Our analysis of the peripheral TRAV14 TCR repertoire strongly suggests that immune regulation is a tissue-specific activity, as the antigen specificity of the protective T reg cell population changes considerably by anatomical location (). These data are consistent with our previous observations of individual T reg TCR–driven proliferation in normal hosts, in which CFSE dilution was more pronounced in certain LNs (7
). Thus, the observation that tissue-specific antigens greatly modify the local T reg TCR repertoire provides direct evidence for a mechanism by which the presence of an organ facilitates T reg cell–mediated tolerance to that organ (9
Another intriguing observation is that the T reg and CD44hi
TCR repertoires showed similar patterns of variability by anatomical location. This suggests that T reg and CD44hi
populations share similar fundamental behaviors governing their interaction with tissue-specific antigens, even though the conditions for their development may be different. These data therefore support previous studies using monoclonal TCR transgenic mice (42
), demonstrating that both T reg and non–T reg cells undergo clonal expansion upon antigen encounter. The expansion and maintenance of T reg cells specific for antigens normally presented in the peripheral self would favor tolerogenic T reg cell responses to those antigens, rather than novel foreign antigens. Thus, we hypothesize that shaping of the T reg cell population to the local antigenic landscape provides another mechanism for self/nonself discrimination by the immune system.
Contrary to previous studies (27
), we did not find that peripheral conversion played a prominent role in the generation of the peripheral T reg cell population (). This difference could be explained in part by our use of Foxp3 as a marker for T reg cells, rather than CD25 (43
), to isolate non–T reg cells for adoptive transfer and analysis of conversion. Nonetheless, the use of mature Foxp3−
thymocytes may still overestimate the role of peripheral conversion because we cannot exclude the presence of rare CD25−
T reg cell precursors in this subset. We can also not be certain that these mature thymocytes are phenotypically identical to recent thymic emigrants in terms of their potential to undergo peripheral conversion. On the other hand, the use of peripheral Foxp3−
T cells may underestimate the role of peripheral conversion because we can only assess the fraction of Foxp3−
cells capable of converting at steady state, and those T cells with the greatest propensity to undergo peripheral conversion are therefore likely to be underrepresented. Thus, we believe our estimate that peripheral conversion contributes ~4–7% of the normal adult peripheral T reg cell population represents the best currently available figure.
We also studied the process of peripheral conversion during the immune response of Foxp3−
cells transferred into αβT cell–deficient hosts, which may mimic the lymphopenic and T reg cell–deficient conditions during the early neonatal period. We found that peripheral conversion occurred much more readily in this environment than in a lymphoreplete host, and this made it technically feasible to perform TCR usage analysis. Interestingly, we found that peripheral conversion in polyclonal populations did not mirror previous experiments using monoclonal T cell populations, where a portion (~20%) of cells became Foxp3+
after noninflammatory antigen encounter in both nonlymphopenic and lymphopenic models (23
). Rather, we observed no consistent patterns governing peripheral conversion during T cell expansion, finding that individual TCRs ranged from being highly skewed toward or against peripheral T reg cell development ( and Fig. S8). Thus, the analysis of polyclonal TCR repertoires from both lymphopenic and nonlymphopenic environments does not support a model in which peripheral conversion is an automatic consequence of T cell activation, but rather one in which peripheral conversion is highly dependent on TCR specificity.
Although our data do not directly allow us to determine whether peripheral conversion occurs upon recognition of nonself-antigens, we hypothesize that many TCRs that facilitate conversion are reactive to self-antigens. This is strictly based on the observation that several TCRs from converted T reg cells in lymphopenic hosts can also be found in the normal thymic T reg cell subset ( and Fig. S9). As previously argued, we believe that thymic T reg cell development occurs via recognition of self- (38
) rather than nonself-antigens (20
). Although we cannot exclude the possibility of peripheral nonself-antigen presentation in the thymus, nor the possibility that different peptides are recognized in the thymus and periphery, the most straightforward explanation is that the same self-reactivity that induces T reg cell development in the thymus also does so in the periphery. Although future experiments will be required to prove that the observed bias of converted TCRs toward the thymic T reg TCR repertoire is caused by the recognition of the same self-antigens, these data suggest that some cells that escape thymic T reg cell development express self-reactive TCRs that facilitate a second chance for T reg cell development in the periphery.
Peripherally converted T reg cells may develop directly from naive T cells or instead differentiate from memory/effector T cells (41
). In vivo data suggests that an appreciable frequency of Foxp3+
cells requires 1 wk or more to develop (23
). This time frame favors a multistep process but may also reflect a need for postconversion expansion. In contrast, in vitro studies have suggested that TGF-β–mediated induction of Foxp3 is considerably more efficient in naive than in memory T cells (45
). In fact, it has been reported that TGF-β is unable to induce Foxp3 in previously in vitro–differentiated T cells (46
). Because in vitro TCR stimulation is used in the retroviral transduction protocol, our data directly demonstrate that in vivo, peripheral conversion from previously activated T cells can still occur in the context of an appropriate TCR specificity (Fig. S7), hinting at possible differences between in vitro and in vivo conversion.
It is clear, however, that conversion alone is insufficient to maintain immune homeostasis, as shown by the development of colitis and other autoimmune manifestations upon transfer of T reg cell–depleted CD4+
T cells (3
). However, conversion may diminish the severity of disease (25
). The inability to prevent disease may be caused by the limited T reg TCR repertoire generated by peripheral T reg cell development (Fig. S7), which has been suggested to permit autoimmunity in lymphopenic mice (48
). Alternatively, it may be that peripheral conversion occurs too late to fully prevent autoimmune pathology. One interesting hypothesis is that autoimmune disease may also be exacerbated by self-reactive Foxp3−
T cells expressing T reg TCRs that escape into the periphery (7
) but are unable to undergo peripheral conversion because of their antigen specificity ().
These data demonstrate a critical role for TCR specificity in the decision to undergo peripheral conversion. One potential mechanism by which this occurs is that the “nature” of the TCR–ligand interaction itself could generate a unique signal that results in Foxp3 expression. This has been suggested by an αβTCR × cognate antigen double transgenic model of thymic T reg cell development (4
). In this model, the high affinity ligand induced both deletion and T reg cell development, whereas a slightly different, lower affinity ligand could only induce deletion. However, a mechanism dependent on unique TCR signals would be unable to distinguish the context of T cell activation in the periphery, making it teleologically less appealing. An alternative, nonmutually exclusive hypothesis is that the TCR specificity results in activation within an environment that facilitates peripheral T reg cell development. We favor this latter model, in which antigen presentation can occur on APCs and/or within microenvironments that determine whether a given T cell will become a regulatory or effector cell. Based on current data, we suspect that a microenvironment promoting peripheral conversion would contain TGF-β and potentially retinoic acid (14
). Therefore, the balance of antigen presentation between two different microenvironments would determine the pathological or tolerogenic outcome of the immune response to an antigen.