Control of Immunoregulatory Molecules by miRNAs in T Cell Activation

MiRNA targeting of key immunoregulatory molecules fine-tunes the immune response. This mechanism boosts or dampens immune functions to preserve homeostasis while supporting the full development of effector functions. MiRNA expression changes during T cell activation, highlighting that their function is constrained by a specific spatiotemporal frame related to the signals that induce T cell-based effector functions. Here, we update the state of the art regarding the miRNAs that are differentially expressed during T cell stimulation. We also revisit the existing data on miRNA function in T cell activation, with a special focus on the modulation of the most relevant immunoregulatory molecules.


INTRODUCTION
MiRNAs are small (∼19-24 nucleotides) single-stranded non-coding RNA species that act as posttranscriptional modulators; they control gene expression, either by promoting mRNAs degradation or repressing their translation (1). More than 2,500 human mature miRNA sequences have been already listed in MirBase (2) although the total amount of miRNAs is likely up to 10 times higher (3). Friedman et al. (4) estimated that miRNAs could modulate around 60% of protein-coding genes, indicating the relevance of these regulatory pathways in gene expression.
The miRNA repertoire changes upon T cell activation (5)(6)(7)(8)(9)(10)(11). Figure 1 summarizes miRNA species described to be either upregulated or downregulated upon T cell stimulation. Different studies have yielded data that may appear contradictory, likely due to T cell subset differences, the origin of the sample (murine or human) and the strategy of stimulation. Additional differences stem from the strategy used to evaluate miRNA expression, being arrays the most commonly employed technique, together with RT-qPCR and Northern Blot.
In addition to variations in miRNA expression, it would be essential to consider the total abundance of each miRNA in the cell. Interestingly, only 7 miRNAs accounted for around 60% of the total sequencing reads in CD8 + T cells (8).
Beyond individual miRNA changes, it is important to highlight that miRNAs undergo a global downregulation upon stimulation. In this regard, almost three times higher total miRNA array hybridization signal has been detected in mouse CD8 + naive T cells compared to activated cells (8); similarly, an independent study found a significant downregulation of the total amount of miRNA in stimulated mouse and human CD4 + T cells compared to non-stimulated controls (5).

LESSONS FROM MIRNA-DEFICIENT MODELS
Dicer is an RNase III endonuclease that controls miRNA biogenesis. It processes precursor miRNA (pre-miRNA) into mature miRNA forms (12)(13)(14). Constitutive Dicer KO mice display embryonic lethality (15), indicating the relevance of this enzyme in development. Lineage-specific Dicer-deficient models were therefore required to study the consequences of reduced miRNA function in a tissue-specific manner.
Dicer-deficient CD4 + T cells were hyper-responsive to TCR stimulation and produced IL-2 in the absence of costimulation (16). After activation, CD4 + Dicer-deficient mice showed reduced proliferation, higher levels of apoptosis and a bias towards Th1 differentiation and IFN-γ release (17). In Th1 differentiation, IFN-γ production and a decline in IL-2 secretion occurred earlier in Dicer-deficient than in wild-type CD4 + T cells (17). Th2 cells presented reduced levels of GATA3 mRNA and failed to suppress IFN-γ expression (17). Consistently, similar phenotypes were observed in T cells lacking Drosha or its RNA-binding cofactor DGCR8, which form a complex responsible for primary miRNA transcript processing. Droshadeficient naïve CD4 + T cells differentiated into Th1 and Th2, but expressed higher levels of IFN-γ than control cells (18). Similarly, DGCR8-deficient T lymphocytes showed reduced proliferation and an increase in IFN-γ secretion (19). A number of very comprehensive reports have addressed the role of miRNAs in T cell differentiation (20)(21)(22)(23)(24). In this review, immunoregulatory molecules responsible for differentiation have been discussed when closely related to T cell activation events.
CD4-specific Dicer deficiency also affects the regulatory T cell compartment, impairing Tregs development in the thymus and reducing their numbers in peripheral lymphoid organs (25). In addition, deficient naïve CD4 + T cells activated in the presence of TGF-β expressed significantly less FOXP3 than control cells (25). Besides, several studies have demonstrated that miRNA disruption in Treg cells leads to autoimmune diseases (18,26,27).
Dicer-deficient CD8 + T lymphocytes responded more rapidly to activation in vitro, as indicated by faster CD69 up-regulation and an earlier proliferative response, although their survival was reduced after 2 days (28). CD8 + Dicer KO cells also showed a delay in CD69 down-regulation after removal of the TCRactivating stimulus, suggesting a sustained activation of cytotoxic lymphocytes in the absence of miRNAs (28). Furthermore, CD8 + Dicer-deficient cells failed to produce an efficient in vivo effector response, including lower proliferation and impaired cytokine production (IFN-γ and TNF-α) (28).
Models with impaired miRNA synthesis machinery highlight the importance of miRNAs as positive (booster) and/or negative (brake) regulators of T cell development and function, which is a major focus of this review (Figure 2).

IMMUNOREGULATORY MOLECULES AS MIRNA TARGETS
T cell activation requires that the TCR recognizes a specific antigen bound to the MHC on the surface of an APC in the presence of co-stimulation. PI3K, AKT and mTOR are crucial mediators of T cell activation. Their positive signaling, downstream the TCR, is counter-balanced by negative regulators such as PTEN and BIM. Costimulatory signals are provided by surface receptors expressed on T lymphocytes that interact with specific ligands on APCs, and can be either activating (such as CD28 and ICOS) or inhibitory (like CTLA-4 and PD-1). These activating and inhibitory events are integrated into a net response that triggers the activation and/or repression of transcription factors (NFAT, AP-1, NF-κB, and others). Their nuclear localization promotes the synthesis of immune effector molecules, e.g., cytokines. MiRNAs also control the activation and integration of these pathways to support T cell effector functions while maintaining immune homeostasis. Herein, we review the miRNA-mediated regulation of key molecules involved in T cell activation.

BIM
The balance between BIM and BCL-2 molecules is essential for the fate of T lymphocytes, and their expression is tightly regulated by miRNAs, promoting either apoptosis or survival. BIM is a pro-apoptotic regulator and tumor suppressor downstream  (11). Whenever more than one detection method was used, only consistent data obtained with at least two techniques was selected (8). Most studies evaluated miRNA expression with miRNAs arrays, some together with RT-qPCR and Northern Blot, as indicated (x).

FIGURE 2 |
Overview of miRNA modulation on positive and negative immune-regulator molecules. Signaling coming from TCR and costimulatory molecules is integrated by the T lymphocyte promoting cell survival, proliferation and production of effector molecules, such as cytokines. This complex network is fine-tuned by miRNAs that target key immunoregulatory molecules, supporting either T cell activation (booster) or inhibition (brake). MiRNAs exert their function by targeting the mRNA 3 ′ UTR in the cytoplasm, although for simplicity sake some have been depicted in the nucleus, close to their targeted immunoregulators. In PI3K, C and R designated the catalytic and regulatory subunits, respectively. of AKT3, an important mediator of TCR signaling (36,37). It destabilizes mitochondrial membrane, inducing CASPASE-9 activation and apoptosis. Within the miR-17∼92 cluster, miR-19 and miR-92 target BIM 3 ′ UTR mRNA (38). MiR-148a is upregulated in mouse Th1 cells after sustained activation (39). It also targets BIM, promoting cell survival (39). MiR-155 indirectly regulates BIM by targeting SHIP-1, which is a phosphatase that reduces AKT activity (40). In turn, AKT represses FOXO3, which is a transcription factor that promotes BIM expression, thus miR-155 limits BIM expression (40). Conversely, miR-150 promotes apoptosis by downregulating AKT3, which induces the accumulation of BIM (41). Human CD4 + T cells with high levels of miR-150 display reduced proliferation, increased apoptosis and lower T cell activation (41).

Cell Cycle Regulators
Molecules involved in cell cycle progression are essential mediators of T cell proliferation. miR-142-null T cells displayed gross cell cycle alterations, with cells differentially arrested in S and G 2 /M phases (32). Cell-cycle defects were associated to the transcription factors E2F7 and E2F8, which are putative targets for miR-142. MiR-142 is likely responsible of maintaining low levels of both molecules in resting T-cells and limiting their increase upon activation. Treatment of mice with miR-142 antagomir markedly increased survival and reduced clinical symptoms in a murine GVHD model, suggesting a potential new therapeutic strategy (32).
Cyclins are also directly targeted by miRNAs. Several miRNAs (miR-27b, miR-29b, miR-150, and miR-223) promote CYCLIN T1 downregulation in human resting CD4 + T cells. The levels of these miRNAs decrease upon activation, correlating with an upregulation of CYCLIN T1 (46). MiR-16 downregulates CYCLIN E1 in mouse CD4 + T cells (43). Another molecule involved in cell cycle progression is CDK4, a target of miR-491 in mouse CD8 + T cells (47). MYC is a transcription factor involved in cell cycle and proliferation, is targeted by let-7 in mouse CD8 + T cells (48) and by miR-451 in both mouse (49) and human (50) CD4 + T cells.
mTOR Mammalian Target Of Rapamycin (mTOR) is a metabolic regulator that promotes protein synthesis and cell growth during the onset of T lymphocyte function (51). mTOR kinase and Raptor are part of the complex mTORC1, while mTORC2 includes mTOR and Rictor. Both miR-16 and let-7c target the 3 ′ UTR of mTOR and RICTOR (16). Elevated mTOR activity in Dicer-deficient CD4 + T cells and the subsequently increased AKT phosphorylation is associated with a lower activation threshold, overcoming the need of co-stimulation. MiRNAmediated mTOR down-regulation contributes to the correct discrimination of activating and anergic stimuli and prevents costimulation independent IL-2, IFN-γ and TNF-α overproduction (16). mTOR signaling suppression is relevant for Treg induction. In this regard, miR-16 and miR-15b, which are abundantly expressed in Tregs, target RICTOR and mTOR mRNAs (52). Furthermore, miR-150 and miR-99a cooperatively target mTOR, promoting Treg induction (53).

Membrane Receptors: ICOS and CD28
Inducible co-stimulatory (ICOS) molecule and CD28 are surface receptors expressed on T cells that recognize specific ligands on APCs, acting as TCR signaling positive regulators (54). In germinal center responses, miR-146a upregulation in Tfh cells downregulates ICOS by interacting with its ligand on germinal center B cells, facilitating the termination of the immune response (55). MiR-101 is highly represented in human naïve CD4 + T cells and its transfection into the EL4 murine T cell line downregulates ICOS (56). Regarding CD28, miR-181a-5p overexpression in mouse T cells increases its levels (57), whereas miR-150 limits CD28 co-stimulation by targeting the arrestin β-2 protein (ARRB-2), with a subsequent increase in cAMP levels and inhibition of LCK, PI3K and AKT (58).

Cytokines
MiRNA regulation of cytokine expression can be due to direct cytokine mRNA targeting or targeting of transcription factors such as NF-κB, NFAT, or AP-1 or their regulators, often affecting multiple cytokines. For example, miR-146a is induced in mouse CD4 + and CD8 + T cells upon TCR engagement through NF-κB (30). This miRNA provides negative feedback regulation, downregulating NF-κB by targeting TRAF6 and IRAK1 (30,59).
CD69 is an early surface marker of lymphocyte activation (96). Dicer KO CD8 + T cells up-regulated CD69 more rapidly upon stimulation and retained the expression longer after stimuli removal (28), indicating a potential miRNA-based repression of CD69 in naïve stages that restrains activation. MiR-130b and miR-301a increased their levels during CD8 + T cell activation and downregulated CD69 (28). MiR-92, which is downregulated in lamina propria leukocytes from rhesus macaques with chronic simian immunodeficiency virus infection, also targets the 3 ′ UTR of CD69 mRNA (97).

Kinases and Phosphatases
TCR signaling is mediated by downstream kinases and phosphatases, which undergo a tight regulation that ensures functional activation while avoiding hyperreactivity.

TCR Inhibitory phosphatases
Phosphatases downstream the TCR pathway counteract signaling by dephosphorylation. Downregulation of some of these phosphatases by miR-181a-5p generates high levels of phosphorylated intermediates in steady-state (57). MiR-181a-5p targets the phosphatases PTPN22, DUSP5 and DUSP6, which dephosphorylate LCK, ZAP70, and ERK1/2; and SHP-2, which mediates negative costimulatory signals from CTLA-4 (57). Therefore, the expression of this miRNA contributes to reduce the activation threshold, increasing the strength and sensitivity of the T cell to peptides with lower affinity (57). In elderly individuals, reduced expression of miR-181a in CD4 + naïve T cells is a cause of the declined T cell responsiveness associated with age (99).

CONCLUDING REMARKS
MiRNA-mediated modulation of molecules involved in T cell activation remains far from being fully understood, although strides have been made in recent years. There is a need to advance towards a "network study" of miRNA function. Considering more than one miRNA in experimental designs increases its technical complication, but also enables models that simulate the complexity of the physiological scenarios, in which individual miRNAs interact with a set of targets and each target in turn can be regulated by several miRNAs, at different levels, either directly targeting the molecule or indirectly regulating its expression via targeting its receptor and/or transcription factors.
Finally, integrating basic and clinical research (e.g., cancer, autoimmunity, and GVHD) could help to achieve a better understanding of T cell immune-regulation to design new strategies for therapy in T cell related malignancies.

ACKNOWLEDGMENTS
We thank Dr M. Vicente-Manzanares for critical reading of the manuscript and for assistance with English editing. This study was supported by the following grants from the Spanish Ministry of Economy and Competitiveness, (grant SAF2017-82886-R to FSM), CIBER CARDIOVASCULAR and PIE 13.0004-BIOIMID from the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III with co-funding from the Fondo Europeo de Desarrollo Regional; FEDER), Programa de Actividades en Biomedicina de la