Dendritic cells (DCs) are important in mediating negative selection

There are three distinct subsets of thymic DCs. Two DC subsets belong to the conventional DCs (cDCs), i.e. CD8+ cDCs (also known as resident DCs) and signal regulatory protein α (SIRPα)+ _{cDCs (also known as migratory DCs). The third subset is}

plasmacytoid DCs (pDCs) (Klein et al., 2014; Perry and Hsieh, 2016; Wu and Shortman, 2005). The intrathymically developed CD8+ _{cDCs comprise ~50% of all}

thymic DCs, whereas the extra-thymically developed SIRPα+ _{cDCs and plasmacytoid}

DCs that migrate to the thymus account for 20% and 30%, respectively, of all thymic DCs (Oh and Shin, 2015; Wu and Shortman, 2005).

Thymic DCs can acquire antigens from mTECs, from the blood, or from peripheral tissues and home to the thymus (Oh and Shin, 2015). SIRPα+ migratory DCs found in

CMJ perivascular space and around small vessels, have been shown to be able to present blood-borne antigens (Baba et al., 2009). CD8+ _{resident cDCs are found}

predominantly in the thymic medulla (Klein et al., 2014; Lei et al., 2011).Plasmacytoid DCs (and potentially SIRPα+ _{DCs) can transport peripheral (extrathymic) antigens and}

migrate to the thymus to promote tolerance (Bonasio et al., 2006; Hadeiba et al., 2012).

Self-antigens can be presented directly by BM-APCs or mTECs (direct presentation), or indirectly presented via antigen transfer from mTECs to BM-APCs (indirect presentation), to induce tolerance. An early BM chimera experiment showed that superantigen-reactive Vβ17α+ _{T cells were reduced when MHCII I-E expression was}

confined to BM-APCs but not when I-E expression was confined to radioresistant thymic epithelium, suggesting that BM-APCs are important in clonal deletion (Marrack et al., 1988). Later studies showed that MHCII-deficiency on BM-APCs (Hinterberger et al., 2010; van Meerwijk et al., 1997) or in vivo ablation of CD11c-expressing DCs using a diphtheria toxin a chain transgene (Ohnmacht et al., 2009) resulted in increased CD4SP thymocytes. These findings indicate the role of BM-APCs, in particular DCs, in mediating clonal deletion.

Based on TCRα sequencing in mice with a fixed transgenic TCRβ, 20% of CD4SP TCRs were found to depend on MHCII+ _{BM-APCs for deletion (Perry et al., 2014). The}

TCRs dependent on MHCII+ _{BM-APCs for deletion were partly distinct from the TCRs}

that were dependent on high MHCII expression in mature mTECs, revealing the non- redundant roles of BM-APCs and mTECs in inducing negative selection (Perry et al., 2014).

Antigen transfer (also called indirect presentation or antigen handover) from mTECs to BM-APCs is an important antigen-presenting mechanism underlying negative selection in the thymus. APCs purified from the thymus can induce T cells to proliferate in vitro, if they present a peptide that is recognised by the T cells. In such an assay, purified thymic DCs were found to stimulate more T-cell proliferation than purified mTECs, even when the peptide was expressed within mTECs and not DCs (Koble and Kyewski, 2009). This suggests that antigen handover can "amplify" the T-cell stimulatory capacity of mTEC-derived self-antigens. In vivo studies have shown that some mTEC- derived self-antigens require antigen handover in order to induce thymocyte deletion (Aichinger et al., 2013; Hubert et al., 2011; Taniguchi et al., 2012). Antigen handover is also important for T-reg selection in the thymus, as a TCR sequencing approach revealed that almost half of the TCRs that required Aire to undergo T-reg

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differentiation, also required MHCII+ BM-APCs in order to meet this fate (Perry et al., 2014). It is thought that CD8+ _{cDCs can contribute to indirect presentation in the}

thymus. This is because mTECs are the only thymic stromal cells that express the Aire- dependent XC-chemokine ligand 1 (XCL1) and XCL1 is important in localising DCs in the medulla, and CD8+ _{cDCs express the XCL1 receptor (Lei et al., 2011).}

Furthermore, Perry et al. (2014) also showed a role for Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3)-dependent CD8+ cDCs in inducing negative selection by indirectly presenting Aire-induced self-antigens to thymocytes.

The above studies (sections 1.9.2 and 1.9.3) revealed the roles of mTECs and/or BM- APCs in mediating thymic medullary negative selection, either via direct presentation or indirect presentation (antigen handover mechanism). As increases in CD4SP thymocytes were observed when mTECs and/or BM-APCs were defective, it was inferred that these APC types mediate negative selection at or around the DP-SP transition. However, these studies did not directly measure negative selection by using markers of negatively selected thymocytes, such as Helios or Nur77. It remains unclear which thymic APC is important in mediating the large amount of negative selection that occurs at wave 1 and whether different APC types are required to mediate waves 1 and 2 negative selection during thymocyte development.

1.10 Aims

The contributions of thymic APC types to negative selection have largely been focused at the mature CD4SP developmental stage. It remains elusive whether different APC types mediate negative selection at different stages of thymocyte development. Although the proportion of thymocytes undergoing apoptotic deletion has been studied, the proportions of positively selected and negatively selected thymocytes responsive to MHCI or MHCII, remain unclear. Finally, the TCR repertoires that provoke positive or negative selection in response to one MHC class, or in the absence of both MHC classes, have not been defined.

This thesis aimed to dissect thymic negative selection mechanisms by examining: i) the role of thymic APC types (MHCII+ _{BM-APCs versus Aire) in thymic}

negative selection (Chapter 3);

ii) the role of MHC Class I and MHC Class II in inducing TCR signalling and thymic negative selection (Chapter 4); and

iii) the comparison between TCR repertoires in wild-type mice and in mice lacking one or both MHC classes using a TCR sequencing approach (Chapter 5).

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CHAPTER 2