c.v. LECs impact peripheral T cell responses through mechanisms independent of antigen presentation

LIST OF FIGURES

Figure 5. Negative selection and central T cell tolerance

I.6. c.v. LECs impact peripheral T cell responses through mechanisms independent of antigen presentation

T cell immunity comprises the generation of pathogen-specific effector responses in order to protect against a vast range of invaders, without causing any undesired damage to self-tissue.

Naïve T cells scan for their cognate antigen constantly. Nonetheless, this challenging task only takes place into well-organized SLOs, such as LNs, Peyer’s patches (PPs) and the spleen, due to the very low frequency of T cells that are specific for a given peptide/MHC complex (Moon et al., 2007; Obar et al., 2008). Blood-borne and tissue-derived antigens are both contained in SLOs, which facilitates the encounter of naïve T cells with their cognate antigens, and subsequently helps T cell activation and differentiation. The following paragraph describes the different

pathways by which T cell are impacted by LECs, a topic that has been reviewed by Card et al.

(Card et al., 2014).

Delivery of antigens and DC migration to the LNs

As mentioned previously, LNs are linked to lymphatics that drain peripheral fluids derived from tissues. Therefore, LECs facilitate the passive entry of tissue-derived antigens by connecting draining LNs and tissues, those antigens can subsequently be acquired, processed, and presented by LN-resident DCs to T cells, which enter the LNs via HEVs (Roozendaal et al., 2009; Sixt et al., 2005). LN-resident DCs immediately sample soluble antigens, while particles that carry antigens, including micro-vesicles, apoptotic bodies and exosomes, and that have not been uptaken by subcapsular sinus macrophages, flow to the medullary sinuses, where DCs can scan them (Gerner et al., 2015).

In addition, LECs facilitate the migration of tissue-resident DCs into LNs (Russo et al., 2013;

Teijeira et al., 2014). The migration of DCs from tissues to draining LN LVs is a major way for antigen presentation and naïve T cell activation. DCs enter afferent lymphatics independently of integrin-mediated adhesion, via preformed portals (Lammermann et al., 2008; Pflicke and Sixt, 2009). However, the expression of adhesion molecules in LECs can be upregulated upon inflammation, depending on the stimulus, which further favors the access of DCs to LVs (Johnson et al., 2006; Russo et al., 2013; Vigl et al., 2011). Moreover, CC-chemokine ligand (CCL)21 secreted by LECs in afferent lymphatics upon inflammation has been shown to be important, depending on the stimulus, for DC egress from tissue and entry into lymphatics (Russo et al., 2016; Teijeira et al., 2014; Vigl et al., 2011). Moreover, the expression of C-type lectin receptor 2 (CLEC2) by DCs supports their migration towards LNs through lymphatics, thanks to an interaction with its ligand gp38, expressed both by FRCs and LECs (Acton et al., 2012). Finally, it was recently shown that the entry of tissue-resident DCs into lymphatics and transit to the lumen, is dependent on a cell-cell contact enabled by the interaction between hyaluronan and Lyve-1, expressed by DCs and LECs, respectively (Johnson et al., 2017). This mechanism was demonstrated to be crucial for the resolution of inflammation, in a model of myocardial infarction (Vieira et al., 2018).

Regulation of DC functions

Tissue-resident DCs that have captured peripheral antigens migrate via afferent lymphatics into LNs, by a mechanism depending on CC-chemokine receptor (CCR)7. However, not only the lymphatic system supports the migration of DCs from tissues to LNs, but it also induces

functional and phenotypic changes in DCs, due to close interactions between DCs and LECs (Malhotra et al., 2013). Contacts between DCs and tumor necrosis factor (TNF)-α-stimulated LECs induce reduced expression of co-stimulatory molecules by DCs in vitro, which therefore impairs the ability of DCs to induce T cell proliferation (Podgrabinska et al., 2009). In addition, the regulation of DC functions by LECs depends on interactions between intercellular adhesion molecule (ICAM)-1 on LECs and CD11b (Mac-1) on DCs (Podgrabinska et al., 2009). Of note, LECs can inhibit the function of lipopolysaccharide (LPS)-activated DCs, which further suggests a role for LECs in the regulation of the resolution phase of inflammation. A study recently showed that LECs act as reservoirs of PTAs, which are subsequently uptaken by DCs and presented to T cell, inducing their anergy, thus contributing to peripheral T cell tolerance (Rouhani et al., 2015).

T cell Homeostasis

T cell migration in the LNs is conducted by FRC-secreted CCL21 and CCL19 (Luther et al., 2002). Nonetheless, the maintenance of naïve and memory T cells in the SLOs depends to a high degree on IL-7. LECs are an important source of IL-7 in vivo, along with FRCs, and therefore involved in the regulation of T cell homeostasis and their entry into SLOs (Link et al., 2007).

Using IL-7-GFP (green fluorescent protein) knock-in mice, it was shown that IL-7 is highly expressed in LECs from both LNs and tissues, while it is moderately expressed in FRCs from LNs (Hara et al., 2012; Miller et al., 2013). In addition, LECs have been demonstrated to be an important source of IL-7, both in murine and human LNs (Onder et al., 2012). Moreover, in addition to the production of IL-7, LECs also express the IL-7Rα of the IL-7 receptor and CD132, which suggests a potential role for IL-7 as a lymphatic drainage mediator (autocrine). For instance, LECs stimulated with IL-7 in vitro induce lymphangiogenesis in mice cornea, while lymphatic drainage in IL-7Rα^-/- mice is compromised (Iolyeva et al., 2013). In addition, the upregulation of IL-7 by LECs and FRCs is crucial for the reconstruction of LNs and their remodeling, after avascular transplantation or viral infection (Onder et al., 2012). These studies suggest that IL-7 secretion in the LNs after resolution of inflammation may be implicated in the homeostasis of memory T cells. In this regard, IL-7 supports the development, proliferation and survival of memory CD8⁺ T cells (Schluns et al., 2000; Schluns and Lefrancois, 2003).

Egress of T cells from the LNs

The specificities of the lymphatic vasculature that allow T cell trafficking from tissue to tumor-draining lymph nodes (TdLNs), through afferent lymphatics, as well as through efferent LVs to

return to the blood circulation from the LNs have been reviewed by the group of C. Halin (Hunter et al., 2016; Schineis et al., 2018).

T cell egress from the LNs depends on sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) expression. By using mice that selectively lack S1P in LECs, Cyster and colleagues have demonstrated that LECs are an important in vivo source of S1P in the LNs, which allows the egress of T cells from the LNs and PPs (Pham et al., 2010). The expression of S1PR1 is downregulated in lymphocytes circulating in the blood and upregulated in the LNs. Egress from the LNs is promoted by interactions between LECs that produce S1P and T cells that express S1P1R, which overcome CCR7-mediated retention signals (Grigorova et al., 2009; Pham et al., 2008).

Despite the fact that LECs express low levels of S1P in the steady-state, S1P secretion is upregulated in LECs in the medullary sinus upon inflammation mediated by PAMPs or DAMPs.

This suggests that LECs which highly express S1P can support the egress of T cells from the LNs in pathogenic contexts. On the contrary, in situation of sterile - non-infectious - inflammation, LECs that produce moderate levels of S1P might rather decrease T cell effector functions, by promoting the retention of T cells in the LNs

Migration of T cells in lymphatics

In addition to their function of tumor cell transporter, emerging evidence is in favor of important roles played by TA-lymphatics in T cell migration. For instance, the modulation of TA-lymphatic expansion can affect both metastatic and primary tumor progression. In the case of solid tumors, lymph flow from tumors is intense, which drives elevated interstitial flow in the stroma of tumors and enhances the lymphatic drainage from the tumor to the TdLNs (Swartz, 2014; Swartz and Lund, 2012). Therefore, it is possible that, in combination with a suppressive cytokine environment, enhanced drainage of tumor antigens could facilitate tumor antigen-specific T cell dysfunction, such as apoptosis and anergy. Moreover, the lymph supports cells that migrate from tissues, particularly CCR7⁺ DCs, which is critical for the initiation of anti-tumor immune responses (Roberts et al., 2016). T cell infiltration in the tumor is a major step in anti-tumor immunity. The infiltration of CTLs is associated with a good prognosis, while Treg or naïve T cell infiltration correlates with a poor clinical outcome (Fridman et al., 2012; Galon et al., 2006). In this regard, CCL21 expression in tumors facilitates immune escape and tumor progression, which may partly be explained by an increase in the recruitment of naïve T cells (Shields et al., 2010).

Although TCR-transgenic tumor-infiltrating naïve T cells could be activated in situ, it is unlikely that it would induce efficient effector T cell differentiation, due to the suppressive TME

(Thompson et al., 2010). Likewise, whether CCL21-secreting LECs participate in this effect and how they contribute to the TME tolerogenic properties remain to be determined. We have previously shown that VEGF-C, a lymphangiogenic growth factor, which is secreted in the tumor promotes tolerance to melanoma in mice, by inducing the deletion of tumor antigen-specific CD8⁺ T cells (Hirosue et al., 2014; Lund et al., 2012). These results are in favor of a new role for lymphatics played in tumor development, suggesting that tumor lymphatic endothelium could be a target for immunomodulation. A study supporting these hypotheses demonstrated that TdLN-FRCs, after tumor-derived factor exposure, are able to adapt in multiple ways to harbor characteristics that are associated with immunosuppression, including reduced IL-7, CCL19 and CCL21 secretion (Riedel et al., 2016). Whether it is also the case for LECs in the TdLNs is still an open question. On the other hand, using K14-VEGFR3-Ig mice, which lack dermal LVs, Swartz’s group showed that the absence of lymphatics within melanoma tumor leads to decrease leukocyte infiltration and enhanced tumor growth, with potential implications for immunotherapies (Lund et al., 2016). In this regard, lymphangiogenesis blockade using anti-VEGFR-3 antibodies inhibited response to immunotherapy, due to a lack of naïve T cell infiltration into the tumor (Fankhauser et al., 2017). The authors suggested that lymphangiogenesis is important for a favorable response to immunotherapy, by supporting tumor infiltration of naïve T cells, which are subsequently locally activated.

I.6.c.vi. Antigen-presenting abilities of LECs: uptake of exogenous antigens and presentation to T cells

By controlling antigen availability, the lymphatic system constitutes one of the primary immune response checkpoints (Hirosue and Dubrot, 2015; Randolph et al., 2017). Therefore, it is not surprising that LECs display various mechanisms for antigen acquisition and processing, knowing they have early access to any given antigen (Fig. 11). Indeed, recent investigations revealed that antigen trafficking can be observed at various levels, not only the classical concept of LECs as lymph carriers. Complex interactions between DCs and LECs are involved in bidirectional antigen exchanges, which may serve, ultimately, to modulate the immune response magnitude (Fig. 11) (Dubrot et al., 2014; Kedl et al., 2017; Kedl and Tamburini, 2015; Rouhani et al., 2015;

Tamburini et al., 2014). Indeed, LECs have a function of antigen archiving; they are able to capture and archive antigens, which can later be acquired by hematopoietic cells, directly or through LEC apoptosis (Kedl et al., 2017; Kedl and Tamburini, 2015; Tamburini et al., 2014).

DCs also acquire antigens endogenously-expressed by LECs (Rouhani et al., 2015). Conversely, LECs acquire peptide/MHC complexes from DCs (Dubrot et al., 2014).

Acquisition of exogenous antigens

LECs exhibit an active endocytotic capacity (Fruhwurth et al., 2013; Sixt et al., 2005); they can capture exogenous molecules and, depending on their location, process antigens for cross-presentation and cross-priming of antigen-specific CD8⁺T cells (Fig. 11) (Hirosue et al., 2014;

Lund et al., 2012). Of note, LN-LECs loaded with antigens have been shown to be able to cross-prime antigen-specific CD8⁺ T cells, by mechanisms depending on TAP (Hirosue et al., 2014).

Antigen-loaded LECs induce T cell apoptosis, the most probable explanation being the lack of co-stimulatory molecule expression. Indeed, LECs do not express the co-stimulatory molecules CD40, CD80 and CD86 after TLR ligation or in the presence of TNF-α or interferon (IFN)-γ (Dubrot et al., 2014; Tewalt et al., 2012). On the contrary, they upregulate MHC-II and the immune-stimulatory molecules CD48 and herpes virus entry mediator (HVEM) in the presence of these cytokines, as well as the expression of the co-inhibitory molecule PD-L1 (Dubrot et al., 2014; Fletcher et al., 2010; Norder et al., 2012; Tewalt et al., 2012).

In this regard, antigen cross-presentation by LSECs leads to CD8⁺T cell tolerance in the liver, using a mechanism involving the expression of PD-L1 (Diehl et al., 2008; Limmer et al., 2000;

von Oppen et al., 2009). Interestingly, in the absence of inflammation, LSEC-educated T cells that survived had a phenotype of antigen-experienced memory-like cell, in the SLOs (Bottcher et al., 2013). In addition, LSEC-primed memory T cells could be reactivated in an antigen-specific manner in vitro and in vivo, and could contribute to a viral challenge (Bottcher et al., 2013). Studies have reported that LSECs are also able to present MHC-II-restricted antigens to CD4⁺ T cells, leading to different outcomes, including Treg differentiation (Kruse et al., 2009; Wittlich et al., 2017). The antigen-presenting ability of LSECs and its outcome on CD8⁺ and CD4⁺ T cell responses have recently been reviewed by different groups (Lukacs-Kornek, 2016; Mehrfeld et al., 2018; Shetty et al., 2018; Wohlleber and Knolle, 2016).

In the case of LECs, whether they directly participate in anti-viral CD4⁺ T cell responses, by presenting MHC-II-restricted antigens, is still under debate. As described above, LECs act as antigen reservoir upon viral infections (Kedl et al., 2017; Kedl and Tamburini, 2015; Tamburini et al., 2014) (Fig. 11). However, the genetic ablation of MHC-II molecules in LN radioresistant stromal cells resulted in a longer maintenance of antigen-specific CD4⁺ T cells (Abe et al., 2014).

50 Figure 11. Antigen acquisition and presentation by lymphatic endothelial cells.

There are various pathways by which lymphatic endothelial cells (LECs) acquire antigens (Ag) and by which those antigens are loaded onto major histocompatibility complex (MHC) molecules. Complex mechanisms allow the transfer of antigens between dendritic cells (DCs) and LECs, in both directions.

DCs use LECs as antigen reservoirs and uptake those antigens. Conversely, LECs are able to acquire peptide/MHC-II complexes (DC-derived antigen in yellow) at the surface of DCs, in a cell-cell contact-dependent mechanism. Exosomes derived from DCs might also be involved. Peripheral tissue-restricted antigens (PTAs) (pink) that are expressed by LECs can be loaded onto MHC-I molecules. However, the intracellular pathways accounting for PTA degradation remains to be deciphered. In addition, whether PTAs can be incorporated in MHC-II compartments is still debated. LECs are also able to acquire exogenous antigens (lymph-borne and tumor-derived), which can be incorporated in MHC-I pathway, by a mechanism involving transporter associated with antigen processing 1 (TAP-1).

Associated references are depicted: 1. (Tamburini et al., 2014); 2. (Rouhani et al., 2015); 3. (Kedl et al., 2017); 4. (Dubrot et al., 2014); 5. (Hirosue et al., 2014); 6. (Lund et al., 2012).

Adapted from Humbert, Hugues* and Dubrot*, Front Immunol, 2016 [Appendix 1 (Humbert et al., 2016)].

Cellular antigen transfers

As described previously, several cell types, from hematopoietic and non-hematopoietic origins, are able to express MHC-II and to interact with CD4⁺ T cells in the periphery (Duraes et al., 2013; Hirosue and Dubrot, 2015; Kambayashi and Laufer, 2014). LECs are a non-professional APC type expressing MHC-II, under the presence of IFN-γ. For instance, the expression of

MHC-II in LN-LECs has been observed, both at the transcriptional and protein levels (Dubrot et al., 2014; Fletcher et al., 2010; Malhotra et al., 2012). Using transgenic mouse models that lack different CIITA promoters, we have previously shown that the levels of MHC-II molecules on the surface of LECs and other LNSCs, at the steady state, represent a combination of basal activity, which is IFN-γ-inducible, and peptide/MHC-II complexes acquired from DCs (Dubrot et al., 2014). Captured MHC-II molecules were loaded with antigens derived from DCs, licensing LECs to induce anergy and increased antigen-specific CD4⁺ T cell apoptosis (Fig. 11). The absence of measurable efficient T cell responses has for long been a major difficulty that prevented the characterization of the effect of antigen-presentation by LECs on CD4⁺ T cell responses. Similarly to the case of CD8⁺ T cell responses, the lack of costimulatory molecules, including CD80 and CD86, and the constitutive PD-L1 expression by LECs prevent the priming of functional effector CD4⁺ T cells. In this respect, it has been shown in human in vitro that LN-LECs do not possess the ability to induce the proliferation of allogeneic CD4⁺ T cells (naïve or memory), even in the presence of IFN-γ (Norder et al., 2012). As mentioned previously, in the immune system, membrane exchange between cells are not rare (Davis, 2007). Peptide/MHC-I and peptide/MHC-II complexes can be transferred between tumor cells or infected cells and DCs, between mTECs and DCs, and also between DCs (de Heusch et al., 2007; Kyewski et al., 2000; Wakim and Bevan, 2011; Zhang et al., 2008). The transfer of antigens can be a peptide exchange on cell surfaces. Epitopes can directly bind to the cell surface, or to MHC molecules in early endosomes, where MHC-I and MHC-II are receptive to the binding of lymph-borne peptides (Griffin et al., 1997). This is of particular relevance in the context of tolerance to self.

Indeed, recent investigations demonstrated that lymph peptidome in human contains mainly self-peptides, such as ones derived from protein extracellular processing (Clement and Santambrogio, 2013). Exosomes have also been shown to be involved in the transfer of peptide/MHC-II complexes from DCs to LNSCs, and the possibility that they could contribute to alternative antigen-trafficking cannot be excluded (Dubrot et al., 2014) (Fig. 11). However, as mentioned previously, the transfer of antigens between DCs and LECs is bidirectional; in addition to the transfer of antigens captured and archived by LECs, the transfer of LEC endogenously-expressed PTAs to hematopoietic cells has also been described (Kedl et al., 2017; Rouhani et al., 2015;

Tamburini et al., 2014) (Fig. 11). Indeed, LECs endogenously-express PTAs that can be transferred to DCs or directly presented to CD8⁺ T cells, inducing their tolerization, the presentation to and impact on CD4⁺ T cells still being a matter of debate (Humbert et al., 2016;

Rouhani et al., 2015). In the analysis performed by Rouhani and colleagues, neither cytoplasmic nor membrane-bound PTAs were presented by LECs in a direct manner to induce

antigen-52

specific CD4⁺ T cell responses. This has been attributed to the expression of H2-M in LECs, which is lower compared with professional APCs, H2-M being required for the binding of peptides onto the MHC-II groove. However, H2-M expression is upregulated upon inflammation (Dubrot et al., in press¹). Peptides derived from LEC-expressed PTAs are instead loaded onto MHC-II in DCs (Rouhani et al., 2015). Although the mechanisms by which antigen transferred are enabled are still under examination, it has been reported that it does not depend on the recognition of apoptotic cells or the phagocytosis of DCs. Therefore, there is a close relationship and communication between LECs and professional APCs that allows MHC-II presentation.

The role of PTA endogenous expression and direct presentation to T cells by LECs in peripheral tolerance to self will be described in Chapter B (introduction section).

1 Dubrot et al., in press (Life Science Alliance)

Dans le document Modulation of peripheral T cell responses by unconventional antigen-presenting cells: role of plasmacytoid dendritic cells in anti-tumor immunity and lymphatic endothelial cells in autoimmunity (Page 71-80)