b.iii. Plasmacytoid DC functions independent of antigen presentation Activation of pDCs

Plasmacytoid DCs express toll-like receptor (TLR)7 and TLR9, whose expression in human is restricted to B cells and pDCs, while in mice, these receptors are also expressed by other cell types (Gilliet et al., 2008; Rothenfusser et al., 2002). TLR7 and TLR9 recognize nucleic acids in early endosomes; viral ssRNA for TLR7 and bacterial unmethylated CpG DNA for TLR9, both TLR7 and TLR9 also have been shown to sense patterns of the parasite Plasmodium (Bauer and Wagner, 2002; Demaria et al., 2014; Diebold et al., 2004; Gilliet et al., 2008; Lund et al., 2004;

Rothenfusser et al., 2002; Yu et al., 2016). Sensing via TLR7 and TLR9 triggers the MyD88-PI3K-IRF7 signaling pathway (myeloid differentiation primary response gene 88; phosphoinositide 3-kinase; interferon-related factor 7), leading to IFN-I production (Diebold et al., 2004; Gilliet et al., 2008; Guiducci et al., 2008; Lund et al., 2004). It also can lead to maturation of pDCs, which relies on nuclear factor (NF)-κB pathway, with upregulation of MHC-II and co-stimulatory molecules, and secretion of pro-inflammatory cytokines (Gilliet et al., 2008). Synthetic analogs of

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TLR7 and TLR9 ligands have been designed, such as Imiquimod and CpG olygodeoxynucleotides (CpG-ODN), respectively (Gilliet et al., 2008; Hemmi et al., 2002).

Different classes of CpG exist, leading to different outcomes. Class A CpG-ODN (CpG-A) leads to the production of IFN-I by pDCs, while CpG-B induces the secretion of cytokines, including IL-12 and IL-6, and pDC maturation (Gilliet et al., 2008; Honda et al., 2005; Vollmer and Krieg, 2009). CpG-C promotes both IFN-I production and pDC maturation (Vollmer and Krieg, 2009).

TLR7 and TLR9 normally do not sense self-nucleic acids (Demaria et al., 2014; Gilliet et al., 2008). Nonetheless, in certain autoimmune diseases, such as type 1 diabetes, psoriasis and systemic lupus erythematosus, host endogenous factors, such as self-nucleic acid-specific immunoglobulins or antimicrobial peptides, can break innate immune tolerance and enable the entry of self-nucleic acids into endosomes where they can trigger TLR7 or TLR9 signaling (Demaria et al., 2014; Diana et al., 2013; Gilliet et al., 2008; Henault et al., 2016; Lande et al., 2007; Panda et al., 2017; Sakata et al., 2018). In addition, TLR7 and TLR9 can recognize DAMPs.

For instance, TLR9 can sense oxidized mitochondrial DNA, which contains unmethylated CpG repeats (Caielli et al., 2016). Conversely, impaired TLR7 or TLR9 signaling is implicated in persistent viral infections and cancer (Hirsch et al., 2010).

In addition to nucleic acid sensing through TLR7 and TLR9, pDCs can also be activated by cytokines, such as IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Ghirelli et al., 2010; Grouard et al., 1997). Depending on the cytokine used to activate pDCs, the pattern of pDC cytokine production differs (Ghirelli et al., 2010; Grouard et al., 1997).

Finally, pDCs can also be activated by CD40-L, a molecule expressed by activated T cells (Grouard et al., 1997).

Innate pDC functions having no direct link with T cell responses

Plasmacytoid DCs have direct innate antiviral and antitumoral roles, and are also implicated in the activation/regulation of innate and adaptive immune cells (Fig. 9).

Through their secretion of chemokines, pDCs can induce the recruitment of innate immune cells to sites of inflammation, including neutrophils, monocytes and macrophages (Swiecki and Colonna, 2015).

As above-mentioned, pDCs are professional IFN-I producers (Reizis et al., 2011a). They also produce IFN-III (IFN-λ), although the roles and targets of this family of cytokines [reviewed in (Wack et al., 2015)] will not be detailed here.

A crucial faculty of pDCs is to induce an anti-viral state through its production of IFN-I, leading to the expression of IFN-stimulated genes in infected cells and their subsequent apoptosis (Reizis

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et al., 2011a; Swiecki and Colonna, 2015). IFN-I production, as well as that of pro-inflammatory cytokines, by pDCs is also implicated in NK cell activation (Dalod et al., 2003; Hervas-Stubbs et al., 2011; Swiecki and Colonna, 2015). In addition, IFN-I and IL-6 secretion, along with the expression of specific surface markers, by pDCs play a role in B cell activation, plasma cell generation and the secretion of antibodies (Menon et al., 2016; Swiecki and Colonna, 2015).

Due to TRAIL and Granzyme B expression, pDCs have the ability to directly kill tumor cells (Drobits et al., 2012; Lombardi et al., 2015; Tel et al., 2014; Tel et al., 2012a).

Innate pDC functions having an impact on T cell responses

Plasmacytoid DC innate roles can affect cDCs, as well as other APCs, and T cell recruitment and functions, therefore indirectly impacting T cell responses.

On the one hand, by producing chemokines, pDCs can influence the migration of cDCs and T cells (Swiecki and Colonna, 2015).

On the other hand, cDC and T cell functions can be modulated by the innate roles of pDCs.

Plasmacytoid DCs have been found to regulate several cDC functions via the secretion of IFN-I, leading to different outcomes, which are context-dependent (Hervas-Stubbs et al., 2011; Swiecki and Colonna, 2015). For example, pDC IFN-I production modulates cDC maturation and their secretion of cytokines and enhances tolerance mediated by cDCs, in a mouse model of autoimmunity (Dalod et al., 2003; Prinz et al., 2008). Antigen uptake abilities of cDCs can also be regulated by pDCs (Kastenmuller et al., 2011). In addition, pDCs can promote cDC cross-presentation (Brewitz et al., 2017; Rogers et al., 2017; Schiavoni et al., 2013).

Innate pDC functions can also affect T cell directly. For example, the expression of Granzyme B and TRAIL by pDCs contributes to the suppression of T cell expansion, and to the induction of the apoptosis of infected CD4⁺ T cell (Jahrsdorfer et al., 2010; Swiecki and Colonna, 2015). IFN-I production by pDCs has been shown to be important for the survival of CD8⁺ T cells, in a mouse model of viral infection (Swiecki et al., 2010). In addition the production of IFN-I and IL-12 by pDCs drive CD8⁺ T cell activation and the polarization of Th1 cells, while the secretion of TGF-β and IL-6 are involved in Treg and Th17 cell commitment (Bonnefoy et al., 2011; Cella et al., 2000; Gautreau et al., 2011; Hervas-Stubbs et al., 2011; Swiecki and Colonna, 2015).

Therefore, depending on the context, innate pDC functions lead to various outcomes on T cell responses, such as suppression, apoptosis or Treg polarization on the one hand, and activation, survival, proliferation and effector T cell polarization, on the other hand.

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I.6.b.iv. Antigen-presenting functions of pDCs Uptake of exogenous antigens

Whether pDCs can uptake exogenous antigen to cross-present them via MHC-I to CD8⁺ T cells and present them through MHC-II to CD4⁺ T cells is still debated.

In mice, Siglec-H and BST2 (PDCA-1) have been used as endocytic receptors allowing the internalization of exogenous antigens conjugated with anti-Siglec-H or anti-BST2 antibodies (Loschko et al., 2011a; Loschko et al., 2011b; Moffat et al., 2013; Zhang et al., 2006).

In human, different pDC receptors have been used to deliver exogenous antigens to pDCs, such as CD32 (Fcγ receptor II), DEC205, DC immunoreceptor (DCIR) and BDCA-2, leading to successful antigen internalization (Benitez-Ribas et al., 2006; Meyer-Wentrup et al., 2008; Tel et al., 2011; Tel et al., 2013b). In addition, the uptake of exogenous antigen, e.g. encapsulated in microparticles, solubles, or from virus-infected cell material has also been observed in human cells (Di Pucchio et al., 2008; Lui et al., 2009; Ruben et al., 2018; Segura et al., 2013a; Tel et al., 2010; Tel et al., 2012a). Of note, pDCs were found by some groups to have the capacity to internalize exogenous antigens, although their endocytic abilities were lower than that of cDCs (Lui et al., 2009; Tel et al., 2012a). On the contrary, some studies concluded that pDCs could not acquire exogenous antigens (Bonaccorsi et al., 2014; Salio et al., 2004). Using pDCs from human tonsils, Segura and colleagues, however, observed that internalization of soluble exogenous antigens by pDCs was as efficient as for cDCs (Segura et al., 2013a). The observation that pDCs could not internalize exogenous antigens might therefore be due to the use of pDCs from peripheral blood in other investigations, which could have different properties from that of SLOs, which are the ones that present antigens in vivo (Bonaccorsi et al., 2014; Salio et al., 2004;

Segura et al., 2013a). For instance, pDCs were found to be the most abundant subset of DCs in LNs, and to have the capacity to migrate from the periphery to LNs via the afferent lymphatics (Kohli et al., 2016; Segura et al., 2013a).

Finally, human pDCs were shown to have the ability to process synthetic long peptides, which contain both MHC-I-restricted and MHC-II-restricted epitopes (Aspord et al., 2014b). Antigen processing was increased in the presence of TLR7 or TLR9 ligands.

MHC-I-restricted cross-presentation of exogenous antigens to CD8⁺ T cells

With the exception of erythrocytes, all cell types in mouse and human express MHC-I molecules and can present endogenous antigens to CD8⁺ T cells (Neefjes et al., 2011). However, the presentation of exogenous antigens to CD8⁺ T cells, called cross-presentation, is a selective feature of certain types of APCs (Joffre et al., 2012; Kambayashi and Laufer, 2014).

Cross-31

presentation of exogenous antigens to CD8⁺ T cells by pDCs has been observed in human and mice (Nierkens et al., 2013; Villadangos and Young, 2008) (Fig. 9). Nonetheless, in contrary to cDCs that can cross-present exogenous antigens in inflammatory situations but also in the steady-state, pDCs acquire cross-presentation ability only upon activation.

In human, the cross-presentation of exogenous antigens, either soluble, opsonized, encapsulated, cell-associated or from virus-infected cells, has been observed in several studies in vitro (Benitez-Ribas et al., 2006; Di Pucchio et al., 2008; Hoeffel et al., 2007; Lui et al., 2009; Segura et al., 2013a; Tel et al., 2013b; Tel et al., 2012a). Similar observations have been made in different mouse models, in vitro and in vivo, in which antigens were soluble, in particle, or delivered to pDCs via BST2 (Kool et al., 2011; Loschko et al., 2011b; Moffat et al., 2013; Mouries et al., 2008;

Oberkampf et al., 2018). However, in other investigations, the cross-presentation of exogenous antigens by pDCs to CD8⁺ T cells could not be observed (Salio et al., 2004; Sapoznikov et al., 2007). For instance, antigen-targeting to pDCs through BST2 did not lead to CD8⁺ T cell response in vitro (Sapoznikov et al., 2007). Moreover, pDCs could elicit a CD8⁺ T cell response against exogenous peptides but not exogenous antigens, suggesting an issue in exogenous antigen internalization or processing, which was not elucidated (Salio et al., 2004).

Interestingly, Gilliet and colleagues observed an induction of IL-10-producing CD8⁺ Tregs by culturing in vitro naïve human CD8⁺ T cells with allogeneic pDCs activated with CD40-L (Gilliet and Liu, 2002). These CD8⁺ T cells were anergic and had decreased cytotoxic activity. This suggested a potential implication for pDCs in CD8⁺ T cell tolerance.

Discrepancies between the conclusions of distinct analyzes, on whether pDC can cross-present or not, might be due to different pDC maturation state (different stimulators used) or due to the method used for antigen targeting.

The intracellular pathways involved in antigen cross-presentation to CD8⁺ T cells are still unclear.

The processing of exogenous antigens dedicated to cross-presentation is different from the mechanism of endogenous antigen-processing that relies on the proteasome and for instance, multiple cross-presentation pathways exist in cDCs (Joffre et al., 2012; Neefjes et al., 2011). The processing of exogenous antigens in pDCs did not require the transit of antigens to the cytosol, as it was observed to occur in endocytic organelles (Di Pucchio et al., 2008; Hoeffel et al., 2007).

In addition, the processing machinery is dependent on the activation of pDCs through TLR ligands (Kool et al., 2011; Mouries et al., 2008). Finally, an implication of mitochondrial ROS production was recently demonstrated to be important for the ability of pDCs to cross-present (Oberkampf et al., 2018).

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MHC-II-restricted antigen presentation to CD4⁺ T cells

Plasmacytoid DCs express the promoter III (pIII) of CIITA, the master regulator of MHC-II expression, while cDCs express CIITA pI, hence, their pattern of MHC-II expression are differentially regulated (LeibundGut-Landmann et al., 2004; Reith et al., 2005). Unlike cDCs that express MHC-II constitutively, pDC MHC-II expression is upregulated upon activation (Kool et al., 2011; Sadaka et al., 2009; Young et al., 2008). In addition, in contrary to cDCs, the synthesis of MHC-II by pDCs continues long after stimulation and MHC-II molecules accumulate in antigen-loading compartments, therefore making pDC good candidates for prolonged MHC-II-restricted antigen presentation in chronic pathologies (Sadaka et al., 2009; Young et al., 2008). In addition, the endocytosis of MHC-II and antigen internalization also continues to occur after pDC maturation. Therefore, pDCs have the capacity to constantely renew peptide-MHC-II complexes exposed at their surface (Sadaka et al., 2009). Finally, the expression of MHC-II by pDCs is differentially regulated from their function of IFN-I-production; MHC-II expression is independent of IRF7, but relies on NFκB signaling (Sadaka et al., 2009).

Several studies, in human and mouse, have shown that pDCs could present antigens to CD4⁺ T cells via MHC-II molecules (Benitez-Ribas et al., 2006; Kool et al., 2011; LeibundGut-Landmann et al., 2004; Moffat et al., 2013; Sapoznikov et al., 2007; Tel et al., 2010; Tel et al., 2012a) (Fig. 9).

Depending on the immunological context, MHC-II-restricted antigen-presenting functions of pDCs can be either tolerogenic or immunogenic (Guery and Hugues, 2013). On the one hand, pDCs can induce anergy or deletion of CD4+ T cells, as well as pTreg differentiation or expansion of Tregs already differentiated. On the other hand, pDCs can induce the differentiation of naïve T cells into effector CD4⁺ T cells, or the polarization of antigen-experienced CD4⁺ T cells, such as Th1 or Th17 cells (Guery and Hugues, 2013).

In addition, as mentioned previously, heterogeneity has recently been observed among human pDCs and different groups have characterized pDC subpopulations with increased ability to induce CD4⁺ T cell expansion, compared with other pDC subpopulations (Alculumbre et al., 2018b; Rodrigues et al., 2018; Zhang et al., 2017).

In human, activation of pDCs with influenza virus along with CD40L led to the upregulation of MHC-II and co-stimulatory molecule expression by pDCs, and led to the stimulation of CD4⁺ T cells in vitro, with a Th1 polarization due to IL-12 and IFN-I production (Cella et al., 2000).

In mice, Krug and colleagues found that pDCs could promote the expansion of antigen-experienced – non-polarized – CD4⁺ T cells and their polarization towards Th1 cells (Krug et al.,

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2003). However, pDCs were unable to induce naïve T cell proliferation (Krug et al., 2003).

Another study in mice, using in vitro-cultured pDCs or ex vivo splenic pDCs, showed Th1 or Th2 polarization of T cells cultured with these pDCs, depending on TLR stimulation and antigen dose; high or low antigen dose led to Th1 or Th2 polarization, respectively (Boonstra et al., 2003). In addition, in human, Alculumbre et al. characterized three pDC populations, based on the expression of PD-L1 and CD80, after stimulation with influenza virus (Alculumbre et al., 2018b). The population P3 (CD80⁺ PD-L1^-) had increased capacity to induce the expansion of CD4⁺ T cells and led to the differentiation of naïve T cells into Th2 cells.

In a mouse model of graft vs host disease (GvHD), adoptive transfer of antigen-loaded MHC-II-sufficient pDCs into MHC-II-deficient recipient mice led to the priming of IFN-γ⁺ CD8⁺ T cells and to GvHD induction, MHC-II-deficient mice being resistant to the disease (Koyama et al., 2009). Furthermore, antigen-targeting to pDCs through BST2 in mice led to increased IFN-γ production by CD4⁺ T cells, and subsequent antiviral or antitumoral effects (Loschko et al., 2011b). Finally, Mallat’s group, in collaboration with our group, demonstrated a pathogenic role of MHC-II-mediated antigen presentation by pDCs in a mouse model of atherosclerosis (Sage et al., 2014). Indeed, in the absence of MHC-II expression by pDCs, IFN-γ production by pro-atherogenic T cells was decreased, as well as the infiltration of T cells in lesions, showing an immunogenic role of pDC MHC-II-mediated antigen presentation in this context, with a Th1 polarization (Sage et al., 2014).

Immunogenic pDCs can also induce the differentiation or promote the expansion of Th17 cells.

In GvHD patients, Th17 cells and pDC counts were found to be increased in the intestinal mucosa, compared with healthy donors (Bossard et al., 2012). In addition, the number of pDCs in mucosal intestina correlated with clinical score. Although this analysis did not demonstrate that pDCs were directly implicated in the differentiation or expansion of Th17 cells, it suggests a potential link between those cells and a pathogenic role of pDCs in this context (Bossard et al., 2012). Interestingly, pDCs from the peripheral blood of healthy donors induced Th17 cell differentiation in vitro upon TLR7 ligation (Yu et al., 2010).

In experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis, pDC depletion before immunization led to decreased severity and decreased Th17 cell counts, although a direct link between pDCs and Th17 cells was not demonstrated (Isaksson et al., 2009).

Therefore, pDCs were immunogenic in the priming phase of EAE. On the contrary, pDC depletion a week after immunization increased the severity of the disease (Isaksson et al., 2009).

Furthermore, TGF-β-treated pDCs induced the polarization of Th17 cells in vitro, due to increased IL-6 production by TGF-β-treated pDCs compared with non-treated pDCs,

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subsequently leading to CD4⁺ T cells Th17 cells commitment over Tregs (Bonnefoy et al., 2011).

Adoptive transfer of TGF-β-treated pDCs in a mouse model of rheumatoid arthritis increased the clinical score (Bonnefoy et al., 2011). Of note, a study using mice and rats showed pDCs were implicated in the modulation of the Treg/Th17 balance; pDCs can induce the conversion of Tregs into Th17 cells, induced by IL-6 production (Gautreau et al., 2011). Finally, our group showed that subcutaneous injection of the TLR9 ligand CpG-B, along a MHC-II-restricted antigenic peptide, in mice, induced Th17 differentiation in LNs draining the injection site (Guery et al., 2014). Th17 differentiation was abrogated in mice in which pDCs lacked MHC-II expression, showing that MHC-II-antigen presenting functions of pDCs were directly implicated in this mechanism.

Altogether, these mouse and human studies, although not all demonstrating a direct link between MHC-II-restricted antigen presentation by pDCs and effector T cell differentiation or expansion, showed that in certain immunological contexts, or in the presence of specific molecules, pDCs antigen-presenting functions can be immunogenic and be involved in Th1 or Th17 cell polarization, with implications in antiviral and antitumoral immunity, as well as transplantation and autoimmunity.

On the contrary, several human and mouse investigations have observed a tolerogenic role of antigen-presenting pDCs, either by suppressing effector CD4⁺ T cells, or by inducing the differentiation or expansion of Tregs.

Kolhi et al. suggested that intralymphatic injection of ovalbumin (OVA)-loaded pDCs led to tolerance in LNs, by inducing the abortive proliferation of OT-II cells. However, tolerance induction was not direct but mediated by endogenous APCs, since CD4⁺ T cells abortive proliferation was abrogated when antigen-loaded MHC-II-sufficient pDCs were transferred into MHC-II-deficient mice (Kohli et al., 2016). Nonetheless, many studies proposed a direct role for pDCs in the induction of tolerance via their ability of MHC-II-mediated antigen presentation. Of note, Kohli and colleagues used resting pDCs, in steady-state conditions.

Plasmacytoid DCs have been observed to play a tolerogenic role in multiple mouse models, such as EAE, asthma, allergy to food antigen and organ transplant, in which pDCs were involved in the suppression of CD4⁺ T cell responses, by inducing the anergy or deletion of CD4⁺ T cells, by preventing the priming of these cells, or by mechanisms not specified (Chappell et al., 2014; de Heer et al., 2004; Goubier et al., 2008; Liu et al., 2011; Loschko et al., 2011a; Takagi et al., 2011).

For instance, antigen-targeting to pDCs through Siglec-H dampened EAE severity, by decreasing

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CD4⁺ T cell expansion and Th1/Th17 cell polarization, however, not converting these cells into Tregs or other T cells with a regulatory phenotype (Loschko et al., 2011a).

In addition, many studies have shown an implication of pDCs in tolerance induction by promoting the differentiation or expansion of Tregs.

In human, activation of pDCs with CpG-B led to a cell-cell contact-dependent differentiation of naïve T cells into Foxp3⁺ Tregs that inhibited the proliferation of allogeneic CD4⁺ T cells, in vitro (Moseman et al., 2004). Zhang and colleagues characterized a subpopulation of human pDCs (CD2^hi CD5⁺ CD81⁺), with an equivalent population in mice, which has an enhanced capacity to induce the expansion of CD4⁺ T cells, in absence of stimulus, leading to the differentiation of Tregs from naïve T cells (Zhang et al., 2017).

In mouse, pDCs activated with IL-3 and CD40-L, or with thymic stromal lymphopoietin (TSLP)

In mouse, pDCs activated with IL-3 and CD40-L, or with thymic stromal lymphopoietin (TSLP)