Most autoreactive T cells are exposed to self-antigen either in the thymus or the periphery, and high avidity T cells are eliminated in both compartments. In contrast, lower avidity T cells are often found in ongoing autoimmune diseases (Bulek et al., 2012
) and in anti-tumor responses (McMahan and Slansky, 2007
). Thus, expression of a low avidity TCR, allowing autoreactive T cells to bypass elimination, is a major mechanism by which autoreactive T cells escape tolerance (von Herrath et al., 1994
; Nugent et al., 2000
; Zehn and Bevan, 2006
). However, up to this point, we had limited insight into the phenotypic and functional characteristics of these low avidity self-reactive T cells. Thus, it was unclear how self-antigen recognition in the thymus or periphery impacts the function of these T cells and whether such exposure imprinted mechanisms that restrict their activation. The failure to negatively select low avidity self-reactive T cells stimulated us to study how well these T cells respond to self-antigen in the periphery. To address these questions, we needed to establish a well defined experimental system in which we could study T cells expressing a TCR with physiologically relevant functional avidity for a defined self-antigen. We built such a model based on Rip-mOva mice. After identifying what types of Kb
/Ova-reactive T cells can be found in Rip-mOva mice, we generated the novel OT-3 TCR transgenic line to: determine the activation threshold of low avidity autoreactive T cells in the periphery; track the phenotype, cell fate, and function of these low avidity autoreactive T cells; and assess the effect of encountering a ligand at the threshold of negative selection in the thymus for T cell function in the periphery.
We observed that during an infection, T cells respond to a self-antigen that inefficiently induces negative selection when promiscuously expressed by mTec in the thymus. Moreover, even variants of the self-antigen that are weaker in stimulating the OT-3 T cells than Kb/Ova efficiently activate the T cells, induce normal effector and memory T cells, and cause diabetes. Why T cells respond differently to the same antigen in the thymus and the periphery remains unknown. Possibly, the number of peptide–MHC complexes resulting from the ectopic expression of antigen in the thymus is lower than the number of complexes found in an Lm-Ova infection or during presentation of endogenous Ova in the periphery. Thus, a weak ligand that fails to eliminate T cells in the thymus might sufficiently activate T cells in the periphery as the result of a higher epitope abundance. Along with higher ligand density, inflammation, higher levels of co-stimulation, and presentation by activated DC may create an environment that supports the activation of T cells even though they receive only very weak TCR stimulation.
The fact that inflammation and high levels of antigen presentation can activate T cells that survive negative selection appears less significant to us than the observation that the spared T cells induce autoimmunity and effectively destroy peripheral tissue cells. This indicates that there is a discrepancy between the safety margin imprinted by the thymus and what is needed to completely protect peripheral tissues from autoimmunity. Again, the question is why there is such a mismatch. Also in this situation, simply more peptide–MHC complexes could be presented on peripheral cells than during the ectopic expression and presentation in the thymus. Thus, instead of a T cell affinity threshold difference there could be an avidity (epitope abundance) mismatch between the thymus and the periphery. Whether or not such differences apply to all antigens or just a fraction of ectopically expressed TRA remains to be determined. An additional and non–mutually exclusive explanation for why there is a mismatch between thymic tolerance end peripheral T cell activation is that effector T cells might be more sensitive to antigen recognition than thymocytes when they are exposed to TRA in the thymic medulla. Consistent with this, it has been reported that effector T cells are very sensitive to antigen recognition and that sensitivity may increase as cells differentiate into effector T cells (Slifka and Whitton, 2001
). Overall, we interpret our findings to mean that cells which are at the threshold of negative selection pose sufficient reactivity to peripheral tissue antigen to cause disease.
Our observations also raise the question of what is needed to activate low avidity T cells that bypass negative selection. Our experiments shown in indicate that it is rather difficult to expand the endogenous Kb
/Ova-specific T cells in Rip-mOva mice. We considered the possibility that low avidity self-reactive T cells are exposed to antigen either in the thymus or in the periphery and that this exposure might render the cells less responsive to subsequent stimulation. We therefore asked whether Kb
/Ova-exposed OT-3 T cells showed any phenotypic or functional differences compared with OT-3 T cells obtained from control C57BL/6 mice but we found no evidence for such differences. Instead, OT-3 T cells from either source expanded and caused diabetes to a similar extent. This indicates that the impaired expansion of the endogenous population in Rip-mOva mice is not related to tolerance mechanisms but rather to the fact that the cells receive a very low TCR stimulation. In fact, we recently reported that the duration of T cell expansion (i.e., the number of cell divisions) directly correlates with TCR stimulation strength and that weakly stimulated T cells expand much less vigorously than strongly stimulated T cells (Zehn et al., 2009
). Thus, the critical factors determining whether or not autoimmunity develops is not only the ability to activate these T cells but, equally importantly, to expand them to numbers high enough that they cause noticeable tissue destruction.
Applying the latter consideration to the development of autoimmunity, we suggest that pathogens which provide cross-reactive ligands that activate the T cells more strongly than the self-antigen would effectively trigger autoimmunity. This conclusion reinforces the importance of activation of autoreactive T cells via molecular mimicry. Molecular mimicry proposes that T cells responding to a pathogen may cross-react with a self-antigen and thereby cause autoimmunity (Oldstone, 2005
). We mimicked the mechanisms that would occur during molecular mimicry by using pathogens that provide an antigen that is identical to a self-antigen. In a real molecular mimicry situation, a pathogen could introduce a cross-reactive antigen that stimulates autoreactive T cells with higher avidity than in our setup. This would lead to more vigorous expansion of autoreactive T cells and likely lead to higher disease incidence.
The finding that ligands which are weaker than the thymic selection threshold activate T cells in the periphery contrasts with earlier observations showing that T cells are more sensitive to antigen during negative selection in the thymus than they are in the periphery (Pircher et al., 1991
; Sant’Angelo and Janeway, 2002
). The earlier studies investigated selection against ubiquitously expressed antigens, whereas we studied TRA. This has consequences in so far as negative selection to ubiquitously expressed antigen can occur already in the thymus cortex (Baldwin et al., 2005
). Notably, miR-181a, a microRNA which enhances antigen sensitivity, is expressed at higher levels by double-positive thymocytes than by cells in the medulla or in peripheral T cells (Li et al., 2007
). miR-181a–mediated higher antigen sensitivity could enable negative selection against antigen in the thymus cortex, whereas the same antigen might fail to eliminate T cells in the medulla. Thus, exposure to ubiquitous self-antigen in the cortex could indeed impose a safety window, but we propose that this is not the case when T cells are negatively selected against TRA in the medulla. A less stringent selection against TRA than against ubiquitously expressed self-antigen is consistent with the fact that T cell–mediated autoimmune responses often target TRA.
Although high avidity T cells are normally deleted in the thymus (Kisielow et al., 1988
) or the periphery (Kurts et al., 1997
), it has also been shown that these T cells sometimes persist in the periphery (Schietinger et al., 2012
). Interestingly, these high avidity T cells show signs of prior activation and proliferation, they express high levels of CD44, and they are tolerant or anergic. In contrast, low avidity OT-3 T cells in Rip-mOva mice resembled naive T cells both phenotypically and functionally. They were CD44 low and showed no signs of previous antigen exposure. The observed tolerance of high but not low avidity T cells further supports the relevance of low avidity T cells in autoimmune pathology.
Although the induction of islet damage and autoimmune diabetes required two consecutive infections to initiate autoimmune diabetes in Rip-mOva mice, our data clearly demonstrate that severe autoimmunity can occur in normal mice with an unmanipulated TCR repertoire and in the presence of regulatory T cells (). This also indicates that low avidity autoreactive T cells form memory cells and mount a robust secondary response upon antigen reexposure. This is another significant difference from high avidity autoreactive memory T cells which are removed from the T cell repertoire over time (Kreuwel et al., 2002
). Moreover, the low numbers of autoreactive T cells seen after the primary infection implies that formation of memory T cells and repetitive stimulation are likely required to generate sufficient numbers of autoreactive low avidity T cells to cause tissue damage. This is consistent with the proposed relapsing-remitting disease progression for type 1 diabetes (von Herrath et al., 2007
), which proposes that multiple stimulations and increasing tissue damage cause autoimmune diabetes.
In conclusion, we demonstrate that T cells specific for TRA escape negative selection and can cause autoimmunity because of a mismatch between the threshold for negative selection to TRA and the threshold for T cell activation during an infection and the execution of effector function against peripheral tissues. This mismatch allows low avidity T cells to enter the periphery. Moreover, we showed that low avidity T cells, in contrast to high avidity T cells, persist without losing their self-destructive potential. We consider that these dormant autoreactive T cells constitute a significant source for cells which, even in the absence of genetic predisposition to develop autoimmunity, are able to cause organ-specific autoimmune damage.