Intestinal intraepithelial lymphocytes (IELs) are a versatile population of immune cells with both effector and regulatory roles in gut immunity. Although this functional diversity is thought to arise from distinct IEL subpopulations, the heterogeneity of TCRαβ+ and TCRγδ+ IELs have not been well characterized. Using scRNAseq, we identified CD8αα+ T cell subsets with memory-like (Tcf7+) and effector-like (Prdm1+) profiles in both TCRαβ+ and TCRγδ+ IELs. Using CD160 and CD122 as markers of memory-like and effector-like cells, respectively, we found that while effector-like cells dominated the small intestine, memory-like IELs were more prevalent in the large intestine, suggesting a functional specialization of immune responses along the gut. Further transcriptional analysis revealed shared profiles between TCRαβ+ and TCRγδ+ small intestinal IEL subsets, suggesting conserved functional roles across these populations. Finally, our analysis indicated that TCRαβ+ memory-like IELs arise from Tcf7+ double-negative (DN) precursors, and that effector-like IELs subsequently differentiate from the memory-like population. In contrast, TCRγδ+ IELs appear to originate from two distinct precursor populations, one expressing Tcf7 and the other Zeb2, indicating the presence of parallel developmental pathways within this lineage. Overall, our findings reveal that both TCRαβ+ and TCRγδ+ cells contain memory-like and effector-like subsets, which may contribute to the functional heterogeneity of IELs.

Memory CD8 T cells provide long-lasting immunity, but their developmental origins remain incompletely defined. Growing evidence suggests that functional heterogeneity exists within the naïve T cell pool, shaping lineage potential before antigen stimulation. Here, we identify a subpopulation of naïve CD8 T cells expressing death-associated protein-like 1 (Dapl1) that contains preprogrammed precursors biased toward memory differentiation. The differentiation of these precursors is independent of Dapl1 but relies on the transcription factor B-cell lymphoma/leukaemia 11b (Bcl11b), resulting in the generation of Dapl1+ central memory-like CD8 T cells after infection and stem-like memory cells in cancer. Dapl1+ naïve T cells originate among mature thymocytes and gradually appear in the periphery postnatally. Peripheral Dapl1+ and Dapl1- populations show limited plasticity, supporting a thymic-imprinting model. These findings reveal a developmentally imprinted subset of naïve CD8 T cells committed to memory fate, uncovering an alternative pathway for memory T cell generation offering new avenues for therapeutic application.

Memory CD8 T cells play a vital role in providing lasting immune protection, yet their origins remain incompletely understood. Contrary to classical models, emerging evidence suggests that heterogeneity within the naive T cell pool may influence fate decisions prior to antigen encounter. However, the markers of naive T cell heterogeneity have not yet been clearly defined. Here, we describe intraclonal heterogeneity within the naive T cell population marked by the protein Dapl1. Using novel monoclonal antibodies and a reporter-knockout mouse model, we found that Dapl1-positive naive CD8 T cells exhibit distinct phenotypes compared to their Dapl1-negative counterparts. Furthermore, this population includes a subset of pre-programmed precursors biased toward memory lineage fate. The differentiation of these precursors is independent of Dapl1 but relies on the transcription factor Bcl11b, resulting in the generation of Dapl1-positive central memory-like CD8 T cells in response to infection, and stem-like memory cells in response to cancer. Notably, naive Dapl1-positive T cells originate in the thymus among mature thymocytes and gradually appear in the periphery within several days after birth. Our findings suggest that committed memory precursors in the Dapl1-positive population may represent an alternative pathway for memory CD8 T cell generation, offering new avenues for therapeutic application. ### Competing Interest Statement The authors have declared no competing interest.

Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome.

The success of the CD8 T cell-mediated immune response against infections and tumors depends on the formation of a long-lived memory pool, and the protection of effector cells from exhaustion. The advent of checkpoint blockade therapy has significantly improved anti-tumor therapeutic outcomes by reversing CD8 T cell exhaustion, but fails to generate effector cells with memory potential. Here, using in vivo mouse models, we show that let-7 miRNAs determine CD8 T cell fate, where maintenance of let-7 expression during early cell activation results in memory CD8 T cell formation and tumor clearance. Conversely, let-7-deficiency promotes the generation of a terminal effector population that becomes vulnerable to exhaustion and cell death in immunosuppressive environments and fails to reject tumors. Mechanistically, let-7 restrains metabolic changes that occur during T cell activation through the inhibition of the PI3K/AKT/mTOR signaling pathway and production of reactive oxygen species, potent drivers of terminal differentiation and exhaustion. Thus, our results reveal a role for let-7 in the time-sensitive support of memory formation and the protection of effector cells from exhaustion. Overall, our data suggest a strategy in developing next-generation immunotherapies by preserving the multipotency of effector cells rather than enhancing the efficacy of differentiation.

Following activation, CD4 T cells undergo metabolic and transcriptional changes as they respond to external cues and differentiate into T helper (Th) cells. T cells exhibit plasticity between Th phenotypes in highly inflammatory environments, such as colitis, in which high levels of IL-6 promote plasticity between regulatory T (Treg) cells and Th17 cells. Protein Kinase C theta (PKCθ) is a T cell-specific serine/threonine kinase that promotes Th17 differentiation while negatively regulating Treg differentiation. Liver kinase B1 (LKB1), also a serine/threonine kinase and encoded by Stk11, is necessary for Treg survival and function. Stk11 can be alternatively spliced to produce a short variant (Stk11S) by transcribing a cryptic exon. However, the contribution of Stk11 splice variants to Th cell differentiation has not been previously explored. Here we show that in Th17 cells, the heterogeneous ribonucleoprotein, hnRNPLL, mediates Stk11 splicing into its short splice variant, and that Stk11S expression is diminished when Hnrnpll is depleted using siRNA knock-down approaches. We further show that PKCθ regulates hnRNPLL and, thus, Stk11S expression in Th17 cells. We provide additional evidence that exposing induced (i)Tregs to IL-6 culminates in Stk11 splicing downstream of PKCθAltogether our data reveal a yet undescribed outside-in signaling pathway initiated by IL-6, that acts through PKCθ and hnRNPLL to regulate Stk11 splice variants and facilitate Th17 cell differentiation. Furthermore, we show for the first time, that this pathway can also be initiated in developing iTregs exposed to IL-6, providing mechanistic insight into iTreg phenotypic stability and iTreg to Th17 cell plasticity.

The phenomenon of intercellular transfer of cellular material, including membranes, cytoplasm, and even organelles, has been observed for decades. The functional impact and molecular mechanisms of such transfer in the immune system remain largely elusive due to the absence of a robust in vivo model. Here, we introduce a new tumor mouse model, where tumor cells express the soluble ultra-bright fluorescent protein ZsGreen, which allows detection and measurement of intercellular transfer of cytoplasm from tumor cells to infiltrating immune cells. We found that in addition to various types of myeloid lineage cells, a large fraction of T regulatory cells and effector CD8 T cells acquire tumor material. Based on the distribution of tumor-derived ZsGreen, the majority of T cells integrate captured cytoplasm into their own, while most myeloid cells store tumor material in granules. Furthermore, scRNA-seq analysis revealed significant alterations in transcriptomes of T cells that acquired tumor cell cytoplasm, suggesting potential impact on T cell function. We identified that the participation of T cells in intercellular transfer requires cell-cell contact and is strictly dependent on the activation status of T lymphocytes. Finally, we propose to name the described phenomenon of intercellular transfer for tumor infiltrating T cells the “mosquito effect”.

Severe outcomes of COVID-19 are associated with pathological response of the immune system to the SARS-CoV-2 infection. Emerging evidence suggests that an interaction may exist between COVID-19 pathogenesis and a broad range of xenobiotics, resulting in significant increases in death rates in highly exposed populations. Therefore, a better understanding of the molecular basis of the interaction between SARS-CoV-2 infection and chemical exposures may open opportunities for better preventive and therapeutic interventions. We attempted to gain mechanistic knowledge on the interaction between SARS-CoV-2 infection and chemical exposures using an in silico approach, where we identified genes and molecular pathways affected by both chemical exposures and SARS-CoV-2 in human immune cells (T-cells, B-cells, NK-cells, dendritic, and monocyte cells). Our findings demonstrate for the first time that overlapping molecular mechanisms affected by a broad range of chemical exposures and COVID-19 are linked to IFN type I/II signaling pathways and the process of antigen presentation. Based on our data, we also predict that exposures to various chemical compounds will predominantly impact the population of monocytes during the response against COVID-19.

Multiple sclerosis (MS) is a disabling demyelinating autoimmune disorder of the central nervous system (CNS) which is driven by IL-23- and IL-1β-induced autoreactive Th17 cells that traffic to the CNS and secrete proinflammatory cytokines. Th17 pathogenicity in MS has been correlated with the dysregulation of microRNA (miRNA) expression, and specific miRNAs have been shown to promote the pathogenic Th17 phenotype. In the present study, we demonstrate, using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), that let-7 miRNAs confer protection against EAE by negatively regulating the proliferation, differentiation and chemokine-mediated migration of pathogenic Th17 cells to the CNS. Specifically, we found that let-7 miRNAs may directly target the cytokine receptors Il1r1 and Il23r, as well as the chemokine receptors Ccr2 and Ccr5. Therefore, our results identify a novel regulatory role for let-7 miRNAs in pathogenic Th17 differentiation during EAE development, suggesting a promising therapeutic application for disease treatment.

CD8 T cells are the cytotoxic effectors of the adaptive immune response, clearing virally infected and cancerous cells within the host. CD8 T cells acquire their cytotoxic function by differentiating into cytotoxic T lymphocytes (CTLs). Once the CTLs have cleared the antigen, the majority of responding cells will die during contraction, while a small population of the antigen-specific responders will remain, differentiating into long-lived memory cells that provide potent protection to the host upon re-encounter with the antigen. However, during chronic infection and cancer, CD8 T cells are often diverted into the so-called exhausted state, in which CTLs are losing their function. As such, the ability to program the differentiation of CD8 T cells into a particular lineage is an important therapeutic strategy. We have identified a global post-transcriptional mechanism that directs the fate of differentiating CD8 T cells. Specifically, we have found that let-7 miRNAs are downregulated upon T cell activation, allowing T cells to differentiate into CTLs. Moreover, we have found that prolonged expression of these miRNAs programs CD8 T cells to become memory cells, while complete loss induces exhaustion. Thus, these findings may have important implications for the improvement of current immunotherapies targeting CD8 T cell fate.

Autoimmune disorders such as multiple sclerosis (MS) are caused by proinflammatory events mediated by pathogenic Th17 cells. In MS, these cells arise in response to autoantigen recognition and exposure to the cytokines IL-1β and IL-23, migrate to the central nervous system (CNS) by following gradients of CCR2- and CCR5-cognate chemokines, and secrete GM-CSF. GM-CSF is essential for disease development, as it promotes the activation, differentiation, and recruitment of peripheral inflammatory myeloid cells to the CNS that directly demyelinate neurons and damage axons. Th17 cell pathogenicity in MS has been correlated with microRNA (miRNA) dysregulation, which leads to aberrant post-transcriptional regulation of gene expression and enhanced autoreactive phenotype. We found that the lethal-7 (let-7) miRNA family is abundantly expressed in naive CD4+ T cells, but gets dramatically downregulated over time following antigen encounter, indicating that let-7 may control the differentiation of pathogenic Th17 cells. To investigate a potential regulatory role for let-7 in Th17 cell autoreactivity, we used experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Specifically, we demonstrated that let-7 confers protection from EAE by negatively regulating the proliferation, IL-1β/IL-23-dependent differentiation, and CCR2/CCR5-dependent migration of pathogenic Th17 cells to the CNS. Conversely, absence of let-7 led to enhanced Th17 cell autoreactivity and aggravated disease. Therefore, our results identify a novel regulatory role for let-7 miRNAs in pathogenic Th17 differentiation during EAE development, suggesting a promising therapeutic application for the treatment of MS-related autoimmune diseases.

CD8 T cells are among the most vigorous soldiers of the immune system that fight viral infections and cancer. CD8 T cell development, maintenance, activation and differentiation are under the tight control of multiple transcriptional and post-transcriptional networks. Over the last two decades it has become clear that non-coding RNAs (ncRNAs), which consist of microRNAs (miRNAs) and long ncRNAs (lncRNAs), have emerged as global biological regulators. While our understanding of the function of specific miRNAs has increased since the discovery of RNA interference, it is still very limited, and the field of lncRNAs is just starting to blossom. Here we will summarize our knowledge on the role of ncRNAs in CD8 T cell biology, including differentiation into memory and exhausted cells.

Maintaining the diversity and constant numbers of naïve T cells throughout the organism's lifetime is necessary for efficient immune responses. Naïve T cell homeostasis, which consists of prolonged survival, occasional proliferation and enforcement of quiescence, is tightly regulated by multiple signaling pathways which are in turn controlled by various transcription factors. However, full understanding of the molecular mechanisms underlying the maintenance of the peripheral T cell pool has not been achieved. In the present study, we demonstrate that T cell-specific deficiency in let-7 miRNAs results in peripheral T cell lymphopenia resembling that of Dicer1 knockout mice. Deletion of let-7 leads to profound T cell apoptosis while overexpression prevents it. We further show that in the absence of let-7, T cells cannot sustain optimal levels of the pro-survival factor Bcl2 in spite of the intact IL-7 signaling, and re-expression of Bcl2 in let-7 deficient T cells completely rescues the survival defect. Thus, we have uncovered a novel let-7-dependent mechanism of post-transcriptional regulation of naïve T cell survival in vivo.

Future OncologyVol. 14, No. 12 CommentaryOpen AccessOpen Access licenseLet's fight cancer: let-7 is a tool to enhance antitumor immune responsesLeonid A Pobezinsky & Alexandria C WellsLeonid A Pobezinsky*Author for correspondence: Tel.: +1 413 545 2393; Fax: +1 413 545 632; E-mail Address: lpobezinsky@umass.edu Department of Veterinary & Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA & Alexandria C Wells Department of Veterinary & Animal Sciences, University of Massachusetts, Amherst, MA 01003, USAPublished Online:23 Apr 2018https://doi.org/10.2217/fon-2018-0037AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Keywords: cancerCD8 T cellsCTLscytotoxic responseeomeslet-7miRNARNAitumorvaccines/immunotherapyThe ability to program cells into a particular state via the exploitation of global regulators of gene expression has significant therapeutic potential for many diseases, and thus has been a focus of biological research for several years. RNAi mediated by miRNAs is a strong candidate for such applications. MiRNAs are approximately 22 nucleotide (nt) noncoding RNAs that regulate gene expression by targeting specific mRNAs, in a sequence-specific manner, to prevent their translation into protein products. Since their initial discovery, over 2000 miRNA genes have been identified and demonstrated to regulate important cellular processes, including differentiation, proliferation and carcinogenesis. In this review, we will focus on one particular family of miRNAs, let-7, highlighting the role it plays in the differentiation of cytotoxic CD8 T lymphocytes (CTLs), and underscoring its potential for use in enhancing current immunotherapy strategies.During the immune response, antigen-specific CD8 T lymphocytes undergo sequential changes that ultimately result in the acquisition of the cytotoxic function that is responsible for providing robust protection against viral infections and cancer. Before antigen encounter, mature CD8 T cells exist in a naive state, characterized by a quiescent phenotype in which cells use oxidative phosphorylation to generate energy, minimally proliferate, and have no cytotoxic function. Initial antigen recognition through the T-cell receptor, when accompanied by costimulation, leads to the activation of naive T cells. T-cell activation triggers a metabolic switch from oxidative phosphorylation to glycolysis, which supports lymphocytes as they rapidly proliferate and differentiate into CTLs. CTLs acquire the ability to kill target cells by secreting cytolytic effector molecules, such as perforin, granzymes and granulysin (in humans). Following clearance of the pathogenic cells, the majority of CTLs die by apoptosis, an event known as contraction. However, a small population of cells will survive, and will form antigen-specific memory CD8 T cells capable of providing long-term protection to the host, should antigen re-encounter occur [1]. Yet, in the case of chronic viral infection or cancer, these differentiation programs often become disrupted and CD8 T cells are rerouted into the so-called 'exhausted' state, defined by the inability of CD8 T cells to perform their cytotoxic function [2]. Identification of the signals responsible for disturbing the differentiation of CD8 T cells is currently a major focus of T-cell biology.The tumor microenvironment (TME) provides signals that may suppress immune responses, including those mediated by CD8 T cells. The TME is a complex milieu of cells including not only tumor cells and CTLs but also such immunosuppressive cell populations as Tregs, myeloid-derived suppressor cells and subpopulations of tumor-associated macrophages, all of which are supported by the tumor-associated extracellular matrix [3]. In fact, these cellular components of the TME are capable of producing ligands, which lead to CD8 T-cell exhaustion upon recognition. Normally, activated/effector T cells express the receptors for these ligands where receptor/ligand engagement provides inhibitory signals to help prevent overactivation of T cells during the immune response, thus serving as a negative feedback. For this reason, these inhibitory receptors are often referred to as immune checkpoint receptors, of which there are many, the most well-known being PD-1, Tim-3 and CTLA-4 [2]. Inflammatory conditions in the TME further drive the upregulation of the expression of these inhibitory receptors on the resident CTLs, in addition to promoting the expression of their ligands [3]. As a result, tumors provide robust inhibitory signals that drive CD8 T-cell exhaustion.Immune checkpoint blockade therapy addresses this particular type of inhibitory signal by using antibodies to prevent signal transduction through specific inhibitory receptors. Blocking antibodies have been developed against many receptors and their ligands. For example, antibodies that target CTLA-4, the PD-1 receptor and its ligand PD-L1 have been approved by the US FDA and are in clinical use [4]. While this therapy has had immense success in the clinic, it has limitations. Primarily, the success of immune checkpoint blockade therapy is intrinsically tied to CD8 T-cell infiltration into the tumor. It is known that many so-called 'cold' tumors are weakly immunogenic and recruit very few immune cells to the TME [3]. Other immune-based therapies also have been developed to circumvent the constraints imposed by the TME. An important form of adoptive T-cell therapy centers on the isolation of tumor-specific CD8 T cells from the patient, expanding these tumor-infiltrating lymphocytes in vitro, and then transfusing the large number of tumor-specific CD8 T cells back into the patient, thus accounting for the failure to proliferate and expand. Moreover, this approach 'reverses' exhaustion as it removes cells from the suppressive TME and allows them to recover prior to reinfusion [3].The use of these therapies in combination with each other has resulted in the significant improvement of patient responses; however, these improvements are observed in a limited number of patients, and the success rate is dependent on the type of cancer being treated. While the results are remarkable for patients that respond, in two recent clinical trials only 20% of melanoma patients showed durable clinical responses to ipilimumab (anti-CTLA-4 monoclonal antibody), and only 17% of non-small-cell lung cancer patients exhibited an objective response rate with nivolumab (anti-PD-1 monoclonal antibody) [5]. As such, it would be ideal to enhance the currently existing immunotherapies to increase the success rate for all patients, regardless of the type of cancer for which they are being treated. In fact, this is a specific goal of the Cancer Moonshot initiative established to address the prevention, early detection, treatment and curing of cancer [6].Let-7 miRNAs as global regulatorsMiRNAs regulate multiple important biological processes, including cell differentiation, through a post-transcriptional mechanism that is based on sequence-specific inhibition of mRNA translation. Genes-encoding miRNAs are first transcribed as a primary transcript by RNA PolII and PolIII, called primary miRNAs, which may vary in size, but are usually very long. While still in the nucleus, the primary miRNA is processed into the hairpin-like 60–70 nt precursor miRNA (pre-miRNA) by the microprocessor, a complex containing the endonuclease Drosha. Next, the pre-miRNA is exported from the nucleus to the cytoplasm, where the pre-miRNA stem loop is cleaved by the endonuclease Dicer, generating a mature approximately 22 nt double-stranded miRNA. A single strand of this mature miRNA is then loaded into the RNA-induced silencing complex, which can prevent protein synthesis either by stalling the ribosomal machinery or by destabilizing the targeted mRNA [7]. The sequence with which miRNAs bind their RNA targets is known as the 'seed sequence', is typically 6–8 nt long, and is located at the 5′-end of the miRNA. Based on the conservation of this seed sequence, miRNAs are grouped into families, such that all members of one miRNA family have the same target mRNAs.Let-7 is one of the most highly conserved families of miRNAs in the animal kingdom. Having undergone several duplications in evolution, the let-7 family is comprised of multiple paralog genes expressed on different chromosomes, forming the largest miRNA family in mammals. In fact, sequences of mature let-7 miRNAs are often identical although they are derived from different precursors and genes [8]. To indicate the differences in sequence across genes, a letter is placed after let-7 (i.e., let-7a, let-7b), and a number is placed after this letter to indicate that the same sequence of gene is expressed in multiple genomic locations (i.e., let-7c-1, let-7c-2). In humans, there are ten mature let-7 family members generated from 13 precursor sequences, and in mice there are eight mature family members arising from 11 precursor miRNAs.It has been demonstrated that let-7 miRNAs are involved in multiple biological processes including differentiation, cell death and metabolism [8,9]. Specifically, let-7 miRNAs were identified as potent tumor suppressors that directly target mRNAs of genes involved in the cell cycle and in signal transduction pathways that lead to carcinogenesis [8]. Although let-7 miRNAs can be found in many types of cells and tissues, the expression levels vary, revealing complicated regulation. In fact, let-7 miRNA expression is post-transcriptionally regulated by multiple factors that control different stages of let-7 biogenesis. Lin28 and Lin28B are well-studied fetal proteins, that block the generation of mature let-7 miRNAs. Both proteins interfere with Dicer processing by binding to a highly conserved sequence within the stem loop of let-7 miRNA precursors and recruiting the terminal uridylyl transferases, which uridylate the immature miRNA, allowing the exonuclease Dis3I2 to recognize and degrade it [10,11]. This particular mechanism is used to inhibit global let-7 expression during early embryogenesis. Lin28-mediated modulation of let-7 miRNA levels has been implicated during early embryogenesis and in establishing the immune responses of neonates [8,12].Let-7 miRNAs in T-cell responsesWe and others have shown that the let-7 miRNAs are expressed in T-cell progenitors and that their expression is very abundant in mature T lymphocytes, suggesting a potentially important role for these miRNAs in T-cell-mediated immune responses [13,14]. In fact, the regulatory role of let-7 miRNAs has been demonstrated during the activation and differentiation of CD4 T-cell subsets. Specifically, it has been suggested that the let-7 miRNAs are in part responsible for inhibiting the expression of proteins in the mTOR pathway, the major metabolic hub in activated and effector CD4 T cells [15]. Recently, it has been reported that Tregs may even prevent the differentiation of CD4 T-helper cells by secreting let-7 containing exosomes [16]. Thus, the ability of let-7 to modulate CD4 T-cell-mediated responses, including the differentiation of IFN-γ, IL-4 and IL-17-producing CD4 T-helper cells, has become increasingly clear [15,17,18].Although the expression of let-7 miRNAs in CD8 T lymphocytes has been shown earlier, the role of these miRNAs in regulation of CD8 T-cell differentiation was not clear [13]. In our recent paper, we demonstrated the significance of let-7 expression for the homeostasis and function of CD8 T lymphocytes. This work is based on a finding, which is in agreement with previous observations [13] that antigen stimulation of CD8 T cells results in a profound inhibition of let-7 expression. Two important questions followed this observation: why do naive CD8 T cells require high levels of let-7 miRNA expression and why must activated lymphocytes downregulate these miRNAs? These questions were addressed in experiments where let-7 levels in T cells were genetically manipulated. Let-7 depletion was achieved using previously described mice with a T-cell-specific Lin28 transgene while let-7 reexpression was induced in T cells derived from doxycycline inducible let-7 transgenic (let-7Tg) mice [9,19].Analysis of naive CD8 T cells with lin28-mediated knockdown of let-7 expression revealed compromised homeostasis of these cells in vivo. Due to an unknown molecular mechanism, let-7-deficient naive T cells exhibited spontaneous activation and increased proliferation resulting in the loss of the quiescent state. These unexpected results led to the model that let-7 miRNAs function as a molecular brake that prevents spontaneous CD8 T-cell activation, while antigen stimulation releases it by actively reducing let-7 expression and allowing T cells to differentiate into killer cells. In fact, the extent of functional maturation inversely correlates with the levels of let-7 expression in effector cells. CTLs with let-7-deficiency exhibited enhanced cytotoxic function in vitro, while let-7Tg CTLs failed to lyse target cells as compared with their wild-type counterparts. In vivo, let-7Tg mice were unable to mount an efficient antiviral response and failed to reject an allogeneic tumor, a response that typically results in the activation of 1–3% of all CD8 T cells and immediate rejection of the grafted cells. Thus, the reduction of let-7 expression results in enhanced T lymphocyte function while high levels prevent it.We have only begun to understand the complexity of let-7-mediated regulation of CD8 T-cell differentiation, in which the let-7 miRNAs control multiple levels of this process, including the proliferation, metabolism and acquisition of effector functions. Specifically, the let-7 miRNAs inhibit expression of cell-cycle proteins, and the transcription factor Myc, which is responsible for inducing many of the genes that are involved in proliferation and glycolysis [20]. Furthermore, let-7-mediated targeting of the transcription factor Eomes suppresses the effector function of activated CD8 T cells. This work clearly demonstrated that the let-7 miRNAs are important regulators of CD8 T-cell differentiation and function.Importantly, in the absence of the let-7 miRNAs, antigen-specific CTLs exhibited dramatically enhanced cytotoxic function. Thus, manipulating let-7 levels in CTLs may be used as a new approach in T-cell-based therapies, defining let-7 miRNAs as a novel therapeutic target. We expect to significantly improve T-cell performance in chronic viral and antitumor responses by decreasing the levels of let-7 miRNAs and enhancing the killer-function of T lymphocytes. Specifically, this approach could be very useful in improving the performance of existing cancer immunotherapies, which include immune checkpoint blockade, adoptive T-cell transfer and even CAR-T-cell-based therapies. Such manipulations of let-7 levels in vitro and in vivo are already feasible and can be accomplished through cell-targeted biodelivery of agents that can deplete mature let-7 miRNAs in tumor-specific T cells.To successfully translate these findings into clinical practice and use them as a therapeutic tool that enhances antitumor immune responses, more fundamental and preclinical studies will be required. At the moment, it is not clear how the tumor immunosuppressive environment will affect the function and survival of let-7-deficient CTLs in vivo, despite the unexpected and gratifyingly potent performance of these cells in vitro. Furthermore, based on the high conservation of let-7 miRNAs and their targets, the similarity of let-7-mediated effects in mouse and human CD8 T cells can be predicted, but has yet to be shown. Whether the let-7 miRNAs are involved in the formation of memory T cells is unknown. However, the discovery of such an involvement would be of great interest and could have important implications for the development of more effective prophylactic and therapeutic cancer vaccines, thus contributing to cancer prevention and treatment, both of which are identified as goals of the aforementioned 'Cancer Moonshot' program [6]. The development of appropriate tumor models, including humanized mice, will be needed to address these important questions. Taken together, the development of let-7-based therapies could significantly advance the field of immunotherapy by eliminating or improving the limitations of the currently available immunotherapies, while at the same time maintaining the integrity of their most effective features. While let-7 has become a potential therapeutic target, ironically nothing is known about its regulation in T cells. Thus, understanding the molecular mechanisms that control let-7 miRNA expression during CD8 T-cell differentiation is yet another aspect of let-7 immunology that should command urgent attention.In conclusion, the discovery of the let-7-mediated molecular machinery that regulates cytotoxic T-cell function provides information about new levels of control in the immune system and identifies additional targets for the manipulation of T-cell immune responses. While there is still much to learn about the role of let-7 in T cells, and other hematopoietic cells, the potential of let-7 as a therapeutic target is promising and extremely exciting.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/References1 Kaech SM, Cui W. Transcriptional control of effector and memory CD8+ T cell differentiation. Nat. Rev. Immunol. 12(11), 749–761 (2012).Crossref, Medline, CAS, Google Scholar2 Blackburn SD, Shin H, Haining WN et al. Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat. Immunol. 10(1), 29–37 (2009).Crossref, Medline, CAS, Google Scholar3 Joyce JA, Fearon DT. T cell exclusion, immune privilege, and the tumor microenvironment. Science 348(6230), 74–80 (2015).Crossref, Medline, CAS, Google Scholar4 Sharma P, Allison JP. The future of immune checkpoint therapy. Science 348(6230), 56–61 (2015).Crossref, Medline, CAS, Google Scholar5 Topalian SL, Drake CG, Pardoll DM. Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell 27(4), 450–461 (2015).Crossref, Medline, CAS, Google Scholar6 Singer DS, Jacks T, Jaffee E. A U.S. "Cancer Moonshot" to accelerate cancer research. Science 353(6304), 1105–1106 (2016).Crossref, Medline, CAS, Google Scholar7 Ha M, Kim VN. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 15(8), 509–524 (2014).Crossref, Medline, CAS, Google Scholar8 Bussing I, Slack FJ, Grosshans H. let-7 microRNAs in development, stem cells and cancer. Trends Mol. Med. 14(9), 400–409 (2008).Crossref, Medline, Google Scholar9 Zhu H, Shyh-Chang N, Segrè AV et al. The Lin28/let-7 axis regulates glucose metabolism. Cell 147(1), 81–94 (2011).Crossref, Medline, CAS, Google Scholar10 Heo I, Joo C, Kim YK et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 138(4), 696–708 (2009).Crossref, Medline, CAS, Google Scholar11 Faehnle CR, Walleshauser J, Joshua-Tor L. Mechanism of Dis3l2 substrate recognition in the Lin28-let-7 pathway. Nature 514(7521), 252–256 (2014).Crossref, Medline, CAS, Google Scholar12 Wang J, Wissink EM, Watson NB et al. Fetal and adult progenitors give rise to unique populations of CD8+ T cells. Blood 128(26), 3073–3082 (2016).Crossref, Medline, CAS, Google Scholar13 Wu H, Neilson JR, Kumar P et al. miRNA profiling of naive, effector and memory CD8 T cells. PLoS ONE 2(10), e1020 (2007).Crossref, Medline, Google Scholar14 Wells AC, Daniels KA, Angelou CC et al. Modulation of let-7 miRNAs controls the differentiation of effector CD8 T cells. Elife 6, pii: e26398 (2017).Crossref, Google Scholar15 Marcais A, Blevins R, Graumann J et al. microRNA-mediated regulation of mTOR complex components facilitates discrimination between activation and anergy in CD4 T cells. J. Exp. Med. 211(11), 2281–2295 (2014).Crossref, Medline, Google Scholar16 Okoye IS, Coomes SM, Pelly VS et al. MicroRNA-containing T-regulatory-cell-derived exosomes suppress pathogenic T helper 1 cells. Immunity 41(1), 89–103 (2014).Crossref, Medline, CAS, Google Scholar17 Polikepahad S, Knight JM, Naghavi AO et al. Proinflammatory role for let-7 microRNAS in experimental asthma. J. Biol. Chem. 285(39), 30139–30149 (2010).Crossref, Medline, CAS, Google Scholar18 Guan H, Fan D, Mrelashvili D et al. MicroRNA let-7e is associated with the pathogenesis of experimental autoimmune encephalomyelitis. Eur. J. Immunol. 43(1), 104–114 (2013).Crossref, Medline, CAS, Google Scholar19 Pobezinsky LA, Etzensperger R, Jeurling S et al. Let-7 microRNAs target the lineage-specific transcription factor PLZF to regulate terminal NKT cell differentiation and effector function. Nat. Immunol. 16(5), 517–524 (2015).Crossref, Medline, CAS, Google Scholar20 Wang R, Dillon CP, Shi LZ et al. The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation. Immunity 35(6), 871–882 (2011).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByEpstein-Barr Virus BGLF2 commandeers RISC to interfere with cellular miRNA function10 January 2022 | PLOS Pathogens, Vol. 18, No. 1RNA in cancer20 October 2020 | Nature Reviews Cancer, Vol. 21, No. 1Genome-wide profiling and analysis of microRNA expression in buffalo milk exosomesFood Bioscience, Vol. 38Inhibition of let-7b-5p contributes to an anti-tumorigenic macrophage phenotype through the SOCS1/STAT pathway in prostate cancer29 September 2020 | Cancer Cell International, Vol. 20, No. 1Repression of Irs2 by let‐7 mi RNA s is essential for homeostasis of the telencephalic neuroepithelium28 September 2020 | The EMBO Journal, Vol. 39, No. 21Analysis of Let-7 Family miRNA in Plasma as Potential Predictive Biomarkers of Diagnosis for Papillary Thyroid Cancer28 February 2020 | Diagnostics, Vol. 10, No. 3Tumor-derived extracellular vesicles and microRNAs: Functional roles, diagnostic, prognostic and therapeutic optionsCytokine & Growth Factor Reviews, Vol. 51Evaluating prognostic utility of preoperative Neutrophil to Lymphocyte Ratio and hsa-let-7g/c up-regulation in patients with urinary bladder cancerCancer Biomarkers, Vol. 27, No. 1LIN28: A cancer stem cell promoter for immunotherapy in head and neck squamous cell carcinomaOral Oncology, Vol. 98 Vol. 14, No. 12 eToC Sign up Follow us on social media for the latest updates Metrics History Received 15 January 2018 Accepted 2 February 2018 Published online 23 April 2018 Published in print May 2018 Information© 2018 Leonid PobezinskyKeywordscancerCD8 T cellsCTLscytotoxic responseeomeslet-7miRNARNAitumorvaccines/immunotherapyFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/PDF download

The differentiation of naive CD8 T cells into effector cytotoxic T lymphocytes upon antigen stimulation is necessary for successful antiviral, and antitumor immune responses. Here, using a mouse model, we describe a dual role for the let-7 microRNAs in the regulation of CD8 T cell responses, where maintenance of the naive phenotype in CD8 T cells requires high levels of let-7 expression, while generation of cytotoxic T lymphocytes depends upon T cell receptor-mediated let-7 downregulation. Decrease of let-7 expression in activated T cells enhances clonal expansion and the acquisition of effector function through derepression of the let-7 targets, including Myc and Eomesodermin. Ultimately, we have identified a novel let-7-mediated mechanism, which acts as a molecular brake controlling the magnitude of CD8 T cell responses.

Singer and colleagues show that let-7 microRNAs target Zbtb16 mRNA, which encodes the transcription factor PLZF, to modulate PLZF expression during the terminal differentiation of NKT cells into effector subsets.

The lifelong presence of mature T cells in the organism is vital for immune responses. Despite being extensively studied, the molecular mechanisms governing T cell homeostasis are still not fully understood. Here we investigated how microRNAs, acting through post-transcriptional regulation, contribute to the maintenance of mature naïve T cells in the periphery. We found that T cell-specific Dicer ablation caused lymphopenia, due to survival defects in mature T cells. Using miRNA knockout mice, we identified specific miRNAs which, when deleted, resulted in T cell death, and overexpressing these miRNAs in the T cells of transgenic mice provided complete protection. Finally, we characterized the downstream molecular mechanisms responsible for miRNA-induced suppression of cell death. Altogether our findings represent a novel miRNA-mediated mechanism that controls homeostasis of peripheral naïve T cells in vivo.