Quantitative and Qualitative Analysis Of Regulatory T Cells (Tregs) Derived From The Peripheral Blood Of Chronic Lymphocytic Leukemia Patients

T regulatory cells are immunosuppressive cells, which are considered to play an important role in the regulation of immune response to cancer, by restraining autoreactive lymphocytes. Several studies, mostly in solid tumors, revealed that the number of Treg cells increases as the disease progresses and 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. Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy, characterized by both, immunodeficiency and autoimmune disorders. Accumulated data indicate the role of T cells in the pathogenesis and development of CLL and reveal an increased number of Treg cells in CLL patients. The scope of this study is the analysis of the functional role of Tregs derived from the peripheral blood of CLL patients, mainly on B-CLL cells, and its correlation with well known prognostic factors.

Treg cells derived from mononuclear cells of 28 untreated B-cell CLL patients with a median age 62 (44-88) and 17 healthy donors were analyzed through Flow cytometry. Patients were classified according to Rai classification as Rai I:19, Rai II:4, Rai III:5 and according to Binet as Binet A: 24, Binet B:3 and Binet C:1. The following antibodies were used for the fluorescence-activated cell sorter (FACS) analysis:

1. CD45Ro-FITC/CD45RA-PE/CD4-ECD/CD25-PC5/CD127-PC7

2. CD1a-FITC/CD137-PE/CD4-ECD/CD25-PC5/CD127-PC7

3. CD95-FITC/cyCD152-PE/CD4-ECD/CD25-PC5/CD127-PC7

4. beads/FoxP3-PE/CD4-ECD/CD25-PC5/CD127-PC7

5. Annexin V-FITC/CD4-ECD/CD25-PC5/CD127-PC7

Moreover, peripheral blood was obtained from 15 patients with B-cell 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, NB cells, Tregs:Teff in a 4:1 ratio, B-cll:Tregs in 1:20 ratio, B-cll: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.

FACS analysis of the Treg cells resulted at the following observations: (1) The co-expression of the CD45RA-CD45RO markers was significantly higher in patients’ samples than in controls (p=0.047). (2) No significant differences were observed between patients and controls, regarding the expression of the CD1α marker, as well as the expression of CD95 and CD152 markers. (3) 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^- p=0.047, FoxP3⁺ p= 0.036). (4) Annexin V expression in Treg cells from B- CLL patients was significantly lower compared to controls (p=0.027). Following the purification and culturing of T and B cells from B-cell CLL patients’ samples, functional analysis of the different cell populations was performed using the BrdU proliferation assay. We observed that Tregs were able to significantly suppress the proliferation of the Teff cells (p=0.002). After the co-culturing of NB cells (CD5⁺CD19^-)and Tregs (CD4⁺CD25⁺) we found that NB cells seemed to significantly increase the proliferation of Treg cells, compared to the proliferation capacity of the Tregs when cultured alone (p=0.047). Moreover, we observed that Teff (CD4⁺CD25^-) were able to significantly suppress the proliferation of B-CLL cells (CD5⁺CD19⁺), when co-cultured (B-CLL: Teff, 1:20 ratio) (p=0.05).

In B-cell CLL patients, Treg cells are significantly higher and present with lower apoptotic levels compared to healthy donors’ samples. The functional analysis of Treg cells indicates that they can effectively suppress the proliferation of T effector cells. Moreover, T effector cells seem to suppress the proliferation of B-CLL cells, while NB cells increase the proliferation of Treg cells. These observations could probably indicate that at the early stages of the disease, where NB cells are more aberrant, Treg cells’ activity is induced, leading to Teff cells’ suppression and therefore, to an indirect induction of B-CLL cells’ proliferation.

No relevant conflicts of interest to declare.