Central Tolerance: Negative selection in the thymus

Central tolerance refers to the thymic mechanisms permitting the deletion of self-reactive newly generated TCR⁺ thymocytes from the T cell repertoire. These processes are dependent on specialized antigen presenting cells (APCs), the DCs and mTECs, which present a broad panel of peripheral tissue antigens (PTAs) also known as tissue restricted specific antigens (TRAs or TSAs). SP thymocytes reacting to PTAs are either deleted by apoptotic death or will alternatively give rise to regulatory T cells (Treg) depending on the strength of TCR/self-reactive pTAs interactions (Figure I-1). The deletional process is known as negative selection and prevents the survival of potential self-reactive lymphocytes that could lead to autoimmune disorders.

1.2.1. THYMIC APCS ENSURING THE CENTRAL TOLERANCE

Thymocytes displaying TCR with high-affinity for self-peptides MHC complexes undergo negative selection in the thymic cortex and the medulla. Medullary negative selection is driven by interactions of self-reactive SP thymocytes with both mTECs and DCs (Klein et al., 2009) that express a highly diverse set of PTAs (Gotter et al., 2004) presented through MHC molecules by different manners depending on the cell type.

Mature mTECs endogenously express PTAs via an original mechanism, termed promiscuous gene expression (PGE) that is in part controlled by the autoimmune regulator AIRE. AIRE is a nuclear regulatory protein whose binding to unmethylated histone regulates gene expression of PTAs and their presentation by MHC molecules (Peterson et al., 2008). Mutations of AIRE gene in mouse have been shown to affect not only PTAs expression but also clonal deletion (Anderson et al., 2005), medulla organization (Dooley et al., 2008; Milicevic et al., 2010), medullary DC frequency (Lei et al., 2011), maturation of thymocytes (Sha et al., 1988) and

19 | P a g e subsequently results in multi-organ autoimmune disorders (Xing and Hogquist, 2012). In human, mutations of AIRE gene on both alleles are commonly linked to the autoimmune polyglandular syndrome type 1 (APS1) also known as polyendocrinopathy candidiasis ectodermal dystrophy syndrome (APECED). More recently, single AIRE point mutations on one allele have been described and related to an increased susceptibility for autoimmune diseases (Oftedal et al., 2015). Even though AIRE dependent mechanisms leading to PTA expression are not yet fully understood, together with recent findings revealing expression of AIRE-independent PTAs in mTECs (Takaba et al., 2015), AIRE appears as a cornerstone of mTEC mediated T cell tolerance.

Thymic DCs are also competent to present PTAs at their cell surface, but this phenomenon is not mediated through endogenous expression and consequently not directly dependent on AIRE. Three different subsets of DCs are found in the thymus: the intrathymic resident (CD11c⁺CD11b^-CD8a⁺Sirpα^-) conventional DCs (cDCs), the migratory (CD11c⁺CD11b⁺CD8a^- Sirpα⁺) cDCs and the migratory (CD11c^int PDCA1⁺ B220⁺) plasmacytoid DCs (pDCs) (Klein et al., 2009). Intrathymic resident DCs arise from intrathymic precursors and are able to either cross-present Ags provided by apoptotic mTECs (Koble and Kyewski, 2009) or cross-present functional PTAs-MHC complexes acquired from mTECs (Millet et al., 2008). Immature migratory thymic DCs, which present myeloid characteristics, are mainly found in the medulla and enter the thymus after having trafficked in periphery. Consequently they present self-Ags acquired in periphery and therefore increased central tolerance mechanisms by widening the panel of intrathymic presented Ags (Bonasio et al., 2006). Although their role in central tolerance is less described, pDCs are found in human and mouse thymus, and as cDCs are involved in the T cell repertoire. Thymic pDCs arise from the blood circulation (Li et al., 2009) and are mainly localized in the thymic medulla (Bendriss-Vermare et al., 2001) where they present self-Ags

20 | P a g e previously acquired in periphery (Hadeiba et al., 2012). Thymic pDCs are competent at mediating clonal deletion (Hadeiba et al., 2012) and could potentially induce nTreg development (Hanabuchi et al., 2010; Martin-Gayo et al., 2010), this last point remaining to be demonstrated in vivo.

1.2.2. MECHANISMS OF NEGATIVE SELECTION

CLONAL DELETION

The interaction of high affinity TCR displayed by autoreactive thymocytes with self-peptide-MHC complexes presented by mTECs and thymic DCs initiates the negative selection of the involved thymocytes by a process called clonal deletion, resulting in their rapid apoptosis (Figure I-2). Activation of apoptotic pathways dependent on the orphan nuclear receptor Nur77 and the pro-apoptotic member Bim, the antagonist of Bcl2, have been specifically associated with T cell deletion (Baldwin and Hogquist, 2007; Kovalovsky et al., 2010; Moran et al., 2011).

CLONAL DIVERSION

Another tolerogenic mechanism occurring in the thymus is called clonal diversion and results in the generation of Foxp3⁺ Tregs. Tregs are able to suppress immune responses through diverse mechanisms such as direct cell-cell contact, production of anti-inflammatory cytokines and modulation of DC activation state (Li et al., 2015). Thymocytes giving rise to Tregs display self-reactive TCR specificities and positively interact with self-peptide-MHC complexes presented by thymic APCs. However, the mechanisms by which they avoid clonal deletion and acquire suppressive and regulatory functions are still elusive. The strength and the duration of TCR interactions with peptide-MHC complexes could account for the outcome of

self-21 | P a g e reactive thymocytes (Figure I-2). Alternatively, expression of CD28, IL-2 and TGF-β, all important for peripheral Treg differentiation and survival, could be as well linked to clonal diversion (Lio et al., 2010; Liu et al., 2008). On the other hand, it remains controversial whether clonal diversion depends on PTA presentation by mTEC and/or DCs. While Aschenbrenner et al. claimed that it is exclusively supported by mTEC-derived PTAs (Aschenbrenner et al., 2007), others demonstrated that thymic cDCs and pDCs are able to elicit Treg differentiation (Hadeiba et al., 2012; Hanabuchi et al., 2010; Martin-Gayo et al., 2010; Proietto et al., 2008).

Finally, Lei et al. proposed another scenario in which mTEC and DCs interplay together to elicit clonal diversion (Lei et al., 2011).

FIGURE I-2 THYMOCYTE OUTCOME IS DEPENDENT ON THE AFFINITY OF TCR FOR SELF-PEPTIDE-MHC COMPLEXES.

Thymocytes that display low affinity TCR for self-peptides presented by cTECs die by neglect whereas cells with intermediate affinity are positively selected. Thymocytes expressing highly self-reactive TCR can either die by apoptosis via clonal deletion or undergo clonal diversion and differentiate in regulatory T cells. Adapted from (Xing and Hogquist, 2012).

Mature SP thymocytes, having completed both cortical and medullary selection processes, egress from the thymus and are released into the blood circulation as mature naive T lymphocytes (Nitta and Suzuki, 2016).

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