b. Role of self-antigen presentation by LECs in autoimmunity and peripheral T cell tolerance

As mentioned in the general introduction, LECs can modulate T cell responses indirectly, by impacting different mechanisms, as well as directly, by presenting exogenously-acquired antigens to CD8⁺ and CD4⁺ T cells, leading to their dysfunction. For instance, we previously showed that they can acquire peptide/MHC-II complexes from DCs, subsequently inducing cell death and anergy of CD4⁺ T cells (Fig. 18) (Dubrot et al., 2014).

In this section, we will describe how LECs also affect T cell responses by presenting endogenously-express self-antigens, and how this phenomenon is involved in peripheral T cell tolerance and autoimmunity. We have discussed our current knowledge on this topic in a review [Appendix 1 (Humbert et al., 2016)].

IV.1.b.i. Endogenous-expression of peripheral tissue-restricted antigens by LECs The discovery of mTEC ability to ectopically-express peripheral tissue-restricted antigens (PTAs) and present them to T cells was the first example of endogenously-expressed self-antigens presentation to T cells by non-hematopoietic cells (Derbinski et al., 2001; Kyewski et al., 2000).

Intestinal fatty acid-binding protein (iFABP)-tOVA (truncated ovalbumin) and glial fibrillary acidic protein (GFAP)-HA (hemagglutinin) are transgenic mouse models in which tOVA or HA are expressed as self-antigens in mature intestinal epithelial cells (IECs) or enteric glial cells (EGCs), respectively. In those mice, the expression of EGC-associated HA or IEC-associated tOVA proteins have been unexpectedly observed in CD45-negative stromal cells, in addition to EGCs or IECs, and was not restricted to draining (mesenteric) LNs but was found in all LNs (Lee et al., 2007; Magnusson et al., 2008). LNSCs (CD45-negative) had the ability to process endogenously-expressed HA or tOVA into antigenic peptides, and to subsequently present them in SLOs to CD8⁺ T cells. This function of LNSCs can therefore be considered as peripheral counterpart of mTECs in the thymus, i.e. presenting endogenously-expressed PTAs to SP thymocytes during the process of negative selection (Collier et al., 2008; Hirosue and Dubrot, 2015; Lee et al., 2007; Magnusson et al., 2008). In addition, the expression of PTAs and their

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direct presentation to CD8⁺ T cells by LNSCs have been described in non-transgenic mouse models. It was observed that each subtype possesses a distinct but partially redundant PTA expression pattern. Indeed, some PTAs are expressed exclusively by one LNSC subtypes, while other PTAs are expressed in a redundant manner (Cohen et al., 2010; Fletcher et al., 2010) (Fig.

18). For example, LECs are the only LNSC subset that ectopically expresses tyrosinase (Tyr), an antigen whose expression is normally restricted to melanocytes (Cohen et al., 2010; Fletcher et al., 2010; Nichols et al., 2007).

The fact that some PTAs are expressed specifically by one LNSC subset suggests a non-redundant role for the different LNSC subsets regarding the tolerization of a variety of self-specific T cells. Moreover, PTA expression by LECs is compartmentalized subanatomically;

PTAs are only highly expressed in LN medullary sinus LECs (Cohen et al., 2014).

In mTECs, the expression of the vast majority of PTAs is regulated by the transcription factor Aire (Anderson et al., 2002; Derbinski et al., 2005). In the LNs, extrathymic Aire-expressing cells (eTACs), a rare bone-marrow-derived population, have been described to express Aire and named after it. These cells express various PTAs in an Aire-dependent manner (which might therefore be redundant with PTAs expressed by mTECs in the thymus) and present them directly through MHC-I and MHC-II molecules, leading to CD4⁺ T cell anergy and CD8⁺ T cell deletion, respectively (Fig. 18) (Gardner et al., 2008; Gardner et al., 2013). On the other hand, PTAs that are expressed by LNSCs, cells of non-hematopoietic origin, do not depend on Aire but on other transcription factors (Cohen et al., 2010). For example, the regulation of pancreatic polypeptide (Ppy) expression, a pancreatic self-antigen, by LECs in pancreatic LNs is dependent on deformed epidermal autoregulatory factor 1 (DEAF-1), which belongs to the SAND (named after Sp100, AIRE-1, NucP41/75, DEAF-1) gene family, together with Aire (Gibson et al., 1998; Yip et al., 2009). Of note, Deaf1 variant isoforms found in human and in mice display an impaired expression of Ppy and have been linked with autoimmune type I diabetes; decreased DEAF1 function and subsequent loss of eukaryotic translation initiation factor 4 gamma 3 (Eif4g3) expression indeed affects PTA expression (Yip et al., 2013; Yip and Fathman, 2014; Yip et al., 2009). The low overlapping between PTA expression in LNSCs and mTECs may be explained by the fact that Aire is not expressed by LNSCs (Metzger and Anderson, 2011). This, therefore, suggests a complementary contribution of mTECs and LNSCs in inducing and maintaining T cell tolerance. Future work is needed to characterize other transcription factors, commonly or exclusively expressed by the different LNSC subtypes, and promoting a non-redundant expression of PTAs in these cells.

125 Figure 18. Role of antigen-presentation by lymph node stromal cells in peripheral T cell tolerance.

Following negative selection in the thymus, SP thymocytes that successfully passed the selection process, along with thymus-derived regulatory T cells (tTregs), exit from the thymus and reach the periphery. Some self-reactive T cells manage to escape the mechanism of central tolerance and also enter the periphery.

Hence, self antigen-specific T cell tolerance also needs to be maintained in the periphery. Exogenous antigens are acquired from peripheral tissues (yellow) by conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) that subsequently migrate to the LNs where they present self-antigenic peptides to autoreactive T cells.

The antigens expressed by lymphatic endothelial cells (LECs) can also be acquired by cDCs. LECs, blood endothelial cells (BECs) and fibroblastic reticular cells (FRCs) present endogenously-expressed peripheral tissue-restricted antigens (PTAs) (pink) as well as antigenic peptide/major histocompatibility complex (MHC) complexes acquired from cDCs. Therefore, LNSCs contribute to peripheral T cell tolerance through various mechanisms. Extrathymic Aire-expressing cells (eTACs) also present endogenously-expressed PTAs to T cells.

Antigen transfers and cell migration are depicted in dashed and dotted arrows, respectively.

The outcomes regarding antigen presentation by cDCs, pDCs, LNSCs and eTACs on CD4⁺ and CD8⁺ T cell responses are illustrated in the figure. References regarding the contributions of LNSCs to T cell responses are depicted: 1. (Fletcher et al., 2010); 2. (Cohen et al., 2010); 3. (Baptista et al., 2014); 4.

(Gardner et al., 2008); 5. (Gardner et al., 2013); 6. (Rouhani et al., 2015); 7. (Dubrot et al., 2014).

Ags, Antigens; exo Ags, exogenous antigens; PTAs, peripheral tissue-restricted antigens; pTreg, peripherally-induced Treg; thym. cDC, thymus-resident cDC; tTregs, thymus-derived regulatory T cells.

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

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IV.1.b.ii. Impact of LEC presentation of endogenously-expressed PTAs on T cell responses

Not only LNSCs endogenously express PTAs, which can be acquired by DCs from LECs (Rouhani et al., 2015), but LNSCs have the ability to process these antigens and directly present the PTA-derived peptides to CD8⁺ T cells, in the context of MHC-I molecules, leading to their elimination by clonal deletion and to tolerance induction (Fig. 18 and 19) (Lee et al., 2007;

Magnusson et al., 2008; Nichols et al., 2007).

In the iFABP-tOVA and GFAP-HA transgenic models mentioned previously, the lack of presentation of tOVA or HA to tOVA- or HA-specific CD8⁺ T cells by enteric stromal cells was linked with enteric autoimmunity (Lee et al., 2007; Magnusson et al., 2008). LECs, among the other LNSC subtypes, are involved in CD8⁺ T cell clonal deletion and are necessary and sufficient for peripheral tolerance to certain self-antigens, such as Tyr, which has been associated with autoimmune vitiligo, showing a major role for LECs in peripheral tolerance maintenance (Cohen et al., 2010; Fletcher et al., 2010; Fletcher et al., 2011; Yip et al., 2009).

Figure 19. Antigen presentation-dependent role of lymph node stromal cells in peripheral T cell responses.

Fibroblastic reticular cells (FRCs), lymphatic endothelial cells (LECs) and blood endothelial cells (BECs) express peripheral tissue-restricted antigens (PTAs). It has been demonstrated that FRCs and LECs are able to directly present those antigens to CD8⁺ T cells, inducing their deletion. Whether LNSCs can directly present endogenously-expressed PTAs to CD4⁺ T cells and the subsequent outcome on CD4⁺ T cell responses remain a matter of debate.

Adapted from Turley*, Fletcher* and Elpek*, Nat Rev Immunol, 2010 (Turley et al., 2010).

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The capability of LNSCs to present endogenously-expressed PTAs in a direct manner to CD4⁺ T cells, in the context of MHC-II molecules, and whether it is inducing CD4⁺ T cell dysfunction and/or Treg differentiation/maintenance remains under debate (Fig. 18 and 19).

We previously demonstrated that CIITA pIV regulates MHC-II molecules endogenous expression in LECs, BECs and FRCs (Dubrot et al., 2014). A study has shown that the adoptive transfer of the transgenic 6.5 CD4⁺ T cells with a HA-specific TCR in GFAP-HA mice, in which EGCs express HA as an autoantigen, did not dampen enteric autoimmunity (Magnusson et al., 2008). The absence of direct HA peptide presentation by LNSCs to HA-specific CD4⁺ T cells in this model did nonetheless not rule out a potential MHC-II molecule upregulation by LNSCs upon inflammation, and subsequent presentation of the antigenic peptide (Magnusson et al., 2008). Indeed, as mentioned previously, few studies have suggested that the expression of MHC-II-molecules at the surface of LNSCs is upregulated under pro-inflammatory conditions (Dubrot et al., 2014; Malhotra et al., 2012). On the other hand, using models in which endogenously-expressed PTAs were β-galactosidase (β-gal), membrane-bound HA or I-Eα, Engelhard and colleagues concluded that LECs were not able to present these PTAs to CD4⁺ T cells (Rouhani et al., 2015). This outcome was not due to antigen localization, but rather due to the absence of H2-M expression in LECs that could hamper peptide loading onto MHC-II molecules, H2-M being the chaperone that allows antigenic peptide loading onto MHC-II molecules.

Nonetheless, this study was undertaken in steady state conditions, while the expression of CIITA pIV by LECs, BECs and FRCs is IFN-γ-inducible (Reith et al., 2005). Thus, IFN-γ might be required to induce the upregulation of H2-M, as it is the case for the expression of MHC-II expression. Indeed, H2-M and MHC-II molecule expression are co-regulated by CIITA. In addition, Baptista et al. found H2-M mRNA transcripts in LECs, among other MHC-II-related molecules (Baptista et al., 2014). They also observed an unexpected expression of OVA in LECs in K14mOVA transgenic mice, in which OVA is expressed under the control of the keratin 14 promoter. Moreover, OVA⁺ LECs had the ability to present OVA peptides, in the context of MHC-II molecules, to OVA-specific OT-II cells in vitro, which was crucial for the maintenance of Foxp3⁺ OT-II Tregs (Baptista et al., 2014). Baptista and colleagues, by performing LN transplantation experiments, further suggested that endogenously-expressed self-antigen presentation by LNSCs could contribute to Foxp3⁺ CD4⁺ Treg maintenance, in vivo (Fig. 18) (Baptista et al., 2014).

Moreover, another team observed that endogenously-expressed PTA presentation by LNSCs led to CD4⁺ and CD8⁺ T cell hyporesponsiveness/anergy. They used lentiviral vectors that allow the selective transduction of MHC-II-expressing cells of non-hematopoietic origin with MHC-II-

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and MHC-I-restricted HY male-derived epitopes in female mice (Cire et al., 2016). Effector CD4⁺ T cell conversion into CD25⁺ Foxp3⁺ pTregs was found increased in Marylin mice, in which CD4⁺ T cells express a HY-specific transgenic TCR (Cire et al., 2016). However, whether these observations were due to the direct presentation of HY endogenously-expressed by gp38⁺ LNSCs, i.e. LECs and FRCs, to CD4⁺ T cells remains to be investigated. The possibility that (non-DC) hematopoietic cells might contribute to HY antigen presentation cannot be ruled out, because of unwanted transduction and subsequent direct antigen presentation or due to antigen transfer from (non-DC) hematopoietic cells to stromal cells (Cire et al., 2016; Dubrot et al., 2014). It also cannot be excluded that transduced stromal cells could transfer MHC-II-restricted HY peptide to DCs, which could subsequently present the antigen to CD4⁺ T cells (Rouhani et al., 2015). Although the direct antigen presentation by gp38⁺ LNSCs was not fully demonstrated and the contribution of LECs and FRCs could not be distinguished, this study is in agreement with the results found by Baptista et al. (Baptista et al., 2014).

Finally, using K14tgpIV^KO mice, in which MHC-II expression is abrogated in LNSCs, thanks to CIITA pIV deletion, our group very recently showed that elderly K14tgpIV^KO mice present signs of spontaneous systemic autoimmunity (Dubrot et al., in press). This was associated with an impaired Treg compartment, with decreased Treg frequencies and ability to suppress T cell responses, leading to enhanced activation of effector CD8⁺ and CD4⁺ T cell in SLOs. It induced a subsequent increase in T cell infiltration in peripheral tissues, compared with K14tg age-matched controls, along with enhanced production of auto-antibodies. In addition, the adoptive transfer of T cells from LNs of elderly K14tgpIV^KO mice in Rag2^-/- mice, which lack T and B cells, recapitulated the autoimmune phenotype. Moreover, in LNs, the proliferation of Tregs, observed by microscopy, in contact with LECs from WT mice was increased in elderly mice or in IFN-γ-administered mice, due to an enhanced MHC-II expression by LECs in these mice. This effect was abrogated in K14tgpIV^KO mice or in Prox-1Cre^ERT2MHCII^fl/fl mice, a model that will be described later in this chapter, in which MHC-II is selectively abrogated in LECs. These results show the importance of endogenous MHC-II expression by LNSCs in T cell tolerance, as it provides a brake in spontaneous autoimmunity observed in elderly mice in absence of LNSC MHC-II expression, and are in agreement with the studies mentioned previously (Baptista et al., 2014; Cire et al., 2016). Of note, this analysis supports a particular role for LECs in maintaining the Treg compartment through MHC-II-restricted antigen presentation.

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IV.1.b.iii. Molecular pathways implicated in peripheral T cell tolerance mediated by