Cortical thymic epithelial cells
Thymocytes interact with a variety of antigen-presenting cells during the course of their development. DN thymocytes enter the thymus at the corticomedullary junction (CMJ) and traverse outwards towards the subcapsular region where they undergo rearrangement of the TCRβ chain (Fig.
1). As DPs, they migrate randomly though a dense matrix of cortical thymic epithelial cells (cTECs), and their engagement with p/MHC on cTECs is crucial for positive selection [
61]. Whether or not cTECs are also capable of inducing negative selection is less clearly understood. A number of studies have concluded that cTECs are not capable of inducing tolerance to self-antigens [
62‐
66]. However, there is also much data to suggest that cTECs can in fact be tolerogenic [
4,
67‐
75]. This apparent contradiction is likely explained by the observation made by Goldman et al. that studies indicating the thymic epithelium was not capable of inducing tolerance “were done using antigens not normally expressed by thymic epithelium and/or targets derived from other tissues”. They then go on to suggest that cTECs are capable of inducing tolerance to antigens expressed by cTECs, but not to all antigens expressed in the body. In addition, we have shown that cTECs are inefficient at inducing apoptosis of self-reactive thymocytes but that they are ultimately tolerogenic because they prevented the development of mature SP thymocytes [
76]. We were unable to determine if cTECs induced anergy or clonal diversion of these cells, but these observations may also help reconcile the apparent contradictions in previous studies because the induction of tolerance can occur by mechanisms other than clonal deletion.
An intriguing study was recently reported that potentially changes the paradigm of cTECs in positive and negative selection [
77]. In addition to the “standard” proteasome and the “immunoproteasome”, Murata et al. described a novel proteasomal subunit expressed exclusively in cTECs, which they termed β5t. This “thymoproteasome” has unique peptidase activity with reduced chymotrypsin-like activity, which is important for generating peptides with hydrophobic C-termini. Hydrophobic C-termini anchor peptides in the groove of class I MHC. Therefore, it is likely that cTECs not only generate a unique peptide repertoire but also present unstable class I MHC on their cell surface. These observations may explain why cTECs are critical for positive selection, and in fact, β5t-deficient mice had a dramatic defect in generation of CD8 SPs. This unique feature of cTECs also potentially explains why cTECs are incapable of inducing tolerance to antigens expressed throughout the body and their poor ability to induce clonal deletion. It is interesting to speculate that a similar mechanism exists in cTECs for the generation of class II p/mHC complexes, perhaps via cathepsins [
78,
79].
Trafficking to the medulla
The notion that cTECs are not sufficient for induction of clonal deletion is bolstered by the observation that trafficking to the medulla is necessary for achieving complete tolerance [
80,
81] and that this migration is primarily mediated by CCR7-dependant signaling [
82,
83] (See Fig.
1). Indeed, mice with medullar defects also display autoimmune phenotypes (reviewed in [
84]). It is commonly believed that the medulla is a specialized anatomical location for clonal deletion due to the high density of dendritic cells (DC) and the peculiar ability of medullary thymic epithelial cells (mTECs) to ectopically express TRAs (both of which are described in further detail below). However, a defect in clonal deletion may not be sufficient to explain the lack of tolerance in previously mentioned studies. This is because the medulla has also been implicated to be important for the generation of immunoregulatory T cells [
85‐
88] and for conventional T cells to undergo final maturation and adjustment of their TCR sensitivity to self-antigens (a process referred to as “TCR tuning”) [
89]. In addition, Reinhold Förster’s laboratory also questioned whether or not CCR7 was the only chemokine receptor involved in corticomedullar migration [
90]. While there is clearly medullar dysplasia in CCR7-deficient mice and reduced medullar volume, they showed that proportionally normal numbers of CD4 SP thymocytes were found in the small pockets of medulla that remained. They concluded that a redundant mechanism exists for migration to the medulla. Importantly though, it should be noted that the majority of SPs in the medulla were CD4 SPs, and it was impossible to distinguish if these were truly developing thymocytes or another lineage of CD4
+ T cells, such as Foxp3
+ Tregs that also reside in the medulla [
91] and may have an alternate homing mechanism.
Medullary thymic epithelial cells
Despite the complicating factors of Treg development, TCR tuning, and the role of CCR7-mediated chemotaxis, it is clear that developing thymocytes must gain access to the medulla to be screened against a panel of self-antigens uniquely expressed there (See Fig.
1). It has been shown that medullary thymic epithelial cells are capable of expressing the autoimmune regulator (AIRE), which leads to the ectopic expression of tissue-restricted antigens [
92,
93]. This phenomenon of mTECs is crucial for the establishment of central tolerance as AIRE-deficient humans and mice both develop autoimmunity [
94]. It has been shown that mTECs express the costimulatory molecules B7-1 and B7-2 and that direct presentation of TRAs by mTECs is sufficient to induce clonal deletion of antigen-specific thymocytes, although cross-presentation by dendritic cells also occurs [
21] (discussed in further detail below).
The development of mTECs is dependent upon a population of CD4
+ CD3
− lymphoid tissue inducer cells and receptor activator of nuclear factor κB (RANK)–RANK ligand interactions, and their maintenance is, in part, controlled by CD40–CD40L signaling and lymphotoxin-β receptor (LTβR; see Fig.
1) [
95‐
97]. Furthermore, tumor necrosis factor receptor-associated factor 6 (TRAF6), which signals downstream of RANK and CD40, is also needed for mTEC development [
86]. TRAF6 activates the transcription factor NF-κB, which is also required for mTEC development [
98], as is the NF-κB-inducing kinase [
85] and IκB kinase α [
99].
Recent work has also demonstrated that mTECs are a highly dynamic population with high proliferative capacity and a turnover rate of about 2 weeks [
100,
101]. It was observed that the majority of proliferating mTECs were mature, UEA-1
+ MHC class II
hi, and that these mature mTECs are the ones expressing AIRE [
102]. In addition, it appears that AIRE-expressing mTECs are short-lived with rapid turnover [
103]. While AIRE-dependent TRAs are found clustered throughout the genome, implying a role for epigenetics in AIRE function [
104], it has also been shown that individual mTECs express a stochastic battery of TRAs, such that each mTEC is unique with respect to its antigen-presentation profile [
105,
106]. Taken together, these data paint a picture where mTECs are critical for the elimination of self-reactive thymocytes and that individual AIRE-expressing mTECs are highly diverse and turning over rapidly. As loss of tolerance to just one tissue-restricted antigen is sufficient to cause autoimmunity [
107], it becomes clear that thymocytes must have ample opportunity to peruse the entire milieu of the medulla to be adequately screened for self-reactivity. We recently showed that the length of time that an SP thymocyte spends in the medulla is roughly 4–5 days [
108], during which a thymocyte “tunes” its TCR and becomes refractory to apoptosis. Therefore, a short medullary residency time of SP thymocytes, in combination with the diversity and turnover of mTECs expressing TRAs, may be a weak point in the mechanism of central tolerance. Evolution has obviously created a system that prevents overt autoimmunity but bear in mind that T cell-driven autoimmunity is disturbingly common in the human population. Finally, the precise mechanism by which AIRE operates is not entirely understood. AIRE most likely operates as a transcription factor, but as mentioned above with regards to epigenetics, it remains possible that AIRE promotes ectopic expression of TRAs by another mechanism(s) [
109]. In addition, not all TRAs are AIRE-dependent [
104]; thus, another mechanism or molecule, similar to AIRE, must also exist, perhaps involving LTβR [
110].
Dendritic cells
Dogma states that TCR activation and clonal deletion are most efficiently induced by bone marrow-derived cells, and it is widely believed that dendritic cells are the principle mediator of clonal deletion. This is because DC are thought of as “professional” antigen presenting cells with high levels of class II MHC on the cell surface as well as high levels of costimulatory molecules, such as B7-1 and B7-2. Indeed, a direct role for DC in clonal deletion has been demonstrated [
21,
111,
112]. However, the heterogeneity in thymic DC is largely underappreciated and not completely understood. It is unclear if all DC in the thymus are equal in their capacity to induce clonal deletion. Work by Donskoy and Goldschneider has shown that at least two distinct populations of thymic DCs exist: one that develops intrathymically and one that is derived from peripheral DCs migrating to the thymus [
113]. It was suggested that these two populations might have functional differences in their ability to induce deletion of self-reactive thymocytes vs. clonal diversion of immunoregulatory cells. Goldschneider and Cone propose a model where intrathymically derived DC mediate clonal deletion, whereas extrathymically derived cells support the agonist selection of regulatory T cells (see [
114] for a comprehensive review of this argument). It is important to note that the phenotype is unknown of the subset of peripheral DCs that are capable of migrating to the thymus and mediating this selection. In line with this hypothesis, it has been shown in the human thymus that a group of mTECs called Hassall’s corpuscles produces TSLP (thymic stromal lymphopoietin) and this allows DC to induce the proliferation and differentiation of Foxp3
+ Tregs [
115]. Conversely, it was more recently demonstrated that antigen-loaded, splenic DC were capable of homing to the thymus and inducing deletion of antigen-specific thymocytes [
116]. More detailed analysis of the phenotype of the DC migrating to the thymus was also performed, and these DC included all subsets of splenic DC that were present at the time of injection, although LPS-matured DC showed a reduced capacity to home to the thymus. It may be that the ability of peripheral DC to migrate to the thymus and induce deletion vs. agonist selection might not be mutually exclusive and may depend upon the specific environmental and experimental conditions. It is also possible that different subsets of peripheral DC are capable of mediating the different selection outcomes.
As potential support for the latter hypothesis, Ken Shortman’s laboratory has shown that, in addition to intrathymically- and extrathymically derived DCs, at least three other DC subsets can be identified based upon cell surface markers (reviewed in [
117]). Their data showed an initial division of DC into two populations, 35% of which are plasmacytoid DC (pDC) and the remaining 65% are conventional DC (cDC). The distinction between these two populations is based on expression of CD11c and CD45RA, with pDC being CD11c
int and CD45RA
+, and cDC being CD11c
hi and CD45RA
−. The majority of pDC are Ly6c
+, although a small fraction of Ly6c
− can also be found. Similar to pDC in the spleen and lymph node, some thymic pDC can express CD4 and/or CD8α, but unlike peripheral pDC, they express high levels of TLR7 and 9, but only low levels of TLR 2, 3, and 4. Also similar to peripheral pDC, thymic pDC express low levels of MHC class II and costimulatory molecules and can take up antigen by endocytosis but not phagocytosis. Thus, pDC are weak stimulators of T cells. On the other hand, all conventional DC are MHC class II+ and the majority are CD8αα
+. Similar to the minority population of CD8
+ DC in the spleen and lymph node, this population in the thymus is DEC-205
+ (CD205) and CD11b
−. However, unlike peripheral CD8α
+ DC, some thymic CD8α
+ cDC also express BP-1, although no functional difference has yet been shown based upon this distinction. Conventional DC can be further subdivided by their expression of CD8α and Sirpα (CD172a). CD8α
lo Sirpα
+ DCs account for 20% of the thymic cDC population, and similar to splenic CD8α
− DC, this population is also CD11b
int. While thymic cDC express the costimulatory molecules B7-1 and B7-2 at slightly higher levels than their peripheral counterparts, for the most part, they appear to be immature because they are capable of phagocytic or endocytic uptake of antigens and process and present them on MHC class II. In addition, they upregulate expression of MHC class II and costimulatory molecules following activation. It is not entirely clear how this classification of thymic DC aligns with the division created by the Goldschneider laboratory as to the origin of these cell types. It seems that most of CD8α
+ cDC develop intrathymically from a lymphoid prothymocyte precursor, whereas extrathymically derived DC include the CD8α
− DC population, likely of myeloid origin.
Finally, it is well documented that the majority of thymic DCs are found in the medulla, and therefore, it is thought that the anatomical location of clonal deletion is at the CMJ or in the medulla. However, we and others have noted the sparse but distinct presence of DC in the cortex of the thymus (See Fig.
1). This often-overlooked observation raises the possibility that cortical DC may be sufficient to mediate clonal deletion. Indeed, we have recently shown that clonal deletion to ubiquitous self-antigens can occur in the cortex, with no involvement of the medulla [
76]. Furthermore, we found that thymocytes undergoing this process were preferentially in contact with DC present in the cortex and that conditional ablation of DC significantly impaired clonal deletion of antigen-specific cells. The fact that clonal deletion to ubiquitous self-antigens can occur in the cortex does not preclude the requirement of migration to the medulla and clonal deletion to TRAs. It does, however, expand our thinking as to the mechanism underlying clonal deletion. It is unknown if any phenotypic differences exist between cortical and medullar DC, or if any subset(s) of DC discussed previously reside preferentially in the cortex or the medulla. It was recently shown that most cortical DC are found in close association with small blood capillaries that express CCR7 ligands ([
118] and our unpublished data). As positively selected thymocytes also express CCR7, it is interesting to speculate that this exists as an initial mechanism for screening thymocytes for clonal deletion. It also raises the possibility that thymocyte migration to the medulla does not occur randomly but that CCR7
+ cells travel along blood capillaries, which ultimately lead to the CMJ. Finally, given that cTEC are not efficient at inducing clonal deletion and DC are, it is likely that a second signal that can be delivered by a DC but not a cTEC is necessary to induce apoptosis. We have observed that cortical DCs express B7-2, and it is tempting to think that this is the second signal. However, as discussed previously, there is no absolute role for costimulation by B7-2, although it has been shown that complete deletion of superantigen-reactive cells requires B7-1/2 and CD28 interaction [
119]. Therefore, it remains possible that B7-1 and B7-2 play a role in clonal deletion, but it is probable that other costimulatory molecules also exist.
In conclusion, recent advancements have allowed researchers to gain a deeper understanding of the process of clonal deletion. We are finally beginning to understand the molecular pathways that are necessary for the elimination of self-reactive thymocytes, as well as the cellular players that contribute to this process. As the field moves ahead, it is important to develop and use the most physiological tools possible and to consider the intricate anatomical microenvironments of the thymus in order to gain a clear understanding of clonal deletion.