CONCLUDING REMARKS

Modulation of peripheral T cell responses by unconventional APCs

Besides professional APCs, several cell types from the hematopoietic and non-hematopoietic compartments have the ability to cross-present antigens via MHC-I to CD8⁺ T cells and/or to present antigens through MHC-II to CD4⁺ T cells, as unconventional APCs (Kambayashi and Laufer, 2014). Distinct immunological contexts and unconventional APC types lead to various outcomes on T cell responses.

The aim of this thesis was to characterize the role of pDCs and LECs in the modulation of peripheral T cell responses, as unconventional APCs, with a particular focus on MHC-II-restricted antigen presentation.

Role of pDCs in anti-tumor immunity

Plasmacytoid DCs are professional IFN-I-producing cells, which also have the ability to (cross)-present antigens via MHC-I and MHC-II molecules (Alculumbre et al., 2018a; Reizis et al., 2011a;

Reizis et al., 2011b; Swiecki and Colonna, 2015). These cells are extremely plastic and, depending on the immunological context, their MHCII-mediated antigen-presenting functions can be either tolerogenic or immunogenic (Guery and Hugues, 2013).

TA-pDCs harbor a tolerogenic phenotype, due to their immersion in the TME. Nonetheless, the potential of “naïve” pDCs to mount potent tumor responses can be harnessed for anti-cancer therapies (Aspord et al., 2012; Liu et al., 2008a; Loschko et al., 2011b; Tel et al., 2013a; Tel et al., 2012b). In tumor-bearing mice, pDCs in the distal LNs can be activated by a contralateral vaccination with the TLR9 ligand CpG-B along with a model MHC-II-restricted tumor antigenic peptide (Guery et al., 2014). This induces an enhancement of their MHC-II-mediated antigen-presenting functions, leading to anti-tumor immunity through the priming of Th17 cells (Guery et al., 2014).

In the first part of this thesis, we asked the question whether the tolerogenic phenotype of TA-pDCs could be reversed after i.t. administration of CpG-B±MHC-II-restricted peptide.

This local treatment led to tumor growth inhibition. However, TA-pDCs did not undergo a tolerogenic-to-immunogenic reprogramming. These cells were refractory to the treatment and did not contribute to its efficacy. The tumor-derived factors responsible for the induction of a tolerogenic pDC state that is refractory to i.t. CpG-B remain to be elucidated. On the contrary, the working model we propose involves neutrophil, cDC and T cell cooperation, subsequently leading to tumor growth control.

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Role of LECs in autoimmunity

LECs, a LNSC subtype, were long thought to function as simple scaffolds (Turley et al., 2010).

However, new roles have emerged for these cells in the regulation of T cell responses, including implication in antigen presentation, as unconventional APCs (Humbert et al., 2016; Turley et al., 2010).

Whether LNSCs are implicated in the presentation of MHC-II-restricted PTAs and modulate peripheral CD4⁺ T cell responses is not fully understood (Baptista et al., 2014; Cire et al., 2016).

We recently showed that elderly naïve mice, in which MHC-II expression was abrogated in LNSCs, presented signs of spontaneous autoimmunity (Dubrot et al., in press).

The aim of the second part of this thesis was to determine whether LECs contribute to the modulation of autoreactive CD4⁺ T cell responses, as MHC-II-restricted APCs, in EAE.

The selective genetic ablation of MHC-II in LECs exacerbated disease severity, whereas the genetic enforcement of MOG_35-55 presentation via MHC-II by LECs led to delayed onset and dampened EAE severity. MOG_35-55 presentation in LECs induced an important decrease in the spinal cord CD4⁺ T cell infiltrate and a modulation of these cells towards a tolerogenic phenotype. CD4⁺ T cell numbers and phenotype in the LNs draining the site of EAE immunization were unaffected, suggesting that this pathway may inhibit effector T cell responses, rather than impact the priming of naïve T cells in LNs.

Altogether, our findings highlight a role for LECs as unconventional tolerogenic APCs in EAE.

Remarks

Immunological context

Our studies highlight the importance of the immunological context for the function of unconventional APCs. Plasmacytoid DCs are extremely plastic cells and their antigen-presenting functions can be immunogenic or tolerogenic (Guery and Hugues, 2013). However, our results suggest that they may be refractory to reprogramming when they are deeply imprinted in certain environments, such as the TME. Furthermore, LECs function as tolerogenic APCs, but they upregulate MHC-II expression only in the presence of IFN-γ (Dubrot et al., 2014; Reith et al., 2005) (Dubrot et al., in press).

In the steady state, neither pDCs nor LECs function as MHC-II-restricted APCs, as both of these cell types need a stimulus to upregulate the expression of MHC-II (Alculumbre et al., 2018a; Dubrot et al., 2014; Reith et al., 2005). This idea might have been overlooked in studies,

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performed in vitro without stimulus or in vivo in the steady-state, concluding that pDCs or LECs cannot function as MHC-II-restricted APCs (Rouhani et al., 2015; See et al., 2017; Villani et al., 2017).

Professional vs unconventional APCs

Our studies shed light on the unique characteristics of unconventional APCs, compared with professional APCs (Kambayashi and Laufer, 2014). In the first part of this thesis, we observed that tolerogenic pDCs cannot be reprogrammed by i.t. administration of CpG-B, while TA-cDCs were activated following this treatment, suggesting differences in their intrinsic features.

LN-LECs, studied in the second part of this thesis, have been shown to harbor specific characteristics as APCs. Indeed, these cells do not upregulate the expression of co-stimulatory molecules upon inflammation, and rather upregulate the expression of PD-L1 and other co-inhibitory molecules (Dubrot et al., 2014; Fletcher et al., 2010; Norder et al., 2012; Tewalt et al., 2012) (Dubrot et al., in press).

Non-hematopoietic cells in immunity

The second part of this thesis shows the importance of the contribution of non-hematopoietic cells to immune responses. LNSCs, including LECs, as well as other cell types in tissues, can affect immune responses in many ways in indirect manners, such as playing structural roles, or in the draining of immune cells and molecules (Card et al., 2014; Humbert et al., 2016). However, increasing evidence suggest that several non-hematopoietic cells also have the ability to present antigens and/or to express or secrete inflammatory molecules (Duraes et al., 2013; Kambayashi and Laufer, 2014; Reynoso and Turley, 2009; Turley et al., 2010). Therefore, the hematopoietic innate and adaptive immune cells are far from being the only players of immune responses.

Mouse vs human

Caveats in our studies for potential applications in human involve differences in mouse and human. Indeed, in the first part of this thesis, we used a TLR9 ligand, this TLR being expressed exclusively in pDCs and B cells in human, while being expressed by more cell types in mouse (Jarrossay et al., 2001; Takeuchi and Akira, 2010) (Lindau et al., 2013; Puttur et al., 2016;

Schneberger et al., 2013; Xu et al., 2015). In addition, in the second part of this thesis, we investiagted the MHC-II-restricted APC functions of LECs, as previously published work in mouse demonstrated the APC functions of LECs (Cohen et al., 2010; Fletcher et al., 2010;

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Humbert et al., 2016; Turley et al., 2010). However, no evidence, to our knowledge, has demonstrated the ability of human LECs to present MHC-I- or MHC-II-restricted antigens.

Anti-tumor treatments and autoimmunity

A tightly regulated balance between immune activation and tolerance is required in order for the immune system to mount efficient immune responses, for instance required to fight cancers, without inducing adverse effects, such as autoimmunity.

Malignancy treatments, which include immunotherapies, are most of the time administered systemically. Therefore, they are often linked with damaging effects, including autoimmunity.

The first part of this thesis highlights, in accordance with other studies in mouse and human, the potential of local immunotherapy administration as a way that may solve many aspects that limit these treatments (Fransen et al., 2013; Marabelle et al., 2014).

Conclusion

Overall, the two parts of this thesis highlight important contributions of unconventional APCs in shaping peripheral T cell responses. Therefore, these pathways should be considered in the development of therapies aiming at modulating immune responses in disease development.

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VI. APPENDICES

VI.1. Appendix 1