Less than one percent of thymocytes express OX40, and these cells are primarily CD4+CD8− in phenotype. About a quarter of them express CD25 and include FoxP3+ Treg cells and their precursors. Among the remainder, some are the presumptive precursors of a subpopulation of naïve T cells that is distinguished by its gene expression profile, cell surface phenotype, and by its TCR repertoire. The most distinctive characteristic of OX40+ thymocytes is the fact that they are enriched for indications of a strong signaling response to thymic selection ligands. Thus, OX40 is a useful marker for cells that fall at the top end of the distribution of responses to thymic self antigens, and consequently OX40 induction could represent a useful means for identifying cells that are of significance either for suppressing or potentiating autoimmunity.
allele we have used in this study is induced in response to TCR stimulation in a similar fashion to that of OX40. It is more active in CD4+
T cells, and its level of induction is a function of the strength of the TCR stimulus a cell experiences (although it is likely that other signals can also influence its expression (54
)). We show that strongly agonistic ligands of a LACK/I-Ad
-specific TCR induced more Cre recombination than ligands that were weakly agonistic; we have also made similar observations with a panel of ligands for a class I-restricted TCR expressed on CD8+
T cells (data not shown). The relationship between strength of stimulus and amount of Ox40-cre
induction predicted that the few thymocytes expressing OX40 would be enriched for those making strong responses to selecting ligands. Consistent with this, phosphotyrosine and phospho-S6 levels were both elevated in OX40+
cells, and they showed a cell surface phenotype expected of cells that are strongly stimulated.
The analysis of mice that feature constrained TCR repertoires has revealed differences but substantial overlap between regulatory and conventional TCR repertoires (17
). The overlap is inconsistent with commitment to the Treg lineage being solely a consequence of strong reactivity to thymic self ligands. It is, however, permissive of models of Treg cell development in which strong TCR signaling potentiates the Treg fate in a probabilistic fashion and/or in combination with other factors (41
). Conversely, it allows for the formation of naïve T cells from thymocytes that might have experienced the same magnitude of TCR signaling response as Treg precursors. OX40 is a potential marker for the latter class of thymocytes based on its induction as a function of TCR signal strength.
Regulatory T cells are a prominent product of the thymus in mice that carry transgenes encoding TCRs with high affinity for self ligands (14
). This finding is suggestive of a potential instructive role for strong recognition of self in commitment to the regulatory lineage. In support of this, Hsieh et al
. found that TCRs cloned from Treg cells were associated with enhanced capacity to drive T cell expansion in lymphopenic hosts, or to induce autoreactive proliferative responses in vitro
). Although these results have been challenged by other work that failed to show evidence of self-reactivity in the Treg TCR repertoire (18
), they provide a straightforward explanation for the observed differences in the Treg and conventional TCR repertoires (i.e.
, that the former contains more strongly self-reactive specificities than the latter). Moreover, they are consistent with observations presented here that thymocytes marked by OX40 and/or CD25 upregulation are enriched for cells making stronger signaling responses than cells that lack expression of these molecules.
Intrathymic injection experiments have shown that FoxP3−
thymocytes include precursors of Treg cells (41
). Upregulation of FoxP3 in these cells can occur in a fashion that is independent of persistent engagement of TCR ligands, and it correlates with a capacity to signal productively in response to IL-2 (57
). Notably, not all CD25hi
cells induce FoxP3 subsequent to IL-2 treatment, and similarly, a substantial fraction of them do not become FoxP3+
after intrathymic transfer (41
). The majority of CD25hi
thymocytes coexpresses OX40, and therefore should be marked by Ox40-cre
. Hence, at least some of the YFP+
T cells in Ox40-cre
/YFP mice are derived from CD25hi
thymocytes, these likely being the cells that do not become Treg cells following intrathymic transfer (41
Sub-maximal expression of YFP from the ROSA26
-YFP allele identifies thymocytes that have recently undergone Cre-mediated recombination (42
). Our analysis of this population did not reveal clear evidence that OX40 upregulation depends on prior CD25 upregulation or vice versa. We noted that the frequency of CD25hi
cells was higher in the YFPhi
population than in the YFPint
population, an observation that could be consistent with upregulation of CD25 occurring after that of OX40 in some cells. As mentioned, however, there are other possible explanations for this. Intrathymic injection experiments could help to resolve what fraction of OX40+
thymocytes might become CD25+
, but such experiments would not readily address the more compelling question of whether any of the YFP+
cells are derived from OX40+
cells that do not transition through a CD25hi
state. This question is compelling simply because answering it would help to clarify the developmental paths that lead to the YFP+
population and the points at which they might diverge from the path to Treg cells.
The cell surface phenotype of YFP+
T cells was different from that of YFP−
T cells and suggestive of ongoing differences in their responses to self-ligands, and/or a persistent imprint of their distinct thymic experiences. In particular, we noted enrichment for high expression of Ly-6C, which has previously been identified as a marker for naïve T cells that are proficient at providing help for plasma cell differentiation (58
). Ly-6C has been implicated in lymphocyte homing (59
) suggesting that the YFP+
T cells might exhibit different migration patterns in the body from other naïve T cells (although we have not yet detected such differences). Other changes, such as elevated CD69, and decreased CD62L on some of the YFP+
cells were suggestive of partial activation (partial because the cells were low for CD44 expression).
Microarray profiling provided a deeper perspective on differences between the YFP+ and YFP− CD44lo cells, and yielded a substantial collection of differentially expressed genes. This collection was enriched significantly for genes associated with lymphocyte activation consistent with the flow cytometry results just mentioned. Although some of the differentially expressed genes could be attributed to the presence of a small number (~5% of the total) of FoxP3+ cells within the YFP+ population, most of them had a distinct origin and were indicative of true differences between the two populations. In addition to the value of these experiments in identifying differences, they were also of use in assessing the relatedness of the YFP+ CD44lo cells to naïve (i.e., YFP− CD44lo), memory, and regulatory cells. Here again, we noted that the YFP+ cells shared aspects of their gene expression pattern in common with memory and regulatory cells but were nonetheless most closely related to naïve T cells. This was especially true when we focused our analysis on genes that are normally expressed at higher levels in naïve T cells compared to memory cells, and thus are likely to include genes essential for maintaining the naïve state.
In summary, the results presented here show that prior expression of a gene that is normally associated with peripheral T cell activation can be used to identify a distinct subpopulation of naïve T cells. These cells are expected to be enriched for expression of TCRs that have above-average reactivity for thymic self ligands because they induced strong signaling responses during positive selection. They are therefore of potential significance in the context of autoimmunity. The Ox40-cre allele is representative of a class of lineage marking alleles that in principle can be used to split the naïve T cell repertoire on the basis of differential thymic selection experiences. Such alleles should allow for correlations to be drawn between gene expression history and functional properties of T cells, and thus promise a unique perspective on the pathway to healthy and diseased immune responses.