Incomplete depletion and rapid regeneration of Foxp3+ regulatory T cells following anti-CD25 treatment in malaria-infected mice

Investigation of the role of regulatory T cells (Treg) in model systems is facilitated by their depletion using anti-CD25 Abs, but there has been considerable debate about the effectiveness of this strategy. In this study, we have compared the depletion and repopulation of CD4(+)CD25(+)Foxp3(+) Treg in uninfected and malaria-infected mice using 7D4 and/or PC61 anti-CD25 Abs. We find that numbers and percentages of CD25(high) cells, but not Foxp3(+) cells, are transiently reduced after 7D4 treatment, whereas treatment with PC61 alone or in combination with 7D4 (7D4 plus PC61) reduces but does not eliminate Foxp3+ cells for up to 2 wk. Importantly, all protocols fail to eliminate significant populations of CD25(-)Foxp3(+) or CD25(low)Foxp3(+) cells, which retain potent regulatory capacity. By adoptive transfer we show that repopulation of the spleen by CD25high Foxp3(+) cells results from the re-expression of CD25 on peripheral populations of CD25(-)Foxp3(+) but not from the conversion of peripheral Foxp3(-) cells. CD25(high)Foxp3(+) repopulation occurs more rapidly in 7D4-treated mice than in 7D4 plus PC61-treated mice, reflecting ongoing clearance of emergent CD25(+)Foxp3(+) cells by persistent PC61 Ab. However, in 7D4 plus PC61-treated mice undergoing acute malaria infection, repopulation of the spleen by CD25(+)Foxp3(+) cells occurs extremely rapidly, with malaria infection driving proliferation and CD25 expression in peripheral CD4(+)CD25(-)Foxp3(-) cells and/or conversion of CD4(+)CD25(-)Foxp3(-) cells. Finally, we reveal an essential role for IL-2 for the re-expression of CD25 by Foxp3(+) cells after anti-CD25 treatment and observe that TGF-beta is required, in the absence of CD25 and IL-2, to maintain splenic Foxp3(+) cell numbers and a normal ratio of Treg:non-Treg cells.