High Frequency of T Regulatory Cells in Patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL) Is Decreased by Thalidomide and Fludarabine Treatment.
Authors: Giannopoulos Krzysztof , Schmitt Michael , Wlasiuk Paulina , Kowal Malgorzata , Podhorecka Monika , Rolinski Jacek , Dmoszynska Anna 
Published: October 2019

Abstract
Background and Aims: Immunotherapy might represent a novel therapeutical option for patients with B-cell chronic lymphocytic leukemia (B-CLL). Especially patients with limited tumor burden are usually regarded as suitable group for immunotherapy. However the existence of several molecular changes resulting in immunosuppression was reported even in patients with early stages of disease. In current study we assessed the expression of CD25 and FOXP3 in CD4 cells from patients with B-CLL. We also evaluated the influence of immunomodulatory treatment using thalidomide and fludarabine on T regulatory cells (Tregs) population in patients with B-CLL.
Methods: Forty B-CLL patients (mean age: 64,1, range: 37 – 79) were evaluated for the expression of CD4, CD25 as well as specific transcription factor FOXP-3 (Forkhead box protein P3) by FACS analysis. Results were compared to those obtained in healthy volunteers (HV). Mixed lymphocyte cell culture with synthetic peptide (MLPC) that correspond to epitope of tumor associated antigen RHAMM, survivin or fibromodulin were perform to assess antitumor T cell reactivity in B-CLL patients.
Results: We identified subpopulation of CD4+CD25highFOXP3+ T cells that phenotypically corresponds to Tregs in B-CLL patients. Increased levels of Tregs were observed in B-CLL patients. Significantly higher percentages of Tregs were noted in advanced stages of disease, 11.04% in stage 0-II vs 17.84% in stage III and IV according to Rai classification (Fig1a). No correlation between Tregs percentages and ZAP-70 status was shown, interestingly a tendency to higher percentages in ZAP-70 negative patients was observed 9.9% vs. 13.2%. In 9 CLL patients treated with thalidomide and fludarabine significant reduction of absolute number of circulating Tregs after thalidomide was observed (Fig.1b), in 7 this decrease was enhanced by addition of fludarabine to the treatment. First results from MLPC showed no correlation between specific T cell responses against TAA and presence of T regulatory cells.
Conclusion: Increased number of Treg cells in patients with B-CLL suggests that immunosuppression is present in B-CLL patients not only in advanced but also in early stages of disease, and that T cell mediated immune rejection of CLL cell might be hampered by Treg cell. Effective treatment of B-CLL with novel schema combining immunomodulatory drug thalidomide with fludarabine seems to be effective in getting rid of excess of circulating Tregs.
Figure 1. A. T regulatory cells in HV and CLL patients in early (0-II) and advanced (III-IV) stage of disease. B. Absolute number of T regulatory cells (CD4+CD25highFOX3+) during thalidomide + fludarabine treatment. Figure 1. A. T regulatory cells in HV and CLL patients in early (0-II) and advanced (III-IV) stage of disease. B. Absolute number of T regulatory cells (CD4+CD25highFOX3+) during thalidomide + fludarabine treatment.