ISG20L2: an RNA nuclease regulating T cell activation.

ISG20L2, a 3' to 5' exoribonuclease previously associated with ribosome biogenesis, is identified here in activated T cells as an enzyme with a preferential affinity for uridylated miRNA substrates. This enzyme is upregulated in T lymphocytes upon TCR and IFN type I stimulation and appears to be involved in regulating T cell function. ISG20L2 silencing leads to an increased basal expression of CD69 and induces greater IL2 secretion. However, ISG20L2 absence impairs CD25 upregulation, CD3 synaptic accumulation and MTOC translocation towards the antigen-presenting cell during immune synapsis. Remarkably, ISG20L2 controls the expression of immunoregulatory molecules, such as AHR, NKG2D, CTLA-4, CD137, TIM-3, PD-L1 or PD-1, which show increased levels in ISG20L2 knockout T cells. The dysregulation observed in these key molecules for T cell responses support a role for this exonuclease as a novel RNA-based regulator of T cell function.

Immunological synapse-driven transfer of extracellular vesicle microRNAs in primary lymphocytes.

Cell-to-cell communication is necessary to orchestrate effective immune responses against disease-causing agents and in homeostasis. During immune synapsis, transfer of small extracellular vesicles that contain bioactive molecules, including microRNAs, occurs from the T lymphocyte to the antigen-presenting cell. In this chapter, we describe the methodology to identify and validate specific microRNAs shuttled from T lymphocytes to B cells upon immune synapse formation, and to analyze their functional impact on post-synaptic antigen-presenting cells.

Chaperonin CCT controls extracellular vesicle production and cell metabolism through kinesin dynamics.

Cell proteostasis includes gene transcription, protein translation, folding of de novo proteins, post-translational modifications, secretion, degradation and recycling. By profiling the proteome of extracellular vesicles (EVs) from T cells, we have found the chaperonin complex CCT, involved in the correct folding of particular proteins. By limiting CCT cell-content by siRNA, cells undergo altered lipid composition and metabolic rewiring towards a lipid-dependent metabolism, with increased activity of peroxisomes and mitochondria. This is due to dysregulation of the dynamics of interorganelle contacts between lipid droplets, mitochondria, peroxisomes and the endolysosomal system. This process accelerates the biogenesis of multivesicular bodies leading to higher EV production through the dynamic regulation of microtubule-based kinesin motors. These findings connect proteostasis with lipid metabolism through an unexpected role of CCT.

Natural killer (NK) cell-derived extracellular-vesicle shuttled microRNAs control T cell responses.

Natural killer (NK) cells recognize and kill target cells undergoing different types of stress. NK cells are also capable of modulating immune responses. In particular, they regulate T cell functions. Small RNA next-generation sequencing of resting and activated human NK cells and their secreted extracellular vesicles (EVs) led to the identification of a specific repertoire of NK-EV-associated microRNAs and their post-transcriptional modifications signature. Several microRNAs of NK-EVs, namely miR-10b-5p, miR-92a-3p, and miR-155-5p, specifically target molecules involved in Th1 responses. NK-EVs promote the downregulation of GATA3 mRNA in CD4+ T cells and subsequent TBX21 de-repression that leads to Th1 polarization and IFN-γ and IL-2 production. NK-EVs also have an effect on monocyte and moDCs (monocyte-derived dendritic cells) function, driving their activation and increased presentation and costimulatory functions. Nanoparticle-delivered NK-EV microRNAs partially recapitulate NK-EV effects in mice. Our results provide new insights on the immunomodulatory roles of NK-EVs that may help to improve their use as immunotherapeutic tools.

MicroRNAs in T Cell-Immunotherapy.

MicroRNAs (miRNAs) act as master regulators of gene expression in homeostasis and disease. Despite the rapidly growing body of evidence on the theranostic potential of restoring miRNA levels in pre-clinical models, the translation into clinics remains limited. Here, we review the current knowledge of miRNAs as T-cell targeting immunotherapeutic tools, and we offer an overview of the recent advances in miRNA delivery strategies, clinical trials and future perspectives in RNA interference technologies.

MiRNA post-transcriptional modification dynamics in T cell activation.

T cell activation leads to extensive changes in the miRNA repertoire. Although overall miRNA expression decreases within a few hours of T cell activation, some individual miRNAs are specifically upregulated. Using next-generation sequencing, we assessed miRNA expression and post-transcriptional modification kinetics in human primary CD4+ T cells upon T cell receptor (TCR) or type I interferon stimulation. This analysis identified differential expression of multiple miRNAs not previously linked to T cell activation. Remarkably, upregulated miRNAs showed a higher frequency of 3' adenylation. TCR stimulation was followed by increased expression of RNA modifying enzymes and the RNA degrading enzymes Dis3L2 and Eri1. In the midst of this adverse environment, 3' adenylation may serve a protective function that could be exploited to improve miRNA stability for T cell-targeted therapy.

Rapid Visualization of Intracellular Vesicle Events During Synaptic Stimulation.

The immune synapse (IS) enables cell-cell communication between immune cells through close contacts, as well as T-cell activation and vesicle secretion. It is sustained by fine-tuned molecular interactions of receptors at both cell sides of the IS and intracellular cytoskeletal components. The resulting intracellular polarization of different organelles, through cytoskeleton-guided vesicular traffic, is a key player in IS formation and signaling. We describe herein a method to analyze rapid changes of vesicle localization through microscopy analysis upon polarization toward the IS. These vesicles are monitored using the centrosome and its associated microtubular network or the actin-based structures as spatial references during the organization of the IS.

The Swing of Lipids at Peroxisomes and Endolysosomes in T Cell Activation.

The immune synapse (IS) is a well-known intercellular communication platform, organized at the interphase between the antigen presenting cell (APC) and the T cell. After T cell receptor (TCR) stimulation, signaling from plasma membrane proteins and lipids is amplified by molecules and downstream pathways for full synapse formation and maintenance. This secondary signaling event relies on intracellular reorganization at the IS, involving the cytoskeleton and components of the secretory/recycling machinery, such as the Golgi apparatus and the endolysosomal system (ELS). T cell activation triggers a metabolic reprogramming that involves the synthesis of lipids, which act as signaling mediators, and an increase of mitochondrial activity. Then, this mitochondrial activity results in elevated reactive oxygen species (ROS) production that may lead to cytotoxicity. The regulation of ROS levels requires the concerted action of mitochondria and peroxisomes. In this review, we analyze this reprogramming and the signaling implications of endolysosomal, mitochondrial, peroxisomal, and lipidic systems in T cell activation.