Significant evidence demonstrates that normal thymic deletion of autoreactive T cells is an incomplete process. For example, depletion of FoxP3+ regulatory T cells in healthy adult mice unleashes the function of autoreactive T cells that have escaped negative selection, resulting in the development of lethal autoimmunity.44
Also, immunizing healthy mice with autoantigens can break tolerance. By injecting healthy mice with some self antigens, such as myelin basic protein in adjuvant, quiescent autoreactive T cells that have escaped deletion are primed and cause autoimmune attack on the tissues expressing these proteins.45
Marrack et al. argue that “leaky” central tolerance benefits the host by generating a larger T cell repertoire for peripheral immunity to infection. 46
Given that a significant number of autoreactive T cells evade thymic deletion, mechanisms of peripheral tolerance must exist to keep these cells in check.
A novel mechanism of peripheral self tolerance was revealed with studies of Aire in peripheral lymphoid organs.13, 15, 47
Although it was initially assumed that dentritic cells (DCs) were the source of peripheral Aire expression – early studies of Aire in the periphery were focused on DC function48
– current work shows that Aire expression in the lymph nodes and spleen derives from radio-resistant cells in the secondary lymphoid organs and not CD11c+ DCs.47
By generating reporter mice that express GFP under the control of the Aire locus, our lab was able to identify this unique population of extrathymic Aire-expressing cells (eTACs).34
Like Aire expressed in mTECs, Aire in secondary lymphoid organs is required for the expression of many TSAs.34
In order to test the ability of eTACs to induce tolerance to T cells, the islet specific glucose-6-phosphatase (IGRP) antigen was targeted to these cells by engineering a transgene that expressed IGRP under the control of the Aire locus, which was called the Aire-driven IGRP (Adig) mouse model.34
IGRP is not normally expressed in the thymus or secondary lymphoid organs of wild type mice. However, expression of Aire-driven IGRP was detected in Aire+ mTECs and eTACs of the Adig mouse.34
When IGRP-specific CD8+ T cells were transferred into the Adig mouse, eTACs stimulated IGRP-specific CD8+ cell proliferation and death by directly presenting IGRP to these T cells.34
The Adig mouse demonstrates that, like Aire expressing mTECs, eTACs can help to impose tolerance by deleting autoreactive CD8+ T cells.34
Other TSA-expressing, CD45-negative cell subsets in lymph nodes have been described that do not express Aire. For example, a mouse model which expresses a transgene consisting of a truncated OVA (tOVA) antigen under the control of the intestinal fatty acid binding protein (iFABP) promoter, expresses OVA in the intestinal epithelium as well as the stromal cells of peripheral lymph nodes.47, 49
When CD8+ OVA-specific TCR transgenic T cells are injected into iFABP-tOVA mice, the T cells proliferate and are eventually deleted.47
Thus, like eTACs, non-Aire-expressing stromal cell subsets can present TSAs to autoreactive CD8+ T cells, causing the deletion of these potentially harmful cells.
In both the Adig and iFABP-tOVA mice, antigen expression is driven by a transgene. Thus, it is possible that antigen-specific CD8+ cells were deleted due to engagement of non-physiologically high levels of antigen expression. However, Nichols et al. recently showed that a stromal cell subset in lymph nodes expresses the TSA, tyrosinase, and can present this antigen to tyrosinase-specific CD8+ T cells.50
In this model system the endogenous expression level of tyrosinase in these stromal cells was enough to delete tyrosinase-specific CD8+ T cells, providing evidence that physiological expression of TSAs by stromal cells can activate and delete antigen-specific CD8+ T cells, similar to transgenic TSA expression.50
Overall, the existence of different TSA-expressing CD45-negative cell subsets in peripheral lymphoid organs suggests that this class of cells provides a significant survival advantage; the evidence thus far demonstrates that eTACs and TSA-expressing stromal cell subsets play an important role in the maintenance of peripheral self tolerance by deleting autoreactive CD8+ T cells ().
Model of T cell activation by APC under steady state conditions or during a viral infection
There are several properties of both eTACs and non-Aire expressing, TSA-expressing stromal cells which make them well suited to play an important role in peripheral tolerance. First, location of these cells in the lymph nodes is ideal for tolerizing autoreactive T cells. Naïve T cells constitutively traffic through lymph nodes but generally do not traffic through tissues under normal circumstances.51
Thus naïve autoreactive T cells may be tolerized in secondary lymphoid organs and not at the site of the tissue itself. Second, eTACs express a different set of tissue restricted antigens than Aire expressing mTECs; Aire regulates 163 genes in eTACs and 1835 genes in mTECs, but there are only 7 overlapping genes that are controlled by both peripheral and thymic Aire.34
By expressing a different set of TSAs in the periphery, eTACs may tolerize antigen-specific T cells that were not deleted by Aire-expressing mTECs.
The Adig model system demonstrates that eTACs and other TSA expressing stromal cell subsets in peripheral lymphoid organs can function to delete naïve, autoreactive CD8+ T cells in a manner similar to that which Aire-expressing mTECs delete autoreactive thymocytes. Although Aire-expressing mTECs and eTACs present different self antigens, are their functions otherwise redundant? Are there differences between how eTACs and Aire-expressing mTECs delete autoreactive T cells? Thus far, the characterization of eTACs was guided by studies of Aire expressing mTECs, thereby biasing findings to those which are similar to thymic selection. The following is a discussion of features unique to eTACs which may allow this APC population to provide functions distinct from Aire expressing mTECs.
Immunofluorescent staining shows that eTACs are found primarily in the B cell-T cell boundary of T cell zones of peripheral lymphoid organs, in close proximity to CD11c+ APCs.34
Two-photon imaging experiments show that autoreactive T cells can make stable contacts with eTACs in the B cell-T cell boundary of the T cell zone, the same microenvironment in which antigen-specific T cells have been shown to be primed by LPS-activated DCs.34, 52
The proximity of eTACs to sites of T cell priming may allow them to serve a unique function in shaping the fate of TCR-triggered T cells. In other words, perhaps unlike Aire expressing mTECs, eTACs can receive cues from and/or play an important role in local immune responses. In line with this hypothesis, Fletcher et al. have shown that some non-Aire-expressing stromal cell subsets in the lymph node can present TSAs and potentially respond to viral infections through TLR3 signaling.49
Interestingly, one of the TLR3-expressing stromal cell subsets, fibroblastic reticular cells (FRCs), can stimulate and delete autoreactive CD8+ T cells.47, 49
However, upon stimulation with TLR3 agonist, FRCs decrease TSA expression, thus losing the ability to stimulate autoreactive T cell proliferation ().49
It is thought that TLR3 agonist decreases TSA expression on FRCs to reduce the chance of stimulating autoreactive T cells and prevent the development of autoimmunity during clearance of double-stranded RNA viral infections.49
eTACs do not express TLR3, 7, or 849
, but it is not known whether they express other receptors that can detect the presence of foreign microorganisms or endogenous ‘stress’ ligands. That is, studies of infection models could be used to determine whether like FRCs, eTACs can be directly stimulated through innate receptors like TLRs, NLRs, and Rig-like helicases, and/or APC-derived ‘stress’ ligands like inflammatory cytokines, heat-shock proteins, etc. Unlike the thymus, secondary lymphoid organs are well suited for sensing pathogenic processes in the periphery and organizing appropriate immune responses to these processes. Therefore, it would not be surprising if a feature of eTACs that distinguishes them from Aire-expressing mTECs is the ability to somehow adapt to changing environments caused by infection or inflammation.
Although autoreactive CD8+ thymocytes and mature CD8+ T cells are similarly deleted by mTECs and eTACs, respectively,21, 34
our lab has recently found that CD4+ T cells respond differently. While CD4+ thymocytes are deleted or shunted toward the FoxP3+ Treg lineage after interacting with Aire expressing mTECs,21, 24, 25
mature CD4+ T cells are triggered to proliferate and become functionally inert FoxP3-negative cells after interacting with eTACs [Gardner, Metzger, and Anderson, unpublished observations]. The difference in reactivity of CD4+ thymocytes and mature CD4+ T cells to Aire expressing mTECs and eTACs demonstrates that at least some of the functions of thymic and peripheral Aire are not redundant. Further studies of autoreactive CD4+ T cell reactivity to Aire expressing APC may be able to address the following questions: 1) Are the different effects of peripheral vs. thymic Aire on CD4+ T cell function T cell or APC intrinsic? and 2) What are the unique signals that control CD4+ T cell deletion vs. anergy?
A better understanding of the commonalities and differences between Aire-expressing mTECs and eTACs is needed to delineate the evolutionary pressure for peripheral Aire expression. The study of peripheral Aire’s effect on T cells, particularly CD4+ T cells, in infection models will be able to better define the role of eTACs.