The immune system is under strict regulatory control to ensure homeostasis of inflammatory responses, lying dormant when not needed but quick to act when called upon. Small changes in gene expression can lead to drastic changes in lineage commitment, cellular function, and immunity. Conventional assessment of these changes centered on the analysis of mRNA levels through a variety of methodologies, including microarrays. However, mRNA synthesis does not always correlate directly to protein synthesis and downstream functional activity. Work conducted in recent years has begun to shed light on the various posttranscriptional changes that occur in response to a dynamic external environment that a given cell type encounters. We provide a critical review of key posttranscriptional mechanisms (i.e., microRNA) and translational mechanisms of regulation of gene expression in the immune system, with a particular emphasis on these regulatory processes in various CD4+ T cell subsets.
The cells of the immune system possess tightly regulated mechanisms to control the onset, duration, and magnitude of specific beneficial or pathological inflammatory activities in the host. These range from the secretion of inflammatory cytokines in innate cells to the generation of memory T cells for efficient control in reinfection. In T cells, a network of signaling pathways enables them to integrate environmental cues and, in turn, influences their cell fate decisions in lymphoid and nonlymphoid sites. Such timely sensing of environmental signals drives distinct differentiation processes and activates the required transcriptional landscape that drives lineage commitment and effector functions in a variety of immune-mediated diseases.
Regulation of gene-expression events in cells is a complex process that is controlled by transcriptional, posttranscriptional, and posttranslational mechanisms. Several recent studies showed that mRNA translation is a key control point in the regulation of innate and adaptive immune responses. mRNA translational control can be global in nature and applied to most mRNAs or can be transcript specific, in turn, allowing for translation regulation of a limited set of proteins specific to cellular functions or under select contexts. mRNA translational control of gene expression provides several advantages to the host, including rapid protein function, rapid onset or termination of a given response, and use of preformed mRNAs to circumvent the need for de novo nuclear activities, including transcription, mRNA splicing, and transport.
Measuring total mRNA levels within cells (transcriptome) has been widely used to assess gene expression as a means of deciphering divergent or common gene signatures between cell types or functional states. These signatures are often used as references from which protein levels and functions are inferred. However, mRNA levels in cells frequently do not correlate with the levels of proteins in the proteome (1–4), emphasizing the existence of an “expression gap” between the transcriptome and mRNA translation (translatome). Microarray technologies assessing global mRNA levels in cells are only partially valid because posttranscriptional mechanisms dramatically affect protein levels in the proteome. Recent studies showed that ∼30% of protein levels actually correspond to mRNA levels at steady-state, suggesting that mechanisms solely regulating gene transcription or mRNA stability do not necessarily affect protein levels in cells and that changes in mRNA expression profiles frequently do not correspond to those seen in the cell’s proteome (5, 6).
Posttranscriptional mechanisms of gene expression in CD4+ T cell differentiation
The pool of mRNAs available for the proteome depends on a dynamic balance of mRNA synthesis, maintenance, and degradation. Several studies suggest that a significant portion of the variation in mRNA levels is attributed to the stability of the mRNAs (7). One mechanism by which immune cells control mRNA levels is through RNA-binding proteins (RBPs). These proteins bind mRNA molecules with conserved noncoding sequences, such as adenylate-uridylate–rich elements (AREs) or cytosolic polyadenylation elements (CPEs), located in the untranslated regions (UTRs) of mRNA. These motifs were shown to alter translational control of certain genes through alteration of the poly(A) tail. The closed loop formation between the poly(A) tail and the 5′ cap of mRNA is mediated by poly(A)-binding proteins and was shown to increase the translation of closed loop mRNA (8). CPEB1, which recognizes CPE motifs, can function to recruit the PARN deadenylase complex or act as a translational repressor; upon phosphorylation, it can instead recruit CPSF to elongate the poly(A) tails of mRNA, allowing for increased translation (9). The traditional view of mRNA turnover has mRNA deadenylation as an event preceding decapping and subsequent degradation; however, a recent study examining mouse fibroblasts revealed the presence of a large population of mRNAs with shortened poly(A) tails in the cytoplasm (10). This suggests that certain mRNAs can be transcribed without significant translation occurring, allowing for rapid induction of protein synthesis once the poly(A) tails are elongated (11). The presence of ARE motifs on mRNA allows for another level of posttranscriptional control. One example is the inflammatory cytokine TNF-α, whose mRNA is bound by the RBP tristetraprolin (TTP) to mediate degradation of the RNA in resting conditions. However, inflammation-triggered inactivation of TTP allows for a rapid increase in TNF-α (12, 13). Regulatory elements such as these were described in the control of various immunological processes in lymphocytes (14).
Both long noncoding RNA (lncRNA) and shorter sequences, termed microRNA (miRNA), tightly regulate mRNA levels. miRNAs are transcribed primarily by RNA polymerase II into sequences containing the mature miRNA (∼22 nt in length) and variable flanking sequences. RNase III–type proteins like Drosha, in the nucleus, and Dicer, in the cytosol, as well as the cofactor DGCR8, process this precursor molecule. Interaction of this miRNA with the Argonaute family of proteins produces the RNA-induced silencing complex (RISC) (15). RISC is able to influence gene expression at the level of mRNA stability by binding the 3′ UTR and recruiting deadenylases to shorten poly(A) tails, inducing mRNA degradation, or blocking the binding of translational machinery (15). In addition, a component of RISC, GW182, interacts with the poly(A)-binding protein 1 and can interfere with mRNA closed loop formation (16). In contrast, lncRNAs were shown to play a more indirect role in the control of gene expression by functioning as “decoys.” These RNAs prevent DNA–protein interaction by binding DNA-binding motifs or preventing protein–protein interactions (reviewed in Ref. 17). lncRNAs also act as miRNA-binding sites, in turn preventing the action of miRNAs on their target genes (17).
Th1 cell development and function.
Th1 effector cells are a lineage of CD4+ effector T cell (Teff) that promotes cell-mediated immune responses and is required for host defense against intracellular viral and bacterial pathogens. Several posttranscriptional mechanisms, particularly miRNAs, were described to either promote or prevent Th1 commitment.
Growing evidence shows that miRNAs play a role in inhibiting Th1 differentiation. When CD4+ T cells lacking Dicer are exposed to nonpolarizing conditions in vitro, a skew toward Th1 differentiation is observed from the increase in IFN-γ production compared with wild-type cells (18). This could be due to the fact that naive T cells have high expression of miR-125b, which downregulates IFN-γ directly, as well as represses IL-2Rβ, a requirement for Th1 proliferation (19). Another study showed that mice deficient in Eri1, a 3′ to 5′ exo-RNase that degrades miRNA, had reduced levels of IFN-γ–producing Th1 cells in response to viral infection, indicating a role for miRNA in the suppression of immune responses (20). One study elicited that lymphocytes produce mRNAs with shortened 3′ UTRs containing fewer miRNA binding sites upon TCR stimulation (21). This raises the possibility that, although miRNA may function to restrain the inflammatory response in a resting state, a mechanism exists by which the effect of inhibitory miRNAs is reduced when inflammation is required.
Although some miRNAs function to block Th1 differentiation and effector responses, others promote such responses. This can be seen in Th1 cells following activation when they are required to respond to an immune challenge. TCR stimulation results in an increased expression of the miRNA cluster 17–92 (22). This miRNA cluster contains several miRNA that serve to enhance Th1 lineage commitment. miR-17 represses the translation of TGF-βR2, as well as CREB1 (22). In contrast, miR-155 is likely able to promote Th1 differentiation by impeding the Th2-differentiation program by reducing levels of the trans-activator of the IL-4 promoter, c-Maf (23). Paradoxically, another study showed that miR-155 could reduce the expression of IFN-γRα, a characteristic receptor of Th1 cells (24). Another member of the 17–92 cluster, miR-19b, functions to enhance Th1 activity by increasing mTOR signaling and suppressing PTEN (22). This results in global translational changes due to increased activity of eIF4E resulting from the phosphorylation of eIF4E-BP1 and eIF4E-BP2 (25).
Several studies demonstrated a direct role for the IFN-γ/STAT-1 pathway in driving Th1 cell development and Th1-mediated disease processes. Studies showed that T-bet is a critical transcription factor driving Th1 lineage commitment and function (26); however T-bet is now recognized as playing a fundamental role in coordinating type 1 immunity by controlling a largely conserved network of genetic programs that regulate the development and effector functions of many adaptive and innate cell types. It also plays an important role in regulating IFN-γ production. Although naive CD4+ T cells do not express T-bet, IFN-γ was shown to induce T-bet expression, which results in a potential positive-feedback loop during Th1 cell differentiation (26). The activation of STAT-1 in response to IFN-γ causes an increase in the transcription of miR-29, which limits Th1 function by binding and repressing translation of both T-bet and IFN-γ mRNA (27, 28). Thus, the IFN-γ/STAT-1 pathway may also regulate miRNA-mediated control of Th1 differentiation and effector functions.
Th2 cell development and function.
The transcription factor GATA-3 is required for the commitment of CD4+ T cells to the Th2 lineage (29). The hallmark Th2 cytokine, IL-4, is known to increase the generation of GATA-3 mRNA; however, the resulting increase in GATA-3 protein is insufficient to induce Th2 differentiation (30). TCR signaling causes an increase in PI3K/mammalian target of rapamycin (mTOR) signaling. In the context of Th2 responses, this results in an increase in GATA-3 protein levels without affecting the level of GATA-3 mRNA, indicating that Th2 differentiation may be translationally controlled (30). Other translational control mechanisms serve to protect GATA-3 mRNA from degradation to promote Th2 differentiation. For instance, the RBP HuR enhances Th2 lineage commitment by binding to the ARE present on GATA-3 mRNA, blocking the ARE site from being bound by TTP and, subsequently, deadenylated (31).
miRNAs also influence the development of Th2 lymphocytes. As discussed previously, the miRNA landscape is altered in lymphocytes upon activation, and these changes can help drive or impair a Th2 response. For instance, miR-126 is able to promote a Th2 response by enhancing GATA-3 DNA-binding activity. This is accomplished by suppressing an activator of PU.1, a protein that acts to restrict GATA-3 transcriptional activity (32). miRNAs can also play a role in the reduction of Th2 cell numbers. In multiple sclerosis, CD4+ lymphocytes were shown to have increased levels of miR-128 and miR-340 (33). miR-340 reduces IL-4 expression, whereas miR-128 destabilizes the GATA-3 protein by repressing Bmi1, a protein that was shown to decrease ubiquitination and consequently stabilize GATA-3 (32, 34).
The promotion of the Th2 cell lineage and effector function by posttranscriptional mediators can also be detrimental factors leading to disease. Simpson et al. (35) demonstrated that miR-19a expression is elevated in airway-infiltrating lymphocytes during asthma immunopathology. The entire miR17–92 cluster was shown to be very important for Th2 cytokine production, with its absence leading to severely reduced IL-13 production and unchanged Th1 cytokine levels (e.g., IFN-γ). miR-19a promotes Th2 cytokine production and directly propagates proinflammatory signaling during asthma by interacting with PTEN, the signaling inhibitor SOCS1, and the deubiquitinase A20, in turn potentiating airway inflammation.
Follicular helper T cell cell development and function.
Follicular helper T (Tfh) cells are a subset of CD4+ T cells that facilitates the activation of B cells in B cell zones of lymphoid tissues. Tfh cells express high levels of ICOS, which plays an important role in enhancing TCR-mediated signal transduction of CD4+ T cells to facilitate B cell activation and effector functions (36). Recently, Gigoux et al. (36) showed that ICOS assists TCR-mediated signal transduction of mouse splenic CD4+ T cells by activating the PI3K-AKT signaling pathway and, consequently, the mTOR pathway. mTOR activation leads to phosphorylation of P70S6K and 4E-BP1, both of which promote an eIF4E-dependent (5′ cap–dependent) translation of IL-4 mRNA (36). This process is likely dependent on miR17–92, because mice lacking the miRNA cluster 17–92 show a decrease in the amount of Tfh cells, as well as a failure to upregulate the transcriptional repressor Bcl6, a hallmark of Tfh cells (37). Furthermore, mice with T cell–specific expression of a transgene encoding miR-17–92 exhibit an accumulation of Tfh cells, resulting in fatal immunopathology from the overproduction of autoantibodies (38). These studies indicate that ICOS costimulation–dependent translational control may facilitate delivery of IL-4 by T cells to cognate B cells in the germinal center, allowing for the generation of humoral immune responses in germinal centers.
Th17 cell development and function.
Recent years also uncovered how miRNAs play a role in the regulation of Th17 differentiation and effector functions. Th17 is known to play a central role in the severity of experimental autoimmune encephalitis (EAE), an established mouse model for multiple sclerosis. In this model, mice with active disease possess a higher expression of miR-326 compared with healthy controls (39). miR-326 can bind to the ETS-1 transcript, in turn repressing translation of this negative regulator of IL-17 transcription (39). In contrast, miR-21 was shown to facilitate Th17 development through enhancement of the TGF-β signaling pathway by repressing SMAD-7, a negative regulator of TGF-β signaling (40). Other miRNAs were shown to suppress Th17 differentiation. For example, miR-20b was found to be lowered during EAE progression, with overexpression of miR-20b resulting in a decrease in Th17 differentiation and attenuation of disease (41). Importantly, the let-7 miRNA family plays an integral role in Th17 activity. let-7f miRNA was shown to repress translation of the mRNA encoding IL-23R, whose signaling is required for sustained Th17 activity (42). This miRNA is inhibited through the action of Lin28, an RBP that interferes with the processing of the let-7 precursor miRNA into its mature variants (43). Interestingly, AMPK activity is thought to reduce Lin28 expression, suggesting that environmental cues may play a role in regulating Th17 responses (44). External factors that suppress AMPK activity may lead to greater expression of IL-23 and subsequently enhance Th17 differentiation. This notion is supported by studies that showed that an AMPK deficiency results in an increased severity of EAE, whereas metformin, an AMPK activator, can attenuate EAE progression (45, 46).
Regulatory T cell development and function.
Regulatory T cells (Tregs) are critical determinants for maintenance of self-tolerance and control of excessive immune responses. Their differentiation and function are driven by the forkhead winged helix family transcription factor Foxp3. Their development can occur in the thymus or periphery, collectively giving rise to natural/thymic Treg and peripheral/postthymic Treg Foxp3+ Treg pools, respectively (47). Tregs can suppress the activation/maturation, expansion, and differentiation of a variety of immune cell types via several mechanisms, including secretion of anti-inflammatory cytokines like IL-10 and TGF-β1, sequestering IL-2, and CTLA-4–mediated inhibition of costimulation (48, 49). Abrogation of Treg development, function, or homeostasis augments immunity to self-antigens, allergens, transplants, tumors, and pathogens. Genetic alteration in the foxp3 gene in mice or humans provokes multiorgan autoimmune syndromes like Scurfy or immunodysregulation polyendocrinopathy enteropathy X–linked syndrome, respectively (50), conditions that can be prevented by reinfusion of normal Tregs (51).
Foxp3 expression is generally specific to Tregs and is under tight epigenetic control involving an evolutionarily conserved DNA element in the foxp3 locus, known as the Treg-specific demethylated region, which is demethylated in Tregs but heavily methylated in Teffs. Foxp3 functions in a cell-autonomous fashion, and development of Tregs in the thymus and their sustained function in peripheral lymphoid and nonlymphoid tissues require high and sustained Foxp3 expression, because loss of Foxp3 expression levels in Tregs in many inflammatory contexts hinders their functional stability and promotes their reprogramming into pathogenic T cell lineages (e.g., Th1, Th2, Tfh, or Th17). The duration and reversibility of this inflammatory phenotype in reprogramming Tregs, as well as the nature/origin of Treg precursors that are prone to this functional plasticity, remain unknown.
Posttranscriptional regulation exerted by miRNAs also plays a role in the development, maintenance, and effector functions of Tregs. The importance of miRNAs was first highlighted by the fact that mice whose Foxp3+ T cells lacked global miRNA as a result of a conditional deficiency of the dicer gene in Foxp3+ Tregs developed symptoms of autoimmunity due to a progressive loss of Treg suppressive function (52). Moreover, Tregs lacking DGCR8 eventually lose Foxp3 expression and start producing IFN-γ, suggesting that DGCR8 is not involved in the induction of Foxp3 expression but is required to sustain Foxp3 levels in committed Tregs (53).
Individual miRNAs can simultaneously promote the development and lineage commitment of both Teff and Treg subsets. Although miR-155 promotes the generation of Th1 responses, Foxp3 can induce its transcription in Tregs (54). miR-155 functions to increase Treg sensitivity to IL-2 by repressing translation of SOCS1 mRNA (54), resulting in reduced inhibition of STAT5, a transcription factor that can induce transcription of Foxp3 in response to IL-2 signaling (55). In addition, miR-10a, which is also induced by Foxp3, can repress genes like Bcl-6 and Nco2, two important transcription factors in Tfh cell commitment (56). Thus, Foxp3 is capable of promoting Treg differentiation through the induction of miRNA transcription to increase its own expression while blocking the expression of other Th cell lineage regulators.
A variety of Treg effector functions is under posttranscriptional control. For example, IL-10 secretion, which was shown to reduce the production of the inflammatory cytokines IFN-γ and IL-2 in Teffs, is under the control of miR-466l (57). miR-466l stabilizes IL-10 mRNA by binding to the TTP binding site on the IL-10 mRNA, preventing TTP-mediated deadenylation (57). In other cases, the inhibitory effects of miRNAs in Tregs can often serve as a mechanism to suppress inflammation. A recent study showed that Tregs generate exosomes containing miRNAs that are capable of transfecting nearby T cells (58). One of the miRNAs contained within these exosomes, let-7d, suppressed IFN-γ mRNA levels in conventional T cells, and this reduction was abolished when T cells were exposed to Treg-derived exosomes containing let-7d and treated concurrently with a let-7d inhibitor (58). Overall, these results suggest that Tregs promote a microenvironment that favors posttranscriptional control of their gene expression, as well as influence surrounding lymphocytes through posttranscriptional means.
Environmental cues in the regulation of CD4+ lymphocyte activation
T cell activation and differentiation are dependent upon an array of mediators like TCR stimulation, costimulation, interaction with growth factor cytokines (e.g., IL-2), and importantly, changes in metabolism (59). Naive CD4+ T cells generate most of their energy via oxidative phosphorylation, a process dependent on environmental cues, such as IL-7. Signaling through IL-7R allows for the uptake of glucose by promoting the transfer of glucose transporter 1 to the cell surface, regulating glycolysis (60). Moreover, signaling through IL-7R via extrinsic IL-7 leads to inactivation of the proapoptotic protein BIM (61). TCR and costimulatory signals received by the naive T cell lead to expression of miR-17, interfering with IL-7R signaling by targeting JAK1 and altering the T cell metabolic framework (62).
Following TCR-induced proliferation in CD4+ T cells, there is a shift from oxidative phosphorylation to aerobic glycolysis (63). Paradoxically, this process is less efficient at generating ATP than oxidative phosphorylation. However, glycolysis is more rapid, is not oxygen dependent (allowing cell function in hypoxic environments), and does not produce reactive oxygen species that can trigger apoptosis (63, 64). TCR signaling results in activation of the mTOR pathway, causing an increase in the expression of the transcription factor c-Myc (65). c-Myc upregulates the expression of the glutamine antiporter CD98 necessary for maintaining glutamine levels within the cell, which was associated with continued mTOR activity (65, 66). The activity of mTOR upon TCR activation is modulated by miRNAs. Marcais et al. (22) found that Dicer-deficient mice had dysregulated T cell activation/anergy homeostasis due to a lack of let-7 and miR-16 miRNA. let-7 and miR-16 inhibit the mTOR pathway by targeting the mTOR and Rictor mRNAs. Loss of these miRNAs led to excessive AKT phosphorylation and T cell activation, even in the absence of costimulation, suggesting a vital role for miRNAs in controlling T cell activation. Moreover, the PI3K/AKT/mTOR pathway is under posttranscriptional control through several other miRNAs that are upregulated in activated T cells, such as miR-214 in response to CD28 costimulation and the microRNA cluster 17–92 (67, 68). This cluster functions by repressing translation of the protein PTEN, an inhibitor of PI3K-mediated activation of AKT (67, 68). This allows for an increase in mTOR activity, leading to T cell expansion and differentiation.
The IL-2 pathway, necessary for lymphocyte proliferation, is also subject to translational control. In resting T cells, miR-181c represses translation of IL-2 but is repressed upon activation, increasing IL-2 expression (69). IL-2, in turn, induces expression of miR-182, which represses the antiproliferative transcription factor Foxo1 (70). The metabolic machinery used for proliferation in T cells can also regulate IL-2 expression. GAPDH, an enzyme involved in glycolysis, functions as an RBP in the absence of glycolytic substrates (71). It is clear that lymphocytes harness a plethora of mechanisms that allow them to quickly exert changes in gene expression through translational repression with miRNAs and binding proteins, allowing for rapid changes in cellular proliferation and function.
Recent evidence shows that cellular metabolism can dictate the course of CD4+ T cell differentiation. Unlike Teffs, which rely on oxygen for oxidative phosphorylation or external glucose for glycolysis upon activation, Tregs can use a combination of lipid metabolism and cAMP-dependent ATP generation for their energy needs (72). This metabolic state confers a key survival advantage to Tregs in hypoxic and nutrient-deficient environments, such as those seen in tumors (73). This skew toward cAMP metabolism in Tregs can be attributed to the action of Foxp3, which can repress the transcription of miR-142-3p (74). In turn, miR-142-3p represses the translation of adenylyl cyclase 9, which is required for the production of cAMP, further indicating how Foxp3 can induce posttranscriptional changes through miRNAs to allow Tregs to alter their own metabolic landscape (74).
Translational control of gene expression in CD4+ T cell subsets
The cells of the immune system have developed tightly regulated mechanisms to control the balance between specific beneficial or detrimental inflammatory activities to the host. Regulation of gene expression is a complex, multistage process involving transcriptional, posttranscriptional, and posttranslational mechanisms (Fig. 1). Recent studies showed that mRNA translational represents a key control point in the regulation of inflammatory responses. The control of mRNA translation can be global or transcript specific in nature: although global control regulates most mRNAs, transcript-specific control allows translation regulation of a restricted set of proteins for activities in specific cellular processes. Translational regulation of gene expression confers many advantages on the host, including rapid protein activity, timing, and reversibility of cellular responses, and use of pre-existing mRNA transcripts to eliminate the need for de novo nuclear control mechanisms. Translational control allows rapid induction or termination of synthesis of specific proteins required during acute inflammation. Several individual mRNAs encoding proteins engaged in various components of innate or adaptive immunity are regulated at the translational level.
To enable appropriate immune responses in a context-dependent fashion, different CD4+ T cell subsets, each endowed with specialized effector functions, have the capacity to integrate environmental signals with the corresponding gene-expression events. This is particularly true for Foxp3+ Tregs, which must induce a spectrum of mechanisms for rapid and efficient regulation of inflammatory responses. The precise transcriptional landscape that Foxp3 establishes in the process of Treg lineage commitment and effector functions is an area of intense investigation. Microarray transcriptional studies on total cellular RNA established a Treg signature that distinguishes Tregs from Teffs and their related functions in mice. However, these transcriptional profiles, albeit informative, assumed that total RNA levels relate to protein levels. Regulation of gene expression depends on transcriptional, posttranscriptional, and posttranslational mechanisms. Global gene-expression profiling commonly measures changes at the levels of transcription and mRNA stability in total RNA samples.
Posttranscriptional mechanisms dramatically affect protein levels, and changes in mRNA levels often do not correspond to the proteome. Recently, we (75) examined the role of posttranscriptional regulatory mechanisms (i.e., mRNA translational control) in defining the identity and function of CD4+ T cell subsets. More specifically, this study reported the first genome-wide study of mRNA translation control of gene expression in CD4+ Teff or Treg subsets to identify translation signatures, or translatomes, which better correlate with protein levels (75). In this study, a genome-wide polyribosomal approach was taken to identify the translatomes in Foxp3+ Tregs and Foxp3− Teffs directly ex vivo and post-TCR activation in vitro. This novel strategy profiles polyribosome-associated mRNAs (enriched for actively translating mRNAs) that better reflect protein levels compared with those obtained from total RNA (cytosolic) and allows determination of mRNA-specific changes in translational activity. Although some mRNAs showed differences, the levels of polysome-associated mRNAs were largely similar to those of cytosolic mRNAs in Tregs and Teffs directly ex vivo. However, simultaneous measurements of cytosolic and polysome-associated mRNA levels in CD4+ T cell subsets revealed large qualitative and quantitative differences in mRNA-specific translational activity between Tregs and Teffs following TCR activation. These differential translational signatures in activated CD4+ T cell subsets are unique because they represent only 10% of those previously reported for natural/thymic Tregs and peripheral/postthymic Tregs subsets based on analysis of total mRNA pools. Importantly, these translationally regulated mRNAs were not due to transcriptional changes. Polysome-associated mRNA levels are corrected for cytosolic mRNA level to remove confounding variables originating from mRNA transcription, stability, and/or nuclear export and identify truly differentially translated mRNAs. These translationally regulated mRNAs are organized in modules in a T cell subset–specific fashion; each module consists of a group of coregulated genes that are implicated in specific biological functions. Few functions are enriched among translationally active mRNAs in activated Tregs, whereas translationally suppressed mRNAs were highly related to specific processes, including ubiquitination, chromatin modification, and cell cycle. The chromatin modification and ubiquitination modules contained both translationally activated and suppressed mRNAs in activated Tregs compared with activated Teffs.
Most mRNAs in the cell cycle module were translationally suppressed in activated Tregs compared with activated Teffs. Notably, the mRNA encoding eIF4E is translationally silenced in activated Tregs relative to Teffs, with accompanying low eIF4E protein levels, and it regulates a set of mRNAs that constitutes the translational signature of activated T cells enriched for encoded cell cycle proteins. eIF4E is the master mRNA translation initiation factor and component of the eIF4F translation initiation complex and was shown to preferentially allow for the translation of mRNAs containing 5′ cap structures present on genes encoding proteins in cell growth, proliferation, and survival (76, 77). Differential translation of the eIF4E mRNA between Tregs and Teffs correlates with the translation of a subset of eIF4E-sensitive mRNAs and proliferative responses in vitro and in vivo. Consistently, IL-2, a key Treg growth and survival cytokine, induces eIF4E expression in TCR-induced Treg expansion in vitro, and blocking eIF4E activity potently impairs this IL-2–mediated response in TCR-activated Tregs. One striking observation is that inhibition of eIF4E activity in activated Teffs induces Foxp3 expression, suggesting that modulation of eIF4E expression levels may impact effector lineage identity. Thus, the translatomes distinguishing CD4+ T cell subsets differ in mRNAs whose encoded proteins participate in distinct cellular processes underlying the Tregs and Teffs. Thus, TCR activation differentially impacts the translational activity of commonly expressed mRNAs in CD4+ T cell subsets, suggesting that translational control plays an important role in regulating specific gene expression landscapes in Treg and Teff lineages. Single-gene or genome-wide analysis of polyribosome-associated mRNAs provided important insights into gene-expression events, which more closely reflect the protein levels of a cell’s proteome and more likely contribute to the cellular processes underlying inflammatory responses (78, 79).
Although global mRNA translation is substantially augmented following T cell activation, eIF4E mRNA is translationally suppressed in Tregs. Consequently, differential eIF4E protein expression partially imprints CD4+ T cell subsets with a distinct proteome that potentiates responses to proliferative signals. Thus, activation of the mTOR pathway favors the phosphorylation of 4E-BPs and their eventual release from eIF4E, in turn promoting eIF4E-mediated translation. Although mTOR gene deficiency (mTORC−/− mice) or protein inhibition (i.e., rapamycin) abrogates Th1, Th2, or Th17 cell differentiation, similar conditions readily promote Foxp3 expression and the development of a Treg phenotype in CD4+ T cells. Activation of mTOR signaling by various environmental cues may modulate eIF4E activity, regulate the translational activity of eIF4E-sensitive mRNA, and ultimately orchestrate T cell subset–specific responses. Thus, changes in the local inflammatory environment can dynamically regulate eIF4E levels, thereby bridging the extracellular environment, gene expression, and biological responses. Common environmental cues include nutrient deprivation, stress, and hypoxia, as well as inflammatory signals supplied by infiltrating immune cells. Although proproliferative signaling via the PI3K-mTOR pathway is known to induce cap-dependent translation, cellular stress caused by nutrient deprivation or hypoxia can activate alternative modes of translation and, as a result, change the proteome landscape in cells.
When immune responses are generated, the rapid onset and termination of diverse effector mechanisms must be efficiently controlled to prevent the adverse consequences of uncontrolled or excessive inflammation. In this article, we describe an array of posttranscriptional mechanisms of gene regulation (i.e., miRNAs and translational control) that are key in the modulation of T cell differentiation and effector functions. Although transcriptional control is an essential mechanism for the regulation of gene expression, a variety of posttranscriptional mechanisms, including miRNA and translational regulation of gene expression, is advantageous to a cell because it links inflammatory cues with timely and context-dependent protein synthesis and effector responses without the limitation of energy- and time-consuming de novo mRNA synthesis. Recent studies using single-gene or genome-wide approaches highlight how posttranscriptional mechanisms of gene-expression control in various innate and adaptive cell types potentiate a modular regulation of gene expression for a more efficient response to cellular activation and environmental cues.
Although recent years have seen an increase in the amount of work conducted on posttranscriptional mechanisms governing cells of the innate immune system, more work still needs to be done to uncover the mechanisms responsible for the fine regulation of adaptive-immune responses. With T cells, particularly CD4+ T cell subsets, at the focal point of adaptive immunity, uncovering the regulatory processes that underlie gene-expression events during T cell function may shed light onto novel therapeutic applications. Discovering new pathways involved in the proliferation of lymphocytes or the promotion of Treg survival in tumors, as well as those that facilitate the induction of pathogenic T cell lineages in autoimmune disorders, for example, could allow for the development of novel therapies to maintain immunological homeostasis.
This work was supported by Grant MOP 67211 from the Canadian Institute for Health Research.
Abbreviations used in this article:
cytosolic polyadenylation element
experimental autoimmune encephalitis
long noncoding RNA
mammalian target of rapamycin
RNA-induced silencing complex
effector T cell
follicular helper T
regulatory T cell
The authors have no financial conflicts of interest.