To date, most investigators have utilized FOXP3 as a “benchmark” for human Treg despite the newly available information that transient expression of this transcription factor occurs in non-regulatory activated T cells (Ziegler, 2006
; Mottet and Goishayan, 2007
; Roncarolo and Gregori, 2008
; Ahmadzdeh et al, 2008
). Also, FOXP3 is not expressed in all Treg and, more importantly, is not a cell surface molecule. Therefore, it cannot be used for isolation of human Treg. Another surface marker, CD25 (a high affinity IL-2Rα) has been adopted for this purpose, based on the rationale that all Treg are dependent on IL-2 for their function and expansion (Fontenot et al., 2005
; Bayer et al., 2007
). Consequently, CD4+
T-cell subsets have been designated as Treg subsets, which has led to a considerable confusion regarding phenotypic and functional features of Treg in humans. Our results clearly show that the CD4+
subset contains CD4+
effector cells in addition to Treg, and that Treg represent a minority of CD4+
T cells. This finding is consistent with weak suppression mediated by this T-cell subset in the presence of exogenous IL-2. On the other hand, the definition of CD25high
T cells by quantitative flow cytometry has been largely arbitrary and inconsistent, as no definite expression criteria have been so far defined for the “high” CD25 expression level on CD4+
T cells. In view of this unsatisfactory situation, which is exacerbated by the differences that seem to exist between murine and human Treg, there is an urgent need for a more stringent and effective strategy of Treg isolation in humans.
The recent identification of a new surface marker, CD39 ectonucleotidase, for the isolation and characterization of human Treg is a highly desirable development. The advantage of this marker is that it recognizes Treg with suppressor activity mediated via pericellular adenosine, which is the end product of enzymatic degradation of ATP (Robson et al., 2006
). Thus, CD39 defines Treg based not only on the phenotypic but also functional characteristics. Also, CD39 expression on Treg is associated with that of another enzyme, ecto-5″neuleotidase or CD73, which mediates cleavage of AMP to adenosine. Adenosine has been considered to be a strong suppressor of T cell functions (Zarek & Powell, 2007
). However, before this new surface membrane-associated enzyme can be accepted as the Treg marker, it is necessary to consider properties of the T-cell subset it defines relative to those of widely used CD4+
T cell subsets.
T-cell subset represents 7±4% of CD3+
T cells in the circulation of NC and 12±3% in the blood of patients with cancer. The frequency and expression levels of CD39 correlate with those of FOXP3 in CD4+
T cells, especially in the peripheral blood of cancer patients, where nTreg are both more numerous and more functionally active (Strauss et al., 2007a
). They also correlate with the frequency and expression levels of CD25high
receptor. Thus, it appears that CD4+
T cell subset largely but nor completely overlaps with the CD4+
subset, and that single-sorted populations of these cells have similar but not identical phenotypic and functional attributes. Using antibodies specific for CD39 instead of IL-2Rα for nTreg isolation is an advantage, as it allows for capture of the Treg subset with broader and stronger suppressor functions than those mediated by CD4+
We have determined that CD39 allows for the isolation of more than 90% of highly suppressive FOXP3+
Treg in the blood of NC as well as patients with cancer. However, cancer patients have a higher frequency of CD39+
Treg which mediate significantly higher levels of suppression than their counterparts isolated from the circulation of NC. This suggests that chronic antigenic stimulation, such as that existing in patients with cancer, leads to activation and expansion of CD39+
Treg. Similar results were previously reported by us for CD4+
T cells (Strauss et al., 2007a
). The overlapping expression of CD39 and CD25high
markers on Treg in the peripheral circulation of NC and patients with cancer supports the conclusion that these CD4+
T cell subsets, although not identical, represent highly-enriched populations of suppressor T cells.
In aggregate, the presented data indicate that the immuno-bead method for human CD39+ Treg isolation from the peripheral blood of NC or patients with cancer consistently yields Treg with a high purity and strong suppressor function.