Crosstalk between Regnase-1 and -3 shapes mast cell survival and cytokine expression.

Post-transcriptional regulation of immune-related transcripts by RNA-binding proteins (RBPs) impacts immune cell responses, including mast cell functionality. Despite their importance in immune regulation, the functional role of most RBPs remains to be understood. By manipulating the expression of specific RBPs in murine mast cells, coupled with mass spectrometry and transcriptomic analyses, we found that the Regnase family of proteins acts as a potent regulator of mast cell physiology. Specifically, Regnase-1 is required to maintain basic cell proliferation and survival, whereas both Regnase-1 and -3 cooperatively regulate the expression of inflammatory transcripts upon activation, with Tnf being a primary target in both human and mouse cells. Furthermore, Regnase-3 directly interacts with Regnase-1 in mast cells and is necessary to restrain Regnase-1 expression through the destabilization of its transcript. Overall, our study identifies protein interactors of endogenously expressed Regnase factors, characterizes the regulatory interplay between Regnase family members in mast cells, and establishes their role in the control of mast cell homeostasis and inflammatory responses.

Activation of endogenous retroviruses and induction of viral mimicry by MEK1/2 inhibition in pancreatic cancer.

While pancreatic ductal adenocarcinomas (PDACs) are addicted to KRAS-activating mutations, inhibitors of downstream KRAS effectors, such as the MEK1/2 kinase inhibitor trametinib, are devoid of therapeutic effects. However, the extensive rewiring of regulatory circuits driven by the attenuation of the KRAS pathway may induce vulnerabilities of therapeutic relevance. An in-depth molecular analysis of the transcriptional and epigenomic alterations occurring in PDAC cells in the initial hours after MEK1/2 inhibition by trametinib unveiled the induction of endogenous retroviruses (ERVs) escaping epigenetic silencing, leading to the production of double-stranded RNAs and the increased expression of interferon (IFN) genes. We tracked ERV activation to the early induction of the transcription factor ELF3, which extensively bound and activated nonsilenced retroelements and synergized with IRF1 (interferon regulatory factor 1) in the activation of IFNs and IFN-stimulated genes. Trametinib-induced viral mimicry in PDAC may be exploited in the rational design of combination therapies in immuno-oncology.

The mRNA methyltransferase Mettl3 modulates cytokine mRNA stability and limits functional responses in mast cells.

Mast cells are central players in allergy and asthma, and their dysregulated responses lead to reduced quality of life and life-threatening conditions such as anaphylaxis. The RNA modification N6-methyladenosine (m6A) has a prominent impact on immune cell functions, but its role in mast cells remains unexplored. Here, by optimizing tools to genetically manipulate primary mast cells, we reveal that the m6A mRNA methyltransferase complex modulates mast cell proliferation and survival. Depletion of the catalytic component Mettl3 exacerbates effector functions in response to IgE and antigen complexes, both in vitro and in vivo. Mechanistically, deletion of Mettl3 or Mettl14, another component of the methyltransferase complex, lead to the enhanced expression of inflammatory cytokines. By focusing on one of the most affected mRNAs, namely the one encoding the cytokine IL-13, we find that it is methylated in activated mast cells, and that Mettl3 affects its transcript stability in an enzymatic activity-dependent manner, requiring consensus m6A sites in the Il13 3'-untranslated region. Overall, we reveal that the m6A machinery is essential in mast cells to sustain growth and to restrain inflammatory responses.

Eomesodermin-expressing type 1 regulatory (EOMES+ Tr1)-like T cells: Basic biology and role in immune-mediated diseases.

Type 1 regulatory (Tr1) T cells are currently defined all T cells with regulatory functions that lack FOXP3 expression and produce IL-10. Tr1 cells are heterogeneous, and the different reported properties of Tr1-cell populations have caused some confusion in the field. Moreover, understanding the role of Tr1 cells in immune-mediated diseases has been hampered by the lack of a lineage-defining transcription factor. Several independent studies indicated recently that the transcription factor Eomesodermin (EOMES) could act as a lineage-defining transcription factor in a population of IL-10 and IFN-γ co-producing Tr1-like cells, since EOMES directly induces IFN-γ and cytotoxicity, enhances IL-10, and antagonizes alternative T-cell fates. Here, we review the known properties of EOMES+ Tr1-like cells. They share several key characteristics with other Tr1 cells (i.e., "Tr1-like"), namely high IL-10 production, cytotoxicity, and suppressive capabilities. Notably, they also share some features with FOXP3+ Tregs, like downregulation of IL-7R and CD40L. In addition, they possess several unique, EOMES-dependent features, that is, expression of GzmK and IFN-γ, and downregulation of type-17 cytokines. Published evidence indicates that EOMES+ Tr1-like cells play key roles in graft-versus-host disease, colitis, systemic autoimmunity and in tumors. Thus, EOMES+ Tr1-like cells are key players of the adaptive immune system that are involved in several different immune-mediated diseases.

Subcapsular Sinus Macrophages Promote Melanoma Metastasis to the Sentinel Lymph Nodes via an IL1α-STAT3 Axis.

During melanoma metastasis, tumor cells originating in the skin migrate via lymphatic vessels to the sentinel lymph node (sLN). This process facilitates tumor cell spread across the body. Here, we characterized the innate inflammatory response to melanoma in the metastatic microenvironment of the sLN. We found that macrophages located in the subcapsular sinus (SS) produced protumoral IL1α after recognition of tumoral antigens. Moreover, we confirmed that the elimination of LN macrophages or the administration of an IL1α-specific blocking antibody reduced metastatic spread. To understand the mechanism of action of IL1α in the context of the sLN microenvironment, we applied single-cell RNA sequencing to microdissected metastases obtained from animals treated with the IL1α-specific blocking antibody. Among the different pathways affected, we identified STAT3 as one of the main targets of IL1α signaling in metastatic tumor cells. Moreover, we found that the antitumoral effect of the anti-IL1α was not mediated by lymphocytes because Il1r1 knockout mice did not show significant differences in metastasis growth. Finally, we found a synergistic antimetastatic effect of the combination of IL1α blockade and STAT3 inhibition with stattic, highlighting a new immunotherapy approach to preventing melanoma metastasis.

The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells.

T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.

Emerging roles for RNA-binding proteins in T lymphocytes.

RNA-binding proteins (RBPs) are essential effectors in defining and regulating gene expression, and as such their function underlies all cellular processes. Within the immune system in general, and in T lymphocytes in particular, RBPs have been shown to crucially modulate almost every aspect of T cell biology, including differentiation, inflammatory responses and effector functions. However, questions remain regarding the function of many RBPs that have been recently discovered, their regulation, and in general their role within gene regulatory networks that control immune responses. Here, I will focus on unconventional RBPs with an emerging role in T lymphocytes, including proteins with unusual or unknown mode of binding, and proteins displaying enzymatic or regulatory roles in addition to their RNA-binding feature. I will also discuss how in the future distinguishing RBP:mRNA interactions that are functional and biologically relevant from those that have only limited impact will be crucial to fully dissect the intricacies of RBP-mediated regulation in the immune system.

RFX transcription factors control a miR-150/PDAP1 axis that restrains the proliferation of human T cells.

Within the immune system, microRNAs (miRNAs) exert key regulatory functions. However, what are the mRNA targets regulated by miRNAs and how miRNAs are transcriptionally regulated themselves remain for the most part unknown. We found that in primary human memory T helper lymphocytes, miR-150 was the most abundantly expressed miRNA, and its expression decreased drastically upon activation, suggesting regulatory roles. Constitutive MIR150 gene expression required the RFX family of transcription factors, and its activation-induced down-regulation was linked to their reduced expression. By performing miRNA pull-down and sequencing experiments, we identified PDGFA-associated protein 1 (PDAP1) as one main target of miR-150 in human T lymphocytes. PDAP1 acted as an RNA-binding protein (RBP), and its CRISPR/Cas-9-mediated deletion revealed that it prominently contributed to the regulation of T-cell proliferation. Overall, using an integrated approach involving quantitative analysis, unbiased genomics, and genome editing, we identified RFX factors, miR-150, and the PDAP1 RBP as the components of a regulatory axis that restrains proliferation of primary human T lymphocytes.

Ex vivo microRNA and gene expression profiling of human Tr1-like cells suggests a role for miR-92a and -125a in the regulation of EOMES and IL-10R.

Ex vivo gene expression and miRNA profiling of Eomes+ Tr1-like cells suggested that they represent a differentiation stage that is intermediate between Th1-cells and cytotoxic CD4+ T-cells. Several microRNAs were downregulated in Eomes+ Tr1-like cells that might inhibit Tr1-cell differentiation. In particular, miR-92a targeted Eomes, while miR-125a inhibited IFN-g and IL-10R expression.

RNA-binding proteins and RNA methylation in myeloid cells.

RNA-binding proteins (RBPs) regulate all aspects of the life of mRNA transcripts. They are critically important in regulating immune responses, most notably by restraining excessive inflammation that can potentially lead to tissue damage. RBPs are also crucial for pathogen sensing, for instance for the recognition of viral nucleic acids. Concordant with these central regulatory roles, the dysregulated activity of many RBPs can give rise to disease. The expression and function of RBPs are therefore highly controlled by an elaborate network of transcriptional, post-transcriptional and post-translational mechanisms, including the ability of different RBPs to cross-regulate each other's expression. With an emphasis on macrophages and mast cells, we review current knowledge on the role of selected RBPs that have been shown to directly impact the expression of inflammatory transcripts. By focusing specifically on proteins of the Regnase and ZFP36 family, as well as on factors involved in N6 -methyladenosine (m6 A) deposition and recognition, we discuss mechanism of action, regulatory feedback, and impact of these selected proteins on immune responses. Finally, we include examples of the role of m6 A and RBPs in the recognition of viral RNAs. Overall, we provide a general overview of the impact of selected RBPs on the myeloid compartment, followed by a discussion of outstanding questions and challenges for the future.

An optimized workflow for CRISPR-Cas9 deletion of surface and intracellular factors in primary human T lymphocytes.

The appropriate regulation of T lymphocyte functions is key to achieve protective immune responses, while at the same time limiting the risks of tissue damage and chronic inflammation. Deciphering the mechanisms underpinning T cell responses in humans may therefore be beneficial for a range of infectious and chronic diseases. Recently, the development of methods based on CRISPR-Cas9 gene-editing has greatly expanded the available tool-box for the mechanistic studies of primary human T cell responses. While the deletion of a surface protein has become a relatively straightforward task, as long as an antibody for detection is available, the identification and selection of cells lacking an intracellular protein, a non-coding RNA or a protein for which no antibody is available, remain more problematic. Here, we discuss the options currently available to scientists interested in performing gene-editing in primary human T lymphocytes and we describe the optimization of a workflow for the screening and analysis of lymphocytes following gene-editing with CRISPR-Cas9 based on T cell cloning and T7 endonuclease I cleavage assay.

Cell-intrinsic mechanisms to restrain inflammatory responses in T lymphocytes.

A mechanistic understanding of the regulatory circuits that control the effector responses of memory T helper lymphocytes, and in particular their ability to produce pro-inflammatory cytokines, may lead to effective therapeutic interventions in all immune-related diseases. Activation of T lymphocytes induces robust immune responses that in most cases lead to the complete eradication of invading pathogens or tumor cells. At the same time, however, such responses must be both highly controlled in magnitude and limited in time to avoid unnecessary damage. To achieve such sophisticated level of control, T lymphocytes have at their disposal an array of transcriptional and post-transcriptional regulatory mechanisms that ensure the acquisition of a phenotype that is tailored to the incoming stimulus while restraining unwarranted activation, eventually leading to the resolution of the inflammatory response. Here, we will discuss some of these cell-intrinsic mechanisms that control T cell responses and involve transcription factors, microRNAs, and RNA-binding proteins. We will also explore how the same mechanisms can be involved both in anti-tumor responses and in autoimmunity.

Vitamin D and IFN-ß Modulate the Inflammatory Gene Expression Program of Primary Human T Lymphocytes.

IFN-ß treatment is a commonly used therapy for relapsing-remitting multiple sclerosis (MS), while vitamin D deficiency correlates with an increased risk of MS and/or its activity. MS is a demyelinating chronic inflammatory disease of the central nervous system, in which activated T lymphocytes play a major role, and may represent direct targets of IFN-ß and vitamin D activities. However, the underlying mechanism of action of vitamin D and IFN-ß, alone or in combination, remains incompletely understood, especially when considering their direct effects on the ability of T lymphocytes to produce inflammatory cytokines. We profiled the expression of immune-related genes and microRNAs in primary human T lymphocytes in response to vitamin D and IFN-ß, and we dissected the impact of these treatments on cytokine production and T cell proliferation. We found that the treatments influenced primarily memory T cell plasticity, rather than polarization toward a stable phenotype. Moreover, our data revealed extensive reprogramming of the transcriptional output of primary T cells in response to vitamin D and IFN-ß and provide the bases for further mechanistic insights into these commonly used treatments.

A molecular network regulating the proinflammatory phenotype of human memory T lymphocytes.

Understanding the mechanisms that modulate helper T lymphocyte functions is crucial to decipher normal and pathogenic immune responses in humans. To identify molecular determinants influencing the pathogenicity of T cells, we separated ex vivo-isolated primary human memory T lymphocytes on the basis of their ability to produce high levels of inflammatory cytokines. We found that the inflammatory, cytokine-producing phenotype of memory T lymphocytes was defined by a specific core gene signature and was mechanistically regulated by the constitutive activation of the NF-κB pathway and by the expression of the transcriptional repressor BHLHE40. BHLHE40 attenuated the expression of anti-inflammatory factors, including miR-146a, a negative regulator of NF-κB activation and ZC3H12D, an RNase of the Regnase-1 family able to degrade inflammatory transcripts. Our data reveal a molecular network regulating the proinflammatory phenotype of human memory T lymphocytes, with the potential to contribute to disease.

Rebound of disease activity after fingolimod withdrawal: Immunological and gene expression profiling.

Discontinuation of disease-modifying therapy with fingolimod can lead to severe Multiple Sclerosis (MS) rebound activity; however, this phenomenon remains mechanistically incompletely understood, and the short-term impact of a therapy switch on inflammatory gene expression in T lymphocytes is unknown. We present the clinico-radiological and immunological description of a case of rebound activity after fingolimod discontinuation and switching to rituximab treatment in a relapsing-remitting MS patient. After severe rebound, a reduction in the expression of inflammatory cytokines and transcription factors was rapidly observed after administration of methylprednisolone and rituximab. Rituximab led to an effective suppression of inflammatory activity, and at least in this specific case it represented a valid switching approach after fingolimod discontinuation.

DNA (Hydroxy)Methylation in T Helper Lymphocytes.

Upon recognition of an antigen, the differentiation of antigen-inexperienced naïve T lymphocytes into subsets able to effectively coordinate host defense is controlled by a network of transcription factors and regulatory molecules. In the cell nucleus, these factors act in the context of epigenetic modifications that influence DNA accessibility and ultimately gene expression. This review discusses recent findings about the role of DNA methylation (and its oxidized derivatives) in modulating the differentiation and functions of T helper lymphocytes of the adaptive immune system.

The contribution of active and passive mechanisms of 5mC and 5hmC removal in human T lymphocytes is differentiation- and activation-dependent.

In mammals, the 5'-methylcytosine (5mC) modification in the genomic DNA contributes to the dynamic control of gene expression. 5mC erasure is required for the activation of developmental programs and occurs either by passive dilution through DNA replication, or by enzymatic oxidation of the methyl mark to 5-hydroxymethylcytosine (5hmC), which can persist as such or undergo further oxidation and enzymatic removal. The relative contribution of each mechanism to epigenetic control in dynamic biological systems still remains a compelling question. To explore this critical issue, we used primary human T lymphocytes, in which two cellular states can be clearly identified, namely quiescent naïve T cells, which are slowly or rarely proliferating, and rapidly proliferating activated T cells. We found that active mechanisms of methylation removal were selectively at work in naïve T cells, while memory T lymphocytes entirely relied on passive, replication-dependent dilution, suggesting that proliferative capacity influences the choice of the preferential demethylation mechanism. Active processes of demethylation appear to be critical in quiescent naïve T lymphocytes for the maintenance of regulatory regions poised for rapid responses to physiological stimuli.

MicroRNAs as modulators of T cell functions in cancer.

MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression post-transcriptionally. They have emerged as important modulators of T lymphocyte biology, influencing cell activation, differentiation and proliferation in response to environmental signals. Here, we will discuss how miRNAs expressed by T cells can influence two key aspects of tumorigenesis, namely the direct, cell-intrinsic oncogenic transformation of T lymphocytes, as well as the indirect effects on tumor growth mediated by altered immune surveillance. We will specifically focus on three miRNAs that have been shown to regulate different aspects of T cell biology in both physiological and pathological conditions, namely miR-155, miR-146a and miR-181a. We aim at providing examples of the fundamental importance of miRNA-regulated networks in determining the fate of T lymphocytes during oncogenic transformation and in the control of tumor growth.