Inhibitory receptors of plasmacytoid dendritic cells as possible targets for checkpoint blockade in cancer.

Plasmacytoid dendritic cells (pDCs) are the major producers of type I interferons (IFNs), which are essential to mount antiviral and antitumoral immune responses. To avoid exaggerated levels of type I IFNs, which pave the way to immune dysregulation and autoimmunity, pDC activation is strictly regulated by a variety of inhibitory receptors (IRs). In tumors, pDCs display an exhausted phenotype and correlate with an unfavorable prognosis, which largely depends on the accumulation of immunosuppressive cytokines and oncometabolites. This review explores the hypothesis that tumor microenvironment may reduce the release of type I IFNs also by a more pDC-specific mechanism, namely the engagement of IRs. Literature shows that many cancer types express de novo, or overexpress, IR ligands (such as BST2, PCNA, CAECAM-1 and modified surface carbohydrates) which often represent a strong predictor of poor outcome and metastasis. In line with this, tumor cells expressing ligands engaging IRs such as BDCA-2, ILT7, TIM3 and CD44 block pDC activation, while this blocking is prevented when IR engagement or signaling is inhibited. Based on this evidence, we propose that the regulation of IFN secretion by IRs may be regarded as an "innate checkpoint", reminiscent of the function of "classical" adaptive immune checkpoints, like PD1 expressed in CD8+ T cells, which restrain autoimmunity and immunopathology but favor chronic infections and tumors. However, we also point out that further work is needed to fully unravel the biology of tumor-associated pDCs, the neat contribution of pDC exhaustion in tumor growth following the engagement of IRs, especially those expressed also by other leukocytes, and their therapeutic potential as targets of combined immune checkpoint blockade in cancer immunotherapy.

Statin-Sensitive Akt1/Src/Caveolin-1 Signaling Enhances Oxidative Stress Resistance in Rhabdomyosarcoma.

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

Extracellular microRNAs induce dendritic cell-dependent joint inflammation and potentiate osteoclast differentiation via TLR7/8 engagement.

OBJECTIVES: Monocyte-derived dendritic cells (DCs) are key players in the induction of inflammation, autoreactive T cell activation and loss of tolerance in rheumatoid arthritis (RA), but the precise mechanisms underlying their activation remain elusive. Here, we hypothesized that extracellular microRNAs released in RA synovial fluids may represent a novel, physiological stimulus triggering unwanted immune response via TLR8-expressing DC stimulation. METHODS: Human monocyte-derived DCs were stimulated with a mixture of GU-rich miRNAs upregulated in RA tissues and released in synovial fluids (Ex-miRNAs). Activation of DCs was assessed in terms of NF-κB activation by Western blot, cytokine production by ELISA, T cell proliferation and polarization by allogeneic mixed lymphocyte reaction. DC differentiation into osteoclasts was evaluated in terms of tartrate-resistant acid phosphatase production and formation of resorption pits in dentine slices. Induction of joint inflammation in vivo was evaluated using a murine model of DC-induced arthritis. TLR7/8 involvement was assessed by specific inhibitors. RESULTS: Ex-miRNAs activate DCs to secrete TNFα, induce joint inflammation, start an early autoimmune response and potentiate the differentiation of DCs into aggressive osteoclasts. CONCLUSIONS: This work represents a proof of concept that the pool of extracellular miRNAs overexpressed in RA joints can act as a physiological activator of inflammation via the stimulation of TLR8 expressed by human DCs, which in turn exert arthritogenic functions. In this scenario, pharmacological inhibition of TLR8 might offer a new therapeutic option to reduce inflammation and osteoclast-mediated bone destruction in RA.

Modulation of Human Dendritic Cell Functions by Phosphodiesterase-4 Inhibitors: Potential Relevance for the Treatment of Respiratory Diseases.

Inhibitors of phosphodiesterase-4 (PDE4) are small-molecule drugs that, by increasing the intracellular levels of cAMP in immune cells, elicit a broad spectrum of anti-inflammatory effects. As such, PDE4 inhibitors are actively studied as therapeutic options in a variety of human diseases characterized by an underlying inflammatory pathogenesis. Dendritic cells (DCs) are checkpoints of the inflammatory and immune responses, being responsible for both activation and dampening depending on their activation status. This review shows evidence that PDE4 inhibitors modulate inflammatory DC activation by decreasing the secretion of inflammatory and Th1/Th17-polarizing cytokines, although preserving the expression of costimulatory molecules and the CD4+ T cell-activating potential. In addition, DCs activated in the presence of PDE4 inhibitors induce a preferential Th2 skewing of effector T cells, retain the secretion of Th2-attracting chemokines and increase the production of T cell regulatory mediators, such as IDO1, TSP-1, VEGF-A and Amphiregulin. Finally, PDE4 inhibitors selectively induce the expression of the surface molecule CD141/Thrombomodulin/BDCA-3. The result of such fine-tuning is immunomodulatory DCs that are distinct from those induced by classical anti-inflammatory drugs, such as corticosteroids. The possible implications for the treatment of respiratory disorders (such as COPD, asthma and COVID-19) by PDE4 inhibitors will be discussed.

CCRL2 Expression by Specialized Lung Capillary Endothelial Cells Controls NK-cell Homing in Lung Cancer.

Patterns of receptors for chemotactic factors regulate the homing of leukocytes to tissues. Here we report that the CCRL2/chemerin/CMKLR1 axis represents a selective pathway for the homing of natural killer (NK) cells to the lung. C-C motif chemokine receptor-like 2 (CCRL2) is a nonsignaling seven-transmembrane domain receptor able to control lung tumor growth. CCRL2 constitutive or conditional endothelial cell targeted ablation, or deletion of its ligand chemerin, were found to promote tumor progression in a Kras/p53Flox lung cancer cell model. This phenotype was dependent on the reduced recruitment of CD27- CD11b+ mature NK cells. Other chemotactic receptors identified in lung-infiltrating NK cells by single-cell RNA sequencing (scRNA-seq), such as Cxcr3, Cx3cr1, and S1pr5, were found to be dispensable in the regulation of NK-cell infiltration of the lung and lung tumor growth. scRNA-seq identified CCRL2 as the hallmark of general alveolar lung capillary endothelial cells. CCRL2 expression was epigenetically regulated in lung endothelium and it was upregulated by the demethylating agent 5-aza-2'-deoxycytidine (5-Aza). In vivo administration of low doses of 5-Aza induced CCRL2 upregulation, increased recruitment of NK cells, and reduced lung tumor growth. These results identify CCRL2 as an NK-cell lung homing molecule that has the potential to be exploited to promote NK cell-mediated lung immune surveillance.

The RIG-I agonist M8 triggers cell death and natural killer cell activation in human papillomavirus-associated cancer and potentiates cisplatin cytotoxicity.

Although the activation of innate immunity to treat a wide variety of cancers is gaining increasing attention, it has been poorly investigated in human papillomavirus (HPV)-associated malignancies. Because these tumors harbor a severely impaired cGAS-STING axis, but they still retain a largely functional RIG-I pathway, another critical mediator of adaptive and innate immune responses, we asked whether RIG-I activation by the 5'ppp-RNA RIG-I agonist M8 would represent a therapeutically viable option to treat HPV+ cancers. Here, we show that M8 transfection of two cervical carcinoma-derived cell lines, CaSki and HeLa, both expressing a functional RIG-I, triggers intrinsic apoptotic cell death, which is significantly reduced in RIG-I KO cells. We also demonstrate that M8 stimulation potentiates cisplatin-mediated cell killing of HPV+ cells in a RIG-I dependent manner. This combination treatment is equally effective in reducing tumor growth in a syngeneic pre-clinical mouse model of HPV16-driven cancer, where enhanced expression of lymphocyte-recruiting chemokines and cytokines correlated with an increased number of activated natural killer (NK) cells in the tumor microenvironment. Consistent with a role of RIG-I signaling in immunogenic cell killing, stimulation of NK cells with conditioned medium from M8-transfected CaSki boosted NK cell proliferation, activation, and migration in a RIG-I-dependent tumor cell-intrinsic manner. Given the highly conserved molecular mechanisms of carcinogenesis and genomic features of HPV-driven cancers and the remarkably improved prognosis for HPV+ oropharyngeal cancer, targeting RIG-I may represent an effective immunotherapeutic strategy in this setting, favoring the development of de-escalating strategies.

Dendritic cell subsets in cancer immunity and tumor antigen sensing.

Dendritic cells (DCs) exhibit a specialized antigen-presenting function and play crucial roles in both innate and adaptive immune responses. Due to their ability to cross-present tumor cell-associated antigens to naïve T cells, DCs are instrumental in the generation of specific T-cell-mediated antitumor effector responses in the control of tumor growth and tumor cell dissemination. Within an immunosuppressive tumor microenvironment, DC antitumor functions can, however, be severely impaired. In this review, we focus on the mechanisms of DC capture and activation by tumor cell antigens and the role of the tumor microenvironment in shaping DC functions, taking advantage of recent studies showing the phenotype acquisition, transcriptional state and functional programs revealed by scRNA-seq analysis. The therapeutic potential of DC-mediated tumor antigen sensing in priming antitumor immunity is also discussed.

LRRK2 Kinase Inhibition Attenuates Astrocytic Activation in Response to Amyloid ß1-42 Fibrils.

Intracerebral accumulation of amyloid-ß in the extracellular plaques of Alzheimer's disease (AD) brains represents the main cause of reactive astrogliosis and neuroinflammatory response. Of relevance, leucine-rich repeat kinase 2 (LRRK2), a kinase linked to genetic and sporadic Parkinson's disease (PD), has been identified as a positive mediator of neuroinflammation upon different inflammatory stimuli, however its pathogenicity in AD remains mainly unexplored. In this study, by using pharmacological inhibition of LRRK2 and murine primary astrocytes, we explored whether LRRK2 regulates astrocytic activation in response to amyloid-ß1-42 (Aß1-42). Our results showed that murine primary astrocytes become reactive and recruit serine 935 phosphorylated LRRK2 upon Aß1-42 fibril exposure. Moreover, we found that pharmacological inhibition of LRRK2, with two different kinase inhibitors, can attenuate Aß1-42-mediated inflammation and favor the clearance of Aß1-42 fibrils in astrocytes. Overall, our findings report that LRRK2 kinase activity modulates astrocytic reactivity and functions in the presence of Aß1-42 deposits and indicate that PD-linked LRRK2 might contribute to AD-related neuroinflammation and pathogenesis.

SARS-CoV-2-Associated ssRNAs Activate Human Neutrophils in a TLR8-Dependent Fashion.

COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of proinflammatory genes, similar to that promoted by R848. Furthermore, by using CU-CPT9a, a TLR8-specific inhibitor, we found that SCV2-RNA2 stimulates neutrophils exclusively via TLR8-dependent pathways. In sum, our study proves that single-strand RNAs from the SARS-CoV-2 genome potently activate human neutrophils via TLR8, thus uncovering a potential mechanism whereby neutrophils may contribute to the pathogenesis of severe COVID-19 disease.

Inhibition of Class I Histone Deacetylase Activity Blocks the Induction of TNFAIP3 Both Directly and Indirectly via the Suppression of Endogenous TNF-α.

Histone deacetylase inhibitors (HDIs) are promising drugs for the treatment of inflammatory diseases. However, their therapeutical exploitation is slowed down by severe adverse manifestations that can hardly be foreseen, mainly due to incomplete knowledge of how HDIs impact the delicate balance of inflammatory mediators. In this work, we characterized the effects of the HDI trichostatin A (TSA) on the expression of TNFAIP3, which is a crucial inhibitor of the classical NF-kB pathway and an LPS-induced negative feedback regulator. The accumulation of TNFAIP3 mRNA after LPS stimulation showed biphasic behavior, with one wave within the first hour of stimulation and a second wave several hours later, which were both reduced by TSA. By using inhibition and knockdown approaches, we identified two temporally and mechanistically distinct modes of action. The first wave of TNAIP3 accumulation was directly blunted by the histone deacetylase (HDAC) blockade. By contrast, the second wave was decreased mainly because of the lack of endogenous TNF-α induction, which, in turn, depended on the intact HDAC activity. In both cases, class I HDACs appeared to play a nonredundant role, with HDAC3 required, but not sufficient, for TNF-α and TNFAIP3 induction. In addition to TNFAIP3, TNF-α is known to induce many response genes that orchestrate the inflammatory cascade. Thus, suppression of TNF-α may represent a general mechanism through which HDIs regulate a selected set of target genes.

The PDE4 inhibitor tanimilast shows distinct immunomodulatory properties associated with a type 2 endotype and CD141 upregulation.

BACKGROUND: Tanimilast is a novel and selective inhaled inhibitor of phosphodiesterase-4 in advanced clinical development for chronic obstructive pulmonary disease (COPD). Tanimilast is known to exert prominent anti-inflammatory activity when tested in preclinical experimental models as well as in human clinical studies. Recently, we have demonstrated that it also finely tunes, rather than suppressing, the cytokine network secreted by activated dendritic cells (DCs). This study was designed to characterize the effects of tanimilast on T-cell polarizing properties of DCs and to investigate additional functional and phenotypical features induced by tanimilast. METHODS: DCs at day 6 of culture were stimulated with LPS in the presence or absence of tanimilast or the control drug budesonide. After 24 h, DCs were analyzed for the expression of surface markers of maturation and activation by flow cytometry and cocultured with T cells to investigate cell proliferation and activation/polarization. The regulation of type 2-skewing mediators was investigated by real-time PCR in DCs and compared to results obtained in vivo in a randomized placebo-controlled trial on COPD patients treated with tanimilast. RESULTS: Our results show that both tanimilast and budesonide reduced the production of the immunostimulatory cytokine IFN-γ by CD4+ T cells. However, the two drugs acted at different levels since budesonide mainly blocked T cell proliferation, while tanimilast skewed T cells towards a Th2 phenotype without affecting cell proliferation. In addition, only DCs matured in the presence of tanimilast displayed increased CD86/CD80 ratio and CD141 expression, which correlated with Th2 T cell induction and dead cell uptake respectively. These cells also upregulated cAMP-dependent immunosuppressive molecules such as IDO1, TSP1, VEGF-A and Amphiregulin. Notably, the translational value of these data was confirmed by the finding that these same genes were upregulated also in sputum cells of COPD patients treated with tanimilast as add-on to inhaled glucocorticoids and bronchodilators. CONCLUSION: Taken together, these findings demonstrate distinct immunomodulatory properties of tanimilast associated with a type 2 endotype and CD141 upregulation in DCs and provide a mechanistic rationale for the administration of tanimilast on top of inhaled corticosteroids.

The PDE4 Inhibitor Tanimilast Restrains the Tissue-Damaging Properties of Human Neutrophils.

Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001-is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide-a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, degranulation, extracellular DNA traps casting, and cytokine secretion. In contrast, it promoted neutrophil survival by decreasing both spontaneous apoptosis and cell death in the presence of pro-survival factors. The present work suggests that tanimilast can alleviate the severe tissue damage caused by massive recruitment and activation of neutrophils in inflammatory diseases such as COPD.

SARS-CoV-2-associated ssRNAs activate inflammation and immunity via TLR7/8.

The inflammatory and IFN pathways of innate immunity play a key role in the resistance and pathogenesis of coronavirus disease 2019 (COVID-19). Innate sensors and SARS-CoV-2-associated molecular patterns (SAMPs) remain to be completely defined. Here, we identified single-stranded RNA (ssRNA) fragments from the SARS-CoV-2 genome as direct activators of endosomal TLR7/8 and MyD88 pathway. The same sequences induced human DC activation in terms of phenotype and function, such as IFN and cytokine production and Th1 polarization. A bioinformatic scan of the viral genome identified several hundreds of fragments potentially activating TLR7/8, suggesting that products of virus endosomal processing potently activate the IFN and inflammatory responses downstream of these receptors. In vivo, SAMPs induced MyD88-dependent lung inflammation characterized by accumulation of proinflammatory and cytotoxic mediators and immune cell infiltration, as well as splenic DC phenotypical maturation. These results identified TLR7/8 as a crucial cellular sensor of ssRNAs encoded by SARS-CoV-2 involved in host resistance and the disease pathogenesis of COVID-19.

Cisplatin Cytotoxicity in Human Testicular Germ Cell Tumor Cell Lines Is Enhanced by the CDK4/6 Inhibitor Palbociclib.

BACKGROUND: Cisplatin-based chemotherapy is the mainstay of pharmacological treatment of testicular germ cell tumors (TGCTs) that, together with early diagnosis, surgery, and/or radiotherapy, has dramatically improved the prognosis. However, under the pressure of such pharmacological therapy (both classical cytotoxic drugs and targeted therapy), cancer cells may develop resistance. Thus, combination therapy that may include cytotoxic drugs and targeted therapy could offer an advantage to curing cancers. Here, we investigated the in vitro and in vivo antitumor activity of cisplatin, as a single-agent or in combination with palbociclib. PATIENTS AND METHODS: The cell viability of Ntera-2/cl.D1 (NT2/D1) and 833K after exposure to palbociclib and/or cisplatin was evaluated by MTT dye reduction assay and by ATPLite Luminescence Assay. Gene and protein expression was evaluated by quantitative reverse transcription polymerase chain reaction and by western blot. Flow cytometric cell-cycle analysis was performed, as well. The in vivo experiments were conducted on NT2/D1 xenografts in AB zebrafish embryos exposed to the drugs. RESULTS: Palbociclib and cisplatin decreased TGCT cell viability both in vitro and in vivo. This effect was additive when cells were exposed to the drug combination. In the NT2/D1 cell lines, the drug combination also exerted a positive effect with regard to delaying cell recovery after the toxic insult. In the combination experiments, cisplatin-induced cell accumulation in G2/M was predominant compared with the palbociclib effect. CONCLUSIONS: These results could provide the rationale for developing further studies to improve the pharmacological treatment of TGCTs, but they must be demonstrated in a dedicated clinical trial.

The PDE4 Inhibitor Tanimilast Blunts Proinflammatory Dendritic Cell Activation by SARS-CoV-2 ssRNAs.

Phosphodiesterase 4 (PDE4) inhibitors are immunomodulatory drugs approved to treat diseases associated with chronic inflammatory conditions, such as COPD, psoriasis and atopic dermatitis. Tanimilast (international non-proprietary name of CHF6001) is a novel, potent and selective inhaled PDE4 inhibitor in advanced clinical development for the treatment of COPD. To begin testing its potential in limiting hyperinflammation and immune dysregulation associated to SARS-CoV-2 infection, we took advantage of an in vitro model of dendritic cell (DC) activation by SARS-CoV-2 genomic ssRNA (SCV2-RNA). In this context, Tanimilast decreased the release of pro-inflammatory cytokines (TNF-α and IL-6), chemokines (CCL3, CXCL9, and CXCL10) and of Th1-polarizing cytokines (IL-12, type I IFNs). In contrast to ß-methasone, a reference steroid anti-inflammatory drug, Tanimilast did not impair the acquisition of the maturation markers CD83, CD86 and MHC-II, nor that of the lymph node homing receptor CCR7. Consistent with this, Tanimilast did not reduce the capability of SCV2-RNA-stimulated DCs to activate CD4+ T cells but skewed their polarization towards a Th2 phenotype. Both Tanimilast and ß-methasone blocked the increase of MHC-I molecules in SCV2-RNA-activated DCs and restrained the proliferation and activation of cytotoxic CD8+ T cells. Our results indicate that Tanimilast can modulate the SCV2-RNA-induced pro-inflammatory and Th1-polarizing potential of DCs, crucial regulators of both the inflammatory and immune response. Given also the remarkable safety demonstrated by Tanimilast, up to now, in clinical studies, we propose this inhaled PDE4 inhibitor as a promising immunomodulatory drug in the scenario of COVID-19.

Molecular Basis for CCRL2 Regulation of Leukocyte Migration.

CCRL2 is a seven-transmembrane domain receptor that belongs to the chemokine receptor family. At difference from other members of this family, CCRL2 does not promote chemotaxis and shares structural features with atypical chemokine receptors (ACKRs). However, CCRL2 also differs from ACKRs since it does not bind chemokines and is devoid of scavenging functions. The only commonly recognized CCRL2 ligand is chemerin, a non-chemokine chemotactic protein. CCRL2 is expressed both by leukocytes and non-hematopoietic cells. The genetic ablation of CCRL2 has been instrumental to elucidate the role of this receptor as positive or negative regulator of inflammation. CCRL2 modulates leukocyte migration by two main mechanisms. First, when CCRL2 is expressed by barrier cells, such endothelial, and epithelial cells, it acts as a presenting molecule, contributing to the formation of a non-soluble chemotactic gradient for leukocytes expressing CMKLR1, the functional chemerin receptor. This mechanism was shown to be crucial in the induction of NK cell-dependent immune surveillance in lung cancer progression and metastasis. Second, by forming heterocomplexes with other chemokine receptors. For instance, CCRL2/CXCR2 heterodimers were shown to regulate the activation of ß2-integrins in mouse neutrophils. This mini-review summarizes the current understanding of CCRL2 biology, based on experimental evidence obtained by the genetic deletion of this receptor in in vivo experimental models. Further studies are required to highlight the complex functional role of CCRL2 in different organs and pathological conditions.

Human Metapneumovirus Establishes Persistent Infection in Lung Microvascular Endothelial Cells and Primes a Th2-Skewed Immune Response.

Human Metapneumovirus (HMPV) is a major cause of lower respiratory tract infections. HMPV infection has been hypothesized to alter dendritic cell (DC) immune response; however, many questions regarding HMPV pathogenesis within the infected lung remain unanswered. Here, we show that HMPV productively infects human lung microvascular endothelial cells (L-HMVECs). The release of infectious virus occurs for up to more than 30 days of culture without producing overt cytopathic effects and medium derived from persistently HMPV-infected L-HMVECs (secretome) induced monocyte-derived DCs to prime naïve CD4 T-cells toward a Th2 phenotype. Moreover, we demonstrated that infected secretomes trigger DCs to up-regulate OX40L expression and OX40L neutralization abolished the pro-Th2 effect that is induced by HMPV-secretome. We clarified secretome from HMPV by size exclusion and ultracentrifugation with the aim to characterize the role of viral particles in the observed pro-Th2 effect. In both cases, the percentage of IL-4-producing cells and expression of OX40L returned at basal levels. Finally, we showed that HMPV, per se, could reproduce the ability of secretome to prime pro-Th2 DCs. These results suggest that HMPV, persistently released by L-HMVECs, might take part in the development of a skewed, pro-Th2 lung microenvironment.

Functional Role of Dendritic Cell Subsets in Cancer Progression and Clinical Implications.

Dendritic cells (DCs) constitute a complex network of cell subsets with common functions but also with many divergent aspects. All dendritic cell subsets share the ability to prime T cell response and to undergo a complex trafficking program related to their stage of maturation and function. For these reasons, dendritic cells are implicated in a large variety of both protective and detrimental immune responses, including a crucial role in promoting anti-tumor responses. Although cDC1s are the most potent subset in tumor antigen cross-presentation, they are not sufficient to induce full-strength anti-tumor cytotoxic T cell response and need close interaction and cooperativity with the other dendritic cell subsets, namely cDC2s and pDCs. This review will take into consideration different aspects of DC biology, including the functional role of dendritic cell subsets in both fostering and suppressing tumor growth, the mechanisms underlying their recruitment into the tumor microenvironment, as well as the prognostic value and the potentiality of dendritic cell therapeutic targeting. Understanding the specificity of dendritic cell subsets will allow to gain insights on role of these cells in pathological conditions and to design new selective promising therapeutic approaches.

In vitro cytotoxicity of cabazitaxel in adrenocortical carcinoma cell lines and human adrenocortical carcinoma primary cell cultures☆.

Adrenocortical cancer (ACC) is a rare and aggressive malignancy with a poor prognosis. The overall 5-year survival rate of patients with ENS@T stage IV ACC is less than 15%. Systemic antineoplastic therapies have a limited efficacy and new drugs are urgently needed. Human ACC primary cultures and cell lines were used to assess the cytotoxic effect of cabazitaxel, and the role of P-glycoprotein in mediating this effect. Cabazitaxel reduced ACC cell viability, both in ACC cell lines and in ACC primary cell cultures. Molecular and pharmacological targeting of ABCB1/P-gp did not modify its cytotoxic effect in NCI-H295R cells, while it increased the paclitaxel-induced toxicity. Cabazitaxel modified the expression of proteins involved in cellular physiology, such as apoptosis and cell cycle regulation. The drug combination cabazitaxel/mitotane exerted an additive/moderate synergism in different ACC cell experimental models. These results provide a rationale for testing cabazitaxel in a clinical study.