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We and others have reported increased numbers of CD4+CD25+ T cells in peripheral blood of cancer patients as measured by percentage of CD4+ T cells. It has been postulated that this could represent increased natural or inducible regulatory T cell (Treg) subsets that may abrogate anti-tumor immune activity. While natural CD4+ T regulatory cells constitutively bear the α-chain of the IL-2 receptor (CD25), it is unclear whether the increased levels of CD4+CD25+ represent regulatory T cells or activated effector T cells. Since overexpression of the forkhead transcription factor FoxP3 is necessary and sufficient for natural Treg function, we analyzed expression of FoxP3 mRNA levels in CD4+ subsets of human PBMCs. Since CD4+CD25Hi T cells have been postulated to be the suppressive entity in human peripheral blood, we sorted CD4+ cells according to CD25+ expression (CD25Hi, CD25Lo and CD25- subsets) and performed qRT-PCR analyses and co-suppression assays. Herein we report that in the CD4CD25Hi subsets FoxP3 is expressed at 25-30 times the level of CD4+CD25- lymphocytes. FoxP3 was also overexpressed in the CD4+CD25Lo subset, but was only 3-5 times the level measured in CD4+CD25- cells. GITR, which is thought to be another Treg marker, was also highly overexpressed in the CD4+CD25HI population but was also found to be highly expressed after activation, whereas FoxP3 was not. Neuropilin-1 has been reported to be a specific marker for regulatory T cells in mice, but was undetectable in these CD4+ subsets. We also performed co-suppression assays of these subsets and found that the CD4+CD25Hi subset was anergic and suppressed the proliferation of CD4+CD25- cells. Sorted CD4+CD25Lo cells, however, proliferated strongly and suggest that the CD4+CD25Lo subset consists largely of newly activated effector cells. Together these data reinforce the notion that the determination between activated cell and natural Treg is very problematic. Since FoxP3 is overexpressed in the CD4+CD25Lo subset, it remains possible that Treg exhibit various levels of CD25 expression, but are “masked” by large numbers of CD4+CD25Lo effector cells in co-suppression experiments using sorted cells. These data suggest more assays and reagents need to be developed to fully enumerate and isolate the true natural Treg population in peripheral human blood. We are currently developing assays which can quantitate this immune compartment as well as other immune suppressive subsets (such as IL-10 and TGF-β secreting cells) in the blood and lymph nodes of cancer patients.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]