COMPOSICIÓN FORMULA RANGO

LECs are actively involved in immune regulation and inflammation. These interactions can occur within the lymphatic vessels as antigen and immune cells are transported from the periphery to nearby LNs where the dermal immune response is initiated, or when immune cells exit lymphatic sinuses in the LN. LECs are grouped in the category of lymph node stromal cells (LNSCs) which broadly are, non-haematopoietic cells that regulate immunity and self-tolerance (Malhotra et al. 2012), constituting ~1% of LN cellularity. These include: LECs (gp38+ _CD31+_{), fibroblastic reticular cells (FRCs; gp38}+ _CD31-_{), BECs (gp38}+ _CD31+₎

and double-negative cells (DNCs; gp38- _CD31-_{). Transcriptomic analyses of mouse LNSC}

subsets identified that DNCs closely resemble FRCs in terms of global gene expression and cytokine production. Whereas, LECs and BECs have a closer developmental relationship and differ slightly in terms of expression of transcripts. LECs produce IL-7 transcripts whereas BECs do not (Malhotra et al. 2012). In addition, IL-7 was shown to be required for remodelling and homeostasis of the LN microenvironment after viral infection and transplantation (Onder et al. 2012). FRCs, BECs and LECs express TLR-4 and actively respond to the onset of inflammation by upregulation of IFN- and/or TLR-4-inducble genes including chemokine (C-X-C motif) ligand 9 (CXCL9) (Malhotra et al. 2012).

LECs express the chemokine (C-C motif) ligand (CCL21) that attracts and guides the interactions of CCR7-positive T, B and DCs to LNs via the afferent lymphatics (Förster et al. 2008). LN-LECs express different levels of CCL21 forming chemokine gradients that facilitate directional migration into the LNs through an atypical chemokine receptor, CCRL1 (Ulvmar et al. 2014). Pro-inflammatory cytokines such as TNF- activate human dermal LECs (HDLECs) and induce vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and E-selectin, facilitating adherence and transmigration of DCs (Johnson et al. 2006). Furthermore, chemokines secreted by activated HDLECs are significantly increased, including monocyte chemoattractant protein 1 (MCP-1 or CCL2), regulated on activation, normal T cell expressed and secreted (RANTES or CCL5) and macrophage inflammatory protein-3 (MIP-3 or CCL20), compared to resting HDLECs. Furthermore, ICAM-1 expression on inflamed HDLECs can directly regulate DCs via CD11b to suppress the co-stimulatory marker CD86, preventing effective T cell activation (Podgrabinska et al. 2009). Nitric oxide synthase 2 (NOS2 or iNOS) is a short-lived metabolic product that was demonstrated to regulate CD4+ _{and CD8}+ _{T cell proliferation}

(Lukacs-Kornek et al. 2011). The combination of IFN- and TNF- secreted by activated T cells elevated the levels of NOS2 and the immune checkpoint molecule indoleamine 2,3- dioxygenase (IDO) in primary LECs. Co-culture of wild-type LECs and IFN--deficient splenocytes restored T cell proliferation suggesting IFN- is essential for NOS2 production.

29 In addition, co-culture of NOS2-deficient LECs with wild-type splenocytes ablated the immunosuppressive effect.

LECs have been demonstrated to induce peripheral tolerance of CD8+ _{T cells along with}

other lymph node stromal cells (Nichols et al. 2007; Cohen et al. 2010; Lund et al. 2012; Tewalt et al. 2012; Hirosue et al. 2014). VEGF-C-activated LECs in B16 melanoma and LECs under steady-state conditions can scavenge and cross-present exogenous antigen through MHC class I, leading to dysfunctional activation of antigen-specific CD8+ _{T cells}

(Lund et al. 2012; Hirosue et al. 2014). LN-LECs express endogenous peripheral tissue antigens (PTAs) that are independent of autoimmune regulator (AIRE) including tyrosinase (Nichols et al. 2007; Cohen et al. 2010). Direct presentation to tyrosinase-specific CD8+ _T

cells led to their deletion (Nichols et al. 2007; Cohen et al. 2010). It is suggested that viral antigens are “archived” in LECs following viral infection for at least three weeks but are not presented by LECs (Tamburini et al. 2014). These antigens are transferred to DCs for cross- presentation to T cells in order to maintain memory CD8+ _{T cell responses when the}

lymphatic endothelium contracts following lymphoangiogenesis.

Notably, LEC express PD-L1 and induce peripheral deletion of CD8+ _{T cells mediated via}

PD-1/PD-L1 pathway (Fletcher et al. 2010; Cohen et al. 2014; Tewalt et al. 2012; Hirosue et al. 2014; Rouhani et al. 2015). Murine LECs from the LN can express PD-L1 after polyI:C stimulation (Fletcher et al. 2010). PD-L1 is expressed higher in LECs of the LN than peripheral tissues such as the diaphragm, colon or liver (Cohen et al. 2014; Michonneau et al. 2016). Radioresistant LECs could induce deletional tolerance to tyrosinase-specific CD8+ _T

cells through a lack of 4-1BBL (CD137) co-stimulatory molecule expression, which resulted in the upregulation of PD-1 expression (Tewalt et al. 2012). As a consequence, PD-1/PD-L1 engagement blocked upregulation of IL-2R on CD8 T cells, necessary for survival. Blockade of PD-1 rescued CD8+ T cells from deletion. Furthermore, LECs can present endogenous -

galactosidase (-gal) antigen via MHC class I to -gal-specific CD8+ _{T cells and induce}

deletion through the PD-1/PD-L1 pathway (Rouhani et al. 2015).

LECs may act as non-professional antigen-presenting cells, LECs express MHC class II after activation and in some cases carry the capacity to acquire peptide-antigen for cross- presentation (Malhotra et al. 2012; Nörder et al. 2012; Baptista et al. 2014; Dubrot et al. 2014; Rouhani et al. 2015). LPS, ovalbumin and IFN- stimulation of human or murine LECs have been shown to induce MHC class II expression (Malhotra et al. 2012; Nörder et al. 2012). LECs were shown to present self-antigen to both OVA-specific CD8+ _{OT-I and}

CD4+ _{OT-II T cells in vitro which increased CD25 expression (Baptista et al. 2014). In}

30 class II complexes to LECs under steady state conditions in vitro in mainly a cell-cell contact-dependent manner to promote CD4+ _{T cell apoptosis (Dubrot et al. 2014). Additional}

evidence have suggested that LEC indirectly induce CD4+ _{T cell anergy by transfer of PTAs}

to DCs (Rouhani et al. 2015). Moreover, LN-LECs were shown to express MHC class II molecules in vivo either through endogenous expression or from acquisition. LEC MHC class II was demonstrated to directly induce tolerance of -gal-specific CD8+ _{T cells via the}

lymphocyte activation gene 3 (LAG-3)/MHC class II immune checkpoint pathway. LECs were unable to directly present -gal to MHC class II because they lack HLA-DM, which is required for loading of peptide to MHC class II (Rouhani et al. 2015). The mechanism of antigen transfer from LEC to DCs requires further clarification. The transfer of antigens from LECs to DCs could potentially pass through exosomes, gap junctions or DC phagocytosis of excess LEC generated by lymphangiogenesis or undergoing apoptosis (Randolph et al. 2017).

Inflammatory activation of the lymphatic system induces lymphatic remodelling in both peripheral tissues and in LNs (Kim et al. 2014). The main pathways that regulate inflammation-induced lymphangiogenesis include VEGF-C/VEGFR3 and VEGF- A/VEGFR2 signalling (Tammela & Alitalo 2010). Lymphangiogenesis is induced by the upregulation of VEGF-A, -C and -D from macrophages during acute inflammation of the skin or chronic infection of the airway, to facilitate antigen clearance and prevention of lymphedema (Baluk et al. 2005; Kataru et al. 2009). Overexpression of VEGF-C was demonstrated to restore lymphatic vessels during chronic skin inflammation (Huggenberger et al. 2010). In addition, treatment of inflamed skin with VEGF-C initiated LECs to generate anti-inflammatory prostaglandin synthase, leading to increased IL-10 on DCs and suppression of DC maturation (Christiansen et al. 2016). Production of VEGF can be enhanced by B cells in order to facilitate the growth of LN lymphatic vasculature and upregulate the trafficking of DCs to the LN (Angeli et al. 2006). In contrast, T cell production of IFN- can suppress the sprouting of LN lymphatic vasculature in vivo and suppress the expression of key LEC lineage factors PROX1, LYVE-1 and podoplanin in

vitro in a mechanism that is JAK/STAT-dependent (Kataru et al. 2011). IFN-γ knockout

mice express a higher baseline of lymphatic vasculature in the LN. During acute skin inflammation, the levels of PROX1, VEGFR3 and LYVE-1 are suppressed (Vigl et al. 2011; Huggenberger et al. 2011). In human dermal LECs, TNF- stimulation or TGF-β can lead to reduced PROX1 and LYVE-1 expression (Johnson et al. 2007; Oka et al. 2008). In contrast, studies in a murine peritonitis model have indicated that NF-κB induction of PROX1 and VEGFR3, increased the sensitivity of pre-existing lymphatic vessels to leukocytes expressing VEGF-C and VEGF-D (Flister et al. 2010). Moreover, IL-3 can upregulate the

31 expression of PROX1 and podoplanin, which indicates that IL-3 is important for the maintenance of LEC phenotype in vitro (Gröger et al. 2004).