was investigated in cell lysates by western blotting and in cultures supernatants by ELISA. ELISA was also used to measure CHI3L1 in the serum and in the synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients. RESULTS: At mRNA level CHI3L1 was highly expressed by Th17, Th17/Th1, non classic Th1 and even in Th17/Th2 cell clones, whereas it was virtually absent in CD161- classic Th1 and Th2 TCCs. CHI3L1 was also detected in cell culture supernatants of Th17 and Th17-derived cells but not of classic Th1. Moreover CHI3L1 was higher in the SF than in serum of JIA patients, and it positively correlated with the frequency of Th17 and non-classic Th1 cells in SF. CHI3L1 in SF also positively correlated with the C reactive protein (CRP) serum levels, and with the levels of some proinflammatory cytokines, such as IL-6 and p40, which is the common subunit of IL12 and IL23. CONCLUSIONS: Here we describe for the first time CHI3L1 production by T cells owing the Th17 family. Moreover the positive correlation found between the frequency of Th17 and Th17-derived cell subsets and CHI3L1 levels in SF of JIA patients, in agreement with the suggested role of these cells in inflammatory process, candidates CHI3L1 as a possible biological target in JIA treatment.

Perianal Crohn's disease and hidradenitis suppurativa: a possible common immunological scenario.

BACKGROUND: Crohn's disease (CD) and Hidradenitis suppurativa (HS) are both chronic inflammatory diseases. The pathogenesis of these diseases is multifactorial, due to the interaction of genetic and environmental factors leading to a deregulated local immune response where T lymphocytes play a major role. To the best of our knowledge, no previous study has clarified whether the pathogenetic mechanism of perianal CD and HS is the same. We therefore analyzed the cellular expression pattern and the cytokine repertoire in three patients suffering from both perianal CD and HS. METHODS: We evaluated three patients affected by concurrent HS and CD with fistulizing perianal disease. Surgical specimens have been fixed and embedded in paraffin prior to sectioning for histological examination. Inflammatory tissue curettages have been recovered during intervention from perianal fistulas and HS lesions in order to analyze the phenotypic and functional characteristics of infiltrating T cells. In particular we evaluated T cells, by flow cytometry, for cytokine production profile and expression of surface markers. Moreover, analysis of the T cell repertoire was performed by means of spectratyping, in only one patient. RESULTS: A higher frequency of CD4+ CD161+ T lymphocytes has been detected in CD fistulas and in HS lesions than in peripheral blood (PB) samples. In the patient in whom we derived enough cells from the three sources, we found higher frequency of CD4+ IL-17- producing cells in HS lesion and fistula lesion compared to PB. It is noteworthy that the same clonotypes were expanded in this patient in T cells derived from both HS lesion and fistula lesion. CONCLUSION: The presence of numerous CD4+ CD161+ lymphocytes in fistula and HS lesion curettages suggests that these cells may play a pathogenic role, and candidates CD161 as a possible biological target for medical treatment.

Early systemic sclerosis: serum profiling of factors involved in endothelial, T-cell, and fibroblast interplay is marked by elevated interleukin-33 levels.

PURPOSE: To assess the serum profile of factors involved in endothelial, T-cell, and fibroblast interplay in patients with Raynaud's phenomenon (RP) associated with nailfold vodeocapillaroscopy (NVC) scleroderma findings and/or systemic sclerosis (SSc) marker autoantibodies, recently labeled as early SSc patients. METHODS: Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), CCL2, CXCL8, IL-13, IL-33, and transforming growth factor-ß (TGF-ß) were measured in 24 early SSc patients, 48 definite SSc patients, and 24 osteoarthritis/fibromyalgia controls by multiplex suspension immunoassay. All SSc patients were investigated for the presence/absence of preclinical and clinical organ involvement, SSc marker autoantibodies, and NVC abnormalities. RESULTS: Serum sICAM-1, CCL2, CXCL8, and IL-13 were increased in all SSc patients as compared to controls, and paralleled the severity of the disease subset (early SSc < limited cutaneous SSc < diffuse cutaneous SSc; p < 0.0001). Surprisingly, IL-33 was significantly higher in early SSc patients as compared to both controls (p < 0.01) and definite SSc patients (p < 0.05). In early SSc there were no differences in the investigated markers according to the functional and serological features assessed. CONCLUSIONS: Our study suggests that an endothelial, T-cell and fibroblast activation can be present in patients with early SSc and it is associated with a distinct profile of circulating factors involved in the cross-talk of these cells. The marked increase of IL-33 in early SSc patients suggests new routes of investigation of cell-cell dynamics in target tissues predating overt disease manifestations, thus opening to new therapeutic approaches.

The immunoregulatory effect of the TREM2-agonist Sulfavant A in human allogeneic mixed lymphocyte reaction.

Introduction: Sulfavant A (SULF A) is a synthetic derivative of naturally occurring sulfolipids. The molecule triggers TREM2-related maturation of dendritic cells (DCs) and has shown promising adjuvant activity in a cancer vaccine model. Methods: the immunomodulatory activity of SULF A is tested in an allogeneic mixed lymphocyte reaction (MLR) assay based on monocyte-derived dendritic cells and naïve T lymphocytes from human donors. Flow cytometry multiparametric analyses and ELISA assays were performed to characterize the immune populations, T cell proliferation, and to quantify key cytokines. Results: Supplementation of 10 µg/mL SULF A to the co-cultures induced DCs to expose the costimulatory molecules ICOSL and OX40L and to reduce release of the pro-inflammatory cytokine IL-12. After 7 days of SULF A treatment, T lymphocytes proliferated more and showed increased IL-4 synthesis along with downregulation of Th1 signals such as IFNγ, T-bet and CXCR3. Consistent with these findings, naïve T cells polarized toward a regulatory phenotype with up-regulation of FOXP3 expression and IL-10 synthesis. Flow cytometry analysis also supported the priming of a CD127-/CD4+/CD25+ subpopulation positive for ICOS, the inhibitory molecule CTLA-4, and the activation marker CD69. Discussion: These results prove that SULF A can modulate DC-T cell synapse and stimulate lymphocyte proliferation and activation. In the hyperresponsive and uncontrolled context of the allogeneic MLR, the effect is associated to differentiation of regulatory T cell subsets and dampening of inflammatory signals.

Marine natural product lepadin A as a novel inducer of immunogenic cell death via CD91-dependent pathway.

Immunogenic Cell Death (ICD) represents a mechanism of enhancing T cell-driven response against tumor cells. The process is enabled by release of damage-associated molecular patterns (DAMPs) and cytokines by dying cells. Based on molecular studies and clinical marker assessment, ICD can be a new target for cancer chemotherapy hitherto restricted to a few conventional anticancer drugs. In view of the development of small molecules in targeted cancer therapy, we reported the preliminary evidence on the role of the natural product lepadin A (1) as a novel ICD inducer. Here we describe the ICD mechanism of lepadin A (1) by proving the translocation of the protein calreticulin (CRT) to the plasma membrane of human A2058 melanoma cells. CRT exposure is an ICD marker in clinical studies and was associated with the activation of the intrinsic apoptotic pathway in A2058 cells with lepadin A (1). After the treatment, the tumour cells acquired the ability to activate dendritic cells (DCs) with cytokine release and costimulatory molecule expression that is consistent with a phenotypic profile committed to priming T lymphocytes via a CD91-dependent mechanism. The effect of lepadin A (1) was dose-dependent and comparable to the response of the chemotherapy drug doxorubicin (2), a well-established ICD inducer.

Evidence of the transient nature of the Th17 phenotype of CD4+CD161+ T cells in the synovial fluid of patients with juvenile idiopathic arthritis.

OBJECTIVE: To investigate the phenotype and function of CD4+ T cells in synovial fluid (SF) from the affected joints of children with oligoarticular-onset juvenile idiopathic arthritis (JIA), and to establish a possible link with disease activity. METHODS: CD4+ T cells were obtained from the peripheral blood (PB) and SF of 23 children with oligoarticular-onset JIA, as well as from the PB of 15 healthy children. The cells were analyzed for the expression of CXCR3, CCR6, and CD161 and for the production of interferon-γ and interleukin-17A (IL-17A). Spectratyping and clonotype analyses were performed to assess different T cell subsets. RESULTS: The numbers of CD4+CD161+ cells showing either the Th1 or the Th17/Th1 phenotype were higher in the SF than in the PB of children with JIA. The few Th17 cells from JIA SF underwent a spontaneous shift to the Th1 phenotype in vitro, whereas Th17 cells from the PB of healthy children shifted only in the presence of JIA SF; this effect was neutralized by antibody blockade of IL-12 activity. Spectratyping and clonotype analyses showed a similar skewing of the T cell receptor V(ß) repertoire in both CD161+ Th17 cells and CD161+ Th1 cells derived from the SF of the same JIA patient. The frequencies of CD4+CD161+ cells, particularly the Th17/Th1 cells, in the JIA SF positively correlated with the erythrocyte sedimentation rate and levels of C-reactive protein. CONCLUSION: These findings suggest that a shifting of CD4+CD161+ T cells from Th17 to the Th17/Th1 or Th1 phenotype can occur in the SF of children with oligoarticular-onset JIA, and indicate that the accumulation of these cells is correlated with parameters of inflammation. Thus, the results support the hypothesis that these cells may play a role in JIA disease activity.

Identification of the Marine Alkaloid Lepadin A as Potential Inducer of Immunogenic Cell Death.

Natural products and their synthetic analogs and derivatives are a traditional source of bioactive molecules with potential development as drug candidates. In this context, Marine Natural Products (MNPs) represent a rich reservoir of diverse molecular skeletons with potential pharmacological activity that, so far, has been mostly explored in cancer and infectious diseases. Starting from the development of a novel bioassay-guided screening platform for immunomodulatory compounds from an in-house MNPs library, we report the identification of the alkaloid lepadin A as a new model compound for immune-based anticancer activity with characteristics that suggest a possible mechanism as Immunogenic Cell Death inducer. The work describes the molecular-based bioprospecting in the Gulf of Naples together with the bioassay-guided fractionation, the chemical characterization of the alkaloid, and the biological activity in mouse dendritic cells (D1).

Dual inhibition of TGFß and AXL as a novel therapy for human colorectal adenocarcinoma with mesenchymal phenotype.

A subset of colorectal cancer (CRC) with a mesenchymal phenotype (CMS4) displays an aggressive disease, with an increased risk of recurrence after surgery, reduced survival, and resistance to standard treatments. It has been shown that the AXL and TGFß signaling pathways are involved in epithelial-to-mesenchymal transition, migration, metastatic spread, and unresponsiveness to targeted therapies. However, the prognostic role of the combination of these biomarkers and the anti-tumor effect of AXL and TGFß inhibition in CRC still has to be assessed. To evaluate the role of AXL and TGFß as negative biomarker in CRC, we conducted an in-depth in silico analysis of CRC samples derived from the Gene Expression Omnibus. We found that AXL and TGFß receptors are upregulated in CMS4 tumors and are correlated with an increased risk of recurrence after surgery in stage II/III CRC and a reduced overall survival. Moreover, we showed that AXL receptor is differently expressed in human CRC cell lines. Dual treatment with the TGFß galunisertib and the AXL inhibitor, bemcentinib, significantly reduced colony formation and migration capabilities of tumor cells and displayed a strong anti-tumor activity in 3D spheroid cultures derived from patients with advanced CRC. Our work shows that AXL and TGFß receptors identify a subgroup of CRC with a mesenchymal phenotype and correlate with poor prognosis. Dual inhibition of AXL and TGFß could represent a novel therapeutic strategy for patients with this aggressive disease.

T-Cell Proapoptotic and Antifibrotic Activity Against Autologous Skin Fibroblasts in vitro Is Associated With IL-17A Axis Upregulation in Systemic Sclerosis.

Background: Systemic sclerosis (SSc) T cells can induce apoptosis of autologous skin fibroblasts in vitro. Th17 cells have been reported to increase in SSc patients, and interleukin-17A (IL-17A) has a profibrotic function. We used a system based on T-cell-autologous fibroblast co-cultures to further investigate a possible role of IL-17A in SSc. Methods: T cells from diffuse SSc patients were co-cultured with autologous skin fibroblasts. IL17A mRNA was assessed by real-time PCR in co-cultured and control T cells, while IL17RA, CXCL1, CCL2, CCL3, COL1A1, COL3A1, CTGF, TGFBR2, and SMAD3 mRNAs were assessed in co-cultured and control fibroblasts. In subset experiments, co-cultures and control cells were treated with either IL-17A or IL-17A plus anti-IL17 receptor monoclonal antibody (α-IL-17RA mAb). Chemokine and procollagen type I (PCI) production was further investigated at the protein level in cell culture supernatants by multiple suspension immunoassay and sandwich ELISA, respectively. Co-cultured and control fibroblasts were also stained with Annexin V and analyzed by flow cytometry. Results: T cell-fibroblast co-cultures overexpressed IL17A and IL17RA. Furthermore, co-cultured fibroblasts upregulated IL-17A targets CXCL1, CCL2, and CCL3, while COL1A1, COL3A1, CTGF, and two key effectors of the TGF-ß signaling, TGFBR2 and SMAD3, were found downregulated. Consistently, chemokine concentrations were increased in co-culture supernatants, while PCI levels were reduced, especially after stimulation with ectopic IL-17A. Finally, simultaneous α-IL-17RA mAb treatment restored PCI levels and reduced fibroblast apoptosis in IL-17A-stimulated co-cultures. Conclusion: These data suggest that IL-17A upregulation might play a role in modulating T cell-mediated antifibrotic and proapoptotic effects in co-cultured autologous skin fibroblasts.

Baseline IFN-γ and IL-10 expression in PBMCs could predict response to PD-1 checkpoint inhibitors in advanced melanoma patients.

Anti-PD-1 antibodies revolutionized the treatment of advanced melanoma patients. However, one out of three do not respond to this therapy, with an overall poor prognosis. Identification of predictive biomarkers in patients receiving immune-based therapies is necessary for minimizing risk of toxicity and optimizing patient benefit and is still an important unmet clinical need. Recently, many studies have evaluated peripheral blood markers as potential biomarkers, but none so far have been validated. We collected at baseline peripheral blood samples from 18 consecutive advanced melanoma patients treated with anti-PD-1 therapy. Main pro- and anti-inflammatory cytokines were studied in PBMCs from baseline blood samples both evaluating mRNA expression by qRT-PCR and identifying PBMCs subpopulations by FACS analysis. We found that IFN-γ mRNA expression levels were significantly higher in responder patients compared to non-responder ones. Moreover, to better validate its role, we evaluated the IFN-γ/IL-10 ratio. This value was higher in responder patients. FACS analysis confirmed that CD4 + IFN-γ + PBMCs percentage was higher in responders. Our data suggest an interesting correlation between IFN-γ/IL-10 ratio and response to anti-PD-1 therapy in advanced melanoma patients, suggesting a new biomarker that could be easily incorporated in clinical practice.

Oxidized low-density lipoproteins induce tissue factor expression in T-lymphocytes via activation of lectin-like oxidized low-density lipoprotein receptor-1.

AIMS: T-lymphocytes plays an important role in the pathophysiology of acute coronary syndromes. T-cell activation in vitro by pro-inflammatory cytokines may lead to functional tissue factor (TF) expression, indicating a possible contribution of immunity to thrombosis. Oxidized low-density lipoproteins (oxLDLs) are found abundantly in atherosclerotic plaques. We aimed at evaluating the effects of oxLDLs on TF expression in T cells and the role of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). METHODS AND RESULTS: CD3+ cells were isolated from healthy volunteers. Gene, protein, and surface expression of TF, as well as of LOX-1, were assessed at different time-points after oxLDL stimulation. To determine whether oxLDL-induced TF was LOX-1 dependent, T cells were pre-incubated with an LOX-1 inhibiting peptide (L-RBP) or with an anti-LOX-1 blocking antibody. To exclude that TF expression was mediated by reactive oxygen species (ROS) generation, oxLDL-stimulated T cells were pre-incubated with superoxide dismutase + catalase or with 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), an intracellular free radical scavenger. Finally, to determine if the observed findings in vitro may have a biological relevance, the presence of CD3+/TF+/LOX-1+ cells was evaluated by immunofluorescence in human carotid atherosclerotic lesions. oxLDLs induced functionally active TF expression in T cells in a dose- and time-dependent manner, independently on ROS generation. No effect was observed in native LDL-treated T cells. LOX-1 expression was also induced by oxLDLs in a time- and dose-dependent manner. Pre-incubation with L-RBP or anti-LOX-1 antibody almost completely inhibited oxLDL-mediated TF expression. Interestingly, human carotid plaques showed significant infiltration of CD3+ cells (mainly CD8+ cells), some of which were positive for both TF and LOX-1. CONCLUSION: oxLDLs induce functional TF expression in T-lymphocytes in vitro via interaction of oxLDLs with LOX-1. Human carotid atherosclerotic plaques contain CD3+/CD8+cells that express both TF and LOX-1, indicating that also in patients these mechanisms may play an important role.