A Breath of Fresh Aire
Authors: Ruan Zhang, Laurence A. Turka
Journal: Immunity (2013)

The origin and function of extrathymic Aire-expressing cells (eTACs) is incompletely defined. In this issue of Immunity, Gardner et al., 2013Gardner J.M. Metzger T.C. McMahon E.J. Au-Yeung B.B. Krawisz A.K. Lu E. Price J.D. Johannes K.P. Satpathy A.T. Murphy K.M. et al.Immunity. 2013; 39 (this issue): 560-572Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar show that eTACs are a distinct tolerogenic cell population that functionally inactivates CD4+ T cells to induce peripheral tolerance. The origin and function of extrathymic Aire-expressing cells (eTACs) is incompletely defined. In this issue of Immunity, Gardner et al., 2013Gardner J.M. Metzger T.C. McMahon E.J. Au-Yeung B.B. Krawisz A.K. Lu E. Price J.D. Johannes K.P. Satpathy A.T. Murphy K.M. et al.Immunity. 2013; 39 (this issue): 560-572Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar show that eTACs are a distinct tolerogenic cell population that functionally inactivates CD4+ T cells to induce peripheral tolerance. Immunological tolerance is the absence of a pathologic autoimmune response to self-antigens. In the case of T cells, central tolerance occurs during thymic development and peripheral tolerance provides a secondary level of protection against autoreactive lymphocytes that were not deleted or inactivated in the thymus. If one assumes that deletion or inactivation of self-reactive cells requires the presence of self-antigen (to distinguish them), then a paradox of central tolerance is how multiple peripheral antigens are made available in the thymus. Just over a decade ago, the “autoimmune regulator” (Aire) was shown to promote ectopic expression of peripheral tissue-specific antigens (PTAs) in thymic medullary epithelial cells (MECs), which impose central tolerance through negative selection of self-reactive T cells (Anderson et al., 2002Anderson M.S. Venanzi E.S. Klein L. Chen Z. Berzins S.P. Turley S.J. von Boehmer H. Bronson R. Dierich A. Benoist C. Mathis D. Science. 2002; 298: 1395-1401Crossref PubMed Scopus (1864) Google Scholar). In humans, lack of a functional Aire protein results in the development of a multiorgan autoimmune disease known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (Nagamine et al., 1997Nagamine K. Peterson P. Scott H.S. Kudoh J. Minoshima S. Heino M. Krohn K.J. Lalioti M.D. Mullis P.E. Antonarakis S.E. et al.Nat. Genet. 1997; 17: 393-398Crossref PubMed Scopus (1119) Google Scholar), and animal studies from Aire−/− mice recapitulate most of this phenotype (Anderson et al., 2002Anderson M.S. Venanzi E.S. Klein L. Chen Z. Berzins S.P. Turley S.J. von Boehmer H. Bronson R. Dierich A. Benoist C. Mathis D. Science. 2002; 298: 1395-1401Crossref PubMed Scopus (1864) Google Scholar). In addition to expression on thymic MECs, Aire is also expressed extrathymically on eTACs (extrathymic Aire-expressing cells) located in secondary lymphoid tissue (Gardner et al., 2008Gardner J.M. Devoss J.J. Friedman R.S. Wong D.J. Tan Y.X. Zhou X. Johannes K.P. Su M.A. Chang H.Y. Krummel M.F. Anderson M.S. Science. 2008; 321: 843-847Crossref PubMed Scopus (366) Google Scholar). Previously, Gardner et al. reported that eTACs are identified as CD45− stromal cells within the secondary lymphoid organs, capable of interacting with and deleting CD8+ naive autoreactive T cells (Gardner et al., 2008Gardner J.M. Devoss J.J. Friedman R.S. Wong D.J. Tan Y.X. Zhou X. Johannes K.P. Su M.A. Chang H.Y. Krummel M.F. Anderson M.S. Science. 2008; 321: 843-847Crossref PubMed Scopus (366) Google Scholar). Importantly, eTACs express a distinct and diverse array of self-antigens compared to mTECs. However, a number of questions remained about the identity of eTACs and their potential in inducing peripheral tolerance in CD4+ T cells. In this issue of Immunity, Gardner et al., 2013Gardner J.M. Metzger T.C. McMahon E.J. Au-Yeung B.B. Krawisz A.K. Lu E. Price J.D. Johannes K.P. Satpathy A.T. Murphy K.M. et al.Immunity. 2013; 39 (this issue): 560-572Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar provide evidence that eTACs are a bone-marrow-derived antigen-presenting cell (APC) population with phenotypic characteristics distinct from dendritic cells (DCs) and stromal cells. They further demonstrate that eTACs prevent CD4+ T cell-mediated autoimmunity via functionally inactivating this population of cells, providing the first evidence that Aire-mediated expression of PTAs in eTACs can be an important mechanism for CD4+ T cell tolerance (Figure 1). The term “antigen-presenting cell” or “professional APC” is frequently used to define cells that express both major histocompatibility complex (MHC) class I and class II molecules under basal conditions (i.e., in the absence of inflammatory stimuli). Depending upon context and other variables, such cells may play roles in the induction of both productive and tolerogenic T cell responses. Earlier, by using Aire-reporter mice, Gardner et al. had identified eTACs in lymphoid organs as a type of APC contained with the CD45− stromal cell population (Gardner et al., 2008Gardner J.M. Devoss J.J. Friedman R.S. Wong D.J. Tan Y.X. Zhou X. Johannes K.P. Su M.A. Chang H.Y. Krummel M.F. Anderson M.S. Science. 2008; 321: 843-847Crossref PubMed Scopus (366) Google Scholar). Now, to further characterize these cells, Gardner et al. traced the generation of eTACs in bone-marrow chimera mice and found that murine eTACs were not a stromal lineage but rather derived from the bone-marrow compartment and were partially radioresistant. Compared to CD11c+ DCs or B220+ plasmacytoid DCs, eTACs expressed high amount of MHC class II on their surface but very low levels of CD80 and CD86. Furthermore, low expression of CD45, CD11c, and CD11b in eTACs distinguished them from the DC cell lineage. Nevertheless, eTACs did share some common surface markers with DCs such as Zbtb46, a recently described DC marker and the transcription factor Deaf 1. Importantly the authors identified a human eTAC equivalent by immunofluorescent staining of lymph node sections and showed that human eTACs localized to the subcapsular zone between the T cell paracortex and B cell follicle. Human eTACs were uniformly MHCII+, CD45lo, and CD11clo. These data indicate that eTACs are a new type of APC with distinct features from stromal cells and DCs. However, it’s unclear whether eTACs and DCs share common hematopoietic progenitors, and further work will be necessary to determine the origin of eTACs and their relationship with DCs. To study how antigen presentation by eTACs affected CD4+ T cells, Gardner et al. used the BDC2.5 CD4+ T cell receptor (TCR) transgenic mouse model and inserted a BDC mimetope peptide into the MHC class II molecule-associated invariant chain under control of the Aire promoter (AdBDC mice). When BDC2.5 CD4+ T cells were adoptively transferred to wild-type (WT) nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, they proliferated rapidly and caused diabetes. However, in AdBDC mice, the transferred BDC2.5 cells proliferated extensively but ultimately were inactivated and failed to cause diabetes. There are multiple mechanisms by which this type of CD4+ T cell tolerance might occur, including deletion, regulation, and induction of “unresponsiveness” (often termed anergy). Deletion, if it occurred, was clearly incomplete because BDC2.5+ cells were recoverable from AdBDC mice. To study the issue of regulation, the authors deleted CD25+ regulatory T (Treg) cells from the inoculum of BDC2.5 T cells prior to adoptive transfer. Nonetheless, they observed that eTAC stimulation of BDC2.5 cells led to enrichment of Foxp3+ cells (presumably Treg cells). Critically, however, deletion of Foxp3+ cells enabled by the use of Foxp3-diphtheria toxic receptor transgenic mice revealed that prevention of diabetes did not depend on Foxp3+ cells. Additionally, BDC2.5 cells transferred into AdBDC mice cells did not demonstrate dominant-suppressive function to prevent diabetes when they were secondarily cotransferred with naive T cells to WT NOD SCID mice. Thus the presence of BDC2.5+ cells, which did not cause diabetes even in the absence of Treg cells, suggests that unresponsiveness is a major mechanism of tolerance. Confusingly, different terms have been coined to describe distinct states of T cell unresponsiveness. For example, clonal anergy typically refers to the inactive state of T lymphocytes generated in vitro by TCR occupancy in the absence of CD28 costimulation (Schwartz, 1996Schwartz R.H. J. Exp. Med. 1996; 184: 1-8Crossref PubMed Scopus (454) Google Scholar) and when occurring in vivo is often termed adaptive tolerance, a characteristic which persists despite repetitive antigen stimulation (Chiodetti et al., 2006Chiodetti L. Choi S. Barber D.L. Schwartz R.H. J. Immunol. 2006; 176: 2279-2291PubMed Google Scholar). The term exhaustion more commonly refers to the loss of T cell effector function following chronic antigen stimulation, most typically during chronic viral infections or in malignancy (Wherry, 2011Wherry E.J. Nat. Immunol. 2011; 12: 492-499Crossref PubMed Scopus (2425) Google Scholar). To characterize the nature of the unresponsive state induced by eTACs, Gardner et al. performed a number of assays. They noted that CD4+ T cells tolerized by eTACs had high expression of inhibitory receptors such as PD-1 and Lag3, as is typical of exhausted T cells (Wherry, 2011Wherry E.J. Nat. Immunol. 2011; 12: 492-499Crossref PubMed Scopus (2425) Google Scholar). However, blockade of the PD-1:PDL1 interaction did not prevent tolerance induction, suggesting that the unresponsiveness observed is distinct from exhaustion. In contrast, several lines of evidence suggested that the unresponsive state was akin to classical anergy (adaptive tolerance). eTACs expressed high amounts of MHC class II with low expression of the CD28-ligands CD80 and CD86. As well, deliberate activation of costimulatory signals in the form of anti-CD28 in vitro or anti-OX-40 in vivo partially abrogated anergy. This partial prevention of anergy is itself a bit difficult to interpret, because the CD28 activation experiments were only done in vitro, and OX40 exerted in vitro but not in vivo effects. Finally, the tolerized cells were found to harbor defects in calcium influx and mitogen-activated protein kinase phosphorylation, consistent with observations described earlier for adaptive tolerance (Chiodetti et al., 2006Chiodetti L. Choi S. Barber D.L. Schwartz R.H. J. Immunol. 2006; 176: 2279-2291PubMed Google Scholar). As with any provocative study, questions are raised as well as answered. Recently, TCR-mediated induction of E3 ubiquitin ligases such as Cbl-b, Itch, and Grail has been suggested to mediate induction and maintenance of T cell anergy (Mueller, 2004Mueller D.L. Nat. Immunol. 2004; 5: 883-890Crossref PubMed Scopus (185) Google Scholar). It will be worthwhile to determine whether the strong TCR signaling and absence of costimulation provided by eTACs actually lead to the expression of these genes. Also important will be determining whether or not the unresponsiveness induced by eTACs is stable and/or progressive to apoptosis or, alternatively, might be reversible by inflammatory or growth-promoting cytokines (Figure 1). Anergy induced by TCR stimulation in the absence of costimulation might be a metastable event in vivo ultimately leading to cell death (Li et al., 2001Li X.C. Strom T.B. Turka L.A. Wells A.D. Immunity. 2001; 14: 407-416Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Whether this occurred in the case of eTACs could not be determined from the present study, nor is it clear whether delivery of CD28 costimulation in vivo will block tolerance. The answer(s) to these questions will be important if these findings are to be harnessed into a potentially new and “fresh” therapeutic approach for autoimmunity and transplantation. Extrathymic Aire-Expressing Cells Are a Distinct Bone Marrow-Derived Population that Induce Functional Inactivation of CD4+ T CellsGardner et al.ImmunityAugust 29, 2013In BriefThe autoimmune regulator (Aire) is essential for prevention of autoimmunity; its role is best understood in the thymus, where it promotes self-tolerance through tissue-specific antigen (TSA) expression. Recently, extrathymic Aire-expressing cells (eTACs) have been described in murine secondary lymphoid organs, but the identity of such cells and their role in immune tolerance remains unclear. Here we have shown that eTACs are a discrete major histocompatibility complex class II (MHC II)hi, CD80lo, CD86lo, epithelial cell adhesion molecule (EpCAM)hi, CD45lo bone marrow-derived peripheral antigen-presenting cell (APC) population. 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