SIRT1 Modulates the Sensitivity of Prostate Cancer Cells to Vesicular Stomatitis Virus Oncolysis.

Oncolytic virotherapy represents a promising experimental anticancer strategy, based on the use of genetically modified viruses to selectively infect and kill cancer cells. Vesicular stomatitis virus (VSV) is a prototypic oncolytic virus (OV) that induces cancer cell death through activation of the apoptotic pathway, although intrinsic resistance to oncolysis is found in some cell lines and many primary tumors, as a consequence of residual innate immunity to the virus. In the effort to improve OV therapeutic efficacy, we previously demonstrated that different agents, including histone deacetylase inhibitors (HDIs), functioned as reversible chemical switches to dampen the innate antiviral response and improve the susceptibility of resistant cancer cells to VSV infection. In the present study, we demonstrated that the NAD+-dependent histone deacetylase SIRT1 (silent mating type information regulation 2 homolog 1) plays a key role in the permissivity of prostate cancer PC-3 cells to VSVΔM51 replication and oncolysis. HDI-mediated enhancement of VSVΔM51 infection and cancer cell killing directly correlated with a decrease of SIRT1 expression. Furthermore, pharmacological inhibition as well as silencing of SIRT1 by small interfering RNA (siRNA) was sufficient to sensitize PC-3 cells to VSVΔM51 infection, resulting in augmentation of virus replication and spread. Mechanistically, HDIs such as suberoylanilide hydroxamic acid (SAHA; Vorinostat) and resminostat upregulated the microRNA miR-34a that regulated the level of SIRT1. Taken together, our findings identify SIRT1 as a viral restriction factor that limits VSVΔM51 infection and oncolysis in prostate cancer cells.IMPORTANCE The use of nonpathogenic viruses to target and kill cancer cells is a promising strategy in cancer therapy. However, many types of human cancer are resistant to the oncolytic (cancer-killing) effects of virotherapy. In this study, we identify a host cellular protein, SIRT1, that contributes to the sensitivity of prostate cancer cells to infection by a prototypical oncolytic virus. Knockout of SIRT1 activity increases the sensitivity of prostate cancer cells to virus-mediated killing. At the molecular level, SIRT1 is controlled by a small microRNA termed miR-34a. Altogether, SIRT1 and/or miR-34a levels may serve as predictors of response to oncolytic-virus therapy.

Activation of Latent HIV-1 T Cell Reservoirs with a Combination of Innate Immune and Epigenetic Regulators.

The presence of T cell reservoirs in which human immunodeficiency virus (HIV) establishes latency by integrating into the host genome represents a major obstacle to an HIV cure and has prompted the development of strategies aimed at the eradication of HIV from latently infected cells. The "shock-and-kill" strategy is one of the most pursued approaches to the elimination of viral reservoirs. Although several latency-reversing agents (LRAs) have shown promising reactivation activity, they have failed to eliminate the cellular reservoir. In this study, we evaluated a novel immune system-mediated approach to clearing the HIV reservoir, based on a combination of innate immune stimulation and epigenetic reprogramming. The combination of the STING agonist cGAMP (cyclic GMP-AMP) and the FDA-approved histone deacetylase inhibitor resminostat resulted in a significant increase in HIV proviral reactivation and specific apoptosis in HIV-infected cells in vitro Reductions in the proportion of HIV-harboring cells and the total amount of HIV DNA were also observed in CD4+ central memory T (TCM) cells, a primary cell model of latency, where resminostat alone or together with cGAMP induced high levels of selective cell death. Finally, high levels of cell-associated HIV RNA were detected ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from individuals on suppressive antiretroviral therapy (ART). Although synergism was not detected in PBMCs with the combination, viral RNA expression was significantly increased in CD4+ T cells. Collectively, these results represent a promising step toward HIV eradication by demonstrating the potential of innate immune activation and epigenetic modulation for reducing the viral reservoir and inducing specific death of HIV-infected cells.IMPORTANCE One of the challenges associated with HIV-1 infection is that despite antiretroviral therapies that reduce HIV-1 loads to undetectable levels, proviral DNA remains dormant in a subpopulation of T lymphocytes. Numerous strategies to clear residual virus by reactivating latent virus and eliminating the reservoir of HIV-1 (so-called "shock-and-kill" strategies) have been proposed. In the present study, we use a combination of small molecules that activate the cGAS-STING antiviral innate immune response (the di-cyclic nucleotide cGAMP) and epigenetic modulators (histone deacetylase inhibitors) that induce reactivation and HIV-infected T cell killing in cell lines, primary T lymphocytes, and patient samples. These studies represent a novel strategy for HIV eradication by reducing the viral reservoir and inducing specific death of HIV-infected cells.

An optimized retinoic acid-inducible gene I agonist M8 induces immunogenic cell death markers in human cancer cells and dendritic cell activation.

RIG-I is a cytosolic RNA sensor that recognizes short 5' triphosphate RNA, commonly generated during virus infection. Upon activation, RIG-I initiates antiviral immunity, and in some circumstances, induces cell death. Because of this dual capacity, RIG-I has emerged as a promising target for cancer immunotherapy. Previously, a sequence-optimized RIG-I agonist (termed M8) was generated and shown to stimulate a robust immune response capable of blocking viral infection and to function as an adjuvant in vaccination strategies. Here, we investigated the potential of M8 as an anti-cancer agent by analyzing its ability to induce cell death and activate the immune response. In multiple cancer cell lines, M8 treatment strongly activated caspase 3-dependent apoptosis, that relied on an intrinsic NOXA and PUMA-driven pathway that was dependent on IFN-I signaling. Additionally, cell death induced by M8 was characterized by the expression of markers of immunogenic cell death-related damage-associated molecular patterns (ICD-DAMP)-calreticulin, HMGB1 and ATP-and high levels of ICD-related cytokines CXCL10, IFNß, CCL2 and CXCL1. Moreover, M8 increased the levels of HLA-ABC expression on the tumor cell surface, as well as up-regulation of genes involved in antigen processing and presentation. M8 induction of the RIG-I pathway in cancer cells favored dendritic cell phagocytosis and induction of co-stimulatory molecules CD80 and CD86, together with increased expression of IL12 and CXCL10. Altogether, these results highlight the potential of M8 in cancer immunotherapy, with the capacity to induce ICD-DAMP on tumor cells and activate immunostimulatory signals that synergize with current therapies.

Social threat exposure in juvenile mice promotes cocaine-seeking by altering blood clotting and brain vasculature.

Childhood maltreatment is associated with increased severity of substance use disorder and frequent relapse to drug use following abstinence. However, the molecular and neurobiological substrates that are engaged during early traumatic events and mediate the greater risk of relapse are poorly understood and knowledge of risk factors is to date extremely limited. In this study, we modeled childhood maltreatment by exposing juvenile mice to a threatening social experience (social stressed, S-S). We showed that S-S experience influenced the propensity to reinstate cocaine-seeking after periods of withdrawal in adulthood. By exploring global gene expression in blood leukocytes we found that this behavioral phenotype was associated with greater blood coagulation. In parallel, impairments in brain microvasculature were observed in S-S mice. Furthermore, treatment with an anticoagulant agent during withdrawal abolished the susceptibility to reinstate cocaine-seeking in S-S mice. These findings provide novel insights into a possible molecular mechanism by which childhood maltreatment heightens the risk for relapse in cocaine-dependent individuals.

Intratumoral injection of IFN-alpha dendritic cells after dacarbazine activates anti-tumor immunity: results from a phase I trial in advanced melanoma.

BACKGROUND: Advanced melanoma patients have an extremely poor long term prognosis and are in strong need of new therapies. The recently developed targeted therapies have resulted in a marked antitumor effect, but most responses are partial and some degree of toxicity remain the major concerns. Dendritic cells play a key role in the activation of the immune system and have been typically used as ex vivo antigen-loaded cell drugs for cancer immunotherapy. Another approach consists in intratumoral injection of unloaded DCs that can exploit the uptake of a wider array of tumor-specific and individual unique antigens. However, intratumoral immunization requires DCs endowed at the same time with properties typically belonging to both immature and mature DCs (i.e. antigen uptake and T cell priming). DCs generated in presence of interferon-alpha (IFN-DCs), due to their features of partially mature DCs, capable of efficiently up-taking, processing and cross-presenting antigens to T cells, could successfully carry out this task. Combining intratumoral immunization with tumor-destructing therapies can induce antigen release in situ, facilitating the injected DCs in triggering an antitumor immune response. METHODS: We tested in a phase I clinical study in advanced melanoma a chemo-immunotherapy approach based on unloaded IFN-DCs injected intratumorally one day after administration of dacarbazine. Primary endpoint of the study was treatment safety and tolerability. Secondary endpoints were immune and clinical responses of patients. RESULTS: Six patients were enrolled, and only three completed the treatment. The chemo-immunotherapy was well tolerated with no major side effects. Three patients showed temporary disease stabilization and two of them showed induction of T cells specific for tyrosinase, NY-ESO-1 and gp100. Of interest, one patient showing a remarkable long-term disease stabilization kept showing presence of tyrosinase specific T cells in PBMC and high infiltration of memory T cells in the tumor lesion at 21 months. CONCLUSION: We tested a chemo-immunotherapeutic approach based on IFN-DCs injected intratumorally one day after DTIC in advanced melanoma. The treatment was well tolerated, and clinical and immunological responses, including development of vitiligo, were observed, therefore warranting additional clinical studies aimed at evaluating efficacy of this approach. TRIAL REGISTRATION: Trial Registration Number not publicly available due to EudraCT regulations: https://www.clinicaltrialsregister.eu/doc/EU_CTR_FAQ.pdf.

Phase 2 clinical trial of rapamycin-resistant donor CD4+ Th2/Th1 (T-Rapa) cells after low-intensity allogeneic hematopoietic cell transplantation.

In experimental models, ex vivo induced T-cell rapamycin resistance occurred independent of T helper 1 (Th1)/T helper 2 (Th2) differentiation and yielded allogeneic CD4(+) T cells of increased in vivo efficacy that facilitated engraftment and permitted graft-versus-tumor effects while minimizing graft-versus-host disease (GVHD). To translate these findings, we performed a phase 2 multicenter clinical trial of rapamycin-resistant donor CD4(+) Th2/Th1 (T-Rapa) cells after allogeneic-matched sibling donor hematopoietic cell transplantation (HCT) for therapy of refractory hematologic malignancy. T-Rapa cell products, which expressed a balanced Th2/Th1 phenotype, were administered as a preemptive donor lymphocyte infusion at day 14 post-HCT. After T-Rapa cell infusion, mixed donor/host chimerism rapidly converted, and there was preferential immune reconstitution with donor CD4(+) Th2 and Th1 cells relative to regulatory T cells and CD8(+) T cells. The cumulative incidence probability of acute GVHD was 20% and 40% at days 100 and 180 post-HCT, respectively. There was no transplant-related mortality. Eighteen of 40 patients (45%) remain in sustained complete remission (range of follow-up: 42-84 months). These results demonstrate the safety of this low-intensity transplant approach and the feasibility of subsequent randomized studies to compare T-Rapa cell-based therapy with standard transplantation regimens.

Human bone marrow stromal cell confluence: effects on cell characteristics and methods of assessment.

BACKGROUND AIMS: Ex vivo expansion and serial passage of human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) is required to obtain sufficient quantities for clinical therapy. The BMSC confluence criteria used to determine passage and harvest timing vary widely, and the impact of confluence on BMSC properties remains controversial. The effects of confluence on BMSC properties were studied and confluence-associated markers were identified. METHODS: BMSC characteristics were analyzed as they grew from 50% to 100% confluence, including viability, population doubling time, apoptosis, colony formation, immunosuppression, surface marker expression, global gene expression and microRNA expression. In addition, culture supernatant protein, glucose, lactate and pH levels were analyzed. RESULTS: Confluence-dependent changes were detected in the expression of several cell surface markers: 39 culture supernatant proteins, 26 microRNAs and 2078 genes. Many of these surface markers, proteins, microRNAs and genes have been reported to be important in BMSC function. The pigment epithelium-derived factor/vascular endothelial growth factor ratio increased with confluence, but 80% and 100% confluent BMSCs demonstrated a similar level of immunosuppression of mixed lymphocyte reactions. In addition, changes in lactate and glucose levels correlated with BMSC density. CONCLUSIONS: BMSC characteristics change as confluence increases. 100% confluent BMSCs may have compromised pro-angiogenesis properties but may retain their immunomodulatory properties. Supernatant lactate and glucose levels can be used to estimate confluence and ensure consistency in passage and harvest timing. Flow cytometry or microRNA expression can be used to confirm that the BMSCs have been harvested at the appropriate confluence.

15 kDa granulysin causes differentiation of monocytes to dendritic cells but lacks cytotoxic activity.

Granulysin is expressed as two isoforms by human cytotoxic cells: a single mRNA gives rise to 15 kDa granulysin, a portion of which is cleaved to a 9 kDa protein. Studies with recombinant 9 kDa granulysin have demonstrated its cytolytic and proinflammatory properties, but much less is known about the biologic function of the 15 kDa isoform. In this study, we show that the subcellular localization and functions of 9 and 15 kDa granulysin are largely distinct. Nine kilodalton granulysin is confined to cytolytic granules that are directionally released following target cell recognition. In contrast, 15 kDa granulysin is located in distinct granules that lack perforin and granzyme B and that are released by activated cytolytic cells. Although recombinant 9 kDa granulysin is cytolytic against a variety of tumors and microbes, recombinant 15 kDa granulysin is not. The 15 kDa isoform is a potent inducer of monocytic differentiation to dendritic cells, but the 9 kDa isoform is not. In vivo, mice expressing granulysin show markedly improved antitumor responses, with increased numbers of activated dendritic cells and cytokine-producing T cells. Thus, the distinct functions of granulysin isoforms have major implications for diagnosis and potential new therapies for human disease.

Quality controls in cellular immunotherapies: rapid assessment of clinical grade dendritic cells by gene expression profiling.

Cell-based immunotherapies are among the most promising approaches for developing effective and targeted immune response. However, their clinical usefulness and the evaluation of their efficacy rely heavily on complex quality control assessment. Therefore, rapid systematic methods are urgently needed for the in-depth characterization of relevant factors affecting newly developed cell product consistency and the identification of reliable markers for quality control. Using dendritic cells (DCs) as a model, we present a strategy to comprehensively characterize manufactured cellular products in order to define factors affecting their variability, quality and function. After generating clinical grade human monocyte-derived mature DCs (mDCs), we tested by gene expression profiling the degrees of product consistency related to the manufacturing process and variability due to intra- and interdonor factors, and how each factor affects single gene variation. Then, by calculating for each gene an index of variation we selected candidate markers for identity testing, and defined a set of genes that may be useful comparability and potency markers. Subsequently, we confirmed the observed gene index of variation in a larger clinical data set. In conclusion, using high-throughput technology we developed a method for the characterization of cellular therapies and the discovery of novel candidate quality assurance markers.

Leukemia cells induce changes in human bone marrow stromal cells.

BACKGROUND: Bone marrow stromal cells (BMSCs) are multipotent cells that support angiogenesis, wound healing, and immunomodulation. In the hematopoietic niche, they nurture hematopoietic cells, leukemia, tumors and metastasis. BMSCs secrete of a wide range of cytokines, growth factors and matrix proteins which contribute to the pro-tumorigenic marrow microenvironment. The inflammatory cytokines IFN-γ and TNF-α change the BMSC secretome and we hypothesized that factors produced by tumors or leukemia would also affect the BMSC secretome and investigated the interaction of leukemia cells with BMSCs. METHODS: BMSCs from healthy subjects were co-cultured with three myeloid leukemia cell lines (TF-1, TF-1α and K562) using a trans-well system. Following co-culture, the BMSCs and leukemia cells were analyzed by global gene expression analysis and culture supernatants were analyzed for protein expression. As a control, CD34+ cells were also cocultured with BMSCs. RESULTS: Co-culture induced leukemia cell gene expression changes in stem cell pluripotency, TGF-ß signaling and carcinoma signaling pathways. BMSCs co-cultured with leukemia cells up-regulated a number of proinflammatory genes including IL-17 signaling-related genes and IL-8 and CCL2 levels were increased in co-culture supernatants. In contrast, purine metabolism, mTOR signaling and EIF2 signaling pathways genes were up-regulated in BMSCs co-cultured with CD34+ cells. CONCLUSIONS: BMSCs react to the presence of leukemia cells undergoing changes in the cytokine and chemokine secretion profiles. Thus, BMSCs and leukemia cells both contribute to the creation of a competitive niche more favorable for leukemia stem cells.

Simplified process for the production of anti-CD19-CAR-engineered T cells.

BACKGROUND AIMS: Adoptive immunotherapy with the use of chimeric antigen receptor (CAR)-engineered T cells specific for CD19 has shown promising results for the treatment of B-cell lymphomas and leukemia. This therapy involves the transduction of autologous T cells with a viral vector and the subsequent cell expansion. We describe a new, simplified method to produce anti-CD19-CAR T cells. METHODS: T cells were isolated from peripheral blood mononuclear cell (PBMC) with anti-CD3/anti-CD28 paramagnetic beads. After 2 days, the T cells were added to culture bags pre-treated with RetroNectin and loaded with the retroviral anti-CD19 CAR vector. The cells, beads and vector were incubated for 24 h, and a second transduction was then performed. No spinoculation was used. Cells were then expanded for an additional 9 days. RESULTS: The method was validated through the use of two PBMC products from a patient with B-cell chronic lymphoblastic leukemia and one PBMC product from a healthy subject. The two PBMC products from the patient with B-cell chronic lymphoblastic leukemia contained 11.4% and 12.9% T cells. The manufacturing process led to final products highly enriched in T cells with a mean CD3+ cell content of 98%, a mean expansion of 10.6-fold and a mean transduction efficiency of 68%. Similar results were obtained from the PBMCs of the first four patients with acute lymphoblastic leukemia treated at our institution. CONCLUSIONS: We developed a simplified, semi-closed system for the initial selection, activation, transduction and expansion of T cells with the use of anti-CD3/anti-CD28 beads and bags to produce autologous anti-CD19 CAR-transduced T cells to support an ongoing clinical trial.

Differential gene expression profile of first-generation and second-generation rapamycin-resistant allogeneic T cells.

BACKGROUND AIMS: We completed a phase II clinical trial evaluating rapamycin-resistant allogeneic T cells (T-rapa) and now have evaluated a T-rapa product manufactured in 6 days (T-rapa(6)) rather than 12 days (T-Rapa(12)). METHODS: Using gene expression microarrays, we addressed our hypothesis that the two products would express a similar phenotype. The products had similar phenotypes using conventional comparison methods of cytokine secretion and surface markers. RESULTS: Unsupervised analysis of 34,340 genes revealed that T-rapa(6) and T-rapa(12) products clustered together, distinct from culture input CD4(+) T cells. Statistical analysis of T-rapa(6) products revealed differential expression of 19.3% of genes (n = 6641) compared with input CD4(+) cells; similarly, 17.8% of genes (n = 6147) were differentially expressed between T-rapa(12) products and input CD4(+) cells. Compared with input CD4(+) cells, T-rapa(6) and T-rapa(12) products were similar in terms of up-regulation of major gene families (cell cycle, stress response, glucose catabolism, DNA metabolism) and down-regulation (inflammatory response, immune response, apoptosis, transcriptional regulation). However, when directly compared, T-rapa(6) and T-rapa(12) products showed differential expression of 5.8% of genes (n = 1994; T-rapa(6) vs. T-rapa(12)). CONCLUSIONS: Second-generation T-rapa(6) cells possess a similar yet distinct gene expression profile relative to first-generation T-rapa(12) cells and may mediate differential effects after adoptive transfer.

Blood immune cells as potential biomarkers predicting relapse-free survival of stage III/IV resected melanoma patients treated with peptide-based vaccination and interferon-alpha.

Introduction: Despite the recent approval of several therapies in the adjuvant setting of melanoma, tumor relapse still occurs in a significant number of completely resected stage III-IV patients. In this context, the use of cancer vaccines is still relevant and may increase the response to immune checkpoint inhibitors. We previously demonstrated safety, immunogenicity and preliminary evidence of clinical efficacy in stage III/IV resected melanoma patients subjected to a combination therapy based on peptide vaccination together with intermittent low-dose interferon-α2b, with or without dacarbazine preconditioning (https://www.clinicaltrialsregister.eu/ctr-search/search, identifier: 2008-008211-26). In this setting, we then focused on pre-treatment patient immune status to highlight possible factors associated with clinical outcome. Methods: Multiparametric flow cytometry was used to identify baseline immune profiles in patients' peripheral blood mononuclear cells and correlation with the patient clinical outcome. Receiver operating characteristic curve, Kaplan-Meier survival and principal component analyses were used to evaluate the predictive power of the identified markers. Results: We identified 12 different circulating T and NK cell subsets with significant (p ≤ 0.05) differential baseline levels in patients who later relapsed with respect to patients who remained free of disease. All 12 parameters showed a good prognostic accuracy (AUC>0.7, p ≤ 0.05) and 11 of them significantly predicted the relapse-free survival. Remarkably, 3 classifiers also predicted the overall survival. Focusing on immune cell subsets that can be analyzed through simple surface staining, three subsets were identified, namely regulatory T cells, CD56dimCD16- NK cells and central memory γδ T cells. Each subset showed an AUC>0.8 and principal component analysis significantly grouped relapsing and non-relapsing patients (p=0.034). These three subsets were used to calculate a combination score that was able to perfectly distinguish relapsing and non-relapsing patients (AUC=1; p=0). Noticeably, patients with a combined score ≥2 demonstrated a strong advantage in both relapse-free (p=0.002) and overall (p=0.011) survival as compared to patients with a score <2. Discussion: Predictive markers may be used to guide patient selection for personalized therapies and/or improve follow-up strategies. This study provides preliminary evidence on the identification of peripheral blood immune biomarkers potentially capable of predicting the clinical response to combined vaccine-based adjuvant therapies in melanoma.

Exploiting natural antiviral immunity for the control of pandemics: Lessons from Covid-19.

The outbreak of coronavirus disease 2019 (COVID-19), triggered by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the disruptive global consequences in terms of mortality and social and economic crises, have taught lessons that may help define strategies to better face future pandemics. Innate and intrinsic immunity form the front-line natural antiviral defense. They involve both tissue-resident and circulating cells, which can produce anti-viral molecules shortly after viral infection. Prototypes of these factors are type I interferons (IFN), antiviral cytokines with a long record of clinical use. During the last two years, there has been an impressive progress in understanding the mechanisms of both SARS-CoV-2 infection and the cellular and soluble antiviral responses occurring early after viral exposure. However, this information was not sufficiently translated into therapeutic approaches. Insufficient type I IFN activity probably accounts for disease progression in many patients. This results from both the multiple interfering mechanisms developed by SARS-CoV-2 to decrease type I IFN response and various pre-existing human deficits of type I IFN activity, inherited or auto-immune. Emerging data suggest that IFN-I-mediated boosting of patients' immunity, achieved directly through the exogenous administration of IFN-ß early post viral infection, or indirectly following inoculation of heterologous vaccines (e.g., Bacillus Calmette Guerin), might play a role against SARS-CoV-2. We review how recent insights on the viral and human determinants of critical COVID-19 pneumonia can foster clinical studies of IFN therapy. We also discuss how early therapeutic use of IFN-ß and prophylactic campaigns with live attenuated vaccines might prevent a first wave of new pandemic viruses.

Chimeric Antigen Receptor Immunotherapy for Solid Tumors: Choosing the Right Ingredients for the Perfect Recipe.

Chimeric antigen receptor T cell therapies are revolutionizing the clinical practice of hematological tumors, whereas minimal progresses have been achieved in the solid tumor arena. Multiple reasons have been ascribed to this slower pace: The higher heterogeneity, the hurdles of defining reliable tumor antigens to target, and the broad repertoire of immune escape strategies developed by solid tumors are considered among the major ones. Currently, several CAR therapies are being investigated in preclinical and early clinical trials against solid tumors differing in the type of construct, the cells that are engineered, and the additional signals included with the CAR constructs to overcome solid tumor barriers. Additionally, novel approaches in development aim at overcoming some of the limitations that emerged with the approved therapies, such as large-scale manufacturing, duration of manufacturing, and logistical issues. In this review, we analyze the advantages and challenges of the different approaches under development, balancing the scientific evidences supporting specific choices with the manufacturing and regulatory issues that are essential for their further clinical development.

Monocyte-derived DC maturation strategies and related pathways: a transcriptional view.

Ex vivo production of highly stimulator mature dendritic cells (DCs) for cellular therapy has been used to treat different pathological conditions with the aim of inducing a specific immune response. In the last decade, several protocols have been developed to mature monocyte-derived DCs: each one has led to the generation of DCs showing different phenotypes and stimulatory abilities, but it is not yet known which one is the best for inducing effective immune responses. We grouped several different maturation protocols according to the downstream pathways they activated and reviewed the shared features at a transcriptomic level to reveal the potential of DCs matured by each protocol to develop Th-polarized immune responses.

15 kDa Granulysin versus GM-CSF for monocytes differentiation: analogies and differences at the transcriptome level.

BACKGROUND: Granulysin is an antimicrobial and proinflammatory protein with several isoforms. While the 9 kDa isoform is a well described cytolytic molecule with pro-inflammatory activity, the functions of the 15 kDa isoform is less well understood. Recently it was shown that 15 kDa Granulysin can act as an alarmin that is able to activate monocytes and immature dendritic cells. Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) is a growth factor widely used in immunotherapy both for in vivo and ex vivo applications, especially for its proliferative effects. METHODS: We analyzed gene expression profiles of monocytes cultured with 15 kDa Granulysin or GM-CSF for 4, 12, 24 and 48 hours to unravel both similarities and differences between the effects of these stimulators. RESULTS: The analysis revealed a common signature induced by both factors at each time point, but over time, a more specific signature for each factor became evident. At all time points, 15 kDa Granulysin induced immune response, chemotaxis and cell adhesion genes. In addition, only 15 kDa Granulsyin induced the activation of pathways related to fundamental dendritic cell functions, such as co-stimulation of T-cell activation and Th1 development. GM-CSF specifically down-regulated genes related to cell cycle arrest and the immune response. More specifically, cytokine production, lymphocyte mediated immunity and humoral immune response were down-regulated at late time points. CONCLUSION: This study provides important insights on the effects of a novel agent, 15 kDa granulysin, that holds promise for therapeutic applications aimed at the activation of the immune response.

Concomitant detection of IFNα signature and activated monocyte/dendritic cell precursors in the peripheral blood of IFNα-treated subjects at early times after repeated local cytokine treatments.

BACKGROUND: Interferons alpha (IFNα) are the cytokines most widely used in clinical medicine for the treatment of cancer and viral infections. Among the immunomodulatory activities possibly involved in their therapeutic efficacy, the importance of IFNα effects on dendritic cells (DC) differentiation and activation has been considered. Despite several studies exploiting microarray technology to characterize IFNα mechanisms of action, there is currently no consensus on the core signature of these cytokines in the peripheral blood of IFNα-treated individuals, as well as on the existence of blood genomic and proteomic markers of low-dose IFNα administered as a vaccine adjuvant. METHODS: Gene profiling analysis with microarray was performed on PBMC isolated from melanoma patients and healthy individuals 24 hours after each repeated injection of low-dose IFNα, administered as vaccine adjuvant in two separate clinical trials. At the same time points, cytofluorimetric analysis was performed on CD14+ monocytes, to detect the phenotypic modifications exerted by IFNα on antigen presenting cells precursors. RESULTS: An IFNα signature was consistently observed in both clinical settings 24 hours after each repeated administration of the cytokine. The observed modulation was transient, and did not reach a steady state level refractory to further stimulations. The molecular signature observed ex vivo largely matched the one detected in CD14+ monocytes exposed in vitro to IFNα, including the induction of CXCL10 at the transcriptional and protein level. Interestingly, IFNα ex vivo signature was paralleled by an increase in the percentage and expression of costimulatory molecules by circulating CD14+/CD16+ monocytes, indicated as natural precursors of DC in response to danger signals. CONCLUSIONS: Our results provide new insights into the identification of a well defined molecular signature as biomarker of IFNα administered as immune adjuvants, and for the characterization of new molecular and cellular players, such as CXCL10 and CD14+/CD16+ cells, mediating and possibly predicting patient response to these cytokines.

Activation of TNF receptor 2 in microglia promotes induction of anti-inflammatory pathways.

Fine regulation of the innate immune response following brain injury or infection is important to avoid excessive activation of microglia and its detrimental consequences on neural cell viability and function. To get insights on the molecular networks regulating microglia activation, we analyzed expression, regulation and functional relevance of tumor necrosis factor receptors (TNFR) 2 in cultured mouse microglia. We found that microglia upregulate TNFR2 mRNA and protein and shed large amounts of soluble TNFR2, but not TNFR1, in response to pro-inflammatory stimuli and through activation of TNFR2 itself. By microarray analysis, we demonstrate that TNFR2 stimulation in microglia regulates expression of genes involved in immune processes, including molecules with anti-inflammatory and neuroprotective function like granulocyte colony-stimulating factor, adrenomedullin and IL-10. In addition, we identify IFN-γ as a regulator of the balance between pro- and anti-inflammatory/neuroprotective factors induced by TNFR2 stimulation. These data indicate that, through TNFR2, microglia may contribute to the counter-regulatory response activated in neuropathological conditions.