Abstract
The suppressive function of regulatory T cells (Tregs) is critical to the maintenance of immune homeostasis in vivo and yet, the specific identification of Tregs by phenotypic markers is not perfect. Tregs were originally identified in the CD4+CD25+ fraction of T cells, but FoxP3 expression was later included as an additional marker of Tregs as FoxP3 expression was identified as being critical to the development and function of these cells. Intracellular expression of FoxP3 makes it difficult in using to isolate live and not permeabilized cells for functional assays. As such CD4+CD25+ fraction is still frequently used for functional assays of Tregs. Although, the CD4+CD25+ fraction substantially overlaps with the FoxP3+ fraction, the minor mismatch between CD4+CD25+ and FoxP3+ fractions may confound the functional characteristics of Tregs. In this study, we isolated CD4+FoxP3+ as well as CD4+CD25+ fractions from Foxp3 knock-in mice, and compared their proliferative and suppressive activity in the presence or absence of various concentrations of IL-2. Our results showed comparable patterns of proliferative and suppressive responses for both fractions, except that contrary to the CD4+CD25+ fraction the FoxP3+ fraction did not proliferate in an autocrine fashion even in response to a strong stimulation. In presence of exogenous IL-2, both CD4+CD25+ and CD4+FoxP3+ fractions were more sensitive than the CD4+CD25- responder cells in proliferative responsiveness. In addition, a low dose IL-2 enhanced whereas a high dose abrogated the suppressive activities of the CD4+CD25+ and CD4+FoxP3+ fractions. These results may provide an additional understanding of the characteristics of the various fractions of isolated Tregs based on phenotype and function and the role of varying levels of exogenous IL-2 on the suppressive activity of these cells.
Highlights
Regulatory T cells (Tregs) play a key role in the maintenance of immune homeostasis in vivo as they suppress almost every kind of effector cells including CD4+ T cells, CD8+ T cells, B cells, and NK cells [1,2,3,4]
It is not easy to predict what happens upon IL-2 administration in vivo, and we investigated in the present study the proliferative and suppressive activity of Tregs in the presence of various concentrations of exogenous IL-2 in vitro, and compared the differences between CD4+CD25+ and CD4+FoxP3+ fractions in their functional responses to exogenous IL-2
In the absence of exogenous IL-2, CD4+CD25+ and CD4+FoxP3+ cells were hypoproliferative than CD4+CD25- responder cells in separate cultures, but hyperproliferative in the cocultures with the responder cells in vitro
Summary
Regulatory T cells (Tregs) play a key role in the maintenance of immune homeostasis in vivo as they suppress almost every kind of effector cells including CD4+ T cells, CD8+ T cells, B cells, and NK cells [1,2,3,4]. As for isolation markers of these cells, FoxP3 is a marker for these cells that has provided a high level of specificity. The CD4+CD25+ fraction has been documented to have many of the suppressive effects of candidate Tregs and studies have shown that the CD4+CD25+ fraction substantially overlaps with the FoxP3+ fraction; as such CD4+CD25+ fraction is still being used in many functional studies of Tregs, in particular, when the genetically engineered mice are not available or that the studies are from human subjects. One should be mindful of the imperfect match between the CD4+CD25+ and FoxP3+ fractions as investigators try to elucidate the function of the Tregs cells in various assays and that they may draw different conclusions if they use them interchangeably
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