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Transcription factor (TF) network: the Aspen Grove. Subsets of CD4+ T cells and innate lymphoid cells (ILCs) exhibit the same or similar functionalities. Presumably, this is because they employ many of the same TFs working in concert to effect similar programs. As a metaphor for this problem, we considered that though aspen trees appear to separate trees, they are, in fact, connected by a shared root system. We posit that the fundamental network of TFs might function analogously such that the same ‘root system’ (i.e. TF network) is used as cells differentiate from hematopoietic stem cell to the multiplicity of lymphoid cells, some of which express a T-cell receptor and some of which do not. Many TFs act deeply below the surface (Notch, GATA-3, STAT5) being fundamental for generation and maintenance; others are important for acquisition of more specific functions later on. Regardless though, even if it appears that one is looking at different ‘trees’, one does not have to dig very deep to find the common elements. The Pando grove is the largest known aspen grove located in Utah (USA), and comprises more than 100 acres and 43 000 trees. It is estimated to be 80 000 years old.

Transcription factor (TF) network: the Aspen Grove. Subsets of CD4+ T cells and innate lymphoid cells (ILCs) exhibit the same or similar functionalities. Presumably, this is because they employ many of the same TFs working in concert to effect similar programs. As a metaphor for this problem, we considered that though aspen trees appear to separate trees, they are, in fact, connected by a shared root system. We posit that the fundamental network of TFs might function analogously such that the same ‘root system’ (i.e. TF network) is used as cells differentiate from hematopoietic stem cell to the multiplicity of lymphoid cells, some of which express a T-cell receptor and some of which do not. Many TFs act deeply below the surface (Notch, GATA-3, STAT5) being fundamental for generation and maintenance; others are important for acquisition of more specific functions later on. Regardless though, even if it appears that one is looking at different ‘trees’, one does not have to dig very deep to find the common elements. The Pando grove is the largest known aspen grove located in Utah (USA), and comprises more than 100 acres and 43 000 trees. It is estimated to be 80 000 years old.

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The discovery of the specification of CD4+ helper T cells to discrete effector ‘lineages’ represented a watershed event in conceptualizing mechanisms of host defense and immunoregulation. However, our appreciation for the actual complexity of helper T-cell subsets continues unabated. Just as the Sami language of Scandinavia has 1000 different words...

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... TFs can define and/or preserve boundaries among lineages. However, the same TFs can be 'recycled' during differentiation by switching their expression on and off, serving distinct functions at different times. They can be also shared among the different lineages, making the boundaries of lineage-defining TFs blur and dif- ficult to distinguish (Fig. ...

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... The human CD4+ T cell is a heterogeneous population, divided into sub-populations depending on the functions performed. The activities of individual subpopulations result from the type of transcription factors and receptors contained in cells, as well as secreted cytokines, specific for each subpopulation (22). ...
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Lymphocytes play a leading role in regulation of the immune system in lung cancer patients. The recognition of T cells profile may help in prediction of effectiveness of anticancer immunotherapy. The aim of the study was to determine the dominant subpopulation of CD4+ and CD8+ lymphocytes in metastatic and non-metastatic lymph nodes (LNs) of lung cancer patients. LNs aspirates were obtained during EBUS/TBNA procedure and cells were analyzed by flow cytometry. We showed a higher percentage of CD4+ and CD8+ effector memory T cells in the metastatic than in the non-metastatic LNs (28.6 vs. 15.3% and 28.6 vs. 14.0%, p< 0.05). The proportion of CD45RO+ T regulatory cells (CD45RO+ Tregs) was higher in the metastatic LNs than in the non-metastatic ones (65.6 vs. 31%, p< 0.05). We reported the significant differences in T cell subsets depending on the lung cancer metastatic process. We observed that the effector memory T cells were predominant subpopulations in metastatic LNs. Lymphocyte profile in LNs is easy to evaluate by flow cytometry of EBUS/TBNA samples and may reflect the immune status in lung cancer.
... Indeed, the ILC1, ILC2 and ILC3 subsets produce T helper (Th) 1-, Th2-and Th17/22-cytokines, respectively. Furthermore, given their phenotypic, developmental and functional similarities, Natural Killer (NK) cells, the innate counterpart of cytotoxic T lymphocytes, are now grouped together with ILC1s, whereas lymphoid tissue inducer (LTi) cells, belong now to group 3 ILCs (5,7,8). ...
Article
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Innate lymphoid cells (ILCs) were firstly described by different independent laboratories in 2008 as tissue-resident innate lymphocytes mirroring the phenotype and function of T helper cells. ILCs have been subdivided into three distinct subgroups, ILC1, ILC2 and ILC3, according to their cytokine and transcriptional profiles. Subsequently, also Natural Killer (NK) cells, that are considered the innate counterpart of cytotoxic CD8 T cells, were attributed to ILC1 subfamily, while lymphoid tissue inducer (LTi) cells were attributed to ILC3 subgroup. Starting from their discovery, significant advances have been made in our understanding of ILC impact in the maintenance of tissue homeostasis, in the protection against pathogens and in tumor immune-surveillance. However, there is still much to learn about ILC ontogenesis especially in humans. In this regard, NK cell developmental intermediates which have been well studied and characterized prior to the discovery of helper ILCs, have been used to shape a model of ILC ontogenesis. Herein, we will provide an overview of the current knowledge about NK cells and helper ILC ontogenesis in humans. We will also focus on the newly disclosed circulating ILC subsets with killing properties, namely unconventional CD56dim NK cells and cytotoxic helper ILCs, by discussing their possible role in ILC ontogenesis and their contribution in both physiological and pathological conditions.
... The TF EOMES, T-BET, GATA3, and RORγt, mentioned above, are also referred to as lineage =-defining TFs (LDTFs), since these molecules dictate ILC fates and are required for determining the effector functions of mature ILC subsets [26,27]. LDTFs represent the first layer of ILC regulation, although the establishment of specific developmental programs and effector functions is now seen as the result of complex TF networks rather than the effect of one single "master" regulator [28]. ...
Article
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Innate lymphoid cells (ILCs) are innate lymphocytes playing essential functions in protection against microbial infections and participate in both homeostatic and pathological contexts, including tissue remodeling, cancer, and inflammatory disorders. A number of lineage-defining transcription factors concur to establish transcriptional networks which determine the identity and the activity of the distinct ILC subsets. However, the contribution of other regulatory molecules in controlling ILC development and function is also recently emerging. In this regard, noncoding RNA (ncRNAs) represent key elements of the complex regulatory network of ILC biology and host protection. ncRNAs mostly lack protein-coding potential, but they are endowed with a relevant regulatory activity in immune and nonimmune cells because of their ability to control chromatin structure, RNA stability, and/or protein synthesis. Herein, we summarize recent studies describing how distinct types of ncRNAs, mainly microRNAs, long ncRNAs, and circular RNAs, act in the context of ILC biology. In particular, we comment on how ncRNAs can exert key effects in ILCs by controlling gene expression in a cell- or state-specific manner and how this tunes distinct functional outputs in ILCs.
... Most of the initial findings leading to the identification of novel ILC subsets were built upon the observation that "helper" responses could be found in Rag-deficient mice and, in general, were based on the evidence that non-T lymphocytes could produce large amounts of type 2 and type 3 cytokines (Cupedo et al., 2009;Fallon et al., 2006;Fort et al., 2001;Hurst et al., 2002;Moro et al., 2010;Neill et al., 2010;Price et al., 2010;Takatori et al., 2009). The identification of such innate cells able to perform effector functions similar to those observed in T cells, but in a shorter time frame, led scientists to rethink many aspects of the immune response, ranging from protection against pathogens to cancer immunosurveillance and immunostimulation (Cherrier et al., 2018;Mattiola and Diefenbach, 2019;Shih et al., 2014;. In, roughly, the last 10 years, the ILC field has brought to light a variety of subsets and complexity which parallel those of T cells Vivier et al., 2018). ...
Article
Innate lymphoid cells (ILCs) and tissue-resident natural killer (NK) cells ensure immunity at environmental interfaces and help maintain barrier integrity of the intestinal tract. This wide range of innate lymphocytes is able to provide fast and potent inflammatory responses that, when deregulated, have been associated with pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC). While the presence of tumor-infiltrating NK cells is generally associated with a favorable outcome in CRC patients, emerging evidence reveals distinct roles for ILCs in regulating CRC pathogenesis and progression. Advances in next generation sequencing technology, and in particular of single-cell RNA-seq approaches, along with multidimensional flow cytometry analysis, have helped to deconvolute the complexity and heterogeneity of the ILC system both in homeostatic and pathological contexts. In this review, we discuss the protective and detrimental roles of NK cells and ILCs in the pathogenesis of CRC, focusing on the phenotypic and transcriptional modifications these cells undergo during CRC development and progression.
... This could improve the therapeutic effects of other anti-tumor drugs, especially immune checkpoint inhibitors (ICIs). Within the TIME, epigenetic modifications can also be found in tumor-associated immune cells, including myeloid cells, CD4 + T cells, and CD8 + T cells (9)(10)(11). During the differentiation from naïve CD8 + T cells to CD8 + effector T cells, epigenetic changes, such as DNA methylation and histone modifications, are involved in the chromatin accessibility (12,13). ...
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The tumor immune microenvironment (TIME), an immunosuppressive niche, plays a pivotal role in contributing to the development, progression, and immune escape of various types of cancer. Compelling evidence highlights the feasibility of cancer therapy targeting the plasticity of TIME as a strategy to retrain the immunosuppressive immune cells, including innate immune cells and T cells. Epigenetic alterations, such as DNA methylation, histone post-translational modifications, and noncoding RNA-mediated regulation, regulate the expression of many human genes and have been reported to be accurate in the reprogramming of TIME according to vast majority of published results. Recently, mounting evidence has shown that the gut microbiome can also influence the colorectal cancer and even extraintestinal tumors via metabolites or microbiota-derived molecules. A tumor is a kind of heterogeneous disease with specificity in time and space, which is not only dependent on genetic regulation, but also regulated by epigenetics. This review summarizes the reprogramming of immune cells by epigenetic modifications in TIME and surveys the recent progress in epigenetic-based cancer clinical therapeutic approaches. We also discuss the ongoing studies and future areas of research that benefits to cancer eradication.
... Innate lymphoid cells (ILCs) play critical roles in tissue homeostasis, barrier integrity and primary host defense and mirror the functionalities of their effector counterparts in the adaptive immune compartment, CD4 + helper T (Th) and CD8 + cytotoxic T lymphocytes (CTL) (7)(8)(9)(10). The similarities between innate and adaptive lymphocyte programming have dramatically accelerated our understanding of ILC regulation using the knowledge accumulated from studies of T cells (11)(12)(13)(14). ...
... Finally, type 3 immunity is governed by RAR-related orphan receptor gamma, RORgt (encoded by the Rorc gene), which controls ILC3 and Th17 lineage specification and cytokine secretion (37, 38). These LDTFs epigenetically activate and stabilize function-related gene expression and, at the same time, inhibit transcription of genes that contribute to alternative cell fates (8,39). ...
Article
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The precise control of cytokine production by innate lymphoid cells (ILCs) and their T cell adaptive system counterparts is critical to mounting a proper host defense immune response without inducing collateral damage and autoimmunity. Unlike T cells that differentiate into functionally divergent subsets upon antigen recognition, ILCs are developmentally programmed to rapidly respond to environmental signals in a polarized manner, without the need of T cell receptor (TCR) signaling. The specification of cytokine production relies on dynamic regulation of cis-regulatory elements that involve multi-dimensional epigenetic mechanisms, including DNA methylation, transcription factor binding, histone modification and DNA-DNA interactions that form chromatin loops. How these different layers of gene regulation coordinate with each other to fine tune cytokine production, and whether ILCs and their T cell analogs utilize the same regulatory strategy, remain largely unknown. Herein, we review the molecular mechanisms that underlie cell identity and functionality of helper T cells and ILCs, focusing on networks of transcription factors and cis-regulatory elements. We discuss how higher-order chromatin architecture orchestrates these components to construct lineage- and state-specific regulomes that support ordered immunoregulation.
... It is worth noting that most previous studies involved with the mechanism of low-dose decitabine therapy mainly focus on cancer cells and CD8 + T cells rather than CD4 + T cells Topper et al., 2020). Epigenetic modification plays an important role in the differentiation of CD4 + T cells (Shih et al., 2014;Tripathi and Lahesmaa, 2014), while the regulation of CD4 + T cells in anti-tumor activity still needs to be deeply explored. ...
Article
Background: CD4+ T cells play multiple roles in controlling tumor growth and increasing IFN-γ+ T-helper 1 cell population could promote cell-mediated anti-tumor immune response. We have previously showed that low-dose DNA demethylating agent decitabine therapy promotes CD3+ T-cell proliferation and cytotoxicity; however, direct regulation of purified CD4+ T cells and the underlying mechanisms remain unclear. Methods: The effects of low-dose decitabine on sorted CD4+ T cells were detected both in vitro and in vivo. The activation, proliferation, intracellular cytokine production and cytolysis activity of CD4+ T cells were analyzed by FACS and DELFIA time-resolved fluorescence assays. In vivo ubiquitination assay was performed to assess protein degradation. Moreover, phosphor-p65 and IκBα levels were detected in sorted CD4+ T cells from solid tumor patients with decitabine-based therapy. Results: Low-dose decitabine treatment promoted the proliferation and activation of sorted CD4+ T cells, with increased frequency of IFN-γ+ Th1 subset and enhanced cytolytic activity in vitro and in vivo. NF-κB inhibitor, BAY 11-7082, suppressed decitabine-induced CD4+ T cell proliferation and IFN-γ production. In terms of mechanism, low-dose decitabine augmented the expression of E3 ligase β-TrCP, promoted the ubiquitination and degradation of IκBα and resulted in NF-κB activation. Notably, we observed that in vitro low-dose decitabine treatment induced NF-κB activation in CD4+ T cells from patients with a response to decitabine-primed chemotherapy rather than those without a response. Conclusion: These data suggest that low-dose decitabine potentiates CD4+ T cell anti-tumor immunity through enhancing IκBα degradation and therefore NF-κB activation and IFN-γ production.
... Our observation that the TFs Klf4 and Nfkbiz are orthogonally expressed suggests a regulatory mechanism for the specialization of IL-6 and IL-12p40 secretion versus Chi3l3 secretion. Klf4 is an example of a macrophage-specific TF that regulates specialized gene programs 35 . Based on sequence analysis, there is a binding site for Klf4 on the promoter of Nfkbiz 35 , and thus it is possible that Klf4 directly negatively regulates Nfkbiz upon co-stimulation. ...
... Macrophage-secreted cytokines and chemokines are essential for coordinating the immune response in complex tissue microenvironments. We observed nearly orthogonal secretion of IL-4stimulated Chi3l3 and LPS+IFN-γ-stimulated IL-6 and IL-12p40 (Fig. 5a), which are essential cytokines for inducing T-cell polarization 35 . The observed diversification of single macrophages across a range of secretion programs, when presented with opposing cues, may enable macrophages to more quickly adapt to changing environments. ...
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Macrophages are innate immune cells that contribute to fighting infections, tissue repair, and maintaining tissue homeostasis. To enable such functional diversity, macrophages resolve potentially conflicting cues in the microenvironment via mechanisms that are unclear. Here, we use single-cell RNA sequencing to explore how individual macrophages respond when co-stimulated with inflammatory stimuli LPS and IFN-γ and the resolving cytokine IL-4. These co-stimulated macrophages display a distinct global transcriptional program. However, variable negative cross-regulation between some LPS + IFN-γ-specific and IL-4-specific genes results in cell-to-cell heterogeneity in transcription. Interestingly, negative cross-regulation leads to mutually exclusive expression of the T-cell-polarizing cytokine genes Il6 and Il12b versus the IL-4-associated factors Arg1 and Chil3 in single co-stimulated macrophages, and single-cell secretion measurements show that these specialized functions are maintained for at least 48 h. This study suggests that increasing functional diversity in the population is one strategy macrophages use to respond to conflicting environmental cues.
... Thus, there was no evidence for epigenomic "poising" of pre-selection DP thymocytes towards either lineage. Last, we observed epigenomic opening at genes involved in mature T cell effector functions (Shih et al., 2014): it was largely CD4 + -lineage specific at Il21 and Cd40lg (Fig. S3GH), whereas CD8 + -lineage preferential peaks opened at the Ifng and Gzmb loci. ...
Article
αβ lineage T cells, most of which are CD4⁺ or CD8⁺ and recognize MHC I- or MHC II-presented antigens, are essential for immune responses and develop from CD4⁺CD8⁺ thymocytes. The absence of in vitro models and the heterogeneity of αβ thymocytes have hampered analyses of their intrathymic differentiation. Here, combining single-cell RNA and ATAC (chromatin accessibility) sequencing, we identified mouse and human αβ thymocyte developmental trajectories. We demonstrated asymmetric emergence of CD4⁺ and CD8⁺ lineages, matched differentiation programs of agonist-signaled cells to their MHC specificity, and identified correspondences between mouse and human transcriptomic and epigenomic patterns. Through computational analysis of single-cell data and binding sites for the CD4⁺-lineage transcription factor Thpok, we inferred transcriptional networks associated with CD4⁺- or CD8⁺-lineage differentiation, and with expression of Thpok or of the CD8⁺-lineage factor Runx3. Our findings provide insight into the mechanisms of CD4⁺ and CD8⁺ T cell differentiation and a foundation for mechanistic investigations of αβ T cell development.
... 14 Helper ILCs can be classified into ILC1, ILC2, ILC3 and lymphoid tissue inducer (LTi) subtypes based on their cytokine production and transcriptional profiles ( Figure 1): in this regard, ILC1s, ILC2s and ILC3s resemble CD4 + T helper (Th)1, Th2 and Th17/22 cells, respectively. 15,16 LTi cells are indispensable for the initiation of secondary lymphoid organ development during embryonic life. 17 Adult LTi cells are Neuropilin-1+ (NRP1+) ILC3-like cells that are key inducers of ectopic lymphoid aggregate (ELA) formation. ...