Dendritic cell subsets and locations

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Dendritic cell subsets and locations Sreekumar Balana,*, Mansi Saxenaa, Nina Bhardwaja,b a

The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States Parker Institute for Cancer Immunotherapy, San Francisco, CA, United States *Corresponding author: e-mail address: [email protected] b

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Introduction DC biology: A short history and progress DC ontogeny Dendritic cell subsets Conventional dendritic cell subsets 5.1 Human conventional dendritic cell subset 1 5.2 Mouse conventional dendritic cell subset 1 5.3 Key gene signatures in cDC1 5.4 Functions of cDC1 Conventional dendritic cell subsets 2 6.1 Human conventional dendritic cell subset 2 6.2 Mouse cDC2 6.3 Key gene signatures in cDC2 6.4 Functions of cDC2 Plasmacytoid dendritic cell subset 7.1 Human plasmacytoid dendritic cells 7.2 Mouse plasmacytoid dendritic cells 7.3 Key gene signature in plasmacytoid DCs 7.4 Functions of pDC Langerhans cells 8.1 Human Langerhans cells 8.2 Mouse Langerhans cells 8.3 Key gene signatures in LC 8.4 Functions of LC Inflammatory DCs 9.1 Human inflammatory DCs 9.2 Mouse inflammatory DC 9.3 Key gene signature inflammatory DC 9.4 Functions of inflammatory DCs

International Review of Cell and Molecular Biology ISSN 1937-6448 https://doi.org/10.1016/bs.ircmb.2019.07.004

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10. Dendritic cell locations 10.1 Bone marrow 10.2 Blood 10.3 Spleen 10.4 Thymus 10.5 Tonsil 10.6 Skin 10.7 Liver 10.8 Lung 10.9 Intestine 10.10 Lymph node 10.11 DCs locations in pathology 11. Conclusion References

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Abstract Dendritic cells (DCs) are a unique class of immune cells that act as a bridge between innate and adaptive immunity. The discovery of DCs by Cohen and Steinman in 1973 laid the foundation for DC biology, and the advances in the field identified different versions of DCs with unique properties and functions. DCs originate from hematopoietic stem cells, and their differentiation is modulated by Flt3L. They are professional antigenpresenting cells that patrol the environmental interphase, sites of infection, or infiltrate pathological tissues looking for antigens that can be used to activate effector cells. DCs are critical for the initiation of the cellular and humoral immune response and protection from infectious diseases or tumors. DCs can take up antigens using specialized surface receptors such as endocytosis receptors, phagocytosis receptors, and C type lectin receptors. Moreover, DCs are equipped with an array of extracellular and intracellular pattern recognition receptors for sensing different danger signals. Upon sensing the danger signals, DCs get activated, upregulate costimulatory molecules, produce various cytokines and chemokines, take up antigen and process it and migrate to lymph nodes where they present antigens to both CD8 and CD4 T cells. DCs are classified into different subsets based on an integrated approach considering their surface phenotype, expression of unique and conserved molecules, ontogeny, and functions. They can be broadly classified as conventional DCs consisting of two subsets (DC1 and DC2), plasmacytoid DCs, inflammatory DCs, and Langerhans cells.

1. Introduction Dendritic cells (DCs) are professional antigen-presenting cells (APCs) critical for initiation and orchestration of the immune response. DCs are a heterogeneous population of leukocytes that originate from hematopoietic stem cells, and their differentiation heavily depends upon the cytokine Flt3L.

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They act as a link between innate and adaptive immunity and play a critical role in initiating and regulating antigen-specific immune response. DCs are a specialized population of APCs that patrol various anatomical locations and environmental interphases, capture antigens, process and present to the effector cells and initiate the immune response. They are a unique APC population with the ability to educate naı¨ve T cells and B cells as well as regulate the secondary response in an antigen-dependent manner. They not only initiate antigen-specific immune response but also induce tolerance and regulate immune homeostasis. The heterogeneity of DCs provides a platform for their functional specialization critical for protecting the organism from a variety of pathogens and also provides tolerance toward self-antigens. DCs belong to the group of mononuclear phagocytes (MPs) consisting of macrophages, monocytes, and dendritic cells. MPs are critical for controlling infection and inducing an inflammatory response. The role of MPs as APCs and their ability to activate T cells was well known in the early 1960s (Puhr et al., 2015). One of the key characteristics of these cell subsets was their ability to adhere on a glass surface, and this property was used as a tool for isolating MPs for the functional assays. As MPs are a mixed cell population, the exact cell subset responsible for the immune response was unknown until the pioneering study by Ralph Steinman and Zan Cohn in 1973 identified DCs as the primary cell subset required for initiating the immune response (Steinman and Cohn, 1973). In the initial days, these cells were characterized based on their ability to adhere on the glass surface and their typical veiled morphology. Development of new monoclonal antibodies and identification of DC specific cell surface markers defined DCs as a cell subset expressing high levels of major histocompatibility class II (MHC II) (Steinman and Witmer, 1978) and cell surface integrin CD11c (Metlay et al., 1990). These two markers provided a convenient option for identifying DCs among MPs and allowed for detailed characterization of DC phenotype and biology. Discovery of the effect of GM-CSF on DC functions and survival (Witmer-Pack et al., 1987) was a critical observation that helped develop the methodology for in vitro DC generation from mouse or rat bone marrow. This methodology was eventually extrapolated to human monocyte derived DCs (MoDCs), that is, DCs generated by differentiating peripheral blood monocytes in vitro in the presence of GM-CSF and IL-4 (Bender et al., 1996; Sallusto and Lanzavecchia, 1994). Most of our current understanding of human DC biology is based on the information gathered from studying MoDCs.

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All together the ability to phenotype DCs and culture MoDCs made it possible to appreciate the role of DCs as professional APCs, critical for initiating the naı¨ve T cell and B cell mediated immune response. The in vitro generation system helped in establishing the golden standards for functional characterization of DCs and in developing the concept of mature and immature DCs based on the expression of different costimulatory molecules. These concepts in turn helped in exploiting the potential of in vitro generated DCs as cellular vaccine for tumor and infectious disease. In vitro differentiation of DCs from hematopoietic stem cells (CD34+ cells) (Caux et al., 1996a,b, 1997) helped to delineate the DC differentiation pathways and generation of DCs equivalent to MoDCs as well as subsets similar to Langerhans cells identified in 19th century on the human epidermis (Romani et al., 2010).

2. DC biology: A short history and progress The pioneering work from Shortman and colleagues in mice identified the presence of MHC II high and CD11c+ cells in thymus and spleen and introduced the concept of lymphoid DCs. They also reported that murine DCs could be divided into two subgroups based on the expression of classical T cell markers CD8 and CD4 (Vremec et al., 2000). Their study further determined the existence of steady-state DC subsets which are different from murine bone marrow derived DCs (generated in vitro in the presence of GM-CSF and IL4). The early studies of human DC biology were mainly restricted to the in vitro generated MoDCs. In time, different research groups identified the existence of DCs in peripheral blood and other lymphoid tissues. Some of the key discoveries in this field are listed below: • In 1989, two subsets of cells expressing a high level of HLA-DR and lacking the expression of lineage markers were identified based on their ability to adhere to plastic and produce type I IFN upon incubation with cytomegalo virus (CMV) infected target cells. The non-adherent subset was able to produce a high level of IFN whereas the loosely adhered subsets produced a very low level of type I IFN (Chehimi et al., 1989). • In 1993, O’Doherty et al. identified the presence of two sub-types of DCs in the peripheral blood expressing MHC II but either positive or negative for CD11c expression. They found that the CD11c negative subset displayed a typical immature phenotype and depended upon

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monocyte-conditioned media for maturation and acquisition of typical DC characteristics (O’Doherty et al., 1993). • In 1992, the presence of dendritic cells subsets also was identified in the germinal center of lymphoid organs and characterized by their ability to activate B cells upon CD40 ligation. These cells were named follicular DCs (Clark et al., 1992). • In 1996, Grouard et al. identified the presence of CD4+ CD11c+ CD3 DCs in the germinal center of lymphoid organs and their potential role in T cell activation. This cell subset was found to be functionally different from the follicular DCs (Grouard et al., 1996). • In 1997, Grourad et al. also identified the differentiation potential of the CD4+CD11c +CD3-CD45RA+ cells, at that time known as plasmacytoid T cells. This study showed that these cells were able to differentiate as dendritic cells when cultured with monocyte-conditioned media and began the earlier concept of pDCs (Grouard et al., 1997). • Kohrgruber et al. identified the presence of two subsets of MHC II high cells based on CD11c+ and CD11c DC in peripheral blood. The study identified dependency of CD11c CD4+ DCs on IL-3 as well as the antagonistic effect of IL-4 on their survival (Kohrgruber et al., 1999). • In 2002, other studies identified the difference between the CD11c+ DCs in the human blood and the in vitro generated MoDCs. It was reported that while both subsets expressed a similar level of MHC II they differed in expression of costimulatory molecules. Importantly, only MoDCs expressed CD209, an important regulator of immunity on DCs (Osugi et al., 2002). In 2002 the role of CD8α DCs in cross presentation of cell-associated antigen was reported (Schulz and Reis e Sousa, 2002). Characterization of human pDCs and their ability to produce high levels of type I IFN in an infection setting instigated the search for the similar subsets in mouse. Nakano et al. identified the presence of interferon producing cells in murine lymph nodes. They characterized this subset based on the expression of Gr1+ B220+ CD19 CD11c+ and also identified the role of IL-3 for their survival. This study also revealed the role of GM-CSF and IL4 for supporting the survival and maturation of the CD11c+ DCs in culture (Nakano et al., 2001). Overall, these studies paved the way for identification of novel surface markers expressed by blood dendritic cells. These markers are generally known as blood dendritic cell antigens (BDCA). The DCs are classified as BDCA1, BDCA2, or BDCA3 DCs based on the expression of these markers (Dzionek et al., 2000). The BDCA1 and BDCA3 DCs are

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positive for CD11c and negative for CD45RA and CD123. The BDCA2 DCs express a high level of CD123 and CD45RA but are negative for expression of CD11c, representing the type I IFN producing cells in the blood (Dzionek et al., 2000). Functionally, DCs can present antigens to both MHC I or MHC II. The MHC I pathway is generally present in all cell subsets and involves the loading of self or intracellular-pathogen derived antigens (e.g., viral infections) on the MHC I and presenting these antigens to the CD8+ T cells (Lin et al., 2008b; McDonnell et al., 2010). However, some DC subsets have a unique MHC I loading pathway that makes it possible for them to present exogenous antigens (which would normally be presented through MHC II) through MHC I pathway to activate CD8 T cells and generate strong cytotoxic T lymphocyte (Bachem et al., 2010). The cross presentation potential of DC subsets is induced by the expression of specialized gene signatures and depends upon the type of antigen encountered by the DCs. Most DC subsets are able to present externally loaded short or long peptides to the CD8 T cells (Segura et al., 2013a). However, some subsets are better than others at processing and presenting antigens from dying cells as discussed later in the chapter (Bachem et al., 2010; Chiang et al., 2016; Crozat et al., 2010a; Flinsenberg et al., 2012; Jongbloed et al., 2010). The low frequency of DC subsets in human tissues as well as the requirement of laborious techniques for their isolation has limited their functional characterization. Even though the different human DC subsets where identified, as mentioned above, most of the studies heavily depended upon the in vitro generated MoDC model. However, in mice, advances in genetic engineering provided a unique opportunity for developing different transgenic mouse models for studying mouse DC biology and function. The novel tools expanded our understanding of different murine DC subsets including the CD8a+ DCs, CD8a CD4+ CD11b DCs, pDCs, and LCs. The critical role of CD8a+ DCs in cross presentation and tumor immunity was very well demonstrated with transgenic animal models (Hildner et al., 2008). Mouse models as an experimental tool can provide tremendous insight into different aspects of the human immune system. For example, the mouse T cell biology is easily translatable to human T cell biology as mice and humans share similar surface markers and functions. However, lack of orthologous markers between human and mouse DC subsets has made it very hard to draw parallels between mouse and human DC biology. The only homologous subset observed in human and mouse is the pDCs,

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identified due to their unique ability to produce type I IFN upon stimulation with methylated nucleotides or virus infected cells. Therefore human and mouse DC biology was developed on completely different tracks (Mestas and Hughes, 2004). As such, mouse models are of limited use for pre-clinical testing of DC-based vaccinations. This was a major bottleneck and significantly limited the development of DCs as cellular vaccines against infections and tumors. One of the pioneering studies by Robbins et al. exploited the potential of comparative transcriptome analysis for identifying unique gene signatures in different DC subsets in humans and mice, irrespective of the differences in the surface phenotype of the DCs (Robbins et al., 2008). This approach opened new avenues for identifying evolutionarily conserved molecular networks across the species, which are critical for the development of unique DC subsets (Crozat et al., 2010b). This paradigm was extended to other species including pig, goat, and macaque, and studies confirmed that subsets were conserved across the species and those observations from model organisms can be extrapolated to other organisms (Deloizy et al., 2016; Marquet et al., 2014; Vu Manh et al., 2015). The last 2 decades have observed an exponential development in flow cytometry techniques. Advanced machines capable of handling more fluorochromes and availability of non-conventional fluorescent dyes have made it possible to perform high throughput multi-color analysis (Granot et al., 2017; Mair and Prlic, 2018). These advances have helped in high dimensional characterization of DC subsets with multiple markers and rigorously defined the subset specific phenotypes. Comparative transcriptome analysis allows for thorough characterization of different DCs and MP subsets and identifies homologs DC subsets in different species. Advances in transcriptome profiling technologies such as RNA sequencing followed by single cell sequencing approaches like Smart Seq (Picelli et al., 2014), massively parallel single cell mRNA sequencing (MARS-seq) ( Jaitin et al., 2014) and 10  Sequencing (Zheng et al., 2017) have helped in unveiling the heterogeneity within each DC subset and confirmed the existence of different DC subsets (See et al., 2017; Villani et al., 2017). These methods also helped in understanding DC ontogeny and differentiation by identifying different stages of DC development. This was done by using algorithms to cluster groups of cells based on their unique transcriptional profiles and proximity with known DC subsets. The entry of mass cytometry further broadened the spectrum of phenotyping targets and provided an opportunity for analyzing multiple cell subsets from healthy or tumor tissues (Alcantara-Hernandez et al., 2017; Guilliams et al., 2016). Development of computing powers

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and novel bioinformatics tools provided an opportunity for exploiting the potential of high dimensional data by removing the limitations of manual analysis. Moreover, different bioinformatics tools provided options for visual exploration of high dimensional data. Examples include: Principle component analysis (PCA), t-distributed stochastic neighbor embedding (tSNE) (van der Maaten and Hinton, 2008), viSNE (Amir El et al., 2013), connectivity MAP (cMAP) analysis (Lamb et al., 2006), density-based spatial clustering of applications with noise (DBSCAN) (Ester et al., 1996), the Mpath algorithm (Chen et al., 2016), Monocle (Trapnell et al., 2014), Diffusion Map algorithms (Haghverdi et al., 2015), unsupervised isoMAP analysis (Tenenbaum et al., 2000), Phenograph analysis (Levine et al., 2015), spanning-tree progression analysis of density-normalized events (SPADE) (Anchang et al., 2016), FlowSOM (analyzes flow or mass cytometry data using a Self-Organizing Map allowing unbiased analyses and interpretation with rigorous controls) (Van Gassen et al., 2015). Advanced bioinformatics software facilitated development of methods to identify the proximity between different cell clusters identified from high throughput data, made biological sense, and defined their development and ontogeny. Moreover the single cell sequencing approach provided an exciting option for gathering valuable data regarding analyses of DC subsets in different patient tissues even when starting with a very low input of cell numbers. This technology provides a wonderful opportunity for understanding the presence of DCs in the tumor, investigating how DC transcriptome profiles are reprogrammed in the tumor microenvironment and understanding the role of each subset in tumor immunity. Such information is necessary for designing a DC-based vaccine against tumors.

3. DC ontogeny Despite the considerable technological advances, our understanding of DC development is heavily derived from animal models with limited data from human studies. DCs originate from CD34+ cells, and their differentiation process heavily depends upon the cytokine Flt3L. Indeed, mice lacking the Flt3L (McKenna et al., 2000) or Flt3 (Waskow et al., 2008) gene demonstrate a profound absence of steady-state DC subsets. Injections of exogenous Flt3L in mice increase the frequency and diversity of different DC subsets (Maraskovsky et al., 1996). Similarly, injection of Flt3L in healthy human subjects as well as in patients increases the frequency of different DC subsets (Anandasabapathy et al., 2015; Bhardwaj et al., 2016;

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Pulendran et al., 2000). The DC subset frequency was reported to be increased up to 6–16-fold in pDCs, 48-fold in cDC1, and 130-fold in cDC2s following Flt3L treatment. Humanized mouse models also demonstrated increased frequency of different human DC subsets upon FLT3L treatment highlighting the critical role of Flt3L on DC subsets differentiation and proliferation (Ding et al., 2014). GM-CSF is another important cytokine for DC survival, Vremec et al. demonstrated that while GM-CSF null mice had a similar level of steady lymphoid resident DCs as wild type mice, the GM-CSF receptor (GMCSF-R) null mice had a threefold reduction in frequency of steady-state DCs. It should be taken into account that this phenotype in GMCSF-R null mice could be due to the lack of common beta chain shared with IL3 or IL-5 receptors. Overexpression of GM-CSF increased the DC frequency in spleen, thymus, and lymph node (Vremec et al., 1997). The CD34+ stem cells differentiate into lymphoid and myeloid progenitors (LMPs) that have the potential for developing into both myeloid and lymphoid lineages (Doulatov et al., 2010; Lee et al., 2017). LMPs further differentiate into common myeloid progenitors (CMPs) and serve as a source of different myeloid cells (Doulatov et al., 2010; Lee et al., 2017). The CMPs differentiate into granulocytes, monocytes, and dendritic cell precursors (GMDPs). These precursors have the potential for generating the granulocytes, monocytes, and dendritic cells. The next layer of hierarchy is the monocyte and dendritic cell precursor (MDPs) with restricted differentiation potential into either monocytes or dendritic cells (Lee et al., 2017). Common dendritic cell progenitors (CDPs) are the first known direct precursors with a committed potential for generating the three DC subsets. The CDPs give rise to the pre-cDCs which eventually differentiate into conventional DCs (See et al., 2017) or pDCs. DCs are identified by lack of expression of well-known lineage specific markers of T (CD3), B (CD19, CD20), NK (CD56), monocytes (CD14, CD16), granulocyte and neutrophils (CD66b), or stem cells (CD34). DCs express a high level of major histocompatibility class II and human DCs are generally characterized by the expression of human leukocyte antigen-DR (HLA-DR) (Sabado et al., 2017). HLA-DR is important for class-IImediated presentation of exogenous antigens to the CD4 T cells. Another important molecule is CD11c, expressed by all the mouse DCs and some subsets of human DCs. CD11c is a transmembrane protein known as integrin alpha X. While its functions in DCs are not very well characterized, Wu et al. reported the potential role of CD11c in binding and uptake

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of CD47 negative cells (Wu et al., 2018). DCs are also characterized by the expression of costimulatory molecules, especially in the context of their maturation status. The B7 family of immunoregulatory molecules is involved in the critical function of regulating the T cell activation and survival functions. The two well-known molecules, CD80 (B7.1) and CD86 (B7.2), can bind with the CD28 expressed on T cells. Efficient ligation of CD28 by the costimulatory B7 molecules is critical for the activation, proliferation, cytokine production by T cells, and for overcoming the T cell anergy (Sansom, 2000). The B7 molecules also can bind CTLA4 on T cells, and the outcome is the inhibition of T cell effector functions. This axis may have a critical role in regulating the T cell activation and mouse models that lack CTLA-4 show a strong autoimmune response and lymphoproliferative disorders (Tivol et al., 1995; Waterhouse et al., 1995). Another important costimulatory molecule on DCs is CD40, a transmembrane glycol-protein belonging to the tumor necrosis factor superfamily. The ligand for CD40, CD40L (CD154), is generally expressed by activated CD4 and CD8 T cells (O’Sullivan and Thomas, 2003). The CD40-CD40L interaction upregulates the expression of costimulatory molecules CD80 and CD86 and induces a more mature phenotype in the DCs. The CD40 ligation induces IL-12 production by DCs that leads to Th1 specific skewing in naı¨ve T cells (O’Sullivan and Thomas, 2003). CD83 a glycoprotein member of IgG superfamily, is considered a classical marker representing the maturation status of DCs and is expressed by mature DCs (Lechmann et al., 2002). Interestingly, the soluble form of CD83 has been reported to induce inhibition of DC maturation as well as DC mediated T cell proliferation (Lechmann et al., 2002). However, mouse models deficient for CD83 show a marked reduction in the peripheral blood CD4+ T cells and indicate the critical role of this molecule for T cell development (Fujimoto et al., 2002; Garcia-Martinez et al., 2004).

4. Dendritic cell subsets As mentioned earlier, DCs are heterogeneous cells that are classified into different subsets. In the past when there were no standardized approaches for DC classification, DCs were loosely classified based on factors such as: • Ontogeny and origin (myeloid lineage or lymphoid lineage). • Expression of different surface molecules like CD8a DCs or CD103 DCs. • Tissue locations such as lymphoid resident DCs, “tissue resident DCs “or “migratory DCs” based on their status of location. The tissue resident DCs were mainly the DC subsets found in the lymphoid organs that remained in lymphoid organs for their entire life cycle. These cells were

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also known as lymphoid organ resident DCs. They did not depend on CCR2-mediated migration to the lymphatic tissues and generally expressed an immature phenotype with a lower level of class II expression. • Immature vs. mature DCs based on the expression of different costimulatory molecules. • Tolorogenic or immunogenic DCs based on their functional outcome and cytokine profile. Currently, DCs classification into subsets is based on an integrated approach considering different factors such as phenotype, key gene signature, including critical transcription factors, TLRs, and other functionally relevant molecules including chemokine receptors and C type lectin receptors and ontogeny. We can now broadly classify DCs as steady-state DCs, inflammatory DCs, and LCs as depicted in Fig. 1. Steady-state DCs can be classified as conventional dendritic cells (cDCs) or myeloid DCs (mDCs) and plasmacytoid dendritic cell subsets (pDCs). These subsets are generally identified based on the lack of expression for T cell, B cell, NK cells, or other granulocytes specific markers, high level expression of MHC II and lack of monocyte markers (Collin and Bigley, 2018). All mouse steady-state DC subsets commonly express CD11c, whereas in humans the conventional DCs expresses CD11c, but pDCs lack the expression. The steady-state DCs generally originate from the common DC precursors with the potential for generating different DC subsets. cDCs and pDCs differentiation depends upon cytokine Flt3L (Mildner and Jung, 2014). Injection of Flt3L into mice or humans has confirmed its role in amplifying the different DC subset (Anandasabapathy et al., 2015; Maraskovsky et al., 1996; Pulendran et al., 2000). The inflammatory DCs are generally absent at steady state and appear during an inflammatory response (Segura and Amigorena, 2013). They generally originate from monocyte precursors including the classical CD14+ monocytes or the non-classical CD16+ monocytes. The mouse equivalent subsets originate from the LyC6hi or LyC6low precursors (Segura and Amigorena, 2013).

Fig. 1 Flowchart depicts a simplified classification of dendritic cell subsets.

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LCs are a subset with DC phenotype and functions but with an embryonic origin and self-proliferating capacity (Merad et al., 2013). In vitro experimental models reveal that CD34+ HSCs differentiated in the presence of GM-CSF, IL-4 and TNF-α or TGF-β have the potential for differentiating into a LC like phenotype (Caux et al., 1996a, 1997, 1999; Jaksits et al., 1999). Some of the reports also indicate the potential of cDC2 subsets to acquire an LC like phenotype when treated with GM-CSF, BMP-7, and TGF-β (Milne et al., 2015). However, there are no in vivo validations are available to confirm the different origin of LCs. Other teams have reported that blood cDC2 subsets but not tonsil cDC2 could potentially differentiate into LCs when cultured with a keratinocyte derived cytokine thymic stromal lymphopoietin (TSLP) and TGF-β (Martinez-Cingolani et al., 2014).

5. Conventional dendritic cell subsets Conventional dendritic cell (cDC) subsets consist of two subsets generally classified as conventional DC1 and conventional DC2. The conventional DC1 are characterized by the expression of unique C type lectin receptor Clec9a and chemokine receptor XCR1 and SIRPa is a conserved signature expressed by the cDC2 across the species. The signature is conserved across the species and a useful marker for identifying the homologs subsets.

5.1 Human conventional dendritic cell subset 1 cDC1 cells are a rare subset of DCs, their presence has been confirmed in blood, tonsil, spleen, skin, lung, intestine, ileum, payers patch, liver, and different lymph nodes. A detailed analysis was performed using high dimensional analysis with multi-color flow cytometry, mass cytometry, and transcriptome profiling on different human tissues to determine the tissue presence of cDC1s. The studies confirmed the presence of this rare subset and characterized tissue specific adaptions of the cell subset in different human tissues and organs. The human cDC1s are characterized by a lack of lineage as makers (CD19 CD14 CD16 CD3 ), high MHC II expression, moderate CD11c expression, and high level expression of CD141. CD141, also known as blood dendritic cell antigen 3 (BDCA3) and thrombomodulin, is highly expressed on cDC1 subsets and acts as cofactor for thrombin (Van Der Aa et al., 2015). BDCA3, has been reported to support anti-inflammatory functions (Li et al., 2012). However, its role on DCs is not well known.

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Since CD141 is expressed by other cell subsets as well, special attention should be given to using CD141 as the sole phenotypic identifier of bonafide cDC1 subset. For example, Chu et al. identified a subset of CD141 dermal dendritic cell subset capable of secreting IL-10 (Chu et al., 2012) that were different from the bonafide cDC1 subset. Granot et al. reported that the MHC II (HLADR) expression on cDC1 was found to be low or intermediate upon tissue localization, with the highest class II expression was observed in intestinal cDC1s (Granot et al., 2017). cDC1 subset can be identified by unique expression of C type lectin receptor 9A (Clec9a). Clec9a facilitates the uptake of necrotic cells (Sancho et al., 2009) and may lend an advantage to DC1s in taking-up dying cells in physiological conditions and cross-presenting the antigens to CD8 T cells (Schreibelt et al., 2012). Even though Clec9a is a specific marker for cDC1, its expression varies in a tissue specific manner. For example, Clec9a expression level is reported to be very low on cDC1s in tonsil and skin (Granot et al., 2017). However, it must be noted that this observation may be in part due to use of collagenase in the cell isolation protocol as collagenase treatment has been shown to reduce Clec9a expression on the cDC1 (Boor et al., 2019). cDC1 cells express another unique chemokine receptor X-C Motif Chemokine Receptor 1 (XCR1) (Dorner et al., 2009). XCR1 ligands X-C motif Chemokine Ligand 1 (XCL1), XCL2 are expressed by memory CD8 T cells and NK cells (Crozat et al., 2010a). The XCR1-XCL1/2 axis facilitates cDC1 interaction with these effector cells. Recent reports have highlighted the critical role of this interaction in antitumor immunity (Bottcher et al., 2018). Interestingly, cDC1s seem to exhibit a bimodal expression of XCR1. The fraction of cDC1s that does not acquire the expression of XCR1 is thought to represent the immature stage of cDC1 and is observed in peripheral blood as well as in skin (Balan et al., 2018; Granot et al., 2017). These XCR1 negative cDC1s can proliferate and eventually acquire the expression of XCR1 (Balan et al., 2018). CD11c is also a critical marker for the cDC1; however, its expression level is on cDC1s is moderate compared to cDC2s. As an alternative, CD26 can be a useful marker to identify the cDC1s in different tissues (Guilliams et al., 2016). Another useful marker to identify the cDC1 subset is the cell adhesion molecule 1 (CADM1) expressed by cDC1s in different species. The molecule interacts with class I-restricted T cell-associated molecule (CRTAM), generally expressed on activated NK cells and T cells (Arase et al., 2005). cDC1s in bone marrow, skin, spleen, lung, intestine, and in various draining lymph

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nodes express CD13 which can be a useful marker to identify this subset in these tissues (Granot et al., 2017). cDC1s generally do not express monocyte or MoDC markers such as CD14, CD16, CD209 and Signal Regulatory Protein Alpha (SIRPa/CD172a). B and T Lymphocyte Associated (BTLA or CD272), a member of immunoglobulin superfamily, functions as an inhibitory receptor and can be another marker for identifying the cDC1 subset (Watanabe et al., 2003). cDC1s also express high levels of CD135, the receptor for Flt3L, and an endocytic c type lectin receptor, DEC205 (Crozat et al., 2010b). The cDC1s lack the expression of CD45RA, CD123, and other pDC specific markers such as BDCA2 and BDCA4. Some reports show that CDC1s express CD206 in bone marrow and CD206 (Granot et al., 2017). Furthermore, cDC1s can express the bonafide cDC2 marker CD1, in human skin (Balan et al., 2018; Dzionek et al., 2000; Haniffa et al., 2012).

5.2 Mouse conventional dendritic cell subset 1 Classically mouse cDC1s are identified based on the expression of MHC II, CD11c and CD8a or CD103, and lack of CD11b and B220. CD8a is generally expressed by the murine cDC1 subset in spleen or other lymphoid organs and serves as a marker for lymphoid resident DCs (Shortman and Heath, 2010). CD103 is generally expressed by the cDC1s in skin or other nonlymphoid tissues such as lung, liver, kidney, etc., and generally considered as marker for the migratory DC subset (Bedoui et al., 2009; Del Rio et al., 2007; Edelson et al., 2010; Ginhoux et al., 2009; Sung et al., 2006). Like the human cDC1s, mouse cDC1s also express the unique markers such as CADM1, Clec9a and XCR1 (Crozat et al., 2010b; Shortman and Heath, 2010. However, unlike human cDC1s, in mice, Clec9a is expressed by both the cDC progenitors and pDCs (Schraml et al., 2013). Murine CD1cs do not express the macrophage markers F4/80, CD64, CD11b and SIRPa (CD172a) (Guilliams et al., 2016). These markers can identify the presence of bonafide cDC1 from different tissue. Expression of CD11c can vary in different tissues and organs and additional markers like CD26, CD24 are useful for the rigorous identification of the subset in different tissues and organs (Guilliams et al., 2016).

5.3 Key gene signatures in cDC1 cDC1s originate from the CD34+ HSCs and the differentiation process is modulated by a combination of transcription factors including interferon regulatory factor 8 (IRF8) (Aliberti et al., 2003), inhibitor of DNA binding

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2 (ID2), and Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF3) (Chandra et al., 2017; Grajales-Reyes et al., 2015). Some of the recent studies show the critical role of Spi-1 Proto-Oncogene (SPI1 or PU.1) on influencing another gene DC SCRIPT and modulate cDC1 differentiation (Chopin et al., 2019; Rosa et al., 2018). PU.1 is a critical transcription factor for the development of myeloid and lymphoid lineages and early hematopoiesis. PU.1 is essential for the induction of Flt3 receptors for the Flt3L, a critical cytokine for the DC subset development (Carotta et al., 2010). cDC1s are low for IRF4, Zinc Finger E-Box Binding Homeo box 2 (Zeb2) and express a high level of Zinc Finger and BTB Domain Containing 46 (ZBTB46) (Meredith et al., 2012). Role of Notch signaling in was identified to be critical for human cDC1 differentiation and important for the functional maturation of mouse cDC1s (Balan et al., 2018; Kirkling et al., 2018). Human blood cDC1s express different toll like receptors (TLR) such as TLR1, TLR3, TLR6, TLR8, and TLR10. However, overall, cDC1s express lower levels of TLRs compared to other DC subsets (Hemont et al., 2013). Mouse splenic cDC1s are capable of producing IL-12 in response to TLR3 ligand, Poly I:C stimulation. On the other hand the human cDC1s are poor producers of IL-12 and only respond to the activation of TLR8 with R848 (Balan et al., 2018). It is interesting to note that the murine cDC1s do not express TLR8 but express TLR4 and produce inflammatory cytokines in responds to LPS activation (Merad et al., 2013). cDC1s are capable of detecting the profiling like molecules (STAg) from the protozoan parasites through TLR11 and produce high level of IL-12 in response, thus serving a critical function in controlling the infection (Reis e Sousa et al., 1997; Yarovinsky et al., 2005). NLR Family Pyrin Domain Containing 1 (NLRPs) are well-known components of innate immunity as they facilitate inflammasome-driven inflammatory response. The inflammasome complexes induce activation of caspase 1. The process leads to the maturation of inflammatory cytokines interleukin 1β, IL-18 and induce the apoptosis of the cells called pyroptosis. Interestingly the cDC1s have very low expression of different NLRP components like NLRP1, NLRP4 and NLRP2. Also the DC subsets do not express caspase 1. The lack of pyroptosis inducing molecules on DCs may be an evolutionary advantage developed by DCs to avoid inflammatory cell death to facilitate antigen presentation during infections or inflammatory conditions (Worah et al., 2016). They also express high level of Indoleamine 2,3-Dioxygenase 1 (IDO1) and IDO2 known for their role immune tolerance and generating regulatory T cells (Crozat et al., 2010b). The expression of Cytokine-Dependent

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Hematopoietic Cell Linker (CLNK), WD repeat- and FYVE domaincontaining protein 4 (WDFY4), a number of small RAB GTPase RAB11A, RAB7B, RAB43, SPT3, is conserved in mouse and human cDC1s indicating the conserved role of these molecules in cDC1 functions (Cancel et al., 2019). WDFY4 was recently described to play a role in cross-presentation. molecules on DC biology has not been well characterized as yet (Theisen et al., 2018). Both mouse and human cDC1s express a high level of TLR3 and produce type III IFN upon Poly I:C stimulation or in response to the natural TLR3 ligand, double-stranded RNA (Lauterbach et al., 2010). Even though the mouse and human cDC1s share many common signatures and functions, they exhibit marked differences in expression profiles of these signatures and in their ability to produce different cytokines. These differences should be considered when extrapolating mouse cDC1 biology to human cDC1s. The DC subset also has a specific expression profile of FC receptors and they may play a role on antigen uptake and modulate the immune response. Several in vitro studies show that the FC receptor mediated antigen uptake by DCs promotes cross presentation in cDC1s. The key shared or unique signatures and surface markers expressed by mouse and human subset are depicted in Fig. 2.

Fig. 2 Illustration depicts the common signatures and shared signatures expressed by human and mouse conventional dendritic cell subset 1. The signature includes surface markers, transcription factors and major pattern recognition receptors. The signatures with * marks indicate tissue specific expression.

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5.4 Functions of cDC1 cDC1s are generally known as cross-presenting DCs and various mouse model studies demonstrated their superior ability for antigen cross presentation from dying cells and elicit antitumor immune response (Cancel et al., 2019). The role of human cDC1s in vivo cross-presentation is still controversial and the assumptions are based on the in vitro experiments. There are reports on the similar efficiency of other DC subsets in cross-presenting antigens (Segura et al., 2013a). However, many other teams demonstrated the superior ability of cDC1s for cross-presentation (Bachem et al., 2010; Chiang et al., 2016; Crozat et al., 2010a; Flinsenberg et al., 2012; Jongbloed et al., 2010). The underlying molecular mechanism of antigen cross presentation is not well understood and several theories have been out forth. The cDC1s may have superior ability to uptake the dying cells through Clec9a and may possess specific machineries for the effective processing and presentation to the T cells. They have a favorable endosomal pH that may help with the rapid degradation of antigens. Role of a small GTPases Rac2 is known for their ability for the selective alkalization of the endosome and improves the cross presentation in cDC1 (Savina et al., 2009). The cDC1a express a high level of different Rab proteins, a family of Ras binding GTPase, known for their role in vesicle trafficking in the endosomes. The role of RAB43 on cross presentation in cDC1 has also been demonstrated (Kretzer et al., 2016). Studies in a number of mouse models have demonstrated that mice that lack cDC1 expression globally or locally in the tumor microenvironment fail to mount antitumor responses. A similar response is observed in mice deficient for key cDC1 molecules (Hildner et al., 2008; Theisen et al., 2018). cDC1s express Clec9a, which can uptake dying cells in vivo and present the antigens to the effector cells. Vaccinating with antigens targeted to the Clec9a has been controlled tumor growth in mouse models (Sancho et al., 2008). Additional targeting experiments using Cle9a or DEC205 have clearly demonstrated the unique potential of the subsets to induce strong adaptive response by activating both CD8 T cell and CD4 T cells. They also induce strong humoral response and induce IgG class switching indicating the superior ability of this subset to regulate the immune response. Similar observations were reported with targeting to XCR1 a chemokine receptor specifically expressed by these DC subsets. XCR1-XCL1/2 axis provide a potential immune modulating system for interacting between the effector cells from both adaptive (CD8 T cell) and innate arms (NK cells) and the

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molecules may be critical for antitumor response. They also secrete IL-15, a critical cytokine for NK activation and proliferation indicating the physiologically relevant interaction between these cell subsets. Antigen targeting studies to DEC205 or Clec9a in the absence of adjuvants demonstrated the expansion of tolerogenic CD4 T cells (Bonifaz et al., 2002; Hawiger et al., 2001; Joffre et al., 2010). The cDC1s may be a unique cells subset as they do not express SIRPa, a molecule critical for inducing a “eat me not” signal by dying cancer cells, effectively inhibiting the uptake of these cells and eventually preventing an antigen specific response to the tumor. Absence of the SIRPA and expression of Clec9a may give a unique advantage for cDC1 to take up antigens from the dying cells and effective presentation to the effector cells. Roberts et al. demonstrated the critical role of cDC1 in taking up antigens from live tumor cells, trafficking the tumor antigens to lymph nodes, presenting to the CD8 T cells and inducing antitumor immunity (Roberts et al., 2016). In vitro generated human cDC1s are also capable of taking up antigens from live cells and presenting to T cells (Balan et al., 2014). Human cDC1s express a high level of TLR3, and have a unique ability to produce high level of type III IFN upon poly I:C stimulation. The mouse and human cDC1 are known for their ability to produce high level of type III interferon upon poly I:C stimulation and may play a critical role in antiviral immunity (Lauterbach et al., 2010). Interestingly pDCs are the major immune cells expressing the receptor for IFN-λ (Megjugorac et al., 2009; Yin et al., 2012). Thus cDC1s may be interacting or activating the pDCs through secretion of type III IFNs. Indeed several in vitro studies show that pretreating pDCs with type III IFN can improve their viability and increase the type I IFN production (Finotti et al., 2016). Of note while the mouse splenic cDC1s could produce IL-12 upon TLR3 stimulation, the human cDC1s did not. Human cDC1s can produce low level of IL-12 compared to the cDC2 subsets upon stimulation with TLR8 ligands (Nizzoli et al., 2013). cDC1s play a critical role in induction of protective immunity during bacterial or viral infections. This subset is shown to be critical for mounting immune responses against different bacterial species (Propionibacterium acnes, Staphylococcus aureus, Bacillus Calmette-Guerin (BCG), or E. coli infection models. cDC1s are critical for the recruitment of neutrophils to the site of bacterial infection and controlling the infection ( Janela et al., 2019). The cDC1 subset is also well known for inducing protective CTL response against various viral infection models such a MCMV and vaccinia virus

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(Eickhoff et al., 2015; Heipertz et al., 2014; Iborra et al., 2012; Snyder et al., 2010). Human cDC1s have a unique property of being resistant to productive infection from enveloped viruses such as human immunodeficiency virus and influenza virus. RAB15 has been suggested to play an important role in conferring this resistance to these cells (Silvin et al., 2017). This subset may also play a critical role on controlling the HCV infection by production of antiviral cytokine IFN-λ (Yoshio et al., 2013) as well as amplifying the type I IFN response (Zhang et al., 2013). cDC1s are important for developing tissue resident memory T cells during vaccinia viral infection, promote t-bet expression and retention of CD8 + T cells in the lymph nodes (Iborra et al., 2016). The role of cDC1s on memory CD8T cell recall response and the CXCL9-CXCR3 axis was demonstrated in viral and bacterial pathogens (Alexandre et al., 2016). cDC1s play a critical role in lung immunity and allergic lung inflammation. Acute and chronic models of house dust mite (HDM) allergens demonstrated an exacerbation in Th2 and Th17 responses and dampening of Th1 response (Conejero et al., 2017). cDC1 also play a critical role on control the Listeria and Toxoplasma gondii infection (Edelson, 2012; Mashayekhi et al., 2011). Thus over all, cDC1s have been shown to play several critical roles in mounting both innate and adaptive immune responses to combat tumorigenesis, infections and allergens.

6. Conventional dendritic cell subsets 2 cDC2s are identified by virtue of high level expression of MHC II, CD11c, CD1c and SIRPA. They are negative for the lineage specific markers as described in cDC1 and do not expresses CD45RA, Clec9a, XCR1 or other pDC specific markers.

6.1 Human conventional dendritic cell subset 2 Human cDC2s are generally known as BDCA1 DCs or CD1c+ DCs with characteristic expression of CD1c, CD11c and SIRPa. They can express low levels of CD14, CD123, CD26 and are generally negative for CD209 and BTLA in blood or tissue. There are additional markers restricted to cDC2 including FCERIA, Clec10a and CD115 that can be handy for identifying the subsets in different organs (Heger et al., 2018; Shin and Greer, 2015; Villani et al., 2017). Even though cDC2s originate from the CDP, they are heterogeneous in nature and exhibit a phenotypical and functional similarity with MoDCs. Two subsets of cDC2a named as DC2 and DC3 are identified based on the expression of CD32b, CD36 and CD163

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(Villani et al., 2017). The DC2s express higher level of CD32b and the DC3s express more CD163 and CD36 (Villani et al., 2017). However, the functional specialization of these subdivisions is not well characterized. cDC2s can also express CD1a, low level of CD141 in skin, or in cell cultures. Hence additional care should be taken when isolating cells from the specific organs. cDC2s have low expression of CD13 and CD26 in skin compared to the cDC1 cells. Human cDC2s express CD11b but the expression may not be uniform across different tissues and is mainly observed on cDC2s in skin. The cDC2 subset in the intestine expresses higher level of CD103 compared to other anatomical locations. CD1a is expressed by lung and skin cDC2 but not by the intestinal cDC2. The skin mononuclear phagocytes subsets can be differentiated from the true cDC2s by using CD64 and CD14. The monocyte-macrophage lineage cells may express these markers at a higher level compared to cDC2s. CD206, a macrophage associated marker, can be expressed by the skin and lymphatic cDC2s. DECTIN1 and CD206 are highly expressed by cDC2s in the skin but completely absent in blood. Segura et al. characterized the skin draining lymph node cDC2s and were able to differentiate these from inflammatory DCs based on the expression of CD206. Moreover, the recent study from Alcantara-Hernandez et al. shows the expression of CD206 on the skin cDC2 cells (Alcantara-Hernandez et al., 2017). The cDC2s generated in in vitro cultures also express high levels of CD206 (Balan et al., 2018). Hence, additional care should be taken to distinguish inflammatory DCs from true cDC2s. High dimensional phenotype mapping has demonstrated that the different subsets observed in cDCs are mainly characterized by the expression level of CD172, CD32b and CD163 (Alcantara-Hernandez et al., 2017). A recent report has shown that the heterogeneity in cDC2 subset depends upon the ontogeny and is modulated by a specific set of transcription factors (Cytlak et al., 2019). IRF8 mutation models have identified the critical requirement of IRF8 for the generation of the DC2 subset of cDC2. The DC2 subsets are replaced by the DC3 cDC2 subsets in IRF8 mutated patients and display a close phenotypic and functional similarity to the inflammatory DCs (Cytlak et al., 2019). cDC2s are also identified as two subsets based on the expression of CD5 as being CD1c+CD5high and CD1c+CD5low (Yin et al., 2017). The subsets show differential gene expression, cytokine production and functions. The CD5high cells have a strong IRF4, CCR7, CD207, TLR3 and low level expression of the monocyte signature. This subset also displays a

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stronger migratory potential, CCR7 expression, and induces IL-10 producing regulatory T cells. In contrast, the CD5low subset has been shown to induce the generation of IFNg producing T cells (Yin et al., 2017).

6.2 Mouse cDC2 Mouse cDC2 are phenotyped based on their lack of expression of lineage markers representing lymphocytes, monocytes or granulocytes and the high level expression of MHC II. They have a strong expression of CD11c and CD11b. Similar to human cDC2, mouse cDC2 also express the conserved marker SIRPa. They are negative for any cDC1 specific markers such as CD8a, XCR1, Clec9a, CD24 and can express additional markers CD26 and CD4. cDC2 express CD103 in a tissue specific manner in the gut and express CADM3 in the spleen. The spleen cDC2s can be subdivided as two subsets based on the expression of Clec12A low ESAM high or Clec12A high and ESAM low. The ESAM low DCs show a strong similarity with MoDCs and also exhibit an IRF8-dependent development, which is generally not a characteristic of cDC2s. The mouse cDC2 ESAM subsets are similar to cDC2-CD5 subsets observed in human cDC2 (Lewis et al., 2011). There are no mouse models available for the specific ablation of cDC2 and due to lack of such tools our understanding of cDC2 biology has been impeded and remains incomplete.

6.3 Key gene signatures in cDC2 cDC2 subsets are mainly dependent upon IRF4 and Zeb2 mediated transcriptional regulation compared to the IRF8, BATF3 and ID2 dependency of cDC1s. Several studies have identified a specific enrichment of CEBPB, SPI1, RUNX3, NFKB1, and BHLHE40 in cDC2 compared to cDC1s (Heidkamp et al., 2016). The differentiation of mouse cDC2s depends on Notch 2 whereas the role of Notch2 in human cDC2 is not well characterized. Human cDC2s express most of the TLRs and exhibit a tissue specific TLR profile. Blood cDC2s express a high level of TLR2 and low level of TLR4. TLR3 expression is high on CD5high cDC2 subsets whereas the TLR7 and TLR8 expression is preferentially observed in the CD5 low cDC2 subset (Yin et al., 2017). Human blood cDC2 express high level of TLR1, TLR2, TLR4, TLR5, TLR6, TLR8 and low levels of TLR3 and TLR10 as compared to other blood DC subset (Hemont et al., 2013). Mouse cDC2s express a variety of cytosolic nucleic acid sensors (RIG-I, MDA-5) and Nod-like receptors (NOD1, NLRX1) that enable them to

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sense the cytosolic nucleic acid and elicit an inflammatory response (Macri et al., 2018). Mouse cDC2 also express an array of TLRs and the subsets at different anatomical locations have a specific expression profile of TLRs and spleen cDC2s mainly expresses TLR3 and TLR12 (Macri et al., 2018). Mouse cDC2s are known to express TLR5, TLR7 and TLR 9and able to produce various inflammatory cytokine (Schlitzer et al., 2015). The key signatures and surface markers specifically or commonly expressed by mouse and human cDC2s are depicted in Fig. 3.

6.4 Functions of cDC2 cDC2s are a major population of DC subset present in different human tissues and organs. They express an array of TLRs and able to respond to a variety of danger signal ranging from nucleotides to polysaccharides. They also express high level of NLRPs and other inflammation associated signaling molecules compared to the other steady-state DC subsets indicating a functional specialization for sensing different danger signals (Worah et al., 2016). They generally secrete higher level of inflammatory cytokines such as IL-6, IL-8 and generally possess a mature phenotype in the draining lung lymph nodes (Granot et al., 2017). They are distributed in the B cell region in the lymph nodes and generally associated with presenting antigens to the

Fig. 3 Illustration depicts the common signatures and shared signatures expressed by human and mouse conventional dendritic cell subset 2. The signature includes surface markers, transcription factors and major pattern recognition receptors. The signatures with * marks indicate tissue specific expression.

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CD4 T cells. They are the major sources of IL-12 and IL-10 in human DCs in response to the activation of TLR3 or TLR8 (Nizzoli et al., 2013, 2016). A functionally specialized subset of cDC2 was identified for its role in anaphylaxis. This subset expresses CD11c+CD301b+, is present in the periphery and is proficient in antigen uptake and transporting the antigens to draining lymph nodes (Choi et al., 2018). This perivascular cDC2 subset has been shown to activate the mast cells by discharging the micro vesicles with surface bound allergens and thus playing a critical role in inducing anaphylaxis (Choi et al., 2018). cDC2s are susceptible to being productively infected by different viruses including HIV and influenza. Mouse skin cDC2s are known to induce the retinoic acid mediated differentiation of naı¨ve CD4 T cells into regulatory T cells (Coombes et al., 2007). The blood cDC2 subsets are the major producers of IL-12p70, IL-1β, IL-6, and IL-23 in response to TLR agonists and are capable of inducing Th1 and Th17 induction (Leal Rojas et al., 2017). Human and mouse cDC2 subsets are the major producer of IL23 and promote a Th17 response during fungal infections (Schlitzer et al., 2013). cDC2s are efficient in presenting soluble antigens and long peptides but are less efficient at presenting cellular antigens. The cross-presentation by cDC2s can be improved by inhibition of endosomal acidification with bafilomycin (Chiang et al., 2016). Mouse cDC2s are capable of presenting antigen and activating CD4 T cells. They are also known to be strong inducers of Th-17 immune response as well as regulatory T cell induction in the gut and thymus.

7. Plasmacytoid dendritic cell subset pDCs possess a unique morphology of secretory cells, like antibody secreting plasma cells, and at steady state lack the typical veiled DC morphology. pDCs are the third subset of steady-state DCs, characterized by their unique ability to produce extremely high amounts of type I IFN and the consequent role in antiviral immunity. pDCs were first reported in human lymph node in early 1950s (Swiecki and Colonna, 2015) and were initially named, “interferon producing cells” and “plasma cells” (due to their morphological similarities with B cells) (Reizis, 2019). pDCs are characterized by the presence of high level of rough endoplasmic reticulum providing the unique cell morphology. The cells were first reported in human and later the ortholog was identified in animal models. Both human and murine pDCs are characterized by their ability to produce type I IFN upon activation with methylated DNA or when stimulated with viruses.

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7.1 Human plasmacytoid dendritic cells Human pDCs are MHCII + CD11c-CD123high CD45RA + cells and they express additional markers including CD303 (BDCA2), CD304 (BDCA4 or CLEC4C), BTLA, Leukocyte Immunoglobulin Like Receptor A4 (LILRA4 or ILT7 or CD85g), CD4, DR6, etc. BDCA2 is type II c type lectin and molecule that could play a role in ligand internalization and presentation to T cells. BDCA2 is an inhibitory molecule and ligation can potentially inhibit the type I IFN secretion by pDCS, demonstrated in systemic lupus erythematosus (SLE) (Dzionek et al., 2001; Furie et al., 2019). BDCA2 can bind glycoproteins including immune globulins (Kim et al., 2018a) indicating an antibody mediated BDCA2-dependent immune homeostatic mechanism (Reizis, 2019). BDCA4 is identical to neuropilin-1, a receptor present in neurons, but the specific role of this molecule is not well known in pDCs (Fanning et al., 2006). CD85g is an immunoglobulin like cell surface protein that functions as a negative regulator of TLR7 and TLR9 signaling cascade and downregulates the production of type I IFN and TNF a by pDCs (Cao et al., 2006, 2009; Tavano and Boasso, 2014). Human pDCs also express protein tyrosine phosphatase sigma (PTPRS), inhibitory receptors regulate the ability of IFN production by pDCs. The pDCs that lack PTPRS show a hyper responsiveness and produce high level of type I IFN (Bunin et al., 2015). High-throughput analysis on the CD123high BDCA2high subsets has identified the presence of a subset of population with the potentials for differentiating as conventional DCs. This subset can be identified based on the additional markers including CD33, Siglec6, Siglec1, CD22, CX3CR1 and AXL (See et al., 2017; Villani et al., 2017). Even though some of the research groups have classified these cells as a separate DC subset, others have identified them as precursors for conventional DCs given their potential for differentiation into cDCs (Collin and Bigley, 2018). One of the unique abilities of pDCs is the production of huge amount of type I IFN upon encounter with different viruses like Flu or HIV or TLR ligands targeting TLR7 or TLR9. They can produce large amount of type I IFN and activate the ISG-related genes on other target cells. One of the intriguing aspects of the IFN production by pDCs is that only a fraction of pDCs produce the IFN while the remaining do not produce any IFN (Reizis, 2019). The human pDCs are classified as CD2 high and CD2 low subsets and both the subsets are capable of producing type I IFN. The CD2 high subsets have been reported to produce more IL-12p40 and induce stronger T cell proliferation (Matsui et al., 2009). Recent advances show the contribution of CDP present in the

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CD123+ CD45RA+ BDCA2+ cells for the production of IL-12 and activate T cells (See et al., 2017; Villani et al., 2017). If pDCs are activated with appropriate TLR ligands or maturation stimuli like CD40L, they can induce strong T cell activation and acquire an APC phenotype (Alcantara-Hernandez et al., 2017; Granot et al., 2017). Bryant et al. reported that the CD2 high subsets are stress-resistant against glucocorticoids and express higher level of BCL2 compared to the CD2 low subsets (Bryant et al., 2016). A subset of pDCs identified by virtue of the expression of CD2 high CD5+CD81+ does not produce any type I IFN as compared to the CD5 CD81 pDCs. The CD5+CD81+ subset is capable of induction and proliferation of regulatory T cells as well as potent stimulation of B cells and inducer of antibody production (Zhang et al., 2017).

7.2 Mouse plasmacytoid dendritic cells Mouse pDCs lack the expression of lineage specific markers and display high level expression of MHCII+. Contrary to human pDCs they express CD11c. Other critical markers expressed by murine pDCs are Bone Marrow Stromal Cell Antigen 2 (BST2) and sialic acid binding Ig-like lectin H (SiglecH). They do not express any conventional DC markers including XCR1, SIRPa, CD11b, CD24, CD26, etc. The mouse pDCs in periphery but in not bone marrow express additional markers CCR9, SCA1 and Ly49Q (Swiecki and Colonna, 2015). Siglec H is downregulated upon pDC maturation and some subsets of macrophages also express the same marker. Hence additional consideration should be taken when characterizing activated pDCs. The existence of heterogeneity as observed in human pDC populations is also observed in mouse pDCs and identified based on the expression of CX3CR1 + CD8 + (Bar-On et al., 2010; Lau et al., 2016) or AXL + subset in murine pDC (Dekker et al., 2018). The existence of pDC subsets that differentially express CD4 or CD8 (O’Keeffe et al., 2002) and CCR2 (Sawai et al., 2013) have also been identified. Mouse pDCs also exhibit the high level interferon production potential. Depletion of pDCs during mCVM infection clearly demonstrated the significance of IFN secreted by pDCs for antiviral immunity in mice. Similar to human pDCs, only a subset of murine pDCs actually secrete the IFN. However, the biology behind the functional specialization for the restricted IFN production is unknown. Mouse pDCs demonstrated heterogeneity with respect to CD4 expression and some pDCs in spleen are reported to express low levels of CD8. Mouse pDCs express high level of functional TLR7 and TLR9 and are capable of producing type I IFN in response to the specific agonists. They also express

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high level of TLR1 and TLR2 but poorly respond to the TLR2 ligands. pDC development is Flt3L depended (Schmid et al., 2010) and it has been observed that type I IFN upregulates FLt3 in common lymphoid progenitors and synergizes the pDC differentiation (Chen et al., 2013).

7.3 Key gene signature in plasmacytoid DCs pDC differentiation and function is regulated by the outcome of the interaction between a bunch of critical genes such as IRF8, IRF7, E2.2, Runx2, SpiB, IRF4, BCL11A and ZEB2. However, the relevant role of each transcription factors is not well understood (Reizis, 2019). The origin of pDCs is proposed to be from both myeloid and lymphoid lineages (Reizis, 2019). pDC differentiation is mainly regulated by a balance between E2.2-ID2. Higher expression of E2.2 can skew the differentiation of the precursors toward pDCs. Conditional deletion of E2.2 on mature pDCs results in transdifferentiation of pDCs into a cDC like phenotype and acquisition of the abilities to prime T cells. E2.2 expression is amplified in differentiating pDCs through a BRD protein dependent feedback loop (Grajkowska et al., 2017) and it cooperates with MTG16 (Ghosh et al., 2014) and BCL11A for promoting pDC differentiation (Ippolito et al., 2014; Wu et al., 2013). IRF8 plays an important role in the differentiation of mononuclear phagocytes from the HSCs. People with homozygous IRF8 deficiency (K108E mutation) (Hambleton et al., 2011) or compound heterozygous R83C/R291Q mutation demonstrate a distinct lack of pDCs as well as cDC1 subset (Bigley et al., 2018). Sichien et al. demonstrated that IRF8 is not intrinsically required for the pDC development where as it is important for the function and survival of pDCs and cDC1 (Sichien et al., 2016). Terminally differentiated pDCs that lack IRF8 fail to produce type I IFNs upon TLR stimulation, thus demonstrating the additional role of these transcription factors in pDCs (Sichien et al., 2016). A point mutation in IRF8 (BXH2-mouse model) leads to a deficiency of cDC1 compartment without affecting pDC differentiation (Tailor et al., 2008). However, no human ortholog has been reported. Zeb2 is identified as essential for pDC differentiation and the absence of Zeb2 null mice shows reduced pDC frequency and skewed differentiation toward the cDC1 (Wu et al., 2016). SpiB is a critical transcription factor required for pDCs differentiation and SpiB / mice shows fewer pDCs in bone marrow and increased pDC number in periphery. The mice also show a defective response to TLR7/9 stimulation and production of type I IFN. Conditional mouse models generated from

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BCL11A / fetal liver cells demonstrated the poor pDC differentiation and Runx2 / model shows the accumulation of pDCs in bone marrow and the defect is due to the reduce expression of CCR5, which is critical for the repopulation of pDCs in periphery. A deficiency on Ikaros family zinc finger 1 (IKZF1) is resulted in poor pDC frequency in mouse (Allman et al., 2006). Similar observations are reported in patients with IKZF1 haploinsufficiency shows a relative reduction of pDCs, an increased frequency of cDC1and functional impairment of pDCs to produce cytokines (Cytlak et al., 2018). PU.1 is a critical for the development of conventional DCs and it represses the development of pDCs (Chopin et al., 2019). Other than the common DC differentiation pathway from CDP, a lymphoid lineage contribution is also attributed to pDC (Rodrigues et al., 2018; Upadhaya et al., 2018). pDCs also exhibit a lymphoid lineage signature and the major contribution due to the common target for the E transcription factors and E2A/HEB binding modulated transcription program (Ceribelli et al., 2016; Ghosh et al., 2014) as well as a contribution of cDC signature suppressing transcription factor ETV6 (Lau et al., 2018). Mouse and human pDCs are known to expresses high level of PACSIN1 and the role in pDCS is not well characterized (Crozat et al., 2010b). pDCs are well known to produce high level of type I iFN includes different subsets of IFNα and IFNβ. They are also known to produce type III IFN and other cytokines includes TNF-α (Reizis, 2019). The TNF-α production by pDC is more homogenous, whereas the IFN production is tightly regulated and highly restricted to a fraction of the pDCs. There are few studies attempted identify the difference between IFN producing and non-producing human pDCs and the underlying mechanism with a single cell seq approach (Wimmers et al., 2018). The study identified eight different clusters of pDCs with specific clusters expressing type I IFN and other clusters consist of mature and activated pDCs (Wimmers et al., 2018). A flow cytometry based approach on human pDCs activated with Flu virus identified the PD1 high CD80 low pDCs are the major producers of type I IFN (Alculumbre et al., 2018). More studies are essential to understand the underlying mechanism of restricted type I IFN production in pDCs. pDCs expresses high level of IRF7 at steady state (Barchet et al., 2002; Dalod et al., 2002) and critical for TLR mediated antiviral sensing and type I IFN productions (Honda et al., 2005). Other transcription factors IRF5 (Dai et al., 2011; Yasuda et al., 2007) and IRF8 (Sichien et al., 2016) are also known to be contribute for the function of type I IFN production. Other transcription factors such as SPIB (Sasaki et al., 2012), NFATC3 (Bao et al., 2016), RUNX2 (Chopin et al., 2016) are

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known to positively regulators whereas MYC (Kim et al., 2016) is a negative regulator of IFN production through an interaction with IRF7. Ma et al. identified the role of epigenetic regulator CXXC5 to recruit a DNA demethylase Tet2 for maintaining the hypo methylation for the constitutive expression of IRF7 critical for the production of type I IFN (Ma et al., 2017). Human pDCs from blood express high level of TLR1, TLR6, TLR7, TLR9 and TLR10. The TLR7 and TLR9 expression is highly restricted to pDCs compared to the cDCs in human and capable of producing type I IFNs in response to the TLR agonists (Hemont et al., 2013). pDCs sense different versions of methylated oligos with TLR9 and based on the intracellular localization they can produce type I IFN or an NFkB depended inflammatory cytokines. pDCs can acquire an interferon producing phenotype or an antigen-presenting phenotype depends upon the virus or TLR ligand activation. Activation with CpGA or HIV-1 induce the type I IFN production, whereas encountering Flu or CpGC turns them to upregulate the costimulatory molecules and acquire a DC phenotype. The APC phenotype shows their ability to present antigen to the CD8 T cells upon right stimuli or under critical conditions. Like human counterpart, mouse pDCs also expresses high level of TLR7 and TLR9 and capable of sensing single stranded RNA or methylated nucleotides or viruses; produce high level of type I IFN. Dasgupta et al. identified the immunomodulatory mechanism of pDCs through TLR2 mediated sensing of poly saccharides and induction of IL-10 production by CD4 + T cells (Dasgupta et al., 2014). pDCs express cytosolic DNA sensor cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) and they are critical for sensing the HSV1 infections and production of type I IFN (Li et al., 2013). Similar observations was reported in human pDCs activated with cyclic dinucleotides including cGAMP induced strong type I IFN production and knock down of STING abrogated the response (Bode et al., 2016). Another cytosolic receptor RIG-I also able to induce type I IFN production upon sensing RNA viruses such as yellow fever virus (Bruni et al., 2015) or Newcastle disease virus (NDV) (Kumagai et al., 2009). Human pDCs are low for different NLRP1, NLRP2, NLRP4 and caspase 1, caspase 5, caspase 8, are critical component for initiating an inflammatory response; lack of these molecules may be critical mechanism for pDCs to produce type I IFN upon TLR activation (Macri et al., 2018; Worah et al., 2016). The key signatures and surface markers specifically or commonly expressed by mouse and human pDCs are depicted in Fig. 4.

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Fig. 4 Illustration depicts the common signatures and shared signatures expressed by human and mouse plasmacytoid dendritic cell. The signature includes surface markers, transcription factors and major pattern recognition receptors. The signatures with * marks indicate tissue specific expression.

7.4 Functions of pDC pDCs are identified as interferon producing cell subset when encountered virus infected cells and their ability to produce type I IFN and activate the effector cells may be critical for antiviral immunity. pDCs respond to a number of RNA and DNA viruses including VSVG, HCV, HAV, LCMV, Dengue viruses, west nile virus, Epstein-Barr virus (EBV), HIV, MCMV as comprehensively reviewed by Reizis (2019). The studies conducted with an in vitro infection model demonstrate the critical role of pDCs to initiate antiviral immune response. The role of mouse pDCs controlling viral infections in vivo is slightly different from the in vitro experiments. pDCs are dispensable for the control of MCMV (Swiecki et al., 2010), influenza virus (Wolf et al., 2009) and LCMV (Cervantes-Barragan et al., 2012) infection and contribution of NK cells (Biron et al., 1999; Nguyen et al., 2002) or cDCs are also important (Baranek et al., 2012). pDCs are known to control the systemic infection of LCMV, MCMV, VSV and HSV1 through type I IFN secretion. They are critical to check the infection at early stage and at later stage adaptive immune system may play a major role in controlling the infection (Swiecki and Colonna, 2015). The role of pDC depends on the route of

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infection and the involvement of other type I IFN producing cells whereas they are not dispensable for mouse hepatitis virus and HSV2 infections (Cervantes-Barragan et al., 2007, 2012; Swiecki et al., 2013). pDCs are well studied on the context of HIV infection in human and they are believed to involved in HIV trafficking to lymph node (Reeves et al., 2012). HIV induces the persistent IFN production and induction of TRAIL mediated apoptosis of T cell or an IDO mediated generation of regulatory T cell (Manches et al., 2008). The IDO mediated cross talk of pDCs to T cells are critical for regulating autoimmune inflammation (Lippens et al., 2016). PDCs are important for the control of infection from Respiratory syncytial virus (RSV) (Cormier et al., 2014) and rhino viruses (Barlow-Anacker et al., 2017) in neonates and impaired IFN production by pDCs favors the infection. Mouse pDCs plays an important role in inducing tolerance; antibody mediated depletion of pDCS enhanced an allergic response and abrogated tolerance in mouse models (Bonaccorsi et al., 2010; Cao et al., 2009; Meyer et al., 2018). pDCs plays a critical role in mucosal immunity and colitis models, studies identified the importance of pDCs on IL-10 producing regulatory T cell generation (Dasgupta et al., 2014). pDCs are involved in mucosal B cell response and able induce T cell independent IgA production and provide protection against different viral infections (Tezuka et al., 2011). They are known for inducing TRAIL mediated cell death in tumor (Drobits et al., 2012; Kalb et al., 2012) and HIV infected CD4 T cells (Stary et al., 2009). pDCs are known to induce granzyme B mediated cell death of infected CD4+ T cells and regulate the T cell proliferation (Fabricius et al., 2013; Jahrsdorfer et al., 2010). Experimental autoimmune encephalomyelitis (EAE) mouse model studies identified the critical role of pDCs on IDO mediated cross talk with regulatory T cells and induction of antigen dependent tolerance (Lippens et al., 2016). The role of pDCs on various autoimmune diseases are well documented, they are involved in systemic lupus erythematosus where pDCs sense the nucleotides from dying cell and produce the type I IFN. Similar type I IFN mediated immune activation by pDCs induced by self-nucleotide ( Jin et al., 2010; Scott et al., 2017; Sisirak et al., 2013) and antimicrobial peptide LL-37 complex are considered to be the major contribution for Psoriasis (Ganguly et al., 2009; Nestle et al., 2005). Type I IFN production by pDCs enhances the maturation of cDC2 and improve their ability to produce IL-12p70. The interaction also helps to improve the beneficial effect of NK and NKT cells activation as well as the IFNg production by T cells (Skold et al., 2018). pDCs are proposed to

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be involved in type I diabetes induction based on the human (Allen et al., 2009) and mouse data (Diana et al., 2013) and constitutive depletion of pDCs could significantly reduce the disease incidence (Hansen et al., 2015). Malignant form of pDCs are a form extremely rare tumor and generally known as Blastic plasmacytoid dendritic cell neoplasm. The BPDCN expresses CD56 along with the general pDC markers and generally affects the skin, bone marrow and lymph nodes and the disease is mainly restricted to elderly male (Laribi et al., 2016).

8. Langerhans cells Langerhans cells are first identified in 19th century by Paul Langerhans and later considered as a cell subset belongs to DC lineage (Romani et al., 2010). LCs are specifically restricted to the epidermis, conserved across the species and even reported in vertebrates, birds and reptiles (Romani et al., 2010). Historically LCs are considered as DCs due to their typical DC like feature of migration to lymph node, present antigens and activate T cells (Merad et al., 2013). They are generally restricted to epidermis and closely associated with keratinocytes; LCs are also reported in the stratified epithelia (Kashem et al., 2017). LCs possess a typical veiled morphology and one of the characteristics is the presence of Birbeck granules and the expression of langerin or CD207 (Merad et al., 2013). The latest advancement in the field identifies them as a close cousin of macrophage than a DC subset (Doebel et al., 2017). LCs have an embryonic origin and capable of self-renewal for replenishing their niches (Merad et al., 2013). There are reports on the differentiation of LCs from bone marrow derived HSC under specific circumstances of infection. The chapter describes LCs as part of the DCs and further advances in the field may eventually define the true identity of this cell subset.

8.1 Human Langerhans cells Human LCs is virtually identified by the expression of human leukocyte antigen-DR, the classical MHC II and expression of CD1a and langerin (CD207) and the epithelial cell adhesion molecule (EpCAM) (Merad et al., 2013). Human LCs are negative for the lineage markers (CD14, CD16, CD3, CD56 and CD19) and macrophage marker F4/80. They express the general cDC2 markers including CD1c, low level of CD11c, SirpA, CD11b and CX3CR1 (Guilliams et al., 2016).

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8.2 Mouse Langerhans cells Mouse LCs are identified based on their expression of MHC II, CD207 and EpCAM. They have brighter CD11c expression compared to human LCs, whereas comparatively dim CD11c expression to the mouse cDCs. Mouse LCs express CD24, CD11b, F4/80+, CD205 and does not express CD26 (Guilliams et al., 2016).

8.3 Key gene signatures in LC Unlike steady-state DCs the LC differentiation is mainly regulated by transforming growth factor-beta ((TGF-β) and do not depend on the Flt3-Flt3L mediated ontogeny. They are radiation resistant, possess longer half-life up to 2 months compared to DCs (Merad et al., 2002). LCs in human and mouse expresses CSF1 receptor and studies highlighted the role of alternative ligand IL-34 on LC development and differentiation (Bogunovic et al., 2009; Lin et al., 2008a). Keratinocytes and neurons are the major sources of the IL-34 and LCs are known to closely associate with keratinocytes (Wang et al., 2012). LCs differentiation is mainly regulated by different transcription factors including pU.1, ID2 and RUNX3 and mouse models demonstrated the critical role of PU.1, Id2 and RUNX3 (Merad et al., 2013; Romani et al., 2010). Other factors including p14, BMP7 are also critical for LC differentiation and proliferation (Milne et al., 2015). Addition of BMP7 to human cDC2 in vitro demonstrated their ability to acquire langerin and an LC phenotype. There is no in vitro evidence for such transdifferentiation of cDC2s to LC like phenotype (Martinez-Cingolani et al., 2014, Milne et al., 2015). LCs isolated from healthy human skin expressed mRNA for TLR1, TLR2, TLR3, TLR5, TLR6 and TLR10 and the study also demonstrated that they are able to respond to TLR2 ligand peptidoglycan and produce inflammatory cytokines (Flacher et al., 2006) and LCS are known to respond dsRNA through the TLR3 mediated sensing. Whereas van der Aar et al. demonstrated the selective impaired expression of TLR2, TLR5 and TLR5 in human LCS and functionally they are not responsive against different bacterial ligands (Van Der Aar et al., 2007).

8.4 Functions of LC LCs are strategically located and present at the outermost environmental interphase and part of the first line immune defense. LCs have a typical veiled morphology and long dendrites with that they frequently probe for antigens breaching the stratum corneum of the epidermal layer (Kashem et al., 2017; Kubo et al., 2009). Activated LCs are capable extending the dendrites

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through the tight junctions and acquire foreign antigens (Kubo et al., 2009). LCs migrates to lymph nodes under steady state as well as in inflammation and capable of initiating immune response (Gerner et al., 2012). The process starts with downregulation of molecules like E cadherin helps them to engage with keratinocytes and followed by the upregulation of MHC II. LCs presents the antigens to the T cells and also participates in inducing humoral immunity (Levin et al., 2017). The mechanism may be critical for initiating protective responses for microbial toxins. LCs undergoes homeostatic proliferation under steady state; under inflammatory conditions, upon depletion from epidermis, bone marrow progenitors can replenish the epidermal compartment in a CCR2 depend way (Merad et al., 2002). Once the progenitors enter the epidermis and acquire the typical LC characteristics including the langerin expression. LCs may play an important role in contact hypersensitivity response and the response may depend upon the antigen concentration and they may also have role on inducing tolerance other than eliciting immune response (Igyarto et al., 2009; Kaplan et al., 2005; Kissenpfennig et al., 2005). The role of LCs under viral infections was studied by herpes virus and studies could not convincingly shows the LCs migrate to lymph node upon infection and activate the T cells (Allan et al., 2006). A leishmanial-based studies demonstrated the role of LCs in antigen transportation to lymph node and T cell activation (Moll et al., 1993). OVA antigens based mouse model K5-mOVA and K14-mOva studies as well as OVA expression in keratinocytes demonstrated the potential role of antigen specific tolerance by LCS in steady state (Bianchi et al., 2009). LC mediated induction of tolerance was demonstrated in human and mouse models. Human LCs induce the proliferation of skin resident regulatory T cells at steady state and targeting foreign antigens to the langerin in LCs with imiquimod induced a tolorogenic immune response (Flacher et al., 2014; Seneschal et al., 2012; Stoitzner et al., 2006). LCs are reported to associate with different pathologies such as histiocytosis and atopic dermatitis. LC histiocytosis is rare proliferative disorder characterized the clonal proliferation of LCs. The cell is accumulated in skin, bone and multiple organs or tissues in body. Some of the reports highlight the role of BRAF V600E mutation associated with the proliferation (Allen et al., 2018; El Demellawy et al., 2015; Kobayashi and Tojo, 2018). The LCH is associated with the production of inflammatory cytokine IL-17A a critical cytokine involved in the Th17 response (Coury et al., 2008). LCs are also associated with atopic dermatitis and the inflammatory responses are regulated by the production of thymic stromal lymphopoietin (TSLP); promote the development of proallergic T cell subsets (Ebner et al., 2007; Elentner et al., 2009).

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9. Inflammatory DCs Inflammation is an outcome of infection or injury and subsequent recruitment of neutrophils, monocytes or other leukocytes and associated production of different cytokines or chemokines. Inflammatory dendritic cells are originated from monocyte precursors upon an infection or injury and subsequent events of cytokine or chemokine production, leads to the differentiation of monocyte precursor acquire a DC phenotype. Generally they are indistinguishable from macrophages and expresses high level MHC II and different costimulatory molecules and CD11c. Like other DC subsets they can be found in any tissue upon the inflammatory response and are able to migrate to the draining lymph node. They exhibit a CCR2 depended migration to the lymphoid tissues and also able to present antigens to the T cells and elicit immune response. Inflammatory DCs are one of the major component of MPs. Inflammatory DCs are very similar to macrophages appear during inflammation and enough precautions should be taken for distinguish the cell subsets.

9.1 Human inflammatory DCs Human inf DCs are characterized by the virtue of expression of lack of lineage specific marker s, high level of HLADR and CD11c. Human infDCs expresses many surface markers of cDC2 such as BDCA1, CD1a, CD11c, CD14 and CD172a (Collin and Bigley, 2018). The blood cDC2 does not express the CD206 and have a low level expression of CD14 (Granot et al., 2017). Whereas the tissue derived cDC2s are known for expressing the CD206 (Granot et al., 2017). Additional cDC2 marker like Clec10A or FCERIA may be useful for distinguish the subset from inflammatory DCs (Heger et al., 2018). In vitro generated MoDCs shows a similar transcriptome profile of the infDCs and it may be associated with the potential development of infDC from CD14+ monocytes. The pioneering studies by Randolph et al. demonstrated culturing blood mononuclear cells in monolayer of endothelial cells grown in collagen matrix could identify the difference between monocytes and DC precursors (Randolph et al., 1998). They identified a cell subset with potential to transmigrate and acquire DC phenotype with strong ability to activate T cells. Whereas the monocytes fails to transmigrate and acquire a macrophage phenotype (Randolph et al., 1998). They also demonstrated that the injecting FITC labeled latex bead, the latex bead accumulation in draining lymph nodes are absent in a monocyte

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deficient mouse models and demonstrated the potential role of monocyte derived DC on antigen presentation (Hespel and Moser, 2012). Geissmann et al. identified the presence of two subset of monocytes in human and mice peripheral blood. The subset is classified based on the expression of CX3CR1 high and CX3CR1 lo, the CX3CR1 high subsets were found in blood and other noninflamed tissues (Geissmann et al., 2003). The CX3CR1 low subsets were actively recruited to the inflamed tissues and the migration was independent of the CX3CR1 expression. They also exhibited a shorter life span compared to the CX3CR1 high subsets (Geissmann et al., 2003). Human monocytes consist of three different subsets and classified based on the expression of CD14 and CD16. Majority of the cells are CD14+ CD16 represents the classical monocytes in human counterpart the Ly6chi monocytes in mouse. There is subset of CD14lo CD16high subset and a CD14+ CD16+ subset represents the non-classical monocyte subset (Collin et al., 2013). Kawamura et al. identified a progenitor cell population with strict monocyte differential potential in the conventional granulocytes monocyte precursor (cGMPs) (Kawamura et al., 2017). cGMPS are defined based on the expression of Lin CD34+ CD38+ CD10 CD123loCD135+ CD45RA+ and the cell subset was Clec12Ahi CD64hi subset of the conventional granulocytes monocyte precursor (cGMPs) (Kawamura et al., 2017).

9.2 Mouse inflammatory DC Mouse in fDCs is developed from the classical or non-classical monocyte precursors; classical monocytes express high level Ly6C and non-classical monocytes are low for their expression (Hespel and Moser, 2012). Classical monocytes are generally present in the blood and non-classical monocytes are observed in blood as well as tissues. Mouse infDCs exhibit a similar phenotype of cDC2 with high level of MHC II, CD11b and CD11c. They also express Ly6C and F4/80+ CD206, CD115, CD107b, FCERI and CD64 (Segura and Amigorena, 2013). Distinguishing inf DCs from macrophages is a major challenge; FCERI expression in fDCs may be a useful marker to identify the inf DCs in tissues as well as lymphoid organs. Functionally they are different from macrophages by their ability to migrate to lymph nodes in a CCR7 depended manner and activate the T cells. InfDCs are differentiated from monocytes and their migrations toward lymphoid tissues depend on a chemokine receptor CCR2. The CCR2 null mice shows dramatic reduction of the frequency of infDCs. Whereas the Flt3L null mice does not show any dramatic difference in the frequency of infDCs confirms that they are

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not belongs to the classical DC lineage. One of the models used to study the DC biology was the in vitro culture system using GM-CSF and IL-4. Hence GM-CSF was considered as a critical cytokine for development of inf DCs. Mouse bone marrow chimera model experiments with bone marrow from GMCSF-R KO mice to normal mice show the normal development and frequency of inf DCs. The results convince that GMCSF is dispensable for the development of inf DCs in an inflammatory conditions. Whereas similar studies with MCSFR null mice demonstrated that the frequency of infDCs are compromised during an inflammatory response. A common precursors of mouse monocyte was identified by the Hettinger et al. and the population was characterized as lineage negative, expresses CD117, CD115, Ly6c + and CX3CR1 . The cell subset was negative for CD135, CD11b, CD11 and MHC II expression. Several studies demonstrated the ability of infDCs to present antigens and activate CD4 T cells and the observations were confirmed in CCR2 / models using A. fumigatus infection and there was no difference observed in Flt3L KO model indicating the critical role of inf DCs inducing infections. Inf DCs are capable of producing different cytokines such as IL-12, IL-23, IL1a and IL1b also capable of inducing of both Th1 and Th2 responses. The HSV-1 infection models shows the ability of inf DCs to present antigens to CD8 T cells and even initiate memory response mediated through CD4 activation.

9.3 Key gene signature inflammatory DC Transcriptome profile identifies the close proximity of the inf DCs to monocytes but they shares the signatures of classical cDC2. The inf DC subsets may have potential role of immune suppression and promote a protumor environment (Bakdash et al., 2016; Van Ee et al., 2018). Sander et al. identified the differential gene expression profile of inf DCs and inflammatory macrophages. The inf DCs expresses high level of CCL22, MMP12, CD226, CCR7 whereas the inflammatory macrophages expresses typical macrophage genes MARCO, CCL2, VSIG4 (Sander et al., 2017). Sander et al. also reported the role of NCOR2 on MoDC differentiation and generally used as in vitro model of inf DCs (Sander et al., 2017). Inf DCs generally express TLR2, TLR4, TLR6, TLR8 and TLR9; they respond to peptidoglycans, LPS, and R848 and produce various inflammatory cytokines (Collin and Bigley, 2018; Segura and Amigorena, 2013).

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9.4 Functions of inflammatory DCs Inf DCs are reported to associate with atopic dermatitis, psoriasis, rheumatoid arthritis and play a critical role in microbial infections (Hespel and Moser, 2012; Segura and Amigorena, 2013). Study with Listeria monocytogenes demonstrated the recruitment of DC subsets capable of producing nitric oxide and tumor necrosis factor (Serbina et al., 2012). The cell subset displays expression of different costimulatory molecules CD80, CD86, CD40, high level of MHC II and other DC markers including CD11c and CD11b. The migration was depend upon a CCR2 chemokine receptor and the population was markedly reduced in CCR2 / mice (Serbina and Pamer, 2006). The infDC subsets were also known as TNF-iNOS producing DCs and abbreviated as TIP DCs. The DC subsets plays a critical on antimicrobial immunity and the activation requires a TLR4, TLR9 mediated MyD88 depended activation (Copin et al., 2007). Human infDCs are reported in various pathological situations such as atopic dermatitis, psoriasis, rheumatoid arthritis and tumor ascites (Hespel and Moser, 2012; Segura and Amigorena, 2013). Segura et al. identified the presence of inf DCs in synovial fluid of arthritis patients as well as in tumor ascites; infDCs isolated from arthritis patients or tumor ascites were able to induce a Th-17 response in in vitro culture (Segura et al., 2013b).

10. Dendritic cell locations Dendritic cells can be classified into subsets based on their anatomical locations such a blood DCs, lymphoid resident DCs, migratory DCs and these classifications are not really useful for identifying the role of DC subset. The advances in multi-color flow cytometry and gene expression profiling could confirm the existence of the homology of the subsets in different tissues. They may acquire some tissue specific gene signature but conserve their core gene signatures which distinguish the subset from each other. DCs are rare cell subset ubiquitously present in all the tissues, identified in most of the lymphoid or nonlymphoid tissues with varying frequency.

10.1 Bone marrow Adult HSC self-proliferation and differentiation is high regulated in the bone marrow niche and DCs are HSCs originated from bone marrow

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precursor and migrate to lymphoid or nonlymphoid organs. The frequencies of human DC subsets are similar to peripheral blood compared to lymphoid organs. cDC2s are the major DC subset in BM (Heidkamp et al., 2016) and constitute approximately 1% of the lineage negative HLADR+ cells. cDC1 frequency is extremely low and contribute to the 0.1% of the lineage negative HLADR+ cells (Granot et al., 2017; Heidkamp et al., 2016). A fraction of the bone marrow cDC1 are reported to express the macrophage marker CD206 and the marker is also expressed by progenitor cells. pDCs are present around 0.1% of the lineage negative HLADR+ cells and expresses the characteristic pDC markers (Granot et al., 2017; Heidkamp et al., 2016).

10.2 Blood Blood is the easily accessible tissue and that made it an attractive tissue for most of the humans DC studies. DC subsets are present at a very low frequency in blood, constitutes <1% of the nucleated cells. Blood consist of two subsets of cDCs identified based on the expression of Linneg HLADR+ CD11c+ and other specific markers represent CD141, Clec9a, CADM1 for cDC1 and CD1c, SIRPa and Clec10A, FCERIA for cDC2. pDCs are identified by virtue of Linneg HLADR+ CD11cneg and high level of expression of the specific markers CD123, CD45RA, BDCA2, BDCA4. cDC1 are the are rare, present at extremely low numbers and the average frequency is 0.2% of the Lineage negative HLADR+ cells in blood. cDC2 and pDCs are present at higher frequency compared to cDC1 and constitute around >2% of the Lineage negative HLADR+ cells in peripheral blood (Rovati et al., 2008). The presence of DC subset is identified in mouse peripheral blood (Adachi et al., 2002) and due to the higher frequency and accessibility other organs are mainly utilized for the studies.

10.3 Spleen All the major DC subset are identified in human spleen and cDC2 are the major DC subsets consist of approximately 5% of the Lin HLADR+ cells in spleen (Heidkamp et al., 2016). In contrast to the peripheral blood cDC1s are present at higher frequency in spleen and constitutes approximately 3% of the Lin-HLADR+ cells. pDCs constitute approximately 4% of the Lineage negative HLADR+ cells in spleen (Heidkamp et al., 2016). Mouse spleen consist of different DC subset, at steady state, cDC1 consist of 15% of the splenic DC compartment (Dong et al., 2016). There are two subset of

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cDC2 contributes to 50–60% of the DC compartment in spleen and they are distinguished based on the differential expression of ESAMhighCD11b+ and ESAMloCD11b+ (Lewis et al., 2011; Mildner and Jung, 2014). pDCs are present at lower frequency in spleen and constitutes around 15–20% of the DC compartment and can be grouped as two subsets based on the CD4 expression as CD4+ or CD4 pDCs (Dong et al., 2016; Yang et al., 2005).

10.4 Thymus Dendritic cell compartment in human thymus consist of all the three steadystate DC subsets and thymic DCs may play an important role in establishment of central tolerance and depletion of autoreactive T cells (Brocker et al., 1997; Gao et al., 1990; Martin-Gayo et al., 2010; Watanabe et al., 2005). The presence of different DC subset in thymus is well characterized by different research teams with multi-color flow cytometry and transcriptome analysis (Bendriss-Vermare et al., 2001; Okada et al., 2003; Vandenabeele et al., 2001). pDCs contribute 1–4% and cDCs constitute 6–10% of the of the Lin HLADR+ cells in thymus. The frequency of CD141+ cells is comparatively higher compared to the CD11b+ cells and they possess 70:30 ratios in thymus (Gurka et al., 2015). Martin-Gayo et al. identified a potential mechanism of notch signaling mediated differentiation of early thymic progenitors (ETPs) as different DC subsets in thymus (Martin-Gayo et al., 2017). In contrast to other organs like spleen or lymph nodes, the major DC population in mouse thymes is CD8α+ cDC1 and CD8α CD172a+ (SIRPα) cDC (Wu and Shortman, 2005). The total cDC compartment consist of the 0.3% of the thymic cells and 75% (C57BL/6) to 90% (Balb/c) of the cells are CD8α+ cDC1 depends upon the mouse strains (Wu and Shortman, 2005). pDCs are present approximately 35% of the DC lineage in mouse thymus (Wu and Shortman, 2005) and the existence of a CD4+ and CD4 subset are observed (Wu and Shortman, 2005; Yang et al., 2005).

10.5 Tonsil One of the pioneering study by Summers et al. identified the presence of five different subsets of DCs defined based on the expression of MHC II, CD11c, CD13, and CD123 (Summers et al., 2001). Lindstedt et al. performed a comparative transcriptome analysis of the DC subsets in tonsil with blood and confirmed the presence and homology of cDCs and pDCs in both the tissues (Lindstedt et al., 2005). Human tonsil DC compartment consist of

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all the three steady-state DCs and the pDCs are the major component and present approximately 15% of the Lin HLADR+ cells. The cDC2 consist of approximately 5% and cDC1 are 1% of the Lineage negative HLADR+ cells. Even though the pDC subset from blood and tonsil exhibited a similar transcriptional profile but functionally the thymic pDCs were poor producers of type I IFN (Vangeti et al., 2019).

10.6 Skin Skin is the largest barrier organ and exposed to wide variety of antigens; skin immune system must be specialized to discriminate the diverse antigens encountered and mount selective response to induce protective or tolerogenic outcome. Different cDC subsets and LCs are identified in the skin at steady state and LCs are generally restricted to the epidermis and cDCs are mainly restricted to the dermis (Kim et al., 2018b). Under steady-state conditions in human and mouse, LCs constitute 2–4% of the epidermis (Lipozencic and Ljubojevic, 2004). Mouse LCs can be distinguished from cDC2s by the difference in the expression of CD24 and CD26 (Guilliams et al., 2016). Mouse LCs share different markers with cDCs such as CD11c, CD207, CD24, SIRPa and macrophage marker F4/80. cDC1s can be identified by the classical XCR1 or Clec9a or CD103 expression in skin and the LCs can be distinguished by the CD26loCD24hi expression compared to the CD26hCD24lo expression on cDC2 (Guilliams et al., 2016; Haniffa et al., 2012). The dermis consists of two subsets of cDC1 based on the expression of CD103hior CD103lo; whereas the cDC2 consist of a CD11c lo CD11b lo and CD11bhiCD11chi subsets. Additionally CD26 and CD24 can be useful markers to identify the different subset of cDC1 and cDC2, otherwise overlooked based on the CD11c and CD11b expression (Guilliams et al., 2016). The skin cDC1 and cD2 subsets expressed higher level of CD80, CD83 and CD86 compared to the blood counterpart (Granot et al., 2017). The PD-L1 expression was higher on skin cDC2 compared to the blood cDC as well as skin cDC1 and generally they skin cDC1 exhibit a mature phenotype compared to the blood cDCs (Granot et al., 2017). Human LC in skin expresses high level of CD1a, langerin, low level of CD11c and the cDC2 marker CD32 and CD172a. Analysis of steady-state human skin dermis from healthy donors could identify the presence of both the cDC subsets and could not confirm the presence of pDCs (Granot et al., 2017; Gregorio et al., 2010). The skin cDC1 are generally low or intermediate for CD11c expression, high for CD141, dim for Clec9a and restricted

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expression of XCR1. The cDC2 in skin expresses higher CD1a compared to cDC1 and both subsets does not expresses langerin. In contrary to blood cDC the skin cDCs are shown to expresses high level of dectin1 and CD206 (Granot et al., 2017). Characterization of the skin draining lymph nodes classified the CD1c positive cells as cDC2 or inflammatory DCs based on the expression of CD206 (Segura et al., 2012). Under inflammatory conditions additional subsets are known to infiltrate the inflamed skin includes pDCs and inf DCs (Kashem et al., 2017).

10.7 Liver The presences of different DC subset are identified in liver and play a critical role in regulating the hepatic immune response. The lineage negative HLADR + subsets constitute 1% of the mononuclear cells in liver and pDCs constitute average of 15% of the total DC subsets. The cDCs compartment is unique compared to other tissues and cDC1 constitutes the two by third of the cDCs in liver (Kelly et al., 2014). The frequency of DC subset is altered upon HCV infection, the frequency of cDC1 is significantly depleted and frequency of cDC2 and pDCs were significantly increased (Kelly et al., 2014). The presence of all three DC subsets are identified in mouse liver and the cDC1 can be identified based on the expression of MHC II, CD11c, CD103, langerin and the cDC2 subsets expresses MHC II, CD11c, CD11b, SIRPa, CX3CR1 (Ginhoux et al., 2009). The pDCs constitute almost 12% of the DC compartment in liver and they exhibit an immature phenotype compared to the pDCs in spleen (Kingham et al., 2007).

10.8 Lung Lung is a highly specialized organ orchestrating the function of respiration and always exposed to the external environment. DCs play a critical role in maintaining the immune homeostasis in lung and they are critical for modulating the inflammatory response and eliciting immune response during an infection. Demedts et al. identified the existence of different DC subset in human lung based the expression of MHC II, BDCA1, BDCA2 and BDCA3 (Demedts et al., 2005). The bonafide cDC subset in human lung can be identified in the Lineage negative HLADR+ cells by virtue of the expression of CD11c and CD26. The subset can be subdivided into cDC1 based on the expression of CD141, clec9a, CADM1, Clec9a and cDC2s expresses CD1c and SIRPa (Guilliams et al., 2016). Baharom et al. performed a

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comprehensive analysis on the presence of DC subsets in healthy human lung (Baharom et al., 2016). The presence of DC subsets are tested on tissue samples from endobronchial biopsies [EBBs] and lavages sampling from the proximal (bronchial wash [BW] and distal airways (broncho alveolar lavage [BAL]). cDC1 was constituted approximately 1.5% in BW, 3% in BAL and 0.1% in EBB of the Lineage negative HLADR+ cells (Baharom et al., 2016). The frequency of cDC2 on different locations were, 6% in BW, 11% in BAL and 15% in EBB of the Lineage negative HLADR + cells. pDCs were less frequent compared to cDCs and represent the 0.8% in BW, 1.5% in BAL and 5% in EBB of the Lineage negative HLA DR + cells (Baharom et al., 2016). Sung et al. identified the presence cDC subsets in mouse lungs by the virtue of MHC II hiCD11chiCD11bloCD103hi and MHC II hi CD11chiCD11bhiCD103neg expression. DCs constitute 1% of the total lung digest and cDC1 in lung express langerin a classical marker of LCs (Sung et al., 2006). pDCs in mouse lung exhibited low expression of MHC II compared to cDCs and express high level of B220, Gr-1 and pDCA1. Guillam et al. defined a minimum set of markers to identify the DC subset in lung and the cDCs can be identified based on the expression of CD11c and CD26. The cDC1 can be identified with conserved markers CADM1 and XCR1, whereas the cDC2s expresses CD172a (Guilliams et al., 2016).

10.9 Intestine Intestine is another organ similar to skin and lung with a huge surface area; constantly encounter antigens from different sources such as food as well as commensal and pathogenic microbes. Intestinal DCs are critical for distinguishing the commensal flora from mucosal pathogens and eliciting tolerogenic or inflammatory response, respectively (Bekiaris et al., 2014). Granot et al. identified the frequency of cDC2 ranges from 0.1% to 1% of the CD45+ cells in various tissues. The highest frequency was observed in lung and jejunum compared to other tissues. Jejunum, ileum, colon, peyers patch and appendix (Granot et al., 2017). The intestinal DC subsets in mouse as well as human are characterized by their expression of CD103 and the molecule may help the DCs for E cadherin mediated adhesion of the gastrointestinal epithelial cell and improve the immunosurvilence (Stagg, 2018; Swain et al., 2018). The gut DC subset expresses tissue specific chemokine signature CCR9 and specific enzymes RALDH2 required for retinoic acid generation critical for the immunomodulatory signature of the gut DCs (Stagg, 2018; Zeng et al., 2013).

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10.10 Lymph node Presence of cDC and pDC are observed in hepatic LN, inguinal LN, the cDC1s are expressed the characteristic markers CD141, Clec9a and absent for CD1a, CD206 and CD209 where as a fraction of cDC2 subsets expressed CD1a and CD206 (Granot et al., 2017). The lymphoid resident DC subsets exhibit a specialized localization; the cDC1s are generally restricted to the deeper paracortical regions and the cDC2s are located to the lymphatic sinus proximal regions (Gerner et al., 2012). The frequency of cDC1 was higher in hepatic draining LN compared to the inguinal LN, whereas the cDC2 where similar in both LN. The cDC subset were exhibited an activated phenotype with high level expression of HLADR, CD40, CD86 and PDL1. pDC frequency in hepatic LN was around 0.13% of the CD45+ cells and inguinal LN was around 0.95%. In contrast to the cDCs pDCs had an immature phenotype with low level expression of costimulatory molecules (Boor et al., 2019).

10.11 DCs locations in pathology The DC subsets are identified in most of the healthy as well as malignant tissues. Presence of all DC subsets is reported in breast cancer, ovarian cancer, lung cancer, melanoma and hepato cell carcinomas (Pedroza-Gonzalez et al., 2015). Presence of pDCs is always correlated with bad prognosis and they may have a role on the generation of regulatory T cells and induce a tolorogenic response. PDCs are shown to accumulate in the sentinel and metastatic lymph node in melanoma (Pedroza-Gonzalez et al., 2015) and the expression of CCR6 (Charles et al., 2010) and CXCR4 on pDCs and production of CCL20 and CXCL12 tumor recruits pDCs to the melanoma and pDC accumulation are associated with poor prognosis (Li et al., 2017). pDCs are functionally impaired for type I IFN production in breast cancer tissues due to the of TGF-β and TNF-α in tumor micro environment (Sisirak et al., 2013). The pDCs in tumor niches expresses ICOS–Ligand and interact with the ICOS expressing tumor infiltrating CD4 T cells and induce the expansion of IL-10 producing Tregs (Faget et al., 2013). In ovarian cancers pDCs are known to induce CD8+ regulatory T cells (Wei et al., 2005) and pDCs depletion reduced tumor growth as well as bone metastasis (Sawant et al., 2012). In hepatocellular carcinoma pDCs migrates to the tumor tissues and shows a reduced frequency in peripheral blood. They induce immune suppression through the ICOS-L-ICOS axis on CD4 T cells and induction of IL-10 and regulatory T cell expansion (Beckebaum et al., 2004; PedrozaGonzalez et al., 2015). The presence of pDCs is also identified in lung cancer

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and they the frequency of pDCs are significantly increased and the frequency was correlated with advanced stages of the disease (Shi et al., 2014). pDCs can produce high level of type I IFN and they can in turn activate the NK cells and other APCS in the tumor tissue. But the pDCs are functionally impaired once they migrate to the tumor sites. Presence of cDC2 is not associated with any good prognosis and they are present in various tumors and their frequency was not associated with tumor regression. cDC1s are also reported in various tumors and some of the reports highlighted the functional impairment of cDC1 in patients peripheral blood. Various mouse model studies identified the critical role of cDC1 in tumor immunity. Spranger et al. demonstrated that the presence of cDC1 at the tumor site is critical for antitumor immune response. They also demonstrated that for an effective immune check point inhibition the cDC1s are critical (Spranger et al., 2015). Similar observations were made in the lung cancer models (Salmon et al., 2016). There are few recent reports high light the positive correlation of cDC1 and tumor regression in different tumor types. Bottcher et al. identified the critical role of XCR1-XCL1/XCL2 axis and the role of NK cell to induce the recruitment of cDC1 to the tumor for an effective antitumor immune response. Barry et al. also demonstrated similar observation and the infiltration of cDC1 in tumor correlated with better antitumor response in patients. They clearly demonstrated the presence of cDC2 does not have any positive correlation with tumor rejection and success of immunotherapy. The study also demonstrated the role of NK cells as a major source of Flt3L in mouse and human tumor microenvironment, helps to recruit the cDC1s to the tumor sites and provides a positive correlation with tumor regression. The presence of cDC1 in tumor locations and the correlation with tumor regression was reviewed by Cancel et al. and high light the importance of cDC1 in tumor microenvironment for inducing the strong antitumor immunity (Cancel et al., 2019). All the three DC subsets were identified in Non-small cell lung cancer (NSCLC) and the cDC2s were observed at the highest frequency (Stankovic et al., 2018). There are contradictory reports on the frequency of cDC1 and some reports shows lower frequency of cDCs (Lavin et al., 2017; Shi et al., 2014) whereas others did not observe any drastic difference (Stankovic et al., 2018). A study on the APC compartment in liver draining lymph node, Iliac lymph node and spleen identified the presence of different DC subset. The draining lymph nodes from healthy donors had a reduced number of cDC2 and pDC compared to the inguinal lymph node (Boor et al., 2019; Tanis et al., 2004). Both the lymph nodes had lower frequency of cDC1 compared the frequency observed in spleen. The cDC subsets isolated from liver draining lymph nodes

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exhibited a more mature phenotype compared to the cDCs from inguinal LN, iliacal LN, spleen and liver tissue (Boor et al., 2019). Patients with inflammatory liver disease had reduced number cDC1, cDC2 and increased frequency of pDCs in their liver draining lymph nodes. Meyer et al. reported the selective inhibition of cDC1 differentiation in breast and pancreatic cancer as mechanism for tumor evasion (Meyer et al., 2018). The study identified reduced cDC differentiation on in bone marrow of the breast and pancreatic cancer patients (Meyer et al., 2018). The presence of low frequency of cDC2 and pDCs were identified in Glioblastoma Multiforme (GBM) patients. The cDC2 are immature and impaired in their ability to produce IL-12 (Adhikaree et al., 2019).

11. Conclusion DCs are crucial component of our immune system and critical for the initiation and regulation of adaptive and humoral immune system. They are considered as natural adjuvant and their superior antigen presentation ability made them an attractive source for cell based vaccines against various malignancies. A large number of clinical trials are performed with monocyte derived DCs (MoDC) and the trials show the safety and possibility of DCs as vaccine candidate against various tumors (Garg et al., 2017). MoDC vaccines are safe whereas objective clinical responses are limited and in most cases prolonged survival in patients has not routinely demonstrated. The results confirm the ability of DCs to elicit immune response and the potential opportunities available with the DCs an agent for cellular vaccination. The major reasons for the failure may be due to the inherent difference between the steady-state DCs and MoDCs, their ability to present antigens and elicit strong antitumor immune response (Saxena et al., 2018). DCs are critical for immune homeostasis and it is not clear the exact role or critical contribution of each subset in maintaining the immune homeostasis. The mouse cDC1 and pDC biology was widely studied due to the availability of transgenic mouse models with potential for specific ablation of the subsets (Cancel et al., 2019; Reizis, 2019). Whereas the cDC2 subsets are not well characterized due to the lack of mouse models which can specifically ablate the cDC2; also the heterogeneous nature of the subsets increases the complexity of the problem. The ontogeny of DC subsets are well studied in mouse models and attempts are made to extrapolate the models to human DCs. Extremely low frequency of DC subsets in human tissues as well as other compounding problems like difficulty to access the tissues, requirement of complex and laborious procedures for DC isolation are major bottleneck

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for the progress of human DC biology. The functional specializations of human DC subsets are not well characterized and most of the time the observations are extrapolated from mouse DC biology with limited experimental evidence. Alternative systems for their generation from HSC using in vitro generation protocol or in vivo systems like humanized mouse models may be a critical tool for overcoming the limitation of DC numbers (Balan et al., 2018; Ding et al., 2014). The system may help us the address the questions on DC ontogeny, functional specialization such as cross presentation and extend the information for clinical applications. The alternative system for generating DCs can be a useful tool, but rigorous measures should be taken to make sure the DCs are bonafide DCs and comparable with the in vivo subsets present in human tissue. The in vitro generated DCs can exhibit a different surface phenotype compared to the in vivo counterparts and hence additional care should be taken for defining each subset and rigorous functional assays should be defined for their characterization to ensure the identity of each subset. Various studies identified the key surface markers expressed by human DC subset and performing a rigorous phenotyping with combination of markers are critical for defining the identity of each subset. The ability of DC subsets to exhibit differential response different TLR ligands can be a simple assay for the functional characterization of the human DC subtests. The technological advancement in flow cytometry, transcriptome analysis or the advanced methods like simultaneous analysis of epitope and transcriptome at single cell level (CITE-Seq) may provide a potential opportunity to delineate these rare cells and advance the DC biology.

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