The role of miRNAs in T helper cell development, activation, fate decisions and tumor immunity

T helper (Th) cells are central members of adaptive immunity and comprise the last line of defense against pathogen infection and malignant cell invasion by secreting specific cytokines. These cytokines then attract or induce the activation and differentiation of other immune cells, including antibody-producing B cells and cytotoxic CD8+ T cells. Therefore, the bidirectional communication between Th cells and tumor cells and their positioning within the tumor microenvironment (TME), especially the tumor immune microenvironment (TIME), sculpt the tumor immune landscape, which affects disease initiation and progression. The type, number, and condition of Th cells in the TME and TIME strongly affect tumor immunity, which is precisely regulated by key effectors, such as granzymes, perforins, cytokines, and chemokines. Moreover, microRNAs (miRNAs) have emerged as important regulators of Th cells. In this review, we discuss the role of miRNAs in regulating Th cell mediated adaptive immunity, focusing on the development, activation, fate decisions, and tumor immunity.


Introduction
CD4 + T helper (Th) cells are core members of the adaptive immune system that orchestrate context-and pathogen-specific responses by secreting specific cytokines (1).These cytokines then attract or induce the activation and differentiation of other immune cells, including antibody-producing B cells and cytotoxic CD8 + T cells (2).Th cells regulate cellular and humoral immune responses to a wide range of pathogens or cancer cells and determine the success of vaccines (3).The development of Th cells origins from hematopoietic stem cells (HSC) in the bone marrow, goes through T cell lineage commitment, b-selection, and positive/negative selection stages to generate single-positive CD4 + cells (4).In general, naïve CD4 + T cells are inactive and quiescent.However, upon antigen recognition and co-stimulation, CD4 + T cells are activated and switch from a resting state to clonal expansion.They differentiate into various effector T (Teff) cells to eliminate pathogens or tumorigenic cells, by activating different intracellular signal transduction cascades and master transcription factors.During this process, CD4 + T cells differentiate into Th cells, such as Th1, Th2, Th17, T follicular helper (Tfh) cells, and induced regulatory T (iTreg) cells (3).Finally, most of these Th cells die, while a small number survive to differentiate into memory T cells, which are distributed in the tissue and provide an anatomically pervasive network for immunosurveillance (5).
Owing to the continuous evolution of living organisms, tumor cells have gained the ability to escape immunosurveillance.The tumor microenvironment (TME), especially the tumor immune microenvironment (TIME), is the primary arena through which tumor cells can overcome the immune system (6,7).There are many types of immune cells in the TIME, among which Th cells are the most abundant.Generally, when tumor cells develop, inflammatory activated immune cells infiltrate the TIME to clear the tumor cells; this is known as an immunologically "hot" tumor.However, sometimes tumor cells can re-educate immune cells in the TIME to switch off their immunosurveillance and prevent activated immune cells from entering the TIME; this is known as an immunologically "cold" tumor (7).Patients with "hot" tumors receive more benefits from clinical therapy than patients with "cold" tumors.Given the limited number of naïve T cells (7), the generation, activation and infiltration of activated Teff cells in the TIME are crucial.
Previous research has shown that granzymes, perforins, cytokines and chemokines are key regulators of Th cell development and activation (1), and recent studies, benefiting from the development of RNA-sequencing methods, demonstrated that transcription factors, post-transcriptional regulators, and microRNAs (miRNAs) are also important factors in Th cell fate decisions (2,4,8).miRNAs are small non-coding RNAs (~23nt) that participate in almost all biological processes including Th cell development, differentiation, migration, activation, and cell fate decisions (9).They generally bind to the 3'untranslated region (UTR) of target mRNAs to repress target gene translation at the post-transcriptional level (10).Because transcription factors, cytokines and chemokines are also regulated by miRNAs, they are essential players in Th cell activation and function.In this review, we highlight the miRNAs that play vital roles in Th cell development, differentiation, activation, and fate decisions, as well as tumor immunity.

miRNAs involved in Th cell development and activation 2.1 miRNAs in Th cell development
The thymus is a unique organ involved in T-cell lineage commitment.Based on the expression of co-receptors (CD4 and CD8) and markers (CD25, CD28 and CD44), the development and differentiation of T cells into CD4 + Teff cells or CD8 + cytotoxic T cells can be mainly divided into three stages: the T-cell lineage commitment, b-selection, and positive/negative selection stages (Stages I-III, respectively) (4,11,12).In Stage I, T cells are originally derived from HSCs located in the bone marrow.They progress from multi-potent progenitors (MPPs), into common lymphoid progenitors (CLPs), and are recruited to the thymus (13).These CLPs then differentiate into early T-cell progenitors (ETPs) and double-negative 2a (DN2a) cells via the promotion of Notch signaling within the thymic microenvironment (4,14).In Stage II, the DN2a cells undergo g, d and b T cell receptor (TCR) gene rearrangement, developing into DN2b and DN3a cells.In Stage III, the T cell lineages complete their commitment through TCRa rearrangement to generate double-positive (DP) cells, producing CD4 + or CD8 + T cells via negative or positive selection.This process begins with DN3b or DN4 cell formation, which gradually evolves into immature single-positive (ISP) cells, DP cells, and finally develop into single-positive CD4 + or CD8 + T cells.Although various studies have demonstrated that activated Notch signaling and its associated transcription factors are vital during T cell development (4), recent findings have shown that miRNAs also play essential roles in T cell development (Figure 1).
The first study was reported in 2005, Muljo et al. revealed that Dicer-1 deletion in the mouse thymus reduces the number of CD4 + T cells and aberrant Th cells (15).While Cobb et al. indicated that deletion of Dicer at an early stage of T cell development reduced the survival of TCR a/b lineage cells but did not affect TCR g/d thymocytes, implying that Dicer might be dispensable for CD4/8 lineage commitment (16).
Consistent with these reports, it appears that each T cell development step is affected by miRNAs, especially the beginning of Stage I (Figure 1).For example, miR-22 (17), miR-125a (18), miR-29a (19), and miR-99 (20) control HSC renewal.Eleven miRNAs functioned together to manipulate HSC homeostasis and expansion.miR-10a and miR-335 weaken long-term HSCs (21), whereas miR-221/222 repress the differentiation of long-term HSCs into short-term HSCs by targeting the KIT receptor (22).In shortterm HSCs that transform into multipotent progenitors, miR-196b is the most abundant miRNA and regulated by the HSC transcription factor family mixed lineage leukemia.It modulates HSC homeostasis and lineage commitment possibly by directly targeting homeobox (HOX) (13).Li et al. showed that miR-126 inhibits apoptosis and increases the viability of acute myeloid leukemia cells and enhanced the colony-forming ability of mouse normal bone marrow progenitor cells alone by targeting tumor suppressor polo like kinase 2, which facilitates the development of leukemia (23).While, Shen et al. found that miR-126 is expressed in hematopoietic stem cells and regulates normal hematopoietic cells function by targeting HOXA9, which is validated by using specific bone marrow-derived cell lines (24).
However, only a limited number of miRNAs have been found to regulate Stage II and Stage III (Figure 1).The most representative miRNA is miR-142-3p, which is essential for normal thymic cellularity and peripheral T cells.Mechanistically, it accelerates early T-cell progenitor proliferation from T lineage progenitors to DN4 cells by targeting cyclin-dependent kinase inhibitor 1B and other critical target genes (25).Another important one is miR-150, which is up-regulated during normal T-cell maturation, however, it blocks T cell development.Ghisi

miRNAs in Th cell activation and proliferation
Generally, naïve CD4 + T cell activation and differentiation begin with antigen recognition.In the thymus, TCR, located on the surface of naïve CD4 + T cells, binds to and recognizes pathogenderived peptides.These, in turn, produce inositol-triphosphate and diacylglycerol via phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) (2).Inositol-triphosphate regulates Ca 2+ influx to activate the transcription factor nuclear factor of activated T cells (NFAT), while diacylglycerol activates AP1 and NF-kB, leading to the activation of T cells and expression of interleukin (IL-2), the hallmark cytokine of activated T cells (28,29).During the co-stimulation step, antigen-presenting cells express B7 family members that bind to the CD28 receptor on T cells and activate class I phosphatidyl-inositol 3-kinase (PI3K) to produce phosphatidyl-inositol 3,4,5-triphosphate (PIP3) from PIP2 (2).PIP3 signaling activates many transcription factors, such as NF-kB and mTOR, which promote cell survival, enhance transduction from TCR, and increase cellular metabolic activity.Moreover, phosphatase and tensin homologs (PTEN) negatively regulate PI3K/AKT pathway activation, as they dephosphorylate PIP3 to generate PIP2 (2,30).
Once activated by antigens, Th cells switch from the resting state to clonal expansion.This switch requires the inactivation of the transcription factor Foxo1, which is expressed in resting Th cells, to suppress cell proliferation (36).During this process, miR-182 is induced by IL-2 and, in turn, targets Foxo1 to promote the clone expansion of activated Th1, Th2, and Th17 cells, indicating that miR-182 plays vital roles in the physiological regulation of IL-2-driven Th cell-mediated immune responses (36).A separate study demonstrated that miR-92a directly targets Foxo1 to restrict Treg induction and supports the Th17 response.Through sustaining the imbalance of Th17/Treg cells, miR-92a promotes CNS autoimmunity (37).miR-425 and miR-873 also target Foxo1 to facilitate the differentiation of naïve CD4 + T cells into Th17 cells in inflammatory bowel disease and the pathogenesis of systemic lupus erythematosus, respectively (38,39).Moreover, miR-125b, which is highly expressed in human naïve CD4 + T cells, inhibits T cell differentiation by targeting IFN-g, IL-2 receptor subunit beta, IL-10 receptor subunit alpha, and PR/SET domain 1 (40).miR-146a also negatively regulates T cell activation by inhibiting NF-kB signaling (41) (Figure 1).

miRNAs in Th cell differentiation and fate decision
The cytokine microenvironment affects Th cells in different ways.Activation of different intracellular signal transduction cascades and master transcription factors shapes the differentiation and plasticity of Th cells.This leads to the maturation of five major cell subsets with different functions, classified based on the cytokines they secrete: Th1, Th2, Tfh, Th17, and iTreg (2,3).Here, we discuss the role of miRNAs in Th cell differentiation and cell fate decisions, focusing on master transcription factors and related intracellular signal transduction cascades.

miRNAs in Th1 cell differentiation and fate decision
Th1 cells regulate immune responses against viruses, intracellular pathogens and tumors via predominantly producing IFN-g (3).Th1 cell dysfunction induces autoimmune diseases.Upon IL-12/signal transducer and activator of transcription (STAT) 4 and IFN-g/STAT1 signaling activation, phosphorylated STAT4 and STAT1 bind to the T-bet promoter and accelerate T-bet (a master regulator of Th1 cell) and IFN-g expression (42).CD4 + T cell then differentiates into Th1 cell, which promotes macrophage and cytotoxic T cell activation and function as pro-inflammatory Teff cell (2).
In Dicer-deficient CD4 + T cells, Muljo et al. found increased differentiation into effector cells expressing T-bet and IFN-g (15).Later research showed that T cells lacking Drosha or DGCR8 have a phenotype similar to that of aberrant Th1 cell differentiation (43).Several miRNAs are involved in Th1 cell differentiation (Figure 2).For example, the overexpression of miR-29a and miR-29b reduced aberrantly high IFN-g and T-bet levels (44).Further study indicated that miR-29 not only directly targets IFN-g mRNA to control innate and adaptive immune responses to intracellular bacterial infection (45), but also targets T-bet and Eomesodermin (Eomes) (44) to reduce IFN-g expression, thus limiting Th1 cell differentiation.miR-146a regulates Th1 cell differentiation, either by inhibiting IFN-g expression or targeting STAT1 (46).
Another well-characterized immune cell-expressed miRNA, miR-155, is strongly up-regulated during T cell activation.It contributes to Th1 differentiation in CD4 + T cells by targeting the IFN-g receptor alpha-chain, thus inhibiting IFN-g signaling (47).Guan et al. revealed that the overexpression of miR-140-5p in peripheral blood mononuclear cells suppressed encephalitogenic Th1 cell differentiation by inhibiting STAT1 and downstream T-bet expression, which attenuates the progression of multiple sclerosis (48).Moreover, miR-24 drives the production of IFN-g and IL-17 in T cells at least in part through targeting TCF1, a transcription factor known for its role in limiting Th1 and Th17 immunity (49).miR-210 induces Th17 and Th1 cell differentiation but inhibits Th2 differentiation by repressing STAT6 and LYN expression, thus promotes psoriasis-like inflammation (50).Activated Th1 cell also expresses elevated levels of certain miRNAs that promote their survival.miR-148a, which is specifically upregulated by Twist1 and T-bet in Th1 cell, is essential for Th1 cell survival as it targets proapoptotic gene Bcl2-interacting protein (Bim) expression (51) (Figure 2).
Moreover, Th1 cell differentiation can be indirectly affected by other immune cells via miRNAs (Figure 2).For example, miR-21 is highly expressed in dendritic cells (DCs) and directly targets and reduces IL-12p35 expression in DCs.Lower secreted IL-12 restrains T-bet and IFN-g expression in CD4 + T cells, which represses the proliferation and survival of Th1 cells (52).Wu et al. found that miR-10a is decreased in the inflamed mucosa of inflammatory bowel disease patients, and downregulates mucosal inflammatory response.Further study showed that DC-derived miR-10a blocks Th1/Th17 cell immune responses and facilitates pathogenesis and progression of inflammatory bowel disease by suppressing IL-12/ IL-23p40, subunits of IL-12 (53).
In addition, exosomes and other small extracellular vesicles also play important roles in Th1 cell differentiation (Figure 2).Zhu et al. found that miR-29a-3p contained in exosomes derived from granulocyte-like myeloid-derived suppressor cells restrains Th1 cell differentiation by targeting T-bet, which attenuates collagen-induced arthritis (54).Okoye et al. reported that Treg cell derived exosomes transfer let-7d to Th1 cell and suppresses its proliferation and cytokine secretion, which contributes to the suppression and prevention of systemic disease (55).Jiang et al. found that keratinocytes secrete small extracellular vesicles containing miR-381-3p.This miRNA targets Foxo1 and activates T-bet transcription to promote Th1 cell polarization and differentiation, which accelerates psoriasis development (56).Other miRNAs involved in Th1 cell fate decision are shown in Supplementary Table 2.
Notably, miRNAs expressed in other immune cells also play important roles in regulating Th2 cell fate (Figure 3).For example, human mesenchymal stem cell-derived exosomes inhibited Th2 cell differentiation by regulating the miR-146a-5p/SERPINB2 pathway in patients with allergic rhinitis (70)
In addition, Th17 cell differentiation and activation are influenced by other immune cells, such as DCs and macrophages (Figure 4).miR-29, the only up-regulated miRNA in mature DCs and significantly downregulated in tumor-associated DCs, downregulates IL-23 and antagonizes the Th17 inflammatory response.Further study showed that multiple myeloma reprograms DCs by reducing miR-29b, which facilitates multiple myeloma cell growth and survival (89).In contrast, transfection of miR-10a into human monocytederived dendritic cells led to a decrease in IL-12/IL-23p40 and markedly suppressed Th1 and Th17 cell responses in inflammatory bowel disease (53).Myeloid-derived suppressor cells (MDSC) not only promote Th17 cell differentiation or polarization through the arginase-1/miR-322-5p/TGF-b pathway (129), but also via the miR-542-5p/TGF-b/Smad3 pathway (130), to accelerate systemic lupus erythematosus progression.B7-H3 is the direct target of miR-29c and the transfection of anti-miR-29c into macrophages enhances RORgt and GATA3 expression in co-cultured CD4 + T cells.This regulation, in turn, elevates IL-4 and IL-17 levels, facilitates Th17 cell responses, which provide a new target for treatment of children with allergic asthma (71).Other miRNAs involved in Th17 cell differentiation and activation are summarized in Supplementary Table 4.

miRNAs in Tfh cell differentiation and fate decision
Tfh cells help B cells differentiate into long-lived antibodysecreting plasma cells or memory B-cells via co-localization and interaction with B cells.This is crucial for controlling the development of humoral immunity, the generation of specific high-affinity antibodies, and the maintenance of long-term protective immunity (2,131).Tfh cells are characterized by expression of the master transcription factor Bcl-6, inhibitory receptor programmed cell death protein 1, inducible T-cell costimulatory (ICOS), CXC-chemokine receptor type 5 (CXCR5), CXC-chemokine ligand 13 (CXCL13), and IL-21, which are important for the migration of Tfh cells toward B-cell follicles (132).Upon activation of IL-6/STAT3 signaling, Bcl-6 expression is triggered.IL-21 promotes this process, whereas IL-2/STAT5 signaling acts as a brake to antagonize this process by activating Blimp1, inhibiting Bcl-6 expression (133).
As shown in Figure 5, only a few miRNAs participate in the differentiation and fate decision of Tfh cell.Among these miRNAs, the most common are the miR-17-92 family, miR-155, and miR-146a.Kang et al. reported that the deletion of the miR-17-92 family in T cells results in defects in Tfh cell differentiation, germinal center formation, and antibody responses.Further investigation indicated that this family directly targets the phosphatase PH domain and leucine-rich repeat protein phosphatase 2 to obstruct the migration of CD4 + T cells into B-cell follicles (134).In addition, this cluster inhibits PTEN and the proapoptotic protein Bim, which promotes the accumulation of antigen-experienced T cells and germinal center B cells.And lymphocytes with high miR-17-92 level show more proliferation and less activation-induced cell death, which leads to lymphoproliferative disease and autoimmunity in mice (135).miR-29a-3p (136) and miR-146a (137) directly binds to Icos mRNA and suppresses its translation, thus hampering the co-stimulation process and Tfh cell differentiation and accumulation.Interestingly, in miR-146a -/-mice, Tfh cells accumulate and promote an inflammation response when miR-155 is overexpressed, indicating that miR-155 drives Tfh cell differentiation and fate decision.Mechanistically, miR-155 repressed Fosl2 expression, which negatively regulated Tfh cell differentiation by inhibiting AP-1 expression (138).Liu et al.Other miRNAs and their target genes are involved in Tfh cell function (Figure 5).For example, miR-92a promotes Tfh precursor induction in T1D islet autoimmunity by directly targeting Krüppellike factor 2 (KLF2), thus suppressing PTEN-PI3K-KLF2 signaling, increasing immune infiltration and activation in pancreas (142)
Additionally, Treg cell fate decision is regulated by miRNAs from other immune cells and cancer cells (Figure 6).Ning et al. found that miR-208b in the exosomes of colon cancer cells promotes Treg expansion by targeting programmed cell death factor 4, thus resulting in tumor growth in vivo (169).Malignant pleural effusion is a special metastatic tumor entity in patients with cancer.Single-cell sequencing data showed that exosomes released from macrophages in malignant pleural effusion can promote the differentiation of naïve T cells into Treg cells by upregulating miR-4443, which influences protein kinase B and lipid biosynthetic processes (170).Other miRNAs involved in regulating Treg cell differentiation and fate decision are displayed in Supplementary Table 6.

miRNAs in the Teff/Treg balance
Because many master transcription factors and related intracellular signal transduction cascades overlap among different Th cell subtypes, the balance between Teff and Treg cells largely orchestrates the outcome of immune responses against pathogens or tumor cells.
Additionally, the Treg/Th17 balance is regulated by miRNAs from other immune cells and cancer cells.Exosomal miR-23b-3p derived from human bone marrow mesenchymal stem cells inhibits the activation of the PI3K/Akt/NF-kB signaling pathway.It reduces the number of Th17 cells to maintain the balance of Th17/Treg cells in intracranial aneurysms by targeting KLF5 (176).Ji et al. also indicated that exosomes derived from thymic stromal lymphopoietin-treated dendritic cells enhance RORgt and IL-17 while repressing Foxp3 and IL-10 expression in CD4 + T cells.Further studies have shown that miR-21 is highly expressed in exosomes and suppresses Smad7 expression, thereby disrupting Treg/Th17 differentiation (177).
Although the Treg/Th17 balance is the most studied aspect of the Treg/Teff balance, the Treg/Th1, Treg/Th2, and Treg/Tfh balance can also can be regulated by several miRNAs.For example, Kelada et al. used an in-silico analysis to identify miR-10a and miR-182 as critical miRNAs in Th1-and Th2-associated Treg cells during Schistosome and Leishmania-associated inflammation.Further investigation indicated that the IL-12/IFNg axis reduces miR-10a and its target gene CREB in Th1-associated Treg cells, promoting their function.In contrast, IL-4 regulates miR-182 and c-Maf in Th2-associed Treg cells, which attenuates Th2 function but augments Treg function (178).Another classical example is miR-17-92, which regulates the Th1/Treg balance.Th1 cell response is promoted whereas Treg cell differentiation is restricted by miR-19b/PTEN and miR-17/TGFbRII/CREB1, which alleviates tumor evasion (32).Geng et al. revealed that patients with systemic lupus erythematosus display a unique miRNA, miR-663, in their bone marrow stromal cells.Mechanically, miR-663 impairs the bone marrow stromal cells-mediated downregulation of Tfh cells and upregulation of Treg cells by targeting TGF-b1, thus affecting the Tfh/Treg balance and aggravating lupus (143).

miRNAs in Th cell-mediated tumor immunity
Tumors develop multiple potent and overlapping mechanisms to mediate immune evasion, one of which is increasing Treg cell accumulation or disturbing the Teff/Treg balance in the TME or TIME (179).Previous studies have shown that many miRNAs are involved in the regulation of Treg cells and the Teff/Treg balance; however, only a small number of miRNAs have been shown to play roles in Th cell-mediated tumor immunity.
First, some miRNAs influence Th17-mediated tumor immunity.miR-130b directly targets the IFNAR1/p-STAT1 pathway to recruit Th17 cell and promotes its tumor-suppressive role via the OX40/ OX40L interaction in diffuse large B-cell lymphoma.This regulation alters the TME and facilitates tumor progression (180).Zhou et al. found that tumor-associated macrophages secrete miRNAs such as miR-29-3p and miR-21-5p to increase the Treg/Th17 imbalance, which generates an immune-suppressive microenvironment and facilitates epithelial ovarian cancer progression (94).Interestingly, miR-21 also contributes to the immuno-suppressive microenvironment in multiple myeloma (181).Rossi et al. indicated that the inhibition of miR-21 in naïve T cells impaired Th17 cell differentiation, upregulated STAT1/STAT5a/5b expression, and redirected Th17 to Th1/Th2-like activated/polarized cells, thereby abrogating Th17-mediated multiple myeloma cell proliferation and osteoclast activity.miR-98-5p and miR-451 reshaped the TME in gastric cancer by influencing Th17 cell distribution and differentiation (83,182).In addition, miR-146a attenuates Th17 cell differentiation to promote cervical cancer cell growth and represses its apoptosis through NF−kB signaling by targeting TRAF6 (125).
Second, other miRNAs may affect Treg cell-mediated tumor immunity.Yin et al. revealed that miR-214 expression is elevated in breast cancer, hepatocellular carcinoma, non-small-cell lung cancer, and pancreatic cancer.Further studies indicated that tumor-derived miR-214 efficiently downregulates PTEN and promotes Treg cell expansion, thereby enhancing immune suppression and tumor implantation/growth in mice (179).Wei et al. reported that miR-124 reverses the glioma stem cell-mediated immunosuppression of T cell proliferation and Treg induction to enhance T cell-mediated immune clearance by inhibiting the STAT3 pathway (183).Ye et al. showed the exosomal miR-24-3p is involved in tumor pathogenesis; it mediates T-cell suppression via the repression of FGF11 and may serve as a potential prognostic biomarker in nasopharyngeal carcinoma (184).In addition, miR-448 (185), miR-302a (186), miR-208b (169), miR-192-5p (187), miR-497 (188), and miR-325-3p (189) affect tumor immunity by effectively regulating Treg cell activation or the Treg/Teff cell balance in breast cancer, glioma, colon cancer, gastric cancer, colorectal cancer, and oral squamous cancer.

Discussion
Since first discovered in 1993, miRNAs have been shown to participate in almost all biological processes, including Th cellmediated immune response, through RNA-sequencing (2,3,8,190).In this review, we comprehensively summarize the roles of miRNAs in Th cell development, activation, differentiation, and fate decisions, as well as in tumor immunity (Figures 1-6; Supplementary Tables 1-6).Previous studies have indicated that master transcription factors are the main players in Th cell function (1); however, our review reveals that miRNAs, which target numerous target genes, are also flexible and accurate regulators of Th cell fate decisions and the Teff/Treg balance.This review broadens our understanding of the role of miRNAs in Th cell development, differentiation, activation, fate decisions and tumor immunity.It also emphasizes that miRNAs play vital roles in regulating immune responses against pathogens and cancer cells.
Analyzing of the miRNAs involved in Th cell function revealed some interesting aspects.First, we found that one miRNA can regulate more than one subset of Th cell functions.The miR-17-92 cluster not only controls T cell development in the long-term HSC self-renewal, DN2a to DN2b, and DN4 to DP steps, but also affects all subsets of Th cells by targeting PTEN.This triggers the activation of PI3K/AKT/mTOR signaling (134, 135,[191][192][193], which is essential for cell survival and co-stimulation during Th cell differentiation and activation.Another example is miR-155, which participates in all Th subset cells differentiation by regulating various genes.It directly targets IFN-g receptor alpha thus contributing to Th1 cell differentiation (47).Conditional knockout mice showed that miR-155 in T cells suppressed c-Maf, SOCS1, Fosl2 and Jarid2 expression in Th2 cell, while suppressing C/EBPb in Th17 cell, attenuating Th2 and Th17 cells activation (67).In Tfh cell, miR-155 represses Peli1 expression and increases cellular proliferation and CD40 ligand expression, thus facilitating Tfh cell differentiation (139).miR-155 enhances Treg cell but inhibits Th17 cell by targeting SIRT1 in chronic periodontitis (174).
Third, although most miRNAs are conserved between humans and mice, some have opposing roles in human and mouse Th cells.For example, miR-21 (181) and miR-21-5p (194) promote Th17 activation in mice, but inhibit Th17 activation in humans (177,195) (Supplementary Table 4).As extensive studies on miRNAs function in Th cells have been conducted in mouse models, more efforts should be made to verify their roles in humans.These findings indicate that miRNAs cooperate to concisely and duly control Th cell differentiation and activation depending on the environment.
Four, since miRNAs play vital roles in the development, differentiation, activation and fate decision of Th cells, the dysregulation of these miRNAs facilitates the progression of many inflammatory diseases.Therefore, using antagomirs or mimics of these miRNAs will be a good strategy to increase the therapy responses.For example, when MRL lpr / lpr lupus mice are administrated with miR-7 antagomir or miR-663 inhibitor, the manifestations of systemic lupus erythematosus in mice are efficiently improved by regulating Tfh/Treg imbalance (143,144).He et al. reported that application of antisense miR-301a in mouse colitis model significantly decreases the numbers of Th17 cell and amounts of pro-inflammatory cytokines in inflamed colon, thus releasing the manifestations of inflammatory bowel disease (128).Wu et al. illustrated that both the ablation of miR-210 in mice and inhibition of miR-210 by intradermal injection of antagomir-210 block the immune imbalance and the development of psoriasis-like inflammation in an imiquimod-induced or IL-23-induced psoriasis-like mouse model.Given that the treatment effects of these miRNAs are partly validated in vivo, so more efforts should be taken to clarify the potential roles of these miRNAs on the treatment of inflammatory diseases.
Finally, we noticed that miRNAs loaded in exosomes derived from other immune cells or cancer cells affect the expression of genes involved in Th cell activation, which leads to the dysfunction of Teff/ Treg balance and changes the TME or TIME.In "cold" tumors, Teff cells fail to infiltrate the tumor and activate immune responses.Therefore, exosome-loaded miRNAs that contribute to Teff cells and/or repress Treg cells may be good candidates for future cancer treatment.One promising example is the delivery of anti-miR-214 micro-vesicles into mice implanted with tumors that block Treg expansion and inhibit tumor growth derived from lung cancer and sarcoma (179).Additionally, Ning et al. demonstrated that colon cancer cell-secreted miR-208b is sufficiently delivered into recipient T cells to promote Treg expansion, thus resulting in the tumor growth and oxaliplatin resistance in colon cancer (169).Therefore, targeting miRNAs therapy may be a good approach for immunotherapy.
In a word, although approximately 300 miRNAs have been shown to control Th cell function, only a limited number of miRNAs are verified to regulate Th cell-mediated tumor immunity or inflammatory disease in vivo.We believe that studies on the role of miRNAs in inflammatory disease, in the TIME, and in cancer treatment are ongoing.The remaining questions include which miRNAs should be included and what combination of these miRNAs achieve the best efficacy in the treatment of inflammatory disease and cancer, which warrants further investigation.
et al. revealed that it reduces Notch3 levels in T-cell lines and represses the SP-DP step, thus damaging T-cell development and physiology (26).While, Xiao et al. indicated that c-Myb, a transcription factor controlling multiple steps of lymphocyte development, is targeted by miR-150 (27).Further study showed that miR-150 controls c-Myb expression in a dose-dependent manner, which dramatically affects lymphocyte development and response at the DN3 to DN4 transition.Other miRNAs participated in T cell development are shown in Figure 1; Supplementary
. Using loss-and gain-offunction strategies, Sun et al. found that miR-29c, which is expressed in macrophages of asthmatic children, regulates Th2 cell differentiation by directly targeting the co-stimulatory molecule B7-H3 (71).Coincidentally, Gu et al. indicated that miR-29b, also decreased in asthmatic children and expressed in
uncovered another mechanism by which miR-155 regulates Tfh cell fate decision by targeting Pellino E3 ubiquitin protein ligase 1 (Peli1).Peli1 is a ubiquitin ligase that promotes the degradation of the NF-kB family transcription factor c-Rel, which controls Tfh cell proliferation and CD40L expression (139).miR-153-3p also targets Peli1 to reduce Tfh cell accumulation in patients with systemic lupus erythematosus(140).Meanwhile, Taganov et al. demonstrated that miR-146a expression is up-regulated by the NF-kB pathway and itself directly targets TRAF6 and IRAK1 to create a negative feedback loop to prevent the overactivation of Tfh cell (141).
miR-663 upregulates Tfh cell and downregulates Treg cell by targeting TGF-b1, which induces immune dysregulation in patients with systemic lupus erythematosus (143).Wang et al. demonstrated that miR-7 not only downregulates PTEN/AKT signaling, which promotes B cell differentiation into plasmablasts/plasma cells and spontaneous germinal center formation, but also increases phosphorylated STAT3 and IL-21 expression to facilitate Tfh expansion and promote systemic lupus erythematosus development (144).miR-346 directly targets Bcl-6 to promote Tfh cell differentiation in the pathogenesis of Graves' disease, whereas Bcl-6 binds to the miR-31 promoter to repress its transcription, leading to the release of CD40L and SAP, which contribute to Tfh function.These results identify a miRNA-Bcl-6 positive feedback loop that stabilizes the Tfh cell program (145, 146).However, genome-wide miRNA expression profiling showed that miR-126-3p represses Tfh cell retention in germinal center by inhibiting S1PR2 expression, which is curial for angioimmunoblastic T-cell lymphoma pathobiology (147).miR-15b (148) and miR-192 (149) also affect Tfh cell differentiation and accumulation by directly targeting IRF4 and CXCR5, respectively.Other miRNAs related to Tfh cell differentiation and fate decision are shown in Supplementary Table

Table 1 .
Xu et al. 10.3389/fimmu.2023.1320305Frontiers in Immunology frontiersin.orgmodulates the pathogenesis of multiple sclerosis (157), while loss of Lin28b expression in fetal T cells leads to increased mature let-7, which reduces fetal T cells differentiate into Treg cells (158).Geng et al. illustrated that patients with systemic lupus erythematosus express a specific miRNA, miR-663, in bone marrow-derived mesenchymal stem cells (BMSCs).It inhibits the proliferation and migration of BMSCs and the activation of BMSCs derived Treg cell, which induces immune dysfunction in systemic lupus erythematosus (143) (Figure