MicroRNA regulation of T-lymphocyte immunity: modulation of molecular networks responsible for T-cell activation, differentiation, and development

Abstract

MicroRNAs (miRNA) are a class of small non-coding RNAs that constitute an essential and evolutionarily conserved mechanism for post-transcriptional gene regulation. Multiple miRNAs have been described to play key roles in T-lymphocyte development, differentiation, and function. In this review, we highlight the current literature regarding the differential expression of miRNAs in various models of murine and human T-cell biology. We emphasize mechanistic understandings of miRNA regulation of thymocyte development, T-cell activation, and differentiation into effector and memory subsets. We describe the participation of miRNAs in complex regulatory circuits shaping T-cell proteomes in a context-dependent manner. It is striking that some miRNAs regulate multiple processes, while others only appear in limited functional contexts. It is also evident that the expression and function of specific miRNAs can differ between murine and human systems. Ultimately, it is not always correct to simplify the complex events of T-cell biology into a model driven by only one or two master regulator miRNAs. In reality, T-cell activation and differentiation involve the expression of multiple miRNAs with many mRNA targets; thus, the true extent of miRNA regulation of T-cell biology is likely far more vast than currently appreciated.

Figures

FIGURE 1. A brief overview of miRNA biogenesis and function

miRNA genes are transcribed primarily by RNA Pol II and sometimes RNA Pol III, into 5’ G-capped, polyadenylated, hairpin-containing primary miRNA transcripts (pri-miRNAs). A type III ribonuclease Drosha, assisted by a non-catalytic protein DGCR8 required for pri-miRNA recognition, cleaves the pri-miRNA, leaving ~80nt stem-loops called pre-miRNAs. pre-miRNAs are transported from the nucleus through the nuclear pore complex by Exportin 5 in a RanGTP-dependent manner. In the cytoplasm, another type III ribonuclease, Dicer, cleaves the hairpin into a ~22nt duplex, leaving mono-phosphate overhangs at the 5’ end of each strand. One of the strands of this duplex is loaded into the RNA-induced silencing complex (RISC) and a member of the Argonaute (Ago) protein family catalyzes miRNA binding to its target site within the mRNA 3’ untranslated region. The RISC complex inhibits mRNA translation by interfering with ribosome assembly and/or recruits the miRNA-bound mRNA to cytoplasmic structures called processing bodies (P-bodies), where the mRNA is deadenylated, decapped and subsequently degraded. It has been demonstrated, however, that the macroscopic integrity of the P-body is not necessary for miRNA-mediated mRNA decay and that the individual protein components of the P-bodies are sufficient. RNA Pol II - RNA polymerase II; DGCR8 - DiGeorge syndrome critical region gene 8; Ago - argonaute; RISC - RNA-induced silencing complex; GW182 - glycine-tryptophan protein of 182 kDa; ORF - open reading frame; pri-miRNA - primary miRNA; pre-miRNA - precursor miRNA.

FIGURE 2. Multiple life and death signaling pathways involved in T cell activation are regulated by miRNAs

miRNAs modulate the proliferative response of T cells to activation by antigen at multiple levels. First, miR-101, possibly in collaboration with a ubiquitin ligase Roquin, inhibits the expression of an essential co-stimulatory molecule ICOS, while miR-155 inhibits the expression of an inhibitory co-receptor CTLA4. Second, the miR-17~92 cluster (specifically miR-19b) and miR-214 promote PI3K signaling by targeting the inhibitory phosphatase PTEN. Third, expression of IL-2 is regulated by 3 different miRNAs in human T cells: miR-181d targets IL-2 mRNA directly, miR-184 targets the IL-2 transcriptional activator, NFAT, thus indirectly reducing IL-2 transcription, and miR-31 promotes IL-2 transcription by targeting an inhibitor of NFAT nuclear localization, RhoA. Fourth, miRNAs regulate cell cycle progression in T cells. miR-27b inhibits the cell cycle in human resting T cells by directly inhibiting expression of Cyclin T1 and miR-182 promotes proliferation of activated murine T cells by targeting the inhibitor of cell cycle Foxo1. NFκB activation leads to transcription of cytokines as well as cyclins and growth factors. The miR-17~92 cluster (specifically miR-19a or miR-19b) promotes NFκB-mediated gene expression by targeting CYLD, an inhibitor of NFκB nuclear translocation. miR-17~92 also directly inhibits expression of a cell cycle inhibitor, E2F1, in leukemic cells. Finally, miRNAs inhibit apoptosis in T cells. A let-7 family member, miR-98, targets extrinsic apoptosis-inducing receptor Fas and miR-17~92 likely directly targets an intrinsic apoptosis inducer Bim. Of note, miR-17~92 has several targets involved in regulation of cell proliferation - PTEN, E2F1, CYLD and Bim. Solid boxes indicate miRNAs that promote T cell activation; dashed boxes indicate miRNAs that inhibit T cell activation. TCR - T cell receptor; ICOS - inducible T-cell co-stimulator; CTLA4 - cytotoxic T-lymphocyte-associated protein 4; PI3K - phosphoinositide-3-kinase; PTEN - phosphatase and tensin homolog; mTOR – mammalian target of rapamycin; IL-2 - interleukin 2; NFAT - nuclear factor of activated T-cells; RhoA - ras homolog family member A; NFκB - nuclear factor of kappa light polypeptide gene enhancer in B-cells; CYLD – cylindromatosis; E2F1 - E2F transcription factor 1; Fas - TNF receptor superfamily, member 6; Bim - bcl-2 interacting mediator of cell death.

FIGURE 3. miRNAs regulate the balance between Th1 and Th2 lineage commitment

miR-19b, miR-17, miR-155 and let-7c indirectly promote differentiation of Th1 effector cells. miR-19b and miR-17 are members of the miR-17~92 cluster and promote IFNγ production and Th1 differentiation, but the targeted genes responsible for this regulation are unclear. miR-155 inhibits differentiation of Th2 cells by targeting c-Maf, an IL-4 gene trans-activator, thus indirectly promoting Th1 polarization. IL-10, a Th1 suppressing cytokine produced by Th2 and regulatory T cells is inhibited by let-7c, and thus let-7c also indirectly promotes differentiation of Th1 effector cells. miR-29a and miR-29b inhibit Th1 polarization and Th1 effector cytokine production by targeting T-bet, Eomes and IFNγ. miR-21 inhibits production of the Th1 polarizing cytokine IL-12A by dendritic cells and macrophages. Several miRNAs influence Th2 differentiation by regulating the activity of GATA3. miR-126 promotes Th2 differentiation by targeting POU2F3, an activator of a GATA3 inhibitor PU.1, while miR-27b, miR-128 and miR- 240 have been suggested to inhibit Th2 polarization by directly targeting an enhancer of GATA3 stability Bmi1. miR-34 also directly targets the Th2 effector cytokine IL-4. Solid boxes indicate miRNAs that promote Th or Th2 differentiation or function; dashed boxes indicate miRNAs that inhibit Th1 or Th2 differentiation or function. miR-155 promotes Th1, but inhibits Th2 differentiation so is marked by both types of boxes. Dashed arrows indicate indirect induction. IFNγ - interferon gamma; IL-10 - interleukin 10; T-bet - T-box expressed in T cells; Eomes - eomesodermin; IL-12A - interleukin 12A; GATA3 - GATA binding protein 3; POU2F3 - POU class 2 homeobox 3; PU.1 - hematopoietic transcription factor PU.1; Bmi1 -BMI1 polycomb ring finger oncogene; IL-4 - interleukin 4.

FIGURE 4. miRNAs regulate Th17 effector cell differentiation and IL-17 production

Let-7f inhibits expression of IL23R and is down-regulated in human memory CD4 T cells, poising these cells for higher IL-17 production. miR-155 promotes Th17 differentiation via an undefined mechanism. miR-301a and miR-326 also promote IL-17 production and Th17 differentiation in mice by targeting STAT3 and RORγt inhibitors PIAS3 and Ets-1, respectively. Solid boxes indicate miRNAs that promote Th17 differentiation or function; dashed boxes indicate miRNAs that inhibit Th17 differentiation or function. Dashed arrow indicates indirect induction. IL23R - interleukin 23 receptor; STAT3 - signal transducer and activator of transcription 3; RORγt - RAR-related orphan receptor t; PIAS3 - protein inhibitor of activated STAT, 3; Ets-1 -v-ets erythroblastosis virus E26 oncogene homolog 1.

FIGURE 5. The development and lineage stability of CD4 regulatory T cells is regulated by miRNAs

miR-10a favors the iTreg lineage stability in the gut by suppressing a Tfh master-regulatory transcription factor Bcl-6 and the associated co-repressor Ncor2. miR-155 favors Treg thymic development and peripheral survival by directly inhibiting the expression of SOCS1, an inhibitor of STAT5 and IL-2 signaling. miR-146a compensates for miR-155 induced activation of STAT1 and the downstream IFNγ response by targeting STAT1. On the other hand, the miR-17~92 cluster attenuates the expression of three positive regulators of iTreg induction: miR-19b of the miR-17~92 cluster targets PTEN, and miR-17 targets TGFβRII and CREB1, resulting in inhibition of iTreg development. Solid boxes indicate miRNAs that enhance Treg development or function; dashed boxes indicate miRNAs that inhibit it. iTreg - induced regulatory T cell; Tfh - follicular helper T cell; Bcl-6 - B-cell lymphoma 6 protein; Ncor2 - nuclear receptor co-repressor 2; SOCS1 - suppressor of cytokine signaling 1; STAT5 - signal transducer and activator of transcription 5; STAT1 - signal transducer and activator of transcription 1; PTEN - phosphatase and tensin homolog; TGFβRII - transforming growth factor, beta receptor II; CREB1 - cAMP responsive element binding protein 1; mTOR - mammalian target of rapamycin.

FIGURE 6. Several miRNAs regulate T cell memory formation

The miR-17~92 cluster inhibits the effector to memory CD8 T cell transition by activating mTOR and is down-regulated in memory cells. Conversely, miR-146a may promote CD4 and CD8 memory cell formation and is more highly expressed in memory cells. miR-146a inhibits IL-2 transcription by negatively regulating AP-1 activity and also directly targets FADD, a Fas adaptor molecule, thus preferentially protecting memory cells from AICD. Let-7b may indirectly promote CD62L (L-selectin) expression in memory central T cells, allowing homing to secondary lymphoid organs and formation of the central memory niche. Solid boxes indicate miRNAs that promote memory cell formation and/or maintenance; dashed boxes indicate miRNAs that inhibit memory cell formation and/or maintenance. mTOR - mammalian target of rapamycin; IL-2 - interleukin 2; AP-1 - activator protein 1; FADD - Fas (TNFRSF6)-associated via death domain; AICD - activation-induced cell death.

FIGURE 7. miR-150 and miR-181a regulate thymocyte development at multiple stages

miR-150 regulates thymic development of T cells and invariant natural killer T cells at multiple stages. It impairs progression through the double negative (DN) 3 stage and survival of CD4 single positive (SP) thymocytes in mice by targeting c-Myb, a critical regulator of the DN3 to DN4 transition and SP cell survival. In human thymocytes, miR-150 also inhibits NOTCH3, thus attenuating pre-TCR signaling required for the survival of double positive (DP) thymocytes during the DN to DP transition. miR-181a increases TCR sensitivity in DP thymocytes by directly inhibiting PTPN22, DUSP5/6 and SHP-2, phosphatases that inhibit distinct signaling molecules downstream of the TCR. This is critical for survival of DP thymocytes undergoing positive selection and deletion of DP clones with moderate TCR affinities to self-antigens. miR- 181a may also directly target CD69, thus attenuating egress of mature thymocytes from the thymus. Solid boxes indicate miRNAs that positively regulate thymocyte survival; dashed boxes indicate miRNAs that negatively regulate thymocyte survival. miR-181a promotes survival of DP thymocytes, but also mediates removal of cells with potentially self-reactive TCRs so is marked by both types of boxes. c-Myb - v-myb myeloblastosis viral oncogene homolog; PTPN22 - protein tyrosine phosphatase, non-receptor type 22; DUSP5/6 - dual specificity phosphatase 5/6; SHP-2 - protein tyrosine phosphatase, non-receptor type 11; CD69 - early T-cell activation antigen.