Analysis of CD4+CD25^hi Regulatory T Cells in the Peripheral Blood of Patients with B Cell Chronic Lymphocytic Leukemia (CLL).

Introduction: Naturally occurring regulatory T cells (T_reg) prevent autoimmune and inflammatory diseases and represent one important factor contributing to the inhibition of antitumor immune response in cancer. CLL is a disease that is characterized both by immunodeficiency and sometimes by autoimmunity. We analyzed the frequency of T_reg population in the peripheral blood of patients with B-CLL and correlated them with clinical and laboratory characteristics.

Methods: We analyzed prospectively 49 patients with B-CLL (29 males, 20 females) with median age of 63 and 24 normal healthy volunteers. Freshly isolated peripheral blood mononuclear cells were analysed by flow cytometry using the EPICS XL /MSL cytometer (Beckman Coulter Co). We determined the level of apoptosis by the method of annexin-V and the frequency of T_reg as cells positive for CD4, CD25^hi and intracellular staining of Foxp3 using the PE anti-Human Foxp3 staining set protocol. We recorded clinical information regarding Rai and Binet staging, hematological and biochemical parameters and the presence of autoimmune hemolytic anemia.

Results: The level of apoptosis as determined by the annexin V method was significantly lower on CD19+ cells of patients compared to normal controls (4.7 vs11.34 p=0.02). Moreover patients under treatment had significantly lower apoptosis level vs untreated (4vs6.4, p=0.023). The mean and median values of T_reg cells in patients with CLL were higher but not significantly compared to controls. However the log10 values of the T_reg frequencies were significantly higher in the group of CLL (0.6287vs0.1021, p=0.03). There was not any statistically significant association of T_regs with age, Rai and Binet staging, LDH values, and the level of apoptosis. Patients with levels of T_reg cells>mean control value presented lower median expression of CD38 antigen of borderline significance (3.8vs7, p=0,06). 5/26 patients with T_reg frequency >median control value presented autoimmune hemolytic anemia compared to 0/18 with T_reg <median control value (p=0.06)

Conclusion: The log10 values of Treg frequencies in patients with B-CLL were significantly higher compared to normal controls in accordance with previously published data. We did not observe any significant association with any laboratory or clinical characteristics or the level of apoptosis. Patients with T_reg levels above the mean control values had a lower although not significantly expression of CD38 antigen indicating an association with more indolent disease. Functional study of these cells will help the interpretation of the data and will shed more light on the their exact role in the pathogenesis of the disease.