Advances in understanding epigenetic impacts on dendritic cell regulation and function

Abstract

Dendritic cells (DCs) are the only cells empowered with inducing primary immune response among resting naïve T lymphocytes, hence it is crucial to understand the regulation and function of DCs. During an adaptive immune response, DCs acquire antigens from invasive entities and present the antigens on their own cell surface, hence they are also known as professional antigen‐presenting cells. Epigenetic modifications to the genome play an important role in both the development and the function of DCs. In this direction, significant advancements have been made using high‐throughput methods like ATAC‐seq, ChIP‐seq, RNA‐seq, bisulfite‐seq and so forth to uncover chromatin accessibility landscape, genome‐wide transcription factor (TF) binding, complete transcriptomic profiles, and DNA methylation, respectively. DCs lineage specification and function are determined by TF binding, which is dependent on epigenetic modifications. However, major gaps in knowledge still exist regarding how and why aberrant epigenetic modifications result in defective development and function of DCs leading to an impaired immune system. Hence, this review compiles up‐to‐date literature regarding the exquisite regulation of DCs via epigenetic regulation, to emphasize the potential of further epigenetics research on DCs for addressing current gaps of knowledge in the field. The review also briefly highlights recent findings on DC defects in coronavirus disease 2019 revealed by single‐cell RNA‐seq, and the importance of DCs in clinical trials. Overall, the review highlights how epigenetics‐based state‐of‐the‐art high‐throughput technology can help discoveries on DCs which are crucial to the immune system and pathogenesis, and how DCs are currently targeted for therapeutic developments and clinical trials.

INTRODUCTION

First discovered in the 1970s by Ralph Steinman and Zanvil Cohn at The Rockefeller University in New York, dendritic cells (DCs) have moved their way to the forefront of immunological research in the last half-century. As vital players of the immune system, DCs are responsible for activating white blood cells in response to inflammatory stimuli. The role of DCs is 2-fold; they act as macrophages by engulfing antigenic material, and they act as antigen-presenting cells to present antigens in a way that can be read by lymphocytes, serving as a bridge between innate and adaptive immunity. Typically referred to as ‘accessory cells’ or ‘A cells’, DCs were first named when their branch-like shape was reported by Steinman in the early 1970s. Apart from their physical characteristics, the function of the DC was under extensive investigation.

Their importance was largely overlooked until an experiment by Mosier showed a lack of antigen response in cell culture when a group of cells incorrectly classified with macrophages were omitted (DCs). It wasn't until the close analysis of Mosier's data that DCs, making up 0.1% of the cell population, were identified as a unique cell population that were necessary players in the immune response. These DCs were further differentiated when they were subsequently observed under a microscope.

Currently, DCs are characterized by their predominant involvement in antigen presentation to lymphocytes. However, the understanding of how DCs are regulated, specifically in terms of epigenetics, and how they can be used therapeutically, is largely opaque. Continued research on the mechanisms of epigenetic regulation of DCs is necessary to catapult technological breakthroughs in DC-based therapeutics for precision medicine.

DCs: BACKGROUND AND SIGNIFICANCE

The development and function of DCs are highly regulated processes that are best understood in the context of epigenetics. Epigenetic regulation describes the processes that control gene expression by means other than altering the actual DNA sequence. The addition of a methyl group (-CH3) onto a cytosine nucleotide or the acetylation of a lysine residue (K) on proteins called histones are both examples of epigenetic modifications that alter gene expression. These mechanisms will be further explained in the next section. Within the immune system, epigenetic modifications organize and regulate transcription factor (TF) networks that are involved in determining the lineage, function and plasticity of immune cells. This epigenetic regulation is essential for maintaining a heterogeneous population of immune cells that allows the immune system to orchestrate powerful and diverse responses.

While generally defined as having antigen presentation as a primary functionality, DCs can be divided into subsets with further specialized roles. DCs are first differentiated from common DC progenitors (CDPs) which are initially derived from multiple potent progenitors. The prominent DCs that are generated from CDPs are plasmacytoid DCs (pDCs) and conventional DCs (cDC1s and cDC2s). pDCs are associated with substantial production of type-I interferons (IFNs), a class of IFNs that work to limit the capacity of infectious agents during a viral response. Interferons are molecules that serve as the basis for the innate immune response against viral infections in vertebrate hosts. On the other hand, cDCs are specialized to activate different types of T-cells, thymus-derived lymphocyte subsets that work in the immune system to recognize foreign material, with the type being largely dependent on the differentiation of cDCs into cDC1s or cDC2s. cDC1s are associated with the activation of CD4+ T-cells, a class of T-cells responsible for recognizing peptides from extracellular protein, while cDC2s activate CD8+ T-cells that are responsible for recognizing peptides from relevant proteins. Studies done on transplant patients have demonstrated another pathway of DC differentiation that is modulated by epigenetic regulation. Following transplantation, DCs can either act as activated, inducing immune responses that activate downstream T-cells and other immune cells, or as tolerogenic, acting as immune suppressors. Epigenetic mechanisms are implicated in the decision of monocyte-derived DCs to differentiate into either activated or tolerogenic DCs. The use of immunotherapy treatments with tolerogenic DCs in transplant patients is becoming an increasingly popular alternative to immunosuppressive drugs.

The lineage fate of DCs is controlled by a variety of TFs, some of which are universal to all DCs, and some that are subset specific. For example, both Pu.1 and STAT3 are TFs crucial to the differentiation of progenitor cells into pDCs or cDCs. Specifically, Pu.1 is a transcription activator that binds the PU-box or purine-rich lymphoid-specific enhancer, while the role of STAT3 is described by its full name, signal transducer and activator of transcription 3. The lack of either of these TFs in progenitor cells results in significant defects during DC lineage specification. For differentiation of cDCs into cDC1s, the expression of subset-specific TFs such as BATF3 and IRF8 are particularly important, while cDC2s require the expression of TFs such as IFR4 (interferon regulatory factor 4) and RELB (reticuloendotheliosis viral [v-rel] oncogene related B) for correct differentiation and function. IFR4 is crucial for immune system development and function given its role in DC specification. Similarly, RELB plays important regulatory roles in lymphoid organs within interdigitating DCs, mature B cells, and plasma cells. Thus, each TF is unique and critical for the correct specification of various DCs.

The expression level of various TFs in DCs is dependent on how well each TF binds DNA to transcribe and ultimately translate more protein. However, their binding capacity to their respective DNA binding sites is modulated via epigenetic modifications to the chromatin structure of the cells. Thus, the ability of PDCs to differentiate into specialized subsets and coordinate a productive immune response is largely dependent on epigenetic regulation.

The molecular functions of DCs are transcriptionally regulated, much like the development of DCs themselves. For example, the activation and repression of DCs are regulated at the level of transcription, primarily with the goal of maintaining DC homeostasis. The nuclear factor kappa-light-chain-enhancer of activated B cells or nuclear factor-kappa B (NF-kB) is a TF important for regulating the activation of DCs; in a steady-state, NF-kB represses DC activation by occupying the transactivator promoter of the major histocompatibility protein complex class II, a class of molecules used by DCs to help present antigens on their surface. Additionally, NF-kB will relocate from the transactivator promoter under inflammatory conditions, thus promoting DC activation as well. The exact mechanisms that control NF-kB binding, and whether or not epigenetic modifications to the DC chromatin landscape play a part, are yet to be discovered and need further research. A study by Bluml et al. has investigated another factor involved in dendritic cell regulation, oxidized phospholipids (Ox-Pls). Ox-Pls arise during inflammation and disrupt the phosphorylation of Histone H3, leading to inhibition of the full activation of DCs. This modification works to impede DC function by reducing the DC production of interleukin-12, a proinflammatory cytokine that works to enhance the activity of natural killer cells and cytotoxic T lymphocytes, and by overall limiting their T-cell stimulating capacity. An additional study by Wen et al. looks into further epigenetic modifications that lead to a decrease in IL-2 production in DCs. The study investigated DCs in animals that survived severe sepsis by ChIP-seq and found that certain histone modifications were leading to inhibited IL-2 production, and thus a weakened immune response. Studies discussed in this section provide an excellent foundation for changes and shift in DC subsets based on epigenetic markers which motivate further research for understanding the molecular mechanisms of those epigenetic regulations towards aberrant immune response and pathogenesis.

Research supports the notion that certain TFs regulate DCs. Some TFs have epigenetic roles in the suppression of inflammatory-sensitive genes in a DC steady-state. One of these TFs is Polycomb Group Ring Finger 6, a member of the Polycomb Repressive Complex 1, which promotes the trimethylation of Histone H3 Lysine 9 and the monoubiquitylation of histone H2A. Both are chromatin modifications that repress transcription and subsequent gene expression. Ultimately, the TF networks that govern DC function are responsible for maintaining a steady-state by repressing gene expression in non-inflammatory conditions and relieving those transcriptional restraints to promote activation in the wake of inflammatory stimuli.

While the processes that regulate the development and activity of DCs are widely studied, it is important to also examine the fate of DCs after they successfully transmit antigenic information to a lymphocyte. In response to an inflammatory signal, DCs undergo activation and search for a T-cell to which it can present its antigen, and in return, the T-cell transfers valuable genetic information to the DC; this is known as an immune synapse (IS). The IS denotes a specialized region where T-cell and APC/DC are in intimate contact to decipher, coordinate, and integrate signals for promoting T-cell activation. IS forms when TCR ligates to cognate peptide bound to MHC on surfaces of APC/DC resulting in spatial reorganization of the TCR and accessory receptors. The information passed to the DC during an IS promotes the anti-viral activities of the DC by prolonging the cell's lifespan and by increasing the capability of the DC to activate other types of T-cells. A study by Alcaraz-Serna et al. investigating the epigenomic and transcriptomic changes in postsynaptic DCs showed that when DCs are pulsed (subjected to time-controlled incubation with a peptide that elicits a hypersensitivity response), they result in a productive IS, while non-pulsed DCs result in aberrant IS. When comparing the pulsed versus unpulsed cells, it was found that many regions with differentially accessible chromatin were located adjacent to differentially expressed genes related to the immune response. These studies provide evidence that the information transferred from the T-cell during communication with a DC result in epigenetic modifications to the DC chromatin structure that led to changes in the expression of immune-response related genes.

EPIGENETICS AND CHROMATIN

To understand the epigenetic regulation of DCs, a brief explanation of epigenetics and chromatin structure is warranted. Epigenetics denotes a heritable phenomenon that regulates gene expression without changing underlying DNA sequences. In other words, epigenetic changes are typically characterized by molecular modifications added on top of the DNA itself that change the way that DNA is expressed. DNA is compactly packaged inside a eukaryotic nucleus where it binds octamers comprised of proteins called core histones, namely H2A, H2B, H3 and H4. A histone octamer is a complex of a tetramer, that is, two copies of H3 and H4, and two dimers of H2A-H2B. Epigenetic mechanisms tighten or loosen the DNA that wraps around the histone octamers, impacting gene expression. Generally, tight packaging around histone octamers renders the DNA inaccessible to transcription machinery, thus, the gene is silenced. Conversely, loose packaging means more DNA is exposed to transcription machinery and the gene is activated or expressed. At the molecular level, 146 base pairs of DNA wrap around a histone octamer which is defined as a nucleosome core particle (NCP). A histone called H1 engages with NCPs where DNA enters and exits to render protection to the free linker DNA in between NCPs. Nucleosomes in their entirety give rise to higher-order chromatin structure and are fundamental units of chromatin. Chromatin structure regulates transcription; therefore gene expression is regulated by factors that alter chromatin dynamics, including histone-modifying enzymes and ATP-dependent chromatin remodelers.

DNA methylation, non-coding RNA, and prions are examples of additional modifications that regulate gene expression. Epigenetic regulation is exerted by DNA methylation near gene promoters at regions called CpG islands which are 5’-cytosine and 3’-guanine separated by one phosphate group. 5-methylcytosine and 5-hydroxymethylcytosine belong to the category of DNA methylation that epigenetically regulates gene expression.

In the context of aberrant epigenetic modifications that regulate DCs, immunological processes are disrupted. To understand the epigenetic mechanisms behind the regulation of DCs, high-throughput epigenetic studies on DCs will be necessary. These studies will contribute to therapeutic developments to treat immunological diseases of DCs. Advancements in methods to study epigenetic processes and gene expression throughout an organism or specific cell population have greatly contributed to our understanding of molecular mechanisms at high-resolution levels. For example, the genome-wide landscape of open and closed chromatin is determined by ATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing), providing insight into chromatin shape and gene expression. Further, genome-wide localization of proteins such as TFs onto DNA is detected using ChIP-seq (chromatin immunoprecipitation with sequencing), providing insight into correlations between TF binding and gene expression. Transcriptional readout of the genome is analyzed by RNA-sequencing which shows how the expression of the genome is impacted by epigenetics and other factors. To expand on this, epigenetic and transcriptional studies on DCs are discussed in the next sections.

ANALYSIS OF CHROMATIN ACCESSIBILITY SHOWS NEW FEATURES OF DC REGULATION

pDCs are specialized DCs that are known for their quick and effective response in inhibiting virus replication. This rapid immune response, characterized by the prolific development of type 1 interferons, is effective in eliminating a viral infection, however, is closely associated with the development of inflammatory and autoimmune diseases. pDCs generate high quantities of type I IFN when their toll-like receptors (TLRs) are stimulated. A study by Mann-Nüttel et al. identified several TFs which exhibited binding reactivity with DNA as well as a significant epigenetic modulation. The study identified the entire set of TFs in pDCs which are regulated by TLR9, and the significance of a family of TFs called AP-1 (activator protein 1). In this direction, Mann-Nüttel et al. performed ATAC-seq on cells derived and purified from bone marrow. Following preparation of the ATAC-seq libraries, sequencing and bioinformatics analysis was performed where a genomic signal map was created through simulation, ATAC-seq peaks were identified and false reads were removed. Hence, the study discovered the pool of TFs and chromatin environment in activated pDCs.

Immunotherapy via DCs to treat cancer is a medical practice that researchers continue to study and improve. A study on murine cancer models treated with an inhibitor of histone deacetylase showed that one of the effects of the treatment was the enhanced function of DCs. Recently, a study by Guenther et al. focuses on β2-integrins which are adhesion receptors that regulate the maturation and migration of DCs. β2-integrin was first discovered when left in an inactive state, and cells immediately matured within a culture. Guenther et al. employed murine models where three threonines are mutated, that is, TTT/AAA β2-integrin knock-in (KI), which inactivates all β2-integrins. The expression of such dysfunctional β2-integrins in the bone marrow-derived DCs results in enhanced tumour rejection and spontaneous maturation. From this, several epigenetic processes associated with spontaneous maturation were detected.

Epigenetic processes investigated in the above study were global chromatin accessibility, genome-wide histone modifications, and transcriptome. For chromatin accessibility, ATAC-seq was performed using an alternate method called Omni-ATAC, which refers to the composition of the buffers that were used to improve the data from the assay. Following the preparation of ATAC-seq libraries, sequencing and bioinformatics analysis, ATAC-seq data allowed for a footprinting analysis which provided scores for the estimation of TF binding sites and their interactions with β2-integrin. One of the inferences from this study is that the global elevation in chromatin accessibility, detected by ATAC-seq, associated with spontaneous maturation seen in DCs with β2-integrin KI. The spontaneous maturation also correlated with elevated histone methylation at lysines 4 and 27, and altered transcriptome, as revealed by ChIP-seq and RNA-seq, respectively, which are described in subsequent sections. Overall, the study infers that cancer immunotherapies involving DCs can improve by therapeutically targeting β2-integrins.

Just as an individual's genetics can be predictive of their susceptibility to disease, the epigenome of a particular subset of cells has the potential to do the same. Systemic sclerosis, a disease that causes skin lesions, has been associated with single-nucleotide polymorphisms (SNPs), however, the complete aetiology is idiopathic. Using ATAC-seq, active DNA regulatory elements were investigated in 8 kinds of primary cells in normal skin obtained from healthy controls and clinically affected and unaffected samples of skin obtained from systemic sclerosis patients. Epigenetic profiles of DCs from patients showed modifications most associated with compromised immunity, which was not seen on healthy skin of the same patient. The site which contained the aforementioned SNPs was also highly enriched for DCs within these sites. Therefore, the pathogenesis of the autoimmune disease systemic sclerosis is largely due to epigenetic modifications of DCs coupled with the SNP in the DC genome.

In the above study, ATAC-seq libraries were prepared from nuclei extracts of flow-sorted skin cells, sequenced and bioinformatically analyzed. Mapped reads were then shifted +/-5bp to reflect the specific region of interest and extended over 50 bp centered by this region. Peak was called, filtered and enriched regions were identified. Samples from the same cell type classified under the same clinical condition (normal, unaffected or affected) were grouped for analysis. Hence, the chromatin accessibility landscape of skin cells in patients with systemic sclerosis reveals the association of DCs in the pathogenesis.

Another recent study by Mendes et al. performed ATAC-seq in addition to various other epigenetics methodologies for a well-rounded understanding of epigenome and transcriptome in the context of moDC which are DCs that differentiate from human blood monocytes (MO). One of the epigenetic phenomena investigated in the study was DNA methylation in moDC. DNA methylation was analyzed using whole-genome bisulfite sequencing where bisulfite conversion of non-methylated cytosine residues of DNA and no conversion of methylated cytosines enable the detection of 5-methylcytosine or DNA methylation levels of individual CpGs. Note that, 5-hydroxymethylcytosine was also detected. Differentially methylated regions or DMR in moDC were analyzed. Elegant experiments involved the presentation of ChIP-seq distribution of proteins of interest, ATAC-seq, and DNA methylation signals across thousands of DMR in moDC. Overall, the results indicate that DNA methylation is regulated by a TF called EGR2 in the context of MO differentiation and moDC. DNA methylation and histone methylation in DCs was also investigated in a study on systemic lupus erythematosus patients with lupus nephritis. mDCs and pDCs were analyzed from peripheral blood mononuclear cell (PBMC) of the patients which showed that severe lupus nephritis renders DC DNA to be hypermethylated while early stages of the disease show reduction in H3K4me3 and H3K27me3. Overall, the study reported changes in epigenome and transcriptome in DCs that correlate with chronic renal pathologies in lupus nephritis.

In addition to chromatin accessibility studies as described in this section, studies using genome-wide protein localization further reveal epigenetic regulation of DCs as discussed next. Recent discoveries on the epigenetic landscape associated with DCs as revealed by the ATAC-seq methodology are presented in Figure .

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REGULATION OF DCs IS ELUCIDATED BY CHIP-Seq

In addition to chromatin accessibility, epigenetics is also frequently studied by investigating the genome-wide localization of proteins with chromatin using a method called ChIP-seq. A study by Ippolito et al. identifies a key gene in dendritic cell regulation and several TFs regulated by BCL11A (B-cell lymphoma/leukaemia 11A). Expressed mainly in the brain and hematopoietic tissue, BCL11A is a zinc-finger protein that binds to DNA and represses transcription to regulate the brain, hematopoietic system development, and fetal-to-adult haemoglobin switching. Although its transcripts are seen in murine and human plasmacytoid DCs, and it plays important roles in faetal hematopoietic progenitors development, the role of BCL11A in DCs, especially in adults, is unknown. Although research has been addressed in this direction, several questions remain as to how progenitor cells commit to DC lineage. Hence, Ippolito et al. focused on the role of Bcl11a in murine fetal and adult plasmacytoid DC development. They studied the gene through two populations of mice: control and a knockout population, the latter being the population with an omitted Bcl11A gene. The absence of this gene led to the depletion of immunological function. Genome-wide association of BCL11A with DNA was analyzed using ChIP-Seq with samples harvested from these mice populations. Bcl11a is also a regulator of the TFs, E2-2, ID2, and MTG16, proteins important for DC differentiation. ChIP-seq libraries were prepared, sequenced, and bioinformatically analyzed. Short reads were mapped along with the genome and data sets were obtained based on an individual reads distance from neighbouring nucleotides. The study showed that BCL11A is a necessary lineage-specific factor for pDC development and differentiation from progenitors.

MO and DCs are closely related and initially studied as if they were one and the same until DCs were identified to have unique functions compared to MO. A study by Kurotaki et al. Illustrates how closely these two cells are related and identifies Irf8 which is responsible for the regulation and differentiation of hematopoietic stem and progenitor cells into MO and DCs. Bone marrow cells and splenocytes were harvested from Mice with the Irf8^−/−gene and treated to isolate monocyte-DC progenitors. ChIP-seq libraries were prepared, sequenced, and bioinformatically analyzed to identify ChIP-seq peaks. An enhancer catalogue was constructed. The collected data show that while biologically similar to MO, DCs are unique.

A study by Vandenbon et al. identified the active dynamics of epigenetic modification under lipopolysaccharide (LPS) stimulation and the dendritic cell response. Within DCs, this study identifies a pattern of modifications to the genome when interacting with immune response initiating entities. By collecting samples on a time-based scale, using the ChIP-seq assay, a dynamic model of epigenetic functions during the immune response can be illustrated. ChIP-Seq experiments were run for 10 different time points over 24 h. Cells were stimulated with LPS and then underwent fixation processes prior to ChIP execution. Bioinformatics analysis was performed to map this data along a mouse genome and to filter data to omit low-quality peak reads. Signals of immunoprecipitated samples were separated from non-specific reads of the control samples in order to allow for a more qualitative ChIP data set displaying the changes in signal over time.

Interleukin-10 (IL-10) is a cytokine that participates in inflammatory immune responses. This protein is under strict regulation of TFs that operate specifically alongside DCs and in their presence, the production of IL-10 is limited. Interactions between DCs and DC-SCRIPT or DC-specific TF, result in the production of IL-10 to resolve the immune response and encourage anti-inflammatory conditions. In this direction, ChIP assays were performed on human monocyte-derived DCs derived from PBMCs of three different donors. Overall, the study presents a new mechanism of DC regulation by DC-SCRIPT. Studies have used ChIP-seq to detect histone modifications like H3K4me3 which indicate epigenetic activation of myeloid cell classical or DC1 genes by a cytokine called tumour necrosis factor α (TNFα). On the other hand, a study on pDCs showed that the production of type I IFN, but not TNFα, is restricted to a small subpopulation of pDCs which are stimulated individually and governed by stochastic gene regulation. Figure  presents recent findings on DCs associated with chromatin occupancy of epigenetic marks as determined by the ChIP-seq methodology.

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TRANSCRIPTOMIC ANALYSIS SHEDS NEW INSIGHTS ON THE REGULATION OF DCs

The epigenetic tools outlined above have been widely implicated in research on dendritic cell-based therapeutics and disease mechanisms. Epigenetic modifications impact gene expression, hence the epigenome and transcriptome are often studied as complementations of each other to obtain a well-rounded understanding of molecular mechanisms. This section focuses on select studies investigating the transcriptome. DCs have been known to be involved in generating hyperresponsiveness as part of the innate and adaptive immune responses of allergy-related diseases such as asthma and dermatitis. However, the mechanisms are not fully elucidated. A study by Lee et al. aimed to bridge this gap by investigating the genetic modifications that occur upon stimulation of DCs in patients with allergies. The study compared the results of whole transcriptome RNA-sequencing (RNA-seq) of myeloid DCs (mDCs) sorted from peripheral blood MO in patients with allergies and those without. Their results identified AREG (Amphiregulin) as a key regulator of DC activity during an allergic immune response. AREG encodes amphiregulin, a protein within the epidermal growth factor family that is involved in the regulation of cell survival and proliferation. RNA-seq libraries were prepared from the sorted patient DCs, quantified, and low-quality RNA samples were removed. Libraries were sequenced and analyzed bioinformatically. Overall, the study showed elevated AREG protein expression in patients with allergies, establishing the importance of whole transcriptome profiling of mDCs in discovering genetic regulations associated with allergies.

The term DC has been used to describe a variety of antigen-presenting cells, including classical DCs (cDCs) and those derived from blood MO. Blood MO, resident tissue macrophages, and progenitors have all been classified as part of the mononuclear phagocyte system (MPS) that is present in all tissues of the body, but the extent to which cDCs can be included in the MPS is under-investigated. Previous analyses of large transcriptomic data sets from mice have revealed unique markers that separate MPS cells from other leukocyte lineages but did not define a clear delineation between macrophages and DCs. In a recent study, Summers et al. investigated the question of whether certain transcriptomic markers can distinguish macrophages from DCs by applying network clustering to 466 RNA-seq data sets derived from mouse MPS cells isolated from bone marrow, blood, and tissues. This method revealed that cDC subsets were contained within the MPS cluster. The overall transcriptomic profile showed that DCs did not diverge more than other MPS cells when compared to the divergences among MO and macrophages isolated from various tissues.

Certain smaller clusters of genes showed increased expression in defined isolated cells like DCs, MO, and macrophages. One of them was a generic lymphoid tissue cDC cluster associated with the Ccr7 gene that encodes a receptor responsible for mediating the effects of the Epstein-Barr virus on B-lymphocytes (Gene ID: 1236). The data sets used in this study all varied in methods of RNA isolation, library preparation, and sequencing. The compiled libraries were downsized to 10 million reads five times each and expression levels were re-quantified as the median transcripts per million. Datasets irrelevant to the tissues being studied as well as low-quality samples were excluded. Data were subject to further network analysis to generate a gene-to-gene correlation matrix. In summary, the study showed the vastness of MPS heterogeneity at the single-cell level and their identification using large datasets. In addition to studying the transcriptome of DCs, RNA-seq has been employed to study small RNAs from extracellular vesicles (EV) of DCs and various kinds of non-coding RNA to understand the impact of immune stimuli in the quest for biomarker potentials of EV-RNA.

As mentioned previously, cDCs can be divided into two subsets, cDC1s and cDC2s. Earlier single-cell transcriptomic studies have shown that cDC1s are characterized by their relative homogeneity and are dependent on the IRF8 (interferon regulatory factor 8) and BATF3 (basic leucine zipper ATF-like TF 3) TFs during development. In contrast, cDC2s contain a heterogeneous population of cells with variable dependence on the IRF4 (interferon regulatory factor 4) TF, however, our understanding of this heterogeneity and the transcriptional regulation of cDC2s is limited. A study by Brown et al. examined these unknowns by characterizing the transcriptome of known DC subsets using single-cell RNA sequencing (scRNA-seq). The study revealed two distinct cDC2 subsets that include T-bet (TF T-box) and RORyt (RAR-related orphan receptor gamma), both TFs involved in defining lymphocyte subsets. To accomplish this, DC subsets were sorted from mouse splenic cells and subsequently enriched and purified. scRNA-seq libraries were prepared and subject to subsequent transcription and PCR. The amplified libraries were sequenced using Illumina technology. Read alignment and multi-mapping read solution were performed on the scRNA-seq data to generate a cells x genes count matrix. Data were filtered to remove genes that were expressed in less than 10 cells. The count matrix was normalized and was subject to further bioinformatics analysis. Overall, the study identified two principal lineages of cDC2 characterized by distinct TFs, pathways of development, metabolic and functional programs.

scRNA-seq and ATAC-seq were also employed by Kurotaki et al. to show that cell fate towards DCs is favoured if the TF IRF8 is expressed early in multipotent progenitors since IRF8 alters their chromatin state. scRNA-seq led to the identification of a new subset of DCs which redefines pDCs and DCs in the context of corneal research. With recent technological advances, RNA-seq is employed in a study involving clonal multiomics and CRISPR which revealed that emergency development of pDC and cDC2 are repressed by a corepressor called Bcor. Table  summarizes some highlights from transcriptomic profiling of DCs using RNA-seq. Multiomics approaches continue to be employed in research on DCs. A recent study on DC development used Divi-Seq which allows for single-cell multi-omics profiling to simultaneously analyze cell-division history, the phenotype of surface-marker and the transcriptome of single cells. The study also performed cellular barcoding where cells were tagged with unique heritable DNA barcodes for investigating the count and quantitative contribution of clones towards a specific lineage, and to distinguish if lineages have common or distinct clonal ancestors. In summary, the study shed new insights on the clonal and molecular aspects of emergency DC development induced by a ligand.

TABLE 1 Highlights from transcriptomic profiling of DCs

References	Cells and conditions	Featured observations from transcriptomic analysis
Lee at al.	myeloid DCs (mDCs) sorted from peripheral blood monocytes in humans +/- allergies	AREG (Amphiregulin) regulates DC activity in the allergic immune response. Regulates cell survival and proliferation.
Summers et al.	mouse mononuclear phagocyte system from bone marrow, blood, tissues	Increased expression in generic lymphoid tissue cDC cluster associated with the Ccr7 – receptor for mediating Epstein-Barr virus (EBV) on B-lymphocytes.
Brown et al.	Single-cell RNA-seq on DCs isolated from mouse spleen, human melanoma samples	2 distinct cDC2 subsets, include TFs like T-bet and RORyt, both lymphocyte subsets. Analysis of comparison and conservation between mouse and human DCs.
Kurotaki et al.	Single-cell RNA-seq on sorted lymphoid-primed multipotent progenitors from mice	Genes epigenetically primed by IRF8 are mostly inactive at the LMPP stage but highly expressed in downstream DC lineage populations e.g. CDPs. Early expression of IRF8 alters epigenetics in multipotent progenitors towards DC fate

Owing to their significance in the immune system, abnormalities in DCs have been detected in patients of the ongoing pandemic of COVID-19 (coronavirus disease 2019). scRNA-seq on blood cells of COVID-19 patients showed that antigen-presenting cells in COVID-19 patients have defective interferon responses, pDC effector pathways have multi-process defects, DC subsets have transcriptional alterations, and communication between DCs and T cells in COVID-19 patients is disrupted. Immunophenotyping of DCs from PBMCs of COVID-19 patients show DC deficit several months post-infection, inflammation, and alteration in various markers of DC activation and homing. Other studies have also shown DC deficits, reduced potential for stimulating naïve CD4+ T cells, upregulation of proinflammatory markers, increased proliferative response and delayed regeneration in COVID-19 patients. Lung tissue autopsies of COVID-19 patients revealed impaired DC maturation or homing, mDC accumulation, and decreased migration to lymph nodes leading to deficient T-cell response. Hence, DCs continue to be in focus for COVID-19 research, and clinical trials e.g. NCT04685603, NCT04816760 and NCT04523246. Overall the studies show that DC regulation and function are severely impacted during COVID-19. It is only a matter of time for further discoveries to connect the epigenetic regulations of DCs as one of the contributors to complications associated with COVID-19 like cytokine storm, organ damage, and death.

CONCLUSION

Overall, studies discussed in the above sections establish the importance of epigenetics-based high-throughput analysis of DCs to address existing gaps of knowledge in the field of DC development and function. As discussed in the above sections, ATAC-seq studies revealed the chromatin landscape in DCs which reflects transcription-factor binding during activation and maturation of DCs that impact their function during immune response against pathogenesis. ChIP-seq revealed roles of TFs in DC maturation and differentiation, enhancers associated with DCs, histone modifications, nuances specific to DC subpopulations, etc. RNA-seq studies revealed transcriptomic profiles which help to infer changes in protein levels, analysis of non-coding RNA as potential biomarkers, identification of specific DC lineages with distinct functions, emergency DC development and so forth. DNA-methylation and histone methylation studies provided insights into DC maturation. Hence, further understanding of molecular mechanisms will help towards therapeutic developments and clinical trials involving DCs in situations where aberrant DC biology results in an impaired immune system.

Clinical trials have been designed to investigate the efficacy of DCs against COVID-19 NCT05007496 where a vaccine comprised of autologous DCs and lymphocytes pre-incubated with SARS-CoV-2 spike protein is developed as a potential tool to induce immunity in diverse populations. In a clinical trial against pancreatic cancer NCT03114631, DCs are primed with peptides of human tumour-associated antigens having potential immunomodulatory and antineoplastic properties to kill cancer cells and provide better outcomes for patients. The efficacy of intra-tumour injection of autologous DCs in combination with other therapies is being tested in the clinical trial against Non-Hodgkin Lymphoma NCT03035331. Since immune health is associated with mental health, DC quantification is an important component for a clinical trial on perinatal anxiety NCT03664128. These examples underscore the widespread impact of DCs in therapeutic developments and showcase the need for a better understanding of the epigenetic regulations of DC biology which is yet to be fully explored.

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