The emergence of T reg cells as an essential pathway in maintaining immune tolerance has opened the opportunity for a better understanding of immune homeostasis and the potential for therapeutic intervention. However, unlike the mouse, the human phenotyping of T reg cells has been complex. Typically, investigators have noted that the most suppressive T reg cells coincide with the CD4+
T cells with the brightest CD25 staining. Unfortunately, the ability to accurately gate for CD25 is rather arbitrary as no other cell surface marker can be used to definitively identify the subset. Baecher-Allen has suggested that other markers such as HLA-DR allows for subdividing the CD4+
subset to enrich T reg cell activity even further (41
). However, this additional marker suggests that the number of T reg cells is even less than previously suggested although a recent paper published by this same group showed that DR−
T reg cells are suppressive as well but with a different kinetics and different mechanism (42
). The identification of FoxP3 as a specific transcription factor that marks these cells suggests that there may be a larger population of T reg cells in human peripheral blood than previously appreciated, although this has been controversial as the result of unanticipated expression in several activated CD25−
T cell populations (30
). However, these studies have been compromised by the absence of cell surface markers that can be used to isolate these and other T cell subsets to examine T reg cell activity because FoxP3 cannot be used as a means to purify the cells for function. In this paper, we demonstrate that CD127 expression is an excellent biomarker of human T reg cells, especially when combined with CD25. The combination of these markers identifies T reg cells that account for up to 7–8% of CD4+
T cells, a significantly greater percentage than identified by previous approaches. Moreover, these cells suppress the proliferative response of alloreactive T cells in an MLR and are themselves anergic to the same stimuli, characteristics shared by CD4+
human T reg cells.
These results raise several critical issues. First, because the majority of CD4+FoxP3+ T cells may fall outside the typical gate for human T reg cells, studies used for functional and immunophenotypic analyses are potentially missing a large number of putative T reg cells. This has important implications in determining quantitative differences in patients with a variety of diseases. Second, the fact that CD4+CD127−CD25− T cells suppress an allogeneic MLR calls into question those studies suggesting that FoxP3 is not a “good” marker for T reg cell activity. It may be that FoxP3 is an excellent marker and that the small populations that arise during normal T cell activation are indeed adaptive T reg cells expanding as a consequence of suboptimal or supraoptimal TCR signaling. It should be emphasized, however, that not all FoxP3+ T cells are necessarily T reg cells and their activity may depend on the level of FoxP3 expression and isoforms of the protein expressed. However, these CD4+CD25+CD127lo/−, once isolated, may be treated in vitro with TGFβ or other factors to enhance T reg cell function in these cells. Third, efforts to select T reg cells for in vitro expansion may be hindered by the underestimation of T reg cells in any separation strategy based on CD25 expression. The ability to identify and select a significantly greater number of T reg cells circulating in the peripheral blood of humans, especially those with autoimmune diseases, is likely to make it easier to expand sufficient cell numbers for immunotherapy. Finally, the identification of CD127 as a marker that distinguishes effector/memory from T reg cells may suggest that anti-CD127 therapy might be appropriate for the treatment of autoimmune diseases such as T1D, systemic lupus erythematosis, or multiple sclerosis.
The identification of CD127 as a useful marker was heralded by genetic observations. First, microarray analysis of mRNA from individual T cell subsets showed that CD127 was expressed at significantly lower levels in CD4+CD25hi versus CD4+CD25− T cells. Unlike the majority of activated T cells, which rapidly reexpress CD127 and memory T cells that express high levels of CD127, the T reg cell population remains CD127lo/−. There may be two reasons for this. First, T reg cells may be constantly undergoing antigenic stimulation that is CD28 dependent, resulting in continued signaling that shuts down CD127 mRNA transcription. In this regard, it is interesting to note that activation of naive T cells by anti-CD3 plus anti-CD28 but not anti-CD3 alone led to a rapid down-regulation of CD127 (unpublished data), suggesting that CD28 signals are uniquely involved in regulating CD127 down-regulation. Alternatively, and not mutually exclusively, is the possibility that FoxP3 expression in this T cell subset controls CD127 expression. There are several reasons that this may be the case. As illustrated in the flow cytometric staining profiles, the more FoxP3 expression, the less CD127 (). In addition, overexpression of FoxP3 in transgenic mice results in a uniformly CD127lo/− population of cells with suppressive activity. Finally, data generated using CHiP analysis (first by ChIP-Chip followed by ChIP-qPCR) suggested that the CD127 promoter is a target for FoxP3 binding. It will be critical to determine whether the low expression of CD127 is indeed a consequence of constant antigen exposure in vivo or FoxP3 up-regulation resulting in CD127 gene repression, although these are not mutually exclusive.
T cells suppress quite effectively, although the percentage of FoxP3+
T cells in this subset can be quite variable. These results suggested that the CD127 marker may be useful in identifying different subtypes of T reg cells including Tr1 and TH3 cells. In this regard, there are currently several settings, including the treatment of humans with T1D with anti-CD3 that induces T cells with a regulatory phenotype (11
). These studies, which mimic similar results in T reg cell–deficient mice treated with nonmitogenic anti-CD3 (10
), indicate that it may be possible to identify an “adaptive” T reg cell response using CD127 as a biomarker in addition to lower CD25 expression previously observed on these cells.
One of the more intriguing aspects of the results is the seeming dichotomy in cytokine receptor expression in memory T cells versus T reg cells. Although a high percentage of T reg cells now appear to be IL-7R low and IL-2R positive, memory T cells have the opposite phenotype, expressing high levels of IL-7R and low levels of IL-2R. The theoretical basis for this differential expression is unclear but it may reflect the evolution of distinct pathways for cell survival and expansion of these T cell subsets. For instance, it is possible that T reg cells may play a critical role in normal homeostasis. Thus, the cells attempt to regulate the earliest immune perturbation that may occur in the absence of a pathogenic response. Because IL-2 is an “early cytokine” produced rapidly by activated T cells in the draining lymph nodes, IL-2 may be a critical signal for awakening the T reg cell response that can effectively suppress T cell expansion in these lymphoid tissues (45
). In contrast, IL-7 is commonly produced locally in sites of inflammation leading to increased survival and expansion of effector cells. If this localized IL-7 expression promoted T reg cell expansion, it might be counterproductive. Moreover, avoiding competition for the use of the common γ chain by these receptors would enhance the functionality of the cytokine function. Finally, it should be noted that the situation might be quite distinct in thymus when all the pre–T and immature T cells are CD127+
. At this stage in development, other factors might come into play to determine the differentiation pathways that determine whether a T cell becomes a T reg cell or a naive conventional T cell.
Several studies have examined the number and function of T reg cell cells in humans with autoimmune diseases. In some settings, such as multiple sclerosis, T1D, and autoimmune polyglandular syndrome II, the data, based on the number and function of CD4+
T reg cells, suggest that there are either fewer T reg cells or less functional T reg cells in diseased individuals (24
). However, in T1D and other autoimmune diseases, there have been contradictory results (25
). In the present study, we reevaluated T reg cells in patients with T1D as compared with normal individuals. Using the new markers, FoxP3 and CD127, we analyzed the frequency of CD4+
T cells. In this study, it is clear that human T reg cells as defined by CD4, CD25, CD127, and FoxP3 expression are present within the same range of percentages as control individuals with no autoimmunity (, a and b). Moreover, the functionality of the T reg cells isolated from the patients with T1D cannot be distinguished from healthy control subjects (unpublished data). We cannot explain the basis for differences between our studies and those of others in the T1D field. It has been suggested that the discrepancy might be the result of subtle differences in flow cytometry–based techniques for cell separation or different mAbs used. However, our use of distinct markers that identify the overwhelming bulk of T reg cells in human peripheral blood is likely to be a more definitive assessment of the T reg cell numbers and functional potential in this patient population. Lastly, it is interesting to note that several of the T1D patients had high T reg cell numbers as compared with the bulk of the control and T1D subjects. This is consistent with some studies in other autoimmune diseases in which the frequency of CD4+
T cells was reported to be increased as compared with controls. Moreover, these results fit with mouse studies, demonstrating increased T reg cell number at the time of T1D disease onset as well as other immune disease settings (unpublished data). We hypothesize that rather than a T reg cell deficiency being the cause of disease precipitation, there is actually increased T reg cell activity in an attempt to stem the increasingly aggressive effector cells that may indeed become T reg cell resistant.
In summary, we have determined that CD127 is an excellent marker of T reg cells in human peripheral blood. The cell surface marker is expressed at low levels on an overwhelming majority of T reg cells and distinguishes up to 10% of CD4+ T cells as potential T reg cells. Moreover, the cell surface marker can be used, in the absence of CD25 to separate a suppressive T cell subset and will thus be a useful tool for the selection and expansion of T cell for diagnostics and therapeutic applications.