Reversing vemurafenib-resistance in primary melanoma cells by combined romidepsin and type I IFN treatment through blocking of tumorigenic signals and induction of immunogenic effects.

BRAFV600 mutations are the most common oncogenic alterations in melanoma cells, supporting proliferation, invasion, metastasis and immune evasion. In patients, these aberrantly activated cellular pathways are inhibited by BRAFi whose potent antitumor effect and therapeutic potential are dampened by the development of resistance. Here, by using primary melanoma cell lines, generated from lymph node lesions of metastatic patients, we show that the combination of two FDA-approved drugs, the histone deacetylate inhibitor (HDCAi) romidepsin and the immunomodulatory agent IFN-α2b, reduces melanoma proliferation, long-term survival and invasiveness and overcomes acquired resistance to the BRAFi vemurafenib (VEM). Targeted resequencing revealed that each VEM-resistant melanoma cell line and the parental counterpart are characterized by a distinctive and similar genetic fingerprint, shaping the differential and specific antitumor modulation of MAPK/AKT pathways by combined drug treatment. By using RNA-sequencing and functional in vitro assays, we further report that romidepsin-IFN-α2b treatment restores epigenetically silenced immune signals, modulates MITF and AXL expression and induces both apoptosis and necroptosis in sensitive and VEM-resistant primary melanoma cells. Moreover, the immunogenic potential of drug-treated VEM-resistant melanoma cells results significantly enhanced, given the increased phagocytosis rate of these cells by dendritic cells, which in turn exhibit also a selective down-modulation of the immune checkpoint TIM-3. Overall, our results provide evidence that combined epigenetic-immune drugs can overcome VEM resistance of primary melanoma cells by oncogenic and immune pathways reprogramming, and pave the way for rapidly exploiting this combination to improve BRAFi-resistant metastatic melanoma treatment, also via reinforcement of immune checkpoint inhibitor therapy.

LOX-1 as a natural IFN-alpha-mediated signal for apoptotic cell uptake and antigen presentation in dendritic cells.

The identification of molecules responsible for apoptotic cell (AC) uptake by dendritic cells (DCs) and induction of T-cell immunity against AC-associated antigens is a challenge in immunology. DCs differentiated in the presence of interferon-alpha (IFN-alpha-conditioned DCs) exhibit a marked phagocytic activity and a special attitude in inducing CD8(+) T-cell response. In this study, we found marked overexpression of the scavenger receptor oxidized low-density lipoprotein receptor 1 (LOX-1) in IFN-alpha-conditioned DCs, which was associated with increased levels of genes belonging to immune response families and high competence in inducing T-cell immunity against antigens derived from allogeneic apoptotic lymphocytes. In particular, the capture of ACs by IFN-alpha DCs led to a substantial subcellular rearrangement of major histocompatibility complex class I and class II molecules, along with enhanced cross-priming of autologous CD8(+) T cells and CD4(+) T-cell activation. Remarkably, AC uptake, CD8(+) T-cell cross-priming, and, to a lesser extent, priming of CD4(+) T lymphocytes were inhibited by a neutralizing antibody to the scavenger receptor LOX-1 protein. These results unravel a novel LOX-1-dependent pathway by which IFN-alpha can, under both physiologic and pathologic conditions, render DCs fully competent for presenting AC-associated antigens for cross-priming CD8(+) effector T cells, concomitantly with CD4(+) T helper cell activation.

Tumor-on-a-chip platforms to study cancer-immune system crosstalk in the era of immunotherapy.

Immunotherapy is a powerful therapeutic approach able to re-educate the immune system to fight cancer. A key player in this process is the tumor microenvironment (TME), which is a dynamic entity characterized by a complex array of tumor and stromal cells as well as immune cell populations trafficking to the tumor site through the endothelial barrier. Recapitulating these multifaceted dynamics is critical for studying the intimate interactions between cancer and the immune system and to assess the efficacy of emerging immunotherapies, such as immune checkpoint inhibitors (ICIs) and adoptive cell-based products. Microfluidic devices offer a unique technological approach to build tumor-on-a-chip reproducing the multiple layers of complexity of cancer-immune system crosstalk. Here, we seek to review the most important biological and engineering developments of microfluidic platforms for studying cancer-immune system interactions, in both solid and hematological tumors, highlighting the role of the vascular component in immune trafficking. Emphasis is given to image processing and related algorithms for real-time monitoring and quantitative evaluation of the cellular response to microenvironmental dynamic changes. The described approaches represent a valuable tool for preclinical evaluation of immunotherapeutic strategies.

Type I IFN-dependent antibody response at the basis of sex dimorphism in the outcome of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, induces severe pneumonia mainly in elderly males. Epidemiological data clearly indicate sex-based differences in disease outcomes, with men accounting for about 70 % of deaths, despite similar susceptibility to infection. It is well known that females are endowed with higher capacity to produce antibodies, which correlates with viral clearance and disease resolution in the context of SARS-Cov-2 infection. Many X-linked immune genes escape X inactivation showing biallelic expression in female immune cells, particularly in plasmacytoid dendritic cells (pDCs). PDCs are more active in females and endowed with high capability to induce IFN-α-mediated B cell activation and differentiation into antibody-producing plasma cells throughout epigenetic mechanisms linked to trained immunity. Thus, we hypothesize that following SARS-CoV-2 infection, epigenetic modifications of X-linked genes involved in pDC-mediated type I IFN (IFN-I) signaling occurs more effectively in females, for inducing neutralizing antibody response as an immune correlate driving sex-biased disease outcome.

Potent immune response against HIV-1 and protection from virus challenge in hu-PBL-SCID mice immunized with inactivated virus-pulsed dendritic cells generated in the presence of IFN-alpha.

A major challenge of AIDS research is the development of therapeutic vaccine strategies capable of inducing the humoral and cellular arms of the immune responses against HIV-1. In this work, we evaluated the capability of DCs pulsed with aldrithiol-2-inactivated HIV-1 in inducing a protective antiviral human immune response in SCID mice reconstituted with human PBL (hu-PBL-SCID mice). Immunization of hu-PBL-SCID mice with DCs generated after exposure of monocytes to GM-CSF/IFN-alpha (IFN-DCs) and pulsed with inactivated HIV-1 resulted in a marked induction of human anti-HIV-1 antibodies, which was associated with the detection of anti-HIV neutralizing activity in the serum. This vaccination schedule also promoted the generation of a human CD8+ T cell response against HIV-1, as measured by IFN-gamma Elispot analysis. Notably, when the hu-PBL-SCID mice immunized with antigen-pulsed IFN-DCs were infected with HIV-1, inhibition of virus infection was observed as compared with control animals. These results suggest that IFN-DCs pulsed with inactivated HIV-1 can represent a valuable approach of immune intervention in HIV-1-infected patients.

Autoantibodies Specific to ERα are Involved in Tamoxifen Resistance in Hormone Receptor Positive Breast Cancer.

Tamoxifen resistance is a major hurdle in the treatment of estrogen receptor (ER)-positive breast cancer. The mechanisms of tamoxifen resistance are not fully understood although several underlying molecular events have been suggested. Recently, we identified autoantibodies reacting with membrane-associated ERα (anti-ERα Abs) in sera of breast cancer patients, able to promote tumor growth. Here, we investigated whether anti-ERα Abs purified from sera of ER-positive breast cancer patients could contribute to tamoxifen resistance. Anti-ERα Abs inhibited tamoxifen-mediated effects on cell cycle and proliferation in MCF-7 cells. Moreover, anti-ERα Abs hampered the tamoxifen-mediated reduction of tumor growth in SCID mice xenografted with breast tumor. Notably, simvastatin-mediated disaggregation of lipid rafts, where membrane-associated ERα is embedded, restored tamoxifen sensitivity, preventing anti-ERα Abs effects. In conclusion, detection of serum anti-ERα Abs may help predict tamoxifen resistance and concur to appropriately inform therapeutic decisions concerning hormone therapy in ER-positive breast cancer patients.

Impaired IFN-α-mediated signal in dendritic cells differentiates active from latent tuberculosis.

Individuals exposed to Mycobacterium tuberculosis (Mtb) may be infected and remain for the entire life in this condition defined as latent tuberculosis infection (LTBI) or develop active tuberculosis (TB). Among the multiple factors governing the outcome of the infection, dendritic cells (DCs) play a major role in dictating antibacterial immunity. However, current knowledge on the role of the diverse components of human DCs in shaping specific T-cell response during Mtb infection is limited. In this study, we performed a comparative evaluation of peripheral blood circulating DC subsets as well as of monocyte-derived Interferon-α DCs (IFN-DCs) from patients with active TB, subjects with LTBI and healthy donors (HD). The proportion of circulating myeloid BDCA3+ DCs (mDC2) and plasmacytoid CD123+ DCs (pDCs) declined significantly in active TB patients compared to HD, whereas the same subsets displayed a remarkable activation in LTBI subjects. Simultaneously, the differentiation of IFN-DCs from active TB patients resulted profoundly impaired compared to those from LTBI and HD individuals. Importantly, the altered developmental trait of IFN-DCs from active TB patients was associated with down-modulation of IFN-linked genes, marked changes in molecular signaling conveying antigen (Ag) presentation and full inability to induce Ag-specific T cell response. Thus, these data reveal an important role of IFN-α in determining the induction of Mtb-specific immunity.

Antitumor Effects of Epidrug/IFNα Combination Driven by Modulated Gene Signatures in Both Colorectal Cancer and Dendritic Cells.

Colorectal cancer results from the progressive accumulation of genetic and epigenetic alterations. IFN signaling defects play an important role in the carcinogenesis process, in which the inability of IFN transcription regulatory factors (IRF) to access regulatory sequences in IFN-stimulated genes (ISG) in tumors and in immune cells may be pivotal. We reported that low-dose combination of two FDA-approved epidrugs, azacytidine (A) and romidepsin (R), with IFNα2 (ARI) hampers the aggressiveness of both colorectal cancer metastatic and stem cells in vivo and triggers immunogenic cell death signals that stimulate dendritic cell (DC) function. Here, we investigated the molecular signals induced by ARI treatment and found that this drug combination increased the accessibility to regulatory sequences of ISGs and IRFs that were epigenetically silenced in both colorectal cancer cells and DCs. Likewise, specific ARI-induced histone methylation and acetylation changes marked epigenetically affected ISG promoters in both metastatic cancer cells and DCs. Analysis by ChIP-seq confirmed such ARI-induced epigenetically regulated IFN signature. The activation of this signal endowed DCs with a marked migratory capability. Our results establish a direct correlation between reexpression of silenced ISGs by epigenetic control and ARI anticancer activity and provide new knowledge for the development of innovative combined therapeutic strategies for colorectal cancer. Cancer Immunol Res; 5(7); 604-16. ©2017 AACR.

3D Microfluidic model for evaluating immunotherapy efficacy by tracking dendritic cell behaviour toward tumor cells.

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches.

IFN-α potentiates the direct and immune-mediated antitumor effects of epigenetic drugs on both metastatic and stem cells of colorectal cancer.

Epigenetic alterations, including dysregulated DNA methylation and histone modifications, govern the progression of colorectal cancer (CRC). Cancer cells exploit epigenetic regulation to control cellular pathways, including apoptotic and metastatic signals. Since aberrations in epigenome can be pharmacologically reversed by DNA methyltransferase and histone deacetylase inhibitors, epigenetics in combination with standard agents are currently envisaged as a new therapeutic frontier in cancer, expected to overcome drug resistance associated with current treatments. In this study, we challenged this idea and demonstrated that the combination of azacitidine and romidepsin with IFN-α owns a high therapeutic potential, targeting the most aggressive cellular components of CRC, such as metastatic cells and cancer stem cells (CSCs), via tight control of key survival and death pathways. Moreover, the antitumor efficacy of this novel pharmacological approach is associated with induction of signals of immunogenic cell death. Of note, a previously undisclosed key role of IFN-α in inducing both antiproliferative and pro-apoptotic effects on CSCs of CRC was also found. Overall, these findings open a new frontier on the suitability of IFN-α in association with epigenetics as a novel and promising therapeutic approach for CRC management.

Inter-annual variations of planktonic food webs in the northern Adriatic Sea.

The temporal dynamics of microphytoplankton, microzooplankton and mesozooplankton were monitored over 37 months in the Adriatic Sea in order to identify alterations in the plankton structures, which can lead to, or enhance the production of macro-aggregates, that affected the entire northern basin in summers 2000 and 2002, and to assess any negative effects of mucilage on plankton temporal patterns. Samples were collected monthly, from June 1999 to July 2002, on three transects at 9 stations across the northern and central Adriatic Sea. Besides the high year-to-year variations in abundances and taxonomical composition, plankton communities only showed a clear seasonal succession during 2001, when since April a grazing food web developed and was able to control large sized phytoplankton increase. In spring-summer 2000 and 2002 consumer abundances remained quite low and the dominant mesozooplankton summer species (Penilia avirostris) did not reach its usual summer maximum. The lack of an efficient top control was more evident on the northernmost transect, where generally grazing food web prevails over the microbial one. A large part of the microphytoplankton blooms, although not particularly intense, was exported to the bottom in the particulate phase, where it was processed by bacteria, enhancing the production of refractory dissolved material.

The natural alliance between type I interferon and dendritic cells and its role in linking innate and adaptive immunity.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and thus play a pivotal role in induction of the immune response. Recent studies in both human and mouse models have shown that type I IFN, cytokines originally characterized for their antiviral activity and exerting multiple biologic effects, efficiently promote the differentiation and activation of DCs. These observations, together with the findings that DCs can express biologically relevant levels of type I interferon (IFN) and, in particular, that high amounts of these cytokines are released by specialized DC precursors (i.e., plasmacytoid DCs) in response to viral infections, strongly suggest the existence of a natural alliance between type I IFN and DCs, which is instrumental in ensuring an efficient immune response to both infectious agents and tumors. Further recent knowledge on the interactions between type I IFN and DCs emphasizes the importance of these cytokines in linking innate and adaptive immunity and may lead to new perspectives in their use as vaccine adjuvants as well as in strategies for the development of DC-based vaccines.

IFN-α regulates Blimp-1 expression via miR-23a and miR-125b in both monocytes-derived DC and pDC.

Type I interferon (IFN-I) have emerged as crucial mediators of cellular signals controlling DC differentiation and function. Human DC differentiated from monocytes in the presence of IFN-α (IFN-α DC) show a partially mature phenotype and a special capability of stimulating CD4+ T cell and cross-priming CD8+ T cells. Likewise, plasmacytoid DC (pDC) are blood DC highly specialized in the production of IFN-α in response to viruses and other danger signals, whose functional features may be shaped by IFN-I. Here, we investigated the molecular mechanisms stimulated by IFN-α in driving human monocyte-derived DC differentiation and performed parallel studies on peripheral unstimulated and IFN-α-treated pDC. A specific miRNA signature was induced in IFN-α DC and selected miRNAs, among which miR-23a and miR-125b, proved to be negatively associated with up-modulation of Blimp-1 occurring during IFN-α-driven DC differentiation. Of note, monocyte-derived IFN-α DC and in vitro IFN-α-treated pDC shared a restricted pattern of miRNAs regulating Blimp-1 expression as well as some similar phenotypic, molecular and functional hallmarks, supporting the existence of a potential relationship between these DC populations. On the whole, these data uncover a new role of Blimp-1 in human DC differentiation driven by IFN-α and identify Blimp-1 as an IFN-α-mediated key regulator potentially accounting for shared functional features between IFN-α DC and pDC.

CD2+/CD14+ monocytes rapidly differentiate into CD83+ dendritic cells.

Since denditric cells (DC) represent the main players linking innate and adaptive immunity, their prompt generation from blood cells would be instrumental for an efficient immune response to infections. Consistent with this, CD2+ monocytes were found to express the DC maturation marker CD83, along with acquisition of high antigen-presenting activity, after a surprisingly short time in culture. This rapid process is associated with expression of IFN-alpha/beta genes and secretion of low levels of pro-inflammatory cytokines. Exposure of monocytes to IFN-alpha, but not to IL-4, induced persistence of CD2+/CD83+ cells, which were fully competent in stimulating primary responses by naive T cells. These results unravel the natural pathway by which infection-induced signals rapidly transform pre-armed monocytes into active DC.