Characterization of Treg Subpopulations in Chronic Lymphocytic Leukemia Using 15 Color Flow Cytometry.

Abstract 1644

Poster Board I-670

CD4+ regulatory T cells (Treg) act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Tregs have been shown to be increased in the peripheral circulation of patients with various types of cancer including CLL and have been postulated to play a role in inhibition of anti-tumor immune responses. 15 color staining was performed on mononuclear cells isolated from peripheral blood from 33 healthy controls and 12 patients with chronic lymphoid leukemia (CLL). Treg cells were defined as viable CD45+ CD3+ CD8- CD4+ CD25+ Forkhead box P3+ (FoxP3)+ mononuclear cells. Among the Treg population we defined 4 sub-populations; Naïve Treg (CD45-RA+ CD27+ CD197+), Effector Treg (CD45-RA- CD27- CD197-), Central memory Treg (CD45-RA- CD27+ CD197+) and Effector memory Treg (CD45-RA- CD27+ CD197-). We report level of expression of activation markers CD39, HLA-DR and CD38 and IL-7 receptor CD127 on Treg subsets as defined above. The percentage of Treg was increased significantly in CLL patients compared with healthy controls 12.8±1.3% vs 5.7±0.3% (p<0.0001). In healthy controls, the majority of Treg are CD45-RA- (80.2±2.9 %) whereas in CLL patients, we observed Treg cells in both CD45-RA- and CD45-RA+ populations (62.6±4.9% and 40.4±5% respectively). Inside the CD45-RA+ populations, we showed that there was a significant higher proportion of naïve Treg among CLL patients compare to healthy controls ((83.8±5% and 41.8±5% respectively (p<0.0001)). Among the CD45-RA- populations, there was no significant difference in the proportion of each memory subtypes. We observed a different pattern of activation among the Treg in CLL patients, naïve Treg expressed more CD39 and HLA-DR compare to healthy controls (2x to 3x fold increase, p<0.0001), but less CD38. In central memory subpopulation, CD39 and CD127 expressions were increased in Treg isolated from CLL patients (p=0.04 and p=0.01 respectively), whereas CD38 was decreased (p=0.01). In effector memory population, HLA-DR expression was lower in CLL patients compared to healthy control (3±0.9% and 9.8±0.8% respectively, p<0.0001). Finally, in the effector subpopulation, HLA-DR expression was also decreased in Treg from CLL patients (p<0.0001). In summary, in these studies, we showed proportions of naïve and memory Treg as well as a different activation pattern of Treg cells present in CLL patients compared to healthy controls. The assessment of complex patterns of sub-phenotype expression in diseases vis a vis healthy individuals should expand our ability to understand the intricacies of both adaptive and innate cellular immune responses and their dysregulation in cancer patients.