Quantitative and Qualitative Analysis of Regulatory T Cells (Tregs) in B Cell Chronic Lymphocytic Leukemia (B-CLL)
Authors: Mpakou Vassiliki , Ioannidou Dikea-Eleni , Vikentiou Myrofora , Konsta Eugenia , Spathis Aris , Kontsioti Frieda , Kontos Christos , Vasilatou Diamantina , Bouchla Anthi , Gontopoulos Kostas , Roubakis Christoforos , Stavroulaki Georgia , Papanikolaou Nikos , Atta Maria , Papageorgiou Sotiris , Bazani Efi , Economopoulou Christina , Kokkori Stela , Kyriakou Elias , Dimitriadis George , Pappa Vassiliki 
Published: October 2019

Abstract
Introduction: T regulatory cells are immunosuppressive cells considered to play an important role in cancer biology and autoimmunity by suppressing host immune response and autoreactive lymphocytes respectively. Several studies reveal that Treg cells act by suppressing anti-tumor immune response, through the targeting of other immune cells, such as T cells, B cells and dendritic cells. Accumulated data indicate a significant role of T cell dysfunction in the pathogenesis of CLL.
Aims: The scope of this study is the analysis of numerical and functional abnormalities of Tregs in B-CLL with the view to elucidate their role in the pathogenesis of the disease.
Methods: Treg cells derived from 44 untreated B-CLL patients with a median age 62 and 17 healthy donors were analyzed by Flow cytometry, using the following antibodies: CD45Ro-FITC/CD45RA-PE/CD4-ECD/CD25-PC5/CD127-PC7, CD1a-FITC/CD137-PE/CD4-ECD/CD25-PC5/CD127-PC7, CD95-FITC/cyCD152-PE/CD4-ECD/CD25-PC5/CD127-PC7, beads/FoxP3-PE/CD4-ECD/CD25-PC5/CD127-PC7, Annexin V-FITC/CD4-ECD/CD25-PC5/CD127-PC7. For the functional analysis, peripheral blood was obtained from 20 patients with B-CLL. Mononuclear cells were isolated using Ficoll-Paque gradient centrifugation. CD4+ CD25+ (Treg cells), CD4+ CD25- (T effectοr cells, Teff), CD5+ CD19+ (B-CLL) and CD5- CD19+ (Normal B, NB) cells were separated using magnetic antibody cell sorting. To test the functionality of the assayed Tregs, the isolated cell populations were cultured in a 96-well plate (Tregs, Teff, B-CLL cells, NB cells, B-CLL cells: Tregs in 1:20 ratio, B-CLL cells: Teff in 1:20 ratio, NB cells: Tregs in 1:20 ratio, NB cells: Teff in 1:20 ratio) and their proliferative capacity was measured using the BrdU assay. To further analyze the functional role of Tregs, peripheral blood was obtained from 22 patients with CLL and 22 healthy donors. Mononuclear cells were isolated using Ficoll-Paque gradient centrifugation. CD4+ CD25+ CD127dim/- (Treg cells), CD5+ CD19+ (B-CLL) and CD5- CD19+ (Normal B, NB) cells were separated using magnetic antibody cell sorting and were co-cultured in a 96-well plate in a 1:10 ratio. The apoptosis of B cells was determined by the Annexin V/PI method.
Results: FACS analysis of the Treg cells resulted at the following observations: The Treg absolute cell number (cells/μL), estimated either as the number of CD4+ CD25+ CD127- cells or as the number of CD4+ CD25+ FoxP3+ cells, was statistically significantly higher in patients' samples than in controls (CD127- 21.65 vs 7.35, p=0.001; FoxP3+ 20.42 vs 6.5, p= 0.001). Annexin V expression in Treg cells from BCLL patients was significantly lower compared to controls (3.626 vs 38.615, p=0.003).
The functional analysis of Treg cells through BrdU assay indicated that CLL Tregs were able to suppress the proliferation of Teff cells (p=0.002) and that Teff cells were in turn able to significantly suppress the proliferation of B-CLL cells (p=0.05).
Moreover, FACS analysis through Annexin V/PI method indicated that Treg CLL cells significantly decrease the apoptosis rate of NB cells after their co-culturing, compared to NB cells (p<0,02). On the contrary, healthy donors derived Treg cells significantly increase the apoptosis of B-CLL cells after their co-culturing, compared to B-CLL cells (p<0.025). Interestingly, no significant alterations were observed after culturing NB cells with Tregs from healthy donors and B-CLL cells with Treg CLL cells.
Conclusions: In CLL patients, Treg cells are significantly higher and present with lower apoptotic levels compared to healthy donors. The functional analysis indicates that T effector cells suppress the proliferation of B-CLL cells and T effector cells are suppressed by Tregs indicating that the increased number of Tregs observed in CLL contributes indirectly to the proliferation of the CLL clone. These data are further supported by our observations that CLL derived Treg cells appear rather incapable of inducing apoptosis of both NB cells and B-CLL cells, in contrast to normal Tregs, suggesting an immunoediting effect of B-CLL cells on Tregs which negatively affects the functionality of the latter. Therefore, Treg cells in CLL do not efficiently eliminate the abnormal clone and play an important role in the pathogenesis of the disease. The molecular underlying mechanisms need to be further elucidated.

Disclosures
No relevant conflicts of interest to declare.