T-Cell Tolerance: Central and Peripheral

Home , Central tolerance, Clonal deletion, Peripheral tolerance

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Yan Xing and Kristin A. Hogquist

Center for Immunology, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455 Correspondence: [email protected]

Somatic recombination of TCR genes in immature thymocytes results in some cellswith useful TCR specificities, but also many with useless or potentially self-reactive specificities. Thus thymic selection mechanisms operate to shape the T-cell repertoire. Thymocytes that have a TCR with low affinity for self-peptide–MHC complexes are positively selected to further differentiate and function in adaptive immunity, whereas useless ones die by neglect. Clonal deletion and clonal diversion (Treg differentiation) are the major processes in the thymus that eliminate or control self-reactive T cells. Although these processes are thought to be efficient, they fail to control self-reactivity in all circumstances. Thus, peripheral tolerance processes exist wherein self-reactive T cells become functionally unresponsive (anergy) or are deleted after encountering self-antigens outside of the thymus. Recent advances in mechanistic studies of central and peripheral T-celltolerance are promoting the development of therapeutic strategies to treat autoimmune disease and cancer and improve transplantation outcome.

cells recognize pathogen fragments in the However, not all antigens that T cells need to be Tcontext of surface MHC molecules on host tolerant of are expressed in the thymus, and thus cells. As such, they have the potential to do enor- central tolerance mechanisms alone are insufﬁ- mous damage to healthy tissue when they are cient. Fortunately, additional tolerance mech- not appropriately directed, that is, when they anisms exist that restrain the numbers and or respond to self-antigens as opposed to foreign function of T cells that are reactive to develop- antigens. T lymphocyte tolerance is particularly mental or food antigens, which are not thymi- important, because it impacts B-cell tolerance as cally expressed. These mechanisms act on ma- well, through the requirement of T cell help in ture circulating T cells and are referred to as antibody responses. Thus, failure of T-cell tol- “peripheral tolerance.” erance can lead to many different autoimmune diseases. The tolerance of T cells begins as soon CENTRAL TOLERANCE as a T-cell receptor is formed and expressed on the cell surface of a T-cell progenitor in the thy- T lymphocytes arise from circulating bone-mar- mus. Tolerance mechanisms that operate in the row-derived progenitors that home to the thy- thymus before the maturation and circulation mus. After T lineage commitment and expan- of T cells are referred to as “central tolerance.” sion, T-cell receptor (TCR) gene rearrangement

Editors: Diane J. Mathis and Alexander Y. Rudensky Additional Perspectives on Immune Tolerance available at www.cshperspectives.org Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a006957 Cite this article as Cold Spring Harb Perspect Biol 2012;4:a006957

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Y. Xing and K.A. Hogquist

ensues and gives rise to either gd or ab progen- dergoing secondary gene rearrangement at the itors at the CD4 and CD8 double-negative (DN) TCRa loci, thereby changing the specificity of stage. A small number of ab committed DN the TCR. This process is known as “receptor cells give rise to a large number of CD4 and editing.” Although examples of receptor editing CD8 double-positive (DP) thymocytes, and so- exist for T cells (Wanget al. 1998; McGargill et al. matic recombination of TCR genes results in 2000, 2002; Buch et al. 2002; Santori et al. 2002), a remarkably broad repertoire of distinct ab it is unclear how prominent this mechanism TCRs with random specificity. The TCR affinity is (Holman et al. 2003), and it will not be dis- for self-peptide–major histocompatibilitycom- cussed further here. Finally, a state of unrespon- plex (MHC) determines a thymocyte’s fate from siveness can be induced in self-reactive thymo- this point forward (Fig. 1). DP thymocytes ex- cytes, called “anergy.” Anergy is likely a more pressing TCRs that do not bind self-peptide– prominent tolerance mechanism that operates MHC complexes die by neglect. Those with a in the periphery and is discussed further in low affinity for self-peptide–major histocom- that section. These four processes—clonal dele- patibility complex MHC complexes differenti- tion, clonal diversion, receptor editing, and an- ate to CD4 or CD8 single-positive (SP) thymo- ergy—are the major mechanisms that limit the cytes—so-called positive selection. However, self-reactivity of the T-cell repertoire and are those with high-affinity TCR for self-peptide– crucial for immune health. MHC complexes represent a potential threat to the health of the animal, and various mecha- CLONAL DELETION nisms operate to ensure tolerance to self, including clonal deletion, clonal diversion, receptor In this section, we discuss several fundamental editing, and anergy. questions of clonal deletion, such as: At which In the thymus, one of the main mechanisms stages of development do thymocytes undergo of T-cell tolerance is “clonal deletion,” although deletion? Where does it occur anatomically? the selection of regulatory T cells (“clonal diver- What cell types induce apoptosis? What is the sion”) is also important and is of enormous in- molecular mechanism? terest (see Benoist 2012). Thymocytes express- The thymus is composed of two major an- ing high-affinity TCR for self-peptide–MHC atomical areas—an outer region known as the can avoid the deletion or diversion fates via un- cortex, which contains DN and DP thymocytes,

Death Cells Clonal by Positive deletion neglect selection

Treg

TCR affinity

Figure 1. A model for the relationship between developmental outcome and TCR affinity for self-peptide– MHC. Cells with TCR that have a low affinity for self die by neglect. Those with an intermediate affinity are positively selected. High-affinity self-reactive clones can die via clonal deletion, and the threshold between positive and negative selection is hypothesized to be steep. Regulatory T cells (Treg) (yellow) have more highly self-reactive TCRs than most conventional T cells (purple). Some Tregs may have very highly self-reactive TCRs and are rescued from deletion via cytokine signaling or by virtue of having a second TCR (dotted line).

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T-Cell Tolerance

and an inner region known as the medulla, tified by the observation that superantigens are which contains SP thymocytes (Fig. 2). Positive primarily expressed in the medulla, which is the selection of thymocytes occurs in the cortex. site where SP thymocytes reside. Likewise, tis- However, whether or not clonal deletion occurs sue-specific antigens are often expressed exclu- in the cortex has been controversial. For exam- sively in the medulla (see below for further dis- ple, superantigen studies have suggested that cussion). In contrast, in TCR transgenic models deletion occurs at the SP stage (Kappler et al. where the TCR is specific for ubiquitous self- 1987) (in the thymic medulla), whereas exam- antigens, deletion occurs at the DN-to-DP tran- ination of TCR transgenics and endogenous sition. However, in transgenic models, thymo- self-antigens had suggested that deletion occurs cytes express both TCRb and TCRa chains early at the transition from DN to DP (Kisielow et al. at the DN stage and undergo negative selection 1988; Sha et al. 1988) (in the thymic cortex). prematurely (Baldwin et al. 2005; Egawa et al. This apparent discrepancy can be partially rec- 2008). What is clear is that the nature of TCR

Positive selection, neglect, and clonal deletion Clonal deletion and Treg differentiation

Apoptosis Cortex Medulla (neglect) Treg

SP Apoptosis DP (deletion) Apoptosis (deletion)

DC DC cTEC mTEC

CD28 CD4 DP SP CD4 or CD8 B7 TCR or CD8 CD40L p-MHC TCR RANKL β5i p-MHC LT CD40 Ctss RANK β5t AIRE TSSP LT-βR TSAs B7 Ctsl B7 p-MHC p-MHC CD40 CD40 DC cTEC DC mTEC

Figure 2. Cell types in central tolerance. (Top) T cells are positively selected in the thymic cortex. Negative selection via clonal deletion can also occur in the cortex, but occurs frequently in the medulla. The thymic medulla is also the site for Treg differentiation. (Bottom) Cortical thymic epithelial cells (cTECs) express several unique genes that relate to proteolysis (cathepsin L, Ctsl; thymus-speciﬁc serine protease, TSSP; and b-5t proteasome subunit, b5t), which are essential for positive selection. Tissue-speciﬁc antigens (TSAs) can be directly presented by medullary thymic epithelial cells (mTECs) or cross-presented by DC (dotted line with arrow). RANKL, CD40L, and lymphotoxin (LT) expressed by SP thymocytes interact with their receptors on mTEC to promote the development of mTEC.

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Y. Xing and K.A. Hogquist

and self-antigen expression can dramatically trophy syndrome (APECED) (Anderson and impact the timing of clonal deletion and the Su 2011). Like patients with APECED, AIRE- molecular mechanism by which apoptosis is in- deficient mice also develop spontaneous multi- duced (McCaughtry and Hogquist 2008). It is organautoimmunedisease.InmTECs,theAIRE therefore important to move forward making genepromotesexpression ofawidearrayofTSAs use of the most physiological tools available. (Anderson et al. 2002; Derbinski et al. 2005). Using a TCR transgenic mouse that recapitu- mTECs also express B7 family costimulatory lates the appropriate timing of TCRa expression molecules, and perhaps not surprisingly, they at the DP stage, it was shown that deletion of can be efficient at inducing clonal deletion of T cells specific for ubiquitous antigens occurs T cells reactive to TSAs (Klein et al. 1998; Liston at the DP-to-SP transition (Baldwin et al. 2005). et al. 2003; Anderson et al. 2005). Although di- In terms of physical location, this deletion event rect presentation of TSAs by mTECs was suf- was shown to occur in the cortex (McCaughtry ficient to induce clonal deletion of CD8SP et al. 2008). Thus, in general, polyclonal thymo- thymocytes, it was not for CD4SP thymocytes cytes specific for ubiquitous self-antigens seem (Gallegos and Bevan 2004). However, cross-pre- to be deleted in the thymic cortex, whereas those sentation of mTEC-derived TSAs by DC can also restricted to tissue-specific antigens, some su- occur, and that promotes deletion of both CD4 perantigens, and circulating antigens all occur and CD8 SPs (Gallegos and Bevan 2004; Koble in the thymic medulla. The relative proportion and Kyewski 2009). of the repertoire specific to each of these types of AIRE contains a nuclear-localization signal, antigens has not yet been determined. aproline-rich region, andotherdomainsthat are Multiple aspects of T-cell development in found in other transcription factors. The PHD1 the thymus, including clonal deletion, depend domain of AIRE binds to unmethylated histone crucially on interactions with other cells in the H3 at lysine-4 (H3K4me0) (Org et al. 2008). In thymic microenvironment. Although the thy- addition, coimmunoprecipitation experiments mus is composed of predominantly T-cell pro- showed that AIRE interacts with an unexpected- genitors, there are small numbers of stromal ly large number of binding partners: proteins cells, which include hematopoietic non-T-cell involved in pre-mRNA processing, transcrip- progenitors, such as macrophages and dendritic tion, nuclear transport, and chromatin binding cells (DCs), and non-hematopoietic cells, such andstructure(Abramsonetal.2010).AIREplays as epithelial cells and fibroblasts. Epithelial cells a key function in regulating gene expression of of the thymic cortex (cTECs) are required for TSAs, although how AIRE specifically promotes positive selection of thymocytes, whereas med- the presentation of tissue-specific antigens by ullary thymic epithelial cells (mTECs) and DC MHC molecules remains enigmatic. are more important for Treg differentiation and The role of AIRE in promoting tolerance clonal deletion (Fig. 2). may not be exclusively due to its ability to promote TSA expression. Even when AIRE deficiency did not affect TSA gene expression, it still THYMIC APCs: mTECs affected clonal deletion (Anderson et al. 2005). mTECs play a crucial role in thymic tolerance. AIRE-deficient mice have a disorganized me- They are unique among thymic antigen-pre- dulla (Gillard et al. 2007; Dooley et al. 2008; senting cells (APCs) in that they express a large Yanoet al. 2008; Milicevic et al. 2009), with fewer number of tissue-specific self-antigens (TSAs) medullary DCs (Lei et al. 2011), altered matu- (Derbinski et al. 2001; Gotter et al. 2004). They ration of thymocytes (Sha et al. 1988), and re- also express an interesting nuclear regulatory duced thymic export in the neonatal period protein called autoimmune regulator (AIRE). (Laan etal. 2009). Interestingly,AIREexpression Mutations in the AIRE gene lead to the multi- that was restricted solely to the neonatal period organ syndrome known as autoimmune poly- could prevent autoimmunity in AIRE knockout endocrinopathy candidiasis ectodermal dys- (KO) mice (Guerau-de-Arellano et al. 2009).

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T-Cell Tolerance

Further work will be necessary to fully compre- DCs develop intrathymically from a lymphoid hend how gene deficiency in AIRE causes auto- pro-thymocyte precursor, whereas the CD8a2 immunity in humans. DC population, is thought to be of myeloid or- In the thymus, several members of the TNF igin (Wu and Shortman 2005; Schlenner et al. receptor superfamily, including the receptor 2010). Given that the majority of thymic DCs activator of NF-kB (RANK), CD40, and lym- are found in the medulla, it is thought that the photoxin-b receptor (LTbR), are prominently anatomical location of clonal deletion is gen- expressed on mTECs, whereas their ligands, in- erally in the medulla. However, in one model cluding RANKL, CD40L, and LT, are expressed of clonal deletion to ubiquitous self-antigen, on hematopoietic cells (Hikosaka et al. 2008). cortical DCs were shown to be important Recent observations indicate that positively (McCaughtry and Hogquist 2008; McCaughtry selected thymocytes produce these cytokines, et al. 2008). Interestingly, it has been reported which act on mTECs to regulate their prolifera- that Sirpaþ cDCs are disseminated in the thymic tion and differentiation to form the microenvi- cortex with some of them localized inside peri- ronment of the thymic medulla, which is crucial vascular regions and nearby small vessels in the for the establishment of self-tolerance (Akiyama thymus, and it was suggested that they crucially et al. 2008; Hikosaka et al. 2008; Irla et al. 2008; contribute to central tolerance against blood- Nitta et al. 2011). Both CD40L and RANKL borne antigens (Baba et al. 2009; Atibalentja activate the classical and alternative NF-kB sig- et al. 2011). naling pathways. In addition, studies of gene- A unidirectional transfer of antigens was deficient and mutant mice have clearly sug- shown to occur inwhich thymic DCs specifically gested that these pathways are involved in the acquire self-antigen for cross-presentation from development of mTECs, including: REL-B-defi- mTECs and not from hematopoietic sources cientmice(Weihetal.1995);Nikaly/aly mice(Ka- (Gallegos and Bevan 2004; Koble and Kyewski jiura et al. 2004), which carry the alymphoplasia 2009). A question arises as to how such antigens mutation in NF-kB-inducing kinase (Nik); tu- can load into the MHC Class II presentation mor-necrosisfactor-receptor-associated factor6 pathway. A recent study has implicated auto- (TRAF6)–deficient mice (Akiyama et al. 2005), phagy as a means of antigen transfer, because and NF-kB2-deficient mice (Zhu et al. 2006). All mTECs appear to have autophagosomes and of these mutant mouse strains show reduced lev- blocking autophagy in the thymus is associated els of AIRE, which promotes TSA expression by with autoimmunity (Nedjic et al. 2008). How- mTECs. mTECs might also contribute to central ever, further study is needed to establish the pre- tolerance through their regulated expression of cise roles of distinct thymic DC subsets. costimulatory molecules, such as CD40, CD80 (B7-1), and CD86 (B7-2) (Fig. 2). MOLECULAR MECHANISMS OF CLONAL DELETION THYMIC APCs: DENDRITIC CELLS Thymocytes that recognize self-peptide–MHC Like mTECs, the majority of thymic DCs also with lowaffinity induce positive selection,where- reside in the medulla, although there are some as those with high affinity undergo negative se- DCs in the cortex. Both medullary and cortical lection (Fig. 1). This model, for which there is DCs are potentially important in central toler- extensive experimental support, is known as an ance by inducing the apoptosis of self-reactive “affinity model” of selection (Starr et al. 2003). thymocytes. Thymic DCs are composed of three Thus a key question for understanding clonal major subsets: CD11cþB220þ plasmacytoid deletion is how a TCR can discriminate between DCs (pDCs), CD11cþB2202CD8a2, signal low- and high-affinity ligands. A simple model regulatory protein Sirpaþ conventional DCs would be that low- and high-affinity signals are (cDCs), and CD11cþB2202CD8aþ, Sirpa2 essentially the same, except that the threshold (CD8þ DCs). It is thought that most CD8þ for positive selection is lower than for negative

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Y. Xing and K.A. Hogquist

selection. This seems not to be the case, because death fate of a thymocyte. cTECs are crucial preventing cell death after high-affinity signal- for positive selection, although the reasons for ing in the thymus was not sufficient to yield this remain a mystery (Hogquist and Xing 2010). positive selection (Hu et al. 2009; Kovalovsky cTECs do express several unique genes that re- et al. 2010). Rather, the evidence suggests that late to proteolysis (Ctsl, TSSP, and b5t) and are low- and high-affinity interactions trigger qual- essential for positive selection. These genes like- itatively different responses. A “zipper” model ly contribute to a unique display of self-peptide was recently proposed, which suggeststhat high- ligands by cTECs, compared with other thymic affinity TCR-peptide–MHC interaction induc- APCs (Nakagawa et al. 1998; Honey et al. 2002; es a stable “zippering” between the membrane- Murata et al. 2007; Gommeaux et al. 2009; Nitta proximal domain of CD8b and the connect- et al. 2009; Viret et al. 2011). The fact that cells ing peptide motif of the a-chain of the TCR that support positive selection display distinct (a-CPM), which then causes signal transduc- self-ligands compared with those that support tion leading to negative selection. Conversely, a negative selection may be a crucial part of the low-affinity peptide–HC ligand that interacts process, or it may simply enhance the numbers with a TCR-coreceptor induces incomplete zip- of progenitors that survive the gauntlet of thy- pering and partial CD3 ITAM phosphoryla- mic selection (Fig. 2). tion, consequently triggering positive selection Two genes that are consistently up-regulated (Palmer and Naeher 2009). in cells undergoing clonal deletion and can pro- With respect to the downstream signals, the moteapoptosisareNur77andbim(Baldwinand strength and kinetics of both Ca2þ and extracel- Hogquist 2007). Nur77 is an orphan nuclear re- lular-signal-regulated kinase (ERK) signaling ceptor that was originally suggested to be a key are important in positive versus negative selec- effector molecule in apoptosis (Woronicz et al. tion. For example, negative selection induces a 1994). Overexpression was sufficient to induce rapid and robust ERK activation that is associ- apoptosis in DP thymocytes, and inhibition ated with death, whereas positive selection stim- blocked clonal deletion in TCR transgenic mod- ulates a lower intensity but sustained ERK acti- els (Calnan et al. 1995). Nur77 can act as a tran- vation (McNeil et al. 2005). Further evidence scriptional regulator, but recent work suggests suggests that the cellular location of ERK acti- that the influence of Nur77 in deletion may be vation is also different under conditions of pos- viaitsability tointeractwithBCL-2family mem- itive and negative selection (Daniels et al. 2006). berproteinsinthemitochondria(Thompsonand A key protein, called Themis (thymocyte ex- Winoto 2008), and not via inducing pro-apopto- pressed molecule involved in selection), seems ticgenes.Althoughstronglyexpressedafterhigh- to be involved in determining the strength and affinity signaling in thymocytes, Nur77 is also kinetics of Ca2þ influx and phosphorylation of expressed at low levels in positively selected T ERK (Fu et al. 2009; Johnson et al. 2009; Kaku- cells, suggesting a complex role in survival. gawa et al. 2009; Lesourne et al. 2009; Patrick Bim is a pro-apoptotic member of the BCL- et al. 2009). Its deficiency markedly impairs 2 family that mediates cell death by an intrinsic positive selection of thymocytes. Interestingly, apoptotic pathway. Bim-deficient mice show thymocytes seem to have a unique biochemical impaired deletion of T cells reactive for su- pathway that prevents them from undergoing perantigen (Bouillet et al. 2002; Villunger et death in response to low-affinity signals, which al. 2004), tissue-specific antigen (Moran et al. involves the protein Schnurri-2 (Staton et al. 2011), and ubiquitous self-antigen (Hu et al. 2011). More work is needed to understand how 2009; Kovalovsky et al. 2010). Interestingly, in these differences in proximal TCR signals lead models of ubiquitous self-antigen, where dele- to unique gene expression patterns and the dis- tion occurs at the DP!SP stage in the thymic tinct outcomes of life versus death. cortex, elimination of Bim reduced the apopto- Although important, TCR affinity is clearly sis of self-reactive thymocytes, but did not res- not the only parameter that decides the life/ cue their differentiation. The cells remained at

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T-Cell Tolerance

the immature CD4loCD8lo stage of develop- merous mechanisms including the production ment (Hu et al. 2009; Kovalovsky et al. 2010). of anti-inflammatory cytokines, direct cell–cell These data suggest a mechanistic distinction be- contact, and by modulating the activation state tween positive and negative selection as dis- and function of APCs (Shevach 2009). Muta- cussed above. One implication of this fact is tions in the Forkhead box P3 (FOXP3) gene that failure of tolerance to ubiquitous antigens cause human immunodysregulation, polyen- does not necessarily present athreat to the health docrinopathy and enteropathy, X-linked syn- of the animals, because thymocytes that receive drome (IPEX). In mouse models, Foxp3 is re- high-affinity signals, but do not die, fail to be quired for Treg development and maintenance positively selected and do not become mature of suppression function. Recognition of self-re- autoaggressive T cells. This is not the case for active TCR ligands appears to be a key event to tissue-specific antigens, where deletion occurs initiate the Treg developmental program. Stud- at the medullary or SP stage. Thymocytes spe- ies of transgenic mice with TCR specific to for- cific for TSA have already survived the positive eign antigens show that Treg develop only when selection checkpoint, and when they fail to be the foreign antigen is also expressed in the thy- deleted (in this case, because of bim deficiency), mus (Itoh et al. 1999; Jordan et al. 2001). Fur- autoreactive T cells accumulate (Moran et al. thermore, studies using transgenic mice pro- 2011). Whether this explains the prevalence of duced with TCR genes cloned from naturally tissue-specific over systemic autoimmune dis- occurring Tregs show that self-reactive TCR spe- eases in humans remains to be determined. Al- cificity is required for the instruction of Treg though Bim induction is key to clonal deletion, thymic differentiation in the thymus (Bautista it is not yet clear what TCR signals are required et al. 2009; Leung et al. 2009). Thus, clonal de- for its expression and why it is uniquely up-reg- letion eliminates self-reactive clones from the ulated after high-affinity TCR signaling. Fur- repertoire, whereas clonal diversion imprints thermore, other molecules induced by TCR sig- self-reactive clones with suppressive or regula- naling in thymocytes, such as Nur77, may tory function (Fig. 1). Thus, the former is some- control the efficacy of BCL-2 family members times referred to as “recessive” and the latter such as Bim (Thompson and Winoto 2008). as “dominant” tolerance mechanisms. Because Another class of molecules that contribute both clonal deletion and clonal diversion re- to clonal deletion is TCR costimulatory mole- quire TCR interaction with self-MHC ligands cules. Experiments that used antibody-mediat- in the thymus, an interesting question is what ed costimulation through CD5, CD28, or CD43 distinguishes these processes? in vitro strongly support this idea (Punt et al. In theory, distinct APCs might instruct the 1994; Kishimoto and Sprent 1999). Yet,surpris- two processes. Because most Foxp3 expression ingly, analysis of individual gene-deficient mice is localized to the medulla, interest has focused showed no effect or a mild effect on clonal dele- on SP thymocytes and medullary APCs. Anti- tion. It is possible that multiple costimulatory gen presentation on mTECs was sufficient for molecules act in a redundant fashion to pro- the generation of antigen-specific Tregs, and mote apoptosis. More recent data suggest that antigen presentation on DCs, which could ac- CTLA-4 signaling in thymocytes may diminish quire and cross-present mTEC-derived antigen, the efficacy of clonal deletion (Buhlmann et al. was dispensable (Aschenbrenner et al. 2007). 2003; Takahashi et al. 2005). Other evidence showed that antigen expressed on thymic DCs could elicit Treg differentiation (Proietto et al. 2008; Hanabuchi et al. 2010). Thus, how thymic DCs and mTECs manage CLONAL DIVERSION to elicit clonal deletion as well as clonal diver- Foxp3-expressing CD4 T cells have been well sion remains to be determined and may reflect a characterized as regulatory T cells (Tregs). These complex interplay between both cell types (Lei cells suppress immune responses through nu- et al. 2011).

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Y. Xing and K.A. Hogquist

FACTORS IN THE DISTINCTION pression of the fluorescent reporter than Tregs BETWEEN CLONAL DIVERSION (Moran et al. 2011). However, in addition to AND CLONE DELETION TCR signals, CD28 costimulation signals have an essential cell-intrinsic role in inducing the TCR engagement is essential for both clonal differentiation of Tregs (Tai et al. 2005; Lio deletion and Foxp3 induction during thymic et al. 2010), and IL-2 signaling is required for differentiation of Tregs. TCR signals of distinct the survival of Tregs (Fig. 3) (Burchill et al. strength and duration have been proposed to 2008; Lio and Hsieh 2008; Vang et al. 2008). It explain this paradox, with stronger signals lead- has long been appreciated that the cytokine ing to deletion and weaker signals leading to TGF-b can convert mature T cells into the so- diversion (Fig.1). Experiments in a recently gen- called adaptive Treg lineage in the periphery, erated mouse model showed that reduction of whereas its role in thymic commitment to the MHC class II levels on mTECs through trans- Treg lineage is less well studied. However, the genic expression of a CIITA-specific microRNA combined absence of both TGF-b and IL-2 diminished the efficacy of negative selection and signaling pathways led to a complete absence led to the increased emergence of Tregs (Hin- of thymic Treg (Liu et al. 2008). Interesting re- terberger et al. 2010). Furthermore, a TCR sig- cent work suggests that TGF-b might provide a nal strength reporter mouse showed that cells protective effect to Tregs that are experiencing undergoing clonal deletion showed greater ex- strong TCR signals from self-MHC, and thus be

cTEC DC mTEC

β5t β5i β5i TSSP Ctsl Ctsl Ctsl or

p-MHC p-MHC (low affinity CD4 B7 (high affinity B7 for TCR) or CD8 for TCR) TCR TCR CD28 CD28 Differentiation- Apoptosis- promoting promoting factor Nur77 factor Nur77 Bim Bim TGF-β Differentiation ApoptosisIL-2 Apoptosis

Thymocyte

Foxp3

Treg

Figure 3. Differential TCR signaling in clonal deletion and clonal diversion. TCRengagement of low-afﬁnity self- peptide MHC ligands presented by cTECs transduces a signal that promotes survival and differentiation. TCR engagement with high-afﬁnity self-peptide–MHC results in expression of Nur77 and Bim and apoptotic cell death. However, cytokines (TGF-b and IL-2) can prevent Treg progenitors from undergoing apoptosis and promotes the differentiation of Tregs.

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T-Cell Tolerance

another keysignal in the discrimination between such as IL-2. Subsequent signaling through the clonal deletion and clonal diversion (Ouyang IL-2Rcomplex can fully activate the PI3K/AKT- et al. 2010). Clearly developing thymocytes in- mTOR pathway. However, T-cell activation in tegrate information from multiple inputs when the absence of a second signal induces a state deciding cell fate in the thymus. of long-term hyporesponsiveness in T cells, termed “anergy,” which is characterized by an active repression of TCR signaling and IL-2 ex- PERIPHERAL TOLERANCE pression. Although central-tolerance mechanisms are Initial studies using the mTOR inhibitor ra- efficient, they cannot eliminate all self-reactive pamycin have shown that blocking mTOR acti- lymphocytes, in part because not all self-anti- vation is sufficient to induce anergy in T cells gens are expressed at the primary site of lym- following full activation with anti-CD3 and phocyte development—the thymus. Therefore, anti-CD28. Recent studies suggest that several peripheral-tolerance mechanisms exist, and energy and nutrient-sensing pathways may be these are crucial to control tolerance of lympho- involved in the promotion of T-cell anergy cytes that first encounter their cognate self-an- through inhibiting mTOR activation (Powell tigens outside of the thymus—such as in the and Delgoffe 2010). These include nutrient dep- case of food antigens, developmental antigens, rivation or activation of AMPK, a direct sen- and antigens displayed during chronic infec- sor of ATP deprivation and hypoxa, or mTOR- tion. Both anergy and deletion of self-reactive independent pathways, such as the GCN2 ami- T cells can occur in the periphery (Fig. 4). no acid–sensing pathway and adenosine signals through the A2A receptor (A2AR). Tregs have recently been postulated to promote a hypoxic ANERGY environment via their expression of both the T cells become activated in the presence of a 50-ectonucleotidease CD73 and the ATPase/ TCR signal and a costimulatory signal mediated ADPase CD39 (Sitkovsky 2009; Chappert and by CD28 ligation, and will then secret cytokines Schwartz 2010). CD39 hydrolyzes ATP to AMP,

Active DC Tolerogenic DC Lymph node stroma

Effector or memory TSA PD-L1 p-MHC B7 ? CD28 PD-1 IL-2 TCR IL-2R Egr2 Egr2 Dgkz Dgkz mTormTor Inhibit mTor Cblb Cblb Pdcd1 Pdcd1 Ctla4 Ctla4 Proliferation Anergy Anergy Apoptosis

T cell T cell T cell T cell

Figure 4. Peripheral tolerance. T-cell anergy is induced by inhibiting mTOR pathways or can be induced by tolerogenic DCs. The expression of Egr2, Cblb, Ctla4, DgkZ, and Pdcd1 genes is important in T-cell anergy. Lymph node stromal cells (LNSCs) express tissue-speciﬁc antigens (TSAs) and can mediate the deletion of self- reactive naive T cells.

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Y. Xing and K.A. Hogquist

and CD73 converts AMP into adenosine. Tregs ysis showed significant similarity in the gene may thus regulate T-cell activation by anergy. expression profiles of cells undergoing periph- Costimulatory pathways provide second sig- eral deletion and anergy induction (Parish et al. nals that promote T-cell activation as discussed 2009). above. However, they can also provide negative second signals that inhibit T-cell responses, me- TOLEROGENIC APCs diate T-cell tolerance, and prevent autoimmunity. Key among these is the programmed death Peripheral DCs are inducers of immune re- 1 (PD-1) receptor and its ligands PD-L1 and sponses, but are also crucial regulators of toler- PD-L2 (Keir et al. 2008). Ligation of TCR and ance induction and maintenance. Tolerogenic PD-1 leads to the recruitment of phosphatases DCs present antigen to antigen-specific T cells, SHP-1 and SHP-2, which dephosphorylate but fail to deliver adequate costimulatory sig- proximal signaling molecules and effectively at- nals (or deliver net coinhibitory signals) for T- tenuate the activation of the PI3K and Akt path- cell activation and proliferation (Gallucci et al. ways. The first evidence that PD-1 plays a critical 1999). DC tolerogenicity is not specific to a role in control of self-reactivity came from the single DC subset. Evidence suggests that tole- phenotype of mice lacking PD-1 (Pdcd12/2), rogenic DCs are generated by incomplete mat- which develop a lupus-like autoimmune disease uration. For example, apoptotic cells, unlike (Nishimura et al. 1999). Subsequent studies necrotic cells, are insufficient to trigger DC mat- suggested that PD-1 interactions with its ligands uration (Hawiger et al. 2001) through suppress- PD-L1 and PD-L2 inhibit T-cell effector func- ing the activation of NF-kB pathways medi- tions in an antigen-specific manner. This path- ated by TLR and cytokine receptor. PD-L1 and way can limit the initial phase of activation, the PD-L2 can be expressed on tolerogenic DCs, expansion of self-reactive T cells (Probst et al. providing a means to control the decision be- 2005), or restrict self-reactive T-cell effector tween T-cell activation and tolerance. Tolero- function and target organ injury, possibly via genic DCs can also be induced and maintained regulating dynamic T-cell motility and the by various anti-inflammatory and immunosup- TCR stop signal (Fife and Pauken 2011). PD-1 pressive agents in vitro and in vivo, including signaling can also mediate the conversion of IL-10, TGFb1, corticosteroids, and rapamycin naive T cells to Tregs. (Morelli and Thomson 2007). Targeting and The other costimulatory molecule impor- manipulating tolerogenic DCs has been shown tant in anergy is CTLA-4. CTLA-4 is induced to suppress experimental autoimmune disease late after T-cell activation and binds B7 family and promote improved outcomes of transplan- costimulatory molecules with high avidity. How- tation; this is discussed in greater detail in Reizis ever, it transduces a negative signal and prevents (2012). cell cycle progression. CTLA-4-deficient mice Lymph node stromal cells (LNSCs) were show spontaneous autoimmunity (Tivol et al. once thought to function solely as parenchymal 1995; Waterhouse et al. 1995), and Ctla42/2 support to lymphocytes. However, new evi- CD4 T cells resist anergy induction (Perez et al. dence shows that all types of LNSCs—includ- 1997). Like PD-1, part of the role of CTLA-4 in ing fibroblastic reticular cells (FRCs), follicular peripheral tolerance relates to its role in regula- DCs (FDCs), and lymphatic endothelial cells tory T cells. Clinical approaches that focus on (LECs)—express TSA (Fletcher et al. 2011). Re- costimulatory molecules are discussed in Blue- cently, an extrathymic AIRE-expressing stromal stone (2012). cell population (eTAC)in lymph nodes was iden- Several other genes are uniquely induced in tified that lacks expression of CD80 and CD86 anergic cells (Macian et al. 2002) and are func- (Gardner et al. 2008). Interestingly, microarray tionally important for the anergic state, includ- experimentsshowthatAIRE-dependentTSAsin ing Cbl-b, p27Kip1, Dgkz, Egr2/3, Itch, NFAT1, peripheral eTACs and thymic mTECs are largely Tob1, and Grail. Interestingly, microarray anal- distinct. The ability of eTACs (Gardner et al.

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T-Cell Tolerance

2009; Fletcheret al. 2010) and other LNSCs (Co- CONCLUDING REMARKS hen et al. 2010) to promote tolerance has been In summary, tremendous progress has been shown primarily in transgenic mouse model sys- made in understanding the cellular and molec- tems.Althoughthemechanismsneedtobemore ular basis of central and peripheral tolerance. fully explored, in these models, LNSCs could This research has led to an understanding of directly present antigens to CD8þ T cells, which the pathogenesis of at least two human inherit- were subsequently deleted. ed autoimmune diseases and many promising therapeutic strategies. PERIPHERAL DELETION

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T-Cell Tolerance

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T-Cell Tolerance: Central and Peripheral

Yan Xing and Kristin A. Hogquist

Cold Spring Harb Perspect Biol 2012; doi: 10.1101/cshperspect.a006957

Subject Collection Immune Tolerance

Regulatory T Cells and Immune Tolerance in the Regulatory T Cells and Immune Tolerance in the Intestine Intestine Oliver J. Harrison and Fiona M. Powrie Oliver J. Harrison and Fiona M. Powrie Dendritic Cells: Arbiters of Immunity and Microbiota and Autoimmunity Immunological Tolerance Alexander V. Chervonsky Kanako L. Lewis and Boris Reizis Current and Future Immunomodulation Strategies Treg Cells, Life History, and Diversity to Restore Tolerance in Autoimmune Diseases Christophe Benoist and Diane Mathis Jeffrey A. Bluestone and Hélène Bour-Jordan T-Cell Tolerance: Central and Peripheral Infectious (Non)tolerance−−Frustrated Yan Xing and Kristin A. Hogquist Commensalism Gone Awry? Jesse C. Nussbaum and Richard M. Locksley Central B-Cell Tolerance: Where Selection Begins Historical Overview of Immunological Tolerance Roberta Pelanda and Raul M. Torres Ronald H. Schwartz The Immunogenetic Architecture of Autoimmune Natural Killer Cell Tolerance: Control by Self or Disease Self-Control? An Goris and Adrian Liston Baptiste N. Jaeger and Eric Vivier

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