Emerging Role of Non-Coding RNAs in Regulation of T-Lymphocyte Function.

T-lymphocytes (T cells) play a major role in adaptive immunity and current immune checkpoint inhibitor-based cancer treatments. The regulation of their function is complex, and in addition to cytokines, receptors and transcription factors, several non-coding RNAs (ncRNAs) have been shown to affect differentiation and function of T cells. Among these non-coding RNAs, certain small microRNAs (miRNAs) including miR-15a/16-1, miR-125b-5p, miR-99a-5p, miR-128-3p, let-7 family, miR-210, miR-182-5p, miR-181, miR-155 and miR-10a have been well recognized. Meanwhile, IFNG-AS1, lnc-ITSN1-2, lncRNA-CD160, NEAT1, MEG3, GAS5, NKILA, lnc-EGFR and PVT1 are among long non-coding RNAs (lncRNAs) that efficiently influence the function of T cells. Recent studies have underscored the effects of a number of circular RNAs, namely circ_0001806, hsa_circ_0045272, hsa_circ_0012919, hsa_circ_0005519 and circHIPK3 in the modulation of T-cell apoptosis, differentiation and secretion of cytokines. This review summarizes the latest news and regulatory roles of these ncRNAs on the function of T cells, with widespread implications on the pathophysiology of autoimmune disorders and cancer.

The Immunomodulation Potential of Exosomes in Tumor Microenvironment.

Exosomes are lipid bilayer particles that originated from almost all types of cells and play an important role in intercellular communication. Tumor-derived exosomes contain large amounts of noncoding RNA, DNA, and proteins, which can be transferred into recipient cells as functional components in exosomes. These exosomal functional constituents depend on the originating cells, and it has been proved that types and numbers of exosomal components differ in cancer patients and healthy individuals. This review summarizes the role of tumor-derived exosomes in immunomodulation and discusses the application of exosomes in immunotherapy in cancers. Overall, exosomes isolated from cancer cells are turned out to promote immune evasion and interfere with immune responses in tumors through inducing apoptosis of CD8+ T cells, facilitating generation of Tregs, suppressing natural killer (NK) cell cytotoxicity, inhibiting maturation and differentiation of monocyte, and enhancing suppressive function of myeloid-derived suppressor cells (MDSCs). Mechanistically, exosomal functional components play a significant role in the immunomodulation in cancers. Moreover, based on the existing studies, exosomes could potentially serve as therapeutic delivery vehicles, noninvasive biomarkers, and immunotherapeutic vaccines for various types of cancers.

A prospective study revealing the role of an immune-related eRNA, WAKMAR2, in breast cancer.

Enhancer RNAs (eRNAs) are a subclass of non-coding RNAs that are generated during the transcription of enhancer regions and play an important role in tumourigenesis. In this study, we focused on the crucial eRNAs that participate in immune responses in invasive breast cancer (IBC). We first used The Cancer Genome Atlas and Human enhancer RNA Atlas to screen for tissue-specific eRNAs and their target genes. Through Pearson correlation analysis with immune genes, the eRNA WAKMAR2 was identified as a key candidate involved in IBC. Our further research suggested that WAKMAR2 is crucial in regulating the tumour microenvironment and may function by regulating immune-related genes, including IL27RA, RAC2, FABP7, IGLV1-51, IGHA1, and IGHD. Quantitative reverse transcription-polymerase chain reaction was used to detect the expression of WAKMAR2 in IBC and normal tissues, and the effect of WAKMAR2 on the regulation of downstream genes in MB-231 and MCF7 cells was studied in vitro. WAKMAR2 was found to be highly involved in tumour immunity and was downregulated in IBC tissues. Furthermore, the expression of WAKMAR2 and its target genes was observed at the pan-cancer level. This study provides evidence to suggest new potential targets for the treatment of breast cancer.

Nc886, a Novel Suppressor of the Type I Interferon Response Upon Pathogen Intrusion.

Interferons (IFNs) are a crucial component in the innate immune response. Especially the IFN-ß signaling operates in most cell types and plays a key role in the first line of defense upon pathogen intrusion. The induction of IFN-ß should be tightly controlled, because its hyperactivation can lead to tissue damage or autoimmune diseases. Activation of the IFN-ß promoter needs Interferon Regulatory Factor 3 (IRF3), together with Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Activator Protein 1 (AP-1). Here we report that a human noncoding RNA, nc886, is a novel suppressor for the IFN-ß signaling and inflammation. Upon treatment with several pathogen-associated molecular patterns and viruses, nc886 suppresses the activation of IRF3 and also inhibits NF-κB and AP-1 via inhibiting Protein Kinase R (PKR). These events lead to decreased expression of IFN-ß and resultantly IFN-stimulated genes. nc886's role might be to restrict the IFN-ß signaling from hyperactivation. Since nc886 expression is regulated by epigenetic and environmental factors, nc886 might explain why innate immune responses to pathogens are variable depending on biological settings.

Network- and systems-based re-engineering of dendritic cells with non-coding RNAs for cancer immunotherapy.

Dendritic cells (DCs) are professional antigen-presenting cells that induce and regulate adaptive immunity by presenting antigens to T cells. Due to their coordinative role in adaptive immune responses, DCs have been used as cell-based therapeutic vaccination against cancer. The capacity of DCs to induce a therapeutic immune response can be enhanced by re-wiring of cellular signalling pathways with microRNAs (miRNAs). Methods: Since the activation and maturation of DCs is controlled by an interconnected signalling network, we deploy an approach that combines RNA sequencing data and systems biology methods to delineate miRNA-based strategies that enhance DC-elicited immune responses. Results: Through RNA sequencing of IKKß-matured DCs that are currently being tested in a clinical trial on therapeutic anti-cancer vaccination, we identified 44 differentially expressed miRNAs. According to a network analysis, most of these miRNAs regulate targets that are linked to immune pathways, such as cytokine and interleukin signalling. We employed a network topology-oriented scoring model to rank the miRNAs, analysed their impact on immunogenic potency of DCs, and identified dozens of promising miRNA candidates, with miR-15a and miR-16 as the top ones. The results of our analysis are presented in a database that constitutes a tool to identify DC-relevant miRNA-gene interactions with therapeutic potential (https://www.synmirapy.net/dc-optimization). Conclusions: Our approach enables the systematic analysis and identification of functional miRNA-gene interactions that can be experimentally tested for improving DC immunogenic potency.

Identification of the Cryptic HLA-I Immunopeptidome.

The success of cancer immunotherapy relies on the ability of cytotoxic T cells to specifically recognize and eliminate tumor cells based on peptides presented by HLA-I. Although the peptide epitopes that elicit the corresponding immune response often remain unidentified, it is generally assumed that neoantigens, due to tumor-specific mutations, are the most common targets. Here, we used a mass spectrometric approach to show an underappreciated class of epitopes that accounts for up to 15% of HLA-I peptides for certain HLA alleles in various tumors and patients. These peptides are translated from cryptic open reading frames in supposedly noncoding regions in the genome and are mostly unidentifiable with conventional computational analyses of mass spectrometry (MS) data. Our approach, Peptide-PRISM, identified thousands of such cryptic peptides in tumor immunopeptidomes. About 20% of these HLA-I peptides represented the C-terminus of the corresponding translation product, suggesting frequent proteasome-independent processing. Our data also revealed HLA-I allele-dependent presentation of cryptic peptides, with HLA-A*03 and HLA-A*11 presenting the highest percentage of cryptic peptides. Our analyses refute the reported frequent presentation of HLA peptides generated by proteasome-catalyzed peptide splicing. Thus, Peptide-PRISM represents an important step toward comprehensive identification of HLA-I immunopeptidomes and reveals cryptic peptides as an abundant class of epitopes with potential relevance for novel immunotherapeutic approaches.

Noncoding RNAs: the shot callers in tumor immune escape.

Immunotherapy, designed to exploit the functions of the host immune system against tumors, has shown considerable potential against several malignancies. However, the utility of immunotherapy is heavily limited due to the low response rate and various side effects in the clinical setting. Immune escape of tumor cells may be a critical reason for such low response rates. Noncoding RNAs (ncRNAs) have been identified as key regulatory factors in tumors and the immune system. Consequently, ncRNAs show promise as targets to improve the efficacy of immunotherapy in tumors. However, the relationship between ncRNAs and tumor immune escape (TIE) has not yet been comprehensively summarized. In this review, we provide a detailed account of the current knowledge on ncRNAs associated with TIE and their potential roles in tumor growth and survival mechanisms. This review bridges the gap between ncRNAs and TIE and broadens our understanding of their relationship, providing new insights and strategies to improve immunotherapy response rates by specifically targeting the ncRNAs involved in TIE.

Non-coding RNAs in CD8 T cell biology.

CD8 T cells are among the most vigorous soldiers of the immune system that fight viral infections and cancer. CD8 T cell development, maintenance, activation and differentiation are under the tight control of multiple transcriptional and post-transcriptional networks. Over the last two decades it has become clear that non-coding RNAs (ncRNAs), which consist of microRNAs (miRNAs) and long ncRNAs (lncRNAs), have emerged as global biological regulators. While our understanding of the function of specific miRNAs has increased since the discovery of RNA interference, it is still very limited, and the field of lncRNAs is just starting to blossom. Here we will summarize our knowledge on the role of ncRNAs in CD8 T cell biology, including differentiation into memory and exhausted cells.

Noncoding RNAs as potential mediators of resistance to cancer immunotherapy.

Substantial evolution in cancer therapy has been witnessed lately, steering mainly towards immunotherapeutic approaches, replacing or in combination with classical therapies. Whereas the use of various immunotherapy approaches, such as adoptive T cell therapy, genetically-modified T cells, or immune checkpoint inhibitors, has been a triumph for cancer immunotherapy, the great challenge is the ability of the immune system to sustain long lasting anti-tumor response. Additionally, epigenetic changes in a suppressive tumor microenvironment can pertain to T cell exhaustion, limiting their functionality. Noncoding RNAs (ncRNAs) have emerged over the last years as key players in epigenetic regulation. Among those, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) have been studied extensively for their potential role in regulating tumor immunity through direct regulation of genes involved in immune activation or suppression. In this review, we will provide an overview of contemporary approaches for cancer immunotherapy and will present the current state of knowledge implicating miRNAs and lncRNAs in regulating immune response against human cancer and their potential implications in resistance to cancer immunotherapy, with main emphasis on immune checkpoints regulation.

The Magnitude of IFN-γ Responses Is Fine-Tuned by DNA Architecture and the Non-coding Transcript of Ifng-as1.

Interferon gamma (IFN-γ), critical for host defense and tumor surveillance, requires tight control of its expression. Multiple cis-regulatory elements exist around Ifng along with a non-coding transcript, Ifng-as1 (also termed NeST). Here, we describe two genetic models generated to dissect the molecular functions of this locus and its RNA product. DNA deletion within the Ifng-as1 locus disrupted chromatin organization of the extended Ifng locus, impaired Ifng response, and compromised host defense. Insertion of a polyA signal ablated the Ifng-as1 full-length transcript and impaired host defense, while allowing proper chromatin structure. Transient knockdown of Ifng-as1 also reduced IFN-γ production. In humans, discordant expression of IFNG and IFNG-AS1 was evident in memory T cells, with high expression of this long non-coding RNA (lncRNA) and low expression of the cytokine. These results establish Ifng-as1 as an important regulator of Ifng expression, as a DNA element and transcribed RNA, involved in dynamic and cell state-specific responses to infection.

RIG-I like receptor sensing of host RNAs facilitates the cell-intrinsic immune response to KSHV infection.

The RIG-I like receptors (RLRs) RIG-I and MDA5 are cytosolic RNA helicases best characterized as restriction factors for RNA viruses. However, evidence suggests RLRs participate in innate immune recognition of other pathogens, including DNA viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus and the etiological agent of Kaposi's sarcoma and primary effusion lymphoma (PEL). Here, we demonstrate that RLRs restrict KSHV lytic reactivation and we demonstrate that restriction is facilitated by the recognition of host-derived RNAs. Misprocessed noncoding RNAs represent an abundant class of RIG-I substrates, and biochemical characterizations reveal that an infection-dependent reduction in the cellular triphosphatase DUSP11 results in an accumulation of select triphosphorylated noncoding RNAs, enabling their recognition by RIG-I. These findings reveal an intricate relationship between RNA processing and innate immunity, and demonstrate that an antiviral innate immune response can be elicited by the sensing of misprocessed cellular RNAs.

Oxidative Stress in Mesenchymal Stem Cell Senescence: Regulation by Coding and Noncoding RNAs.

SIGNIFICANCE: Mesenchymal stem cells (MSCs), adult stem cells with the potential of differentiation into mesodermal lineages, play an important role in tissue homeostasis and regeneration. In different organs, a subpopulation of MSCs is located near the vasculature and possibly represents the original source of lineage-committed mesenchymal progenitors. Recent Advances: The plasticity and immune characteristics of MSCs render them a preferential tool for regenerative cell therapy. CRITICAL ISSUES: The culture expansion needed before MSC transplantation is associated with cellular senescence. Moreover, accelerated senescence of the total and perivascular MSC pool has been observed in humans and mouse models of premature aging disorders. MSC dysfunction is acknowledged as a culprit for the aging-associated degeneration of mesodermal tissues, but the underlying epigenetic pathways remain elusive. This article reviews current understanding of mechanisms impinging on MSC health, including oxidative stress, Nrf2-antioxidant responsive element activity, sirtuins, noncoding RNAs, and PKCs. FUTURE DIRECTIONS: We provide evidence that epigenetic profiling of MSCs is utilitarian to the prediction of therapeutic outcomes. In addition, strategies that target oxidative stress-associated mechanisms represent promising approaches to counteract the detrimental effect of age and senescence in MSCs.-Antioxid. Redox Signal. 29, 864-879.

Noncoding RNAs: Master Regulators of Inflammatory Signaling.

Inflammatory signaling underlies many diseases, from arthritis to cancer. Our understanding of inflammation has thus far been limited to the world of proteins, because we are only just beginning to understand the role that noncoding RNAs (ncRNA) might play. It is now clear that ncRNA do not constitute transcriptional 'noise' but instead harbor physiological functions in controlling signaling pathways. In this review, we cover the newly discovered mechanisms and functions of ncRNAs in the regulation of inflammatory signaling. We also describe advances in experimental techniques allowing this field of research to take root. These findings have opened new avenues for putative therapeutic intervention in inflammatory diseases, which may be seen translated into clinical outcomes in the future.

Noncoding RNAs and immune checkpoints-clinical implications as cancer therapeutics.

A major mechanism of tumor development and progression is silencing of the patient's immune response to cancer-specific antigens. Defects in the so-called cancer immunity cycle may occur at any stage of tumor development. Within the tumor microenvironment, aberrant expression of immune checkpoint molecules with activating or inhibitory effects on T lymphocytes induces immune tolerance and cellular immune escape. Targeting immune checkpoint molecules such as programmed cell death protein 1 (PD-1) and its ligand PD-L1 with specific antibodies has proven to be a major advance in the treatment of several types of cancer. Another way to therapeutically influence the tumor microenvironment is by modulating the levels of microRNAs (miRNAs), small noncoding RNAs that shuttle bidirectionally between malignant and tumor microenvironmental cells. These small RNA transcripts have two features: (a) their expression is quite specific to distinct tumors, and (b) they are involved in early regulation of immune responses. Consequently, miRNAs may be ideal molecules for use in cancer therapy. Many miRNAs are aberrantly expressed in human cancer cells, opening new opportunities for cancer therapy, but the exact functions of these miRNAs and their interactions with immune checkpoint molecules have yet to be investigated. This review summarizes recently reported findings about miRNAs as modulators of immune checkpoint molecules and their potential application as cancer therapeutics in clinical practice.

A New RNA-Based Adjuvant Enhances Virus-Specific Vaccine Responses by Locally Triggering TLR- and RLH-Dependent Effects.

Among innovative adjuvants conferring a Th1-shift, RNAdjuvant is a promising candidate. This adjuvant consists of a 547-nt uncapped noncoding ssRNA containing polyU repeats that is stabilized by a cationic carrier peptide. Whereas vaccination of mice with an influenza subunit vaccine induced moderate virus-specific IgG1, vaccination together with RNAdjuvant significantly enhanced this IgG1 and additionally promoted the formation of IgG2b/c, which is indicative of Th1 responses. Furthermore, such sera neutralized influenza virus, whereas this effect was not detected upon vaccination with the subunit vaccine alone. Similarly, upon vaccination with virus-like particles displaying vesicular stomatitis virus G protein, RNAdjuvant promoted the formation of virus-specific IgG2b/c and enhanced neutralizing IgG responses to an extent that mice were protected against lethal virus infection. RNAdjuvant induced dendritic cells to upregulate activation markers and produce IFN-I. Although these effects were strictly TLR7 dependent, RNAdjuvant-mediated augmentation of vaccine responses needed concurrent TLR and RIG-I-like helicase signaling. This was indicated by the absence of the adjuvant effect in vaccinated MyD88-/-Cardif-/- mice, which are devoid of TLR (with the exception of TLR3) and RIG-I-like helicase signaling, whereas in vaccinated MyD88-/- mice the adjuvant effect was reduced. Notably, i.m. RNAdjuvant injection induced local IFN-I responses and did not induce systemic effects, implying good tolerability and a favorable safety profile for RNAdjuvant.

Biological Response Modifier in Cancer Immunotherapy.

Biological response modifiers (BRMs) emerge as a lay of new compounds or approaches used in improving cancer immunotherapy. Evidences highlight that cytokines, Toll-like receptor (TLR) signaling, and noncoding RNAs are of crucial roles in modulating antitumor immune response and cancer-related chronic inflammation, and BRMs based on them have been explored. In particular, besides some cytokines like IFN-α and IL-2, several Toll-like receptor (TLR) agonists like BCG, MPL, and imiquimod are also licensed to be used in patients with several malignancies nowadays, and the first artificial small noncoding RNA (microRNA) mimic, MXR34, has entered phase I clinical study against liver cancer, implying their potential application in cancer therapy. According to amounts of original data, this chapter will review the regulatory roles of TLR signaling, some noncoding RNAs, and several key cytokines in cancer and cancer-related immune response, as well as the clinical cases in cancer therapy based on them.

An emerging role of Alu RNA in geographic atrophy pathogenesis: the implication for novel therapeutic strategies.

It is generally accepted that geographic atrophy (GA), a currently untreatable advanced form of age-related macular degeneration (AMD), is a multifactorial disease resulting in gradual and permanent blindness. Various risk factors are demonstrated to be responsible for its pathogenesis, such as aging, light exposure, and smoking. Molecular components associated with those risk factors form a complex and interwoven network at the confluence of inflammation, highlighting the significance of inflammasome activation in GA progression. Recently, a new type of modification in AMD microenvironment has been discovered, other than extensively-studied complement dysregulation, lipofuscin deposit, and oxidative by-products, to activate inflammasome. The accumulation of Alu RNA, resulting from DICER1 deficiency, is shown capable of triggering the activation of NLRP3 inflammasome and causing caspase-8-activated apoptosis in an IL-18/MyD88-dependent manner, which provides a new source of evidence for the interplay between cell death and inflammasome. In this review, we lay the emphasis on the mechanism by which Alu RNA activates NLRP3 inflammasome and downstream apoptotic proteins, and on its clinical relevance to GA and potential therapeutic approaches. We also point out several possible crosstalks among inflammasome and different acts of cell death which remain to be further investigated in Alu RNA-induced RPE degeneration.

HCV infection, IFN response and the coding and non-coding host cell genome.

HCV is an ideal model to study how the infected cell is altered to allow the establishment of a chronic infection. After infection, the transcriptome of the cell changes in response to the virus or to the antiviral pathways induced by infection. The cell has evolved to sense HCV soon after infection and to activate antiviral pathways. In turn, HCV has evolved to block the antiviral pathways induced by the cell and, at the same time, to use some for its own benefit. In this review, we summarize the proviral and antiviral factors induced in HCV infected cells. These factors can be proteins and microRNAs, but also long noncoding RNAs (lncRNAs) that are induced by infection. Interestingly, several of the lncRNAs upregulated after HCV infection have oncogenic functions, suggesting that upregulation of lncRNAs could explain, at least in part, the increased rate of liver tumors observed in HCV-infected patients. Other lncRNAs induced by HCV infection may regulate the expression of coding genes required for replication or control genes involved in the cellular antiviral response. Given the evolutionary pressure imposed by viral infections and that lncRNAs are specially targeted by evolution, we believe that the study of proviral and antiviral lncRNAs may lead to unexpected discoveries that may have a strong impact on basic science and translational research.

Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes.

Telomeric repeat-containing RNA (TERRA) has been identified as a telomere-associated regulator of chromosome end protection. Here, we report that TERRA can also be found in extracellular fractions that stimulate innate immune signaling. We identified extracellular forms of TERRA in mouse tumor and embryonic brain tissue, as well as in human tissue culture cell lines using RNA in situ hybridization. RNA-seq analyses revealed TERRA to be among the most highly represented transcripts in extracellular fractions derived from both normal and cancer patient blood plasma. Cell-free TERRA (cfTERRA) could be isolated from the exosome fractions derived from human lymphoblastoid cell line (LCL) culture media. cfTERRA is a shorter form (∼200 nt) of cellular TERRA and copurifies with CD63- and CD83-positive exosome vesicles that could be visualized by cyro-electron microscopy. These fractions were also enriched for histone proteins that physically associate with TERRA in extracellular ChIP assays. Incubation of cfTERRA-containing exosomes with peripheral blood mononuclear cells stimulated transcription of several inflammatory cytokine genes, including TNFα, IL6, and C-X-C chemokine 10 (CXCL10) Exosomes engineered with elevated TERRA or liposomes with synthetic TERRA further stimulated inflammatory cytokines, suggesting that exosome-associated TERRA augments innate immune signaling. These findings imply a previously unidentified extrinsic function for TERRA and a mechanism of communication between telomeres and innate immune signals in tissue and tumor microenvironments.