Phenotypic complexity of T regulatory subsets in patients with B-chronic lymphocytic leukemia

Increased numbers of T regulatory (Treg) cells are found in B-chronic lymphocytic leukemia, but the nature and function of these Tregs remains unclear. Detailed characterization of the Tregs in chronic lymphocytic leukemia has not been performed and the degree of heterogeneity of among these cells has not been studied to date. Using 15-color flow cytometry we show that Treg cells, defined using CD4, CD25, and forkhead box P3 (FOXP3), can be divided into multiple complex subsets based on markers used for naïve, memory, and effector delineation as well as markers of Treg activation. Furthermore FOXP3+ cells can be identified among CD4+CD25− as well as CD8+CD4− populations in increased proportions in patients with chronic lymphocytic leukemia compared with healthy donors. Significantly different frequencies of naïve and effector Tregs populations are found in healthy donor controls compared with donors with chronic lymphocytic leukemia. A population of CCR7+CD39+ Tregs was significantly associated with chronic lymphocytic leukemia. This population demonstrated slightly reduced suppressive activity compared with total Tregs or Tregs of healthy donors. These data suggest that FOXP3-expressing cells, particularly in patients with chronic lymphocytic leukemia are much more complex for Treg sub-populations and transitions than previously reported. These findings demonstrate the complexity of regulation of T-cell responses in chronic lymphocytic leukemia and illustrate the use of high-dimensional analysis of cellular phenotypes in facilitating understanding of the intricacies of cellular immune responses and their dysregulation in cancer.