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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 39  |  Issue : 2  |  Page : 68-71

Regulatory T cells in chronic lymphocytic leukemia


1 Hematology Unit, Clinical Pathology Department, Mansoura University Oncology Center, Mansoura Faculty of Medicine, Mansoura, Egypt
2 Medical Oncology Unit, Mansoura University Oncology Center, Mansoura Faculty of Medicine, Mansoura, Egypt
3 Clinical Hematology Unit, Mansoura University Oncology Center, Mansoura Faculty of Medicine, Mansoura, Egypt
4 Internal Medicine Department, Mansoura University Oncology Center, Mansoura Faculty of Medicine, Mansoura, Egypt

Date of Submission20-Dec-2013
Date of Acceptance10-Jan-2014
Date of Web Publication30-Aug-2014

Correspondence Address:
Tawfik Elkhodary
Faculty of Medicine, Medical Oncology Unit, Mansoura University Oncology Center, Mansoura Faculty of Medicine, PO Box 35516, Mansoura
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-1067.139766

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  Abstract 

Background: Controversial results concerning the prognostic value of the Tregs cells percentage in peripheral blood of chronic lymphocytic leukemia (CLL) patients have been reported in the previous studies.
Materials and Methods: This study aimed to estimate the prognostic relevance of Tregs cells in untreated CLL patients at diagnosis.
Results: CLL patients showed significantly higher Tregs percentage and Tregs cell counts as compared that identified in healthy normal controls (p < 0.01 for both). Furthermore, CLL patients with high LDH, β2 microglobulin levels, those positive for CD38% express significantly higher Tregs percentage as compared to those patients with normal LDH, β2 microglobulin levels and negative CD 38%. Also, the percentage of Tregs cells was significantly higher among CLL patients having autoimmune cytopenias.
Conclusion: Tregs cells percentage is higher in CLL patients as compared to normal healthy controls and related to advanced stages as well as poor prognostic markers. Tregs manipulation may represent a future strategy for management of CLL patients.

Keywords: CLL, Treg, Prognosis


How to cite this article:
Aref S, Elkhodary T, Azmy E, Hakim H, El Menshawy N, Ebrahim L. Regulatory T cells in chronic lymphocytic leukemia . Egypt J Haematol 2014;39:68-71

How to cite this URL:
Aref S, Elkhodary T, Azmy E, Hakim H, El Menshawy N, Ebrahim L. Regulatory T cells in chronic lymphocytic leukemia . Egypt J Haematol [serial online] 2014 [cited 2019 Dec 9];39:68-71. Available from: http://www.ehj.eg.net/text.asp?2014/39/2/68/139766


  Introduction Top


Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the western countries. It is characterized by accumulation of monoclonal B lymphocytes in the bone marrow, lymphoid organ, and peripheral blood [1] . The clinical course of CLL can range from months to decades. The status of the immune system of patients with B-cell CLL is not yet sufficiently characterized. Novel biological markers such as CD38 have shown to offer important prognostic information. Now, there are certain evidences suggesting a role of T-cell dysregulation in the pathogenesis and evaluation of CLL [2] . Natural and inducible regulatory T (Treg) cells, subset of T lymphocytes, are able to suppress immune responses by direct interaction with other immune cell types or immune-suppressive cytokines; they appear crucial in maintaining immune homeostasis, mediating peripheral tolerance, and peripheral autoimmunity. Emerging evidences suggest that such Treg cells may also modulate host T-cell activity against tumor-associated antigens, thereby facilitating tumor escape from immunological control [3],[4] .

Treg cells may be defined as CD4 + T cells expressing CD25 (IL-2 receptor) at high levels, cytoplasmic FoxP3, and very low to no CD127 (IL-7 receptor) on their surface [5],[6] . The findings regarding the Treg-cell number in peripheral blood and/or tissue of patients with cancer are contradictory. Furthermore, little is known on the prognostic role of the Treg cells in CLL patients, despite the fact that a general agreement is mentioned about the association of a higher number of such cells with more advanced disease [7] .

This study aimed to assess the clinicopathological impact of Treg-cell percentage on the peripheral blood of B-cell CLL patients.


  Patients and methods Top


The study was carried out on 98 B-cell CLL patients (63 male patients; 35 female patients) before the initiation of therapy. The diagnosis was based on the examination of the peripheral blood and bone marrow smear. The morphological findings were confirmed by immunophenotyping using the following monoclonal antibodies (CD5 + , CD23 + , CD79b - , CD19 + , sIg - , FMC7 - , CD10 - , and CD25 - ). Clinical staging was performed according to the RIA clinical staging criteria. The patients' characteristics are presented in [Table 1]. Twenty normal healthy persons of matched age and sex were taken as the control group.
Table 1: Patients characteristics

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  Methods Top


Peripheral blood samples anticoagulated with EDTA were obtained from CLL patients before therapy and were used to assess blood counts, blood smear morphology as well as flow cytometry analysis.

Flow cytometric analysis

The monoclonal antibodies (mouse anti-human antibody) - fluorescein isothiocyanate-CD25 antibody, phycoerythrin (PE)-CD127 antibody, PE-cy5-CD4 antibody, allophycocyanin-CD3 antibody, PE-cy7-CD8 antibody, and the isotype control antibody - were obtained from Beckman, Mansoura, Egypt.

For analysis of Treg cells, fluorescence-labeled anti-CD3, anti-CD4, anti-CD25, and anti-CD127 monoclonal antibodies were added to 100 μl of whole blood for 15 min, followed by lysis of erythrocytes. Subsequent analysis was performed on Coulter EPICS XL flow cytometry (Coulter Corporation, Miami, Florida, USA). At least 5000 CD3 + /CD4 + T cells were analyzed to ensure reproducible identification of Treg levels within CD3 + /CD4 + T cells [8] . Analysis of the prognostic marker CD38 in CLL was performed according to a protocol described by Tinhofer et al. [9] .

Statistical analysis

The Student t-test and the Mann-Whitney U-test were used to compare between groups concerning the numerical data. Comparison of the data presented as percentage was performed by the χ2 -test. The significance level was at P value less than 0.05.


  Results Top


Clinicopathological characteristics of chronic lymphocytic leukemia patients

The present study included 98 CLL patients. According to the RIA clinical staging criteria, there were 68 stage 0/II and 30 stage III/IV patients; 40 patients were CD38 + and 58 patients were CD38 - ; and 63 and 53 patients had high lactic dehydrogenase (LDH) and high β2 microglobulin, respectively [Table 1].

Regulatory T-cell percentage and counts in chronic lymphocytic leukemia patients versus healthy controls

Treg-cell percentage and counts were significantly higher in CLL patients as compared with normal healthy controls (P < 0.01, [Table 2].
Table 2: Comparison of laboratory characteristics of chronic lymphocytic leukemia patients and healthy controls

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Relationship between regulatory T-cell percentage and other prognostic factors in patients with chronic lymphocytic leukemia

Treg-cell percentage was significantly higher in advanced RIA stages III/IV as compared with earlier stages 0/II (P < 0.01). Moreover, Treg cells were significantly associated with high LDH, high β2 microglobulin, and positive CD38 as compared with those CLL patients with normal LDH, normal β2 microglobulin levels, and negative CD38 expression (P < 0.01 for all). In contrast, there was no significant difference in Treg cells as regards to groups of lymph nodes enlargement (P > 0.05, [Table 3].
Table 3: Relationship between the percentage of regulatory T cells and clinicopathological factors of chronic lymphocytic leukemia patients

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Regulatory T-cell percentage with respect to blood cytopenias in patients with chronic lymphocytic leukemia

The percentage of Treg cells was significantly higher among CLL patients having autoimmune cytopenias (red cells and platelets) [Table 4].
Table 4: Relationship between regulatory T-cell percentage and blood cytopenia

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  Discussion Top


The capacity of Treg cells to suppress antitumor immunity has been established in many in-vitro and mouse model experiments and has been investigated extensively in human cancers [8] . Although previous reports have indicated an increase in Treg in the peripheral blood of patients with CLL compared with healthy controls [10],[11] , their role in disease progression has remained unclear. The aim of the present study was to investigate the possible clinical relevance of Treg-cell percentage in CLL patients.

In the present study, circulating Treg-cell percentage was significantly higher in untreated CLL patients when compared with healthy controls. This finding was similar to that reported by Beyer and Schultzze [11] , Giannopoulos et al. [10] , Weiss et al. [8] , and Biancotto et al. [12] but was not correlated with the finding reported by Jak et al. [13] . These discrepancies could be attributed to the use of different markers employed to define Treg cells by means of flow cytometry (i.e. the use of an immunological gate on CD4 + cells combined only with CD25 + or alternatively with CD127 and CD25 and FoxP3) [14] . Moreover, D'Arena et al. [15] stated that the difference in prognosis might be related to different ethnic origin.

The Treg-cell percentage is higher in advanced RIA stages III/IV as compared with earlier stages 0/II. In addition, high Treg percentage was recorded in CLL patients with high LDH, β2 microglobulin levels as well as in positive CD38 patients as compared with those patients with negative CD38. These findings were in agreement with that reported by D'Arena et al. [16] who stated that higher counts of Treg-cell percentage was reported in high-risk CLL. There are accumulating evidences that Treg percentage play a critical role in immune evasion mechanisms used by tumor cells. Thus, the manipulation of Treg percentage might be a useful tool to contribute to fight cancer [14] . Moreover, Jadidi-Niaragh et al. [17] recommended that the precise biological and regulatory functions of all Treg-cell subsets should be carefully investigated before planning any immunotherapeutic interventions based on targeting of Treg cells. In addition, the results in the present study point out the possible role of Treg cells in the pathogenesis of blood cytopenias accompanying this disease. This finding is in parallel to that reported by Lad et al. [18] .


  Conclusion Top


Treg-cell percentage is higher in CLL patients as compared with normal healthy controls and is related to advanced stages as well as to poor prognostic markers. Treg manipulation may represent a future strategy for management of CLL patients.


  Acknowledgements Top


 
  References Top

1.Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. N Engl J Med 2005; 24;352 :804-815.  Back to cited text no. 1
    
2.Zupo S, Isnardl L, Megna M, Massara R, Malavasi F, Dono M, et al. CD38 expression distinguishes two groups of B-cell chronic lymphocytic leukemias with different responses to anti-IgM antibodies and propensity to apoptosis. Blood 1996; 88 :1365-1374.  Back to cited text no. 2
    
3.Sakaguchi S, Sakaguchi N, Shimizu J, Yamazaki S, Sakihama T, Itoh M, et al. Immunologic tolerance maintained by CD25 + CD4 + regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 2001; 182 :18-32.  Back to cited text no. 3
    
4.Shevach EM. Fatal attraction: tumors beckon regulatory T cells. Nat Med 2004; 10 :900-901.  Back to cited text no. 4
    
5.Baecher-Allan C, Viglietta V, Hafler DA. Human CD4 + CD25 + regulatory T cells. Semin Immunol 2004; 16 :89-98.  Back to cited text no. 5
    
6.Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell 2008; 133 :775-787.  Back to cited text no. 6
    
7.Badoual C, Hans S, Fridman W, Brasnu D, Erdman S. Revisiting the prognostic value of regulatory T cells in patients with cancer. J Clin Oncol 2009; 27 :E5-E6.  Back to cited text no. 7
    
8.Weiss L, Melchardt T, Egle A, Grabmer C, Greil R, Tinhofer I. Regulatory T cells predict the time to initial treatment in early stage chronic lymphocytic leukemia. Cancer 2011; 117 :2163-2169.  Back to cited text no. 8
    
9.Tinhofer I, Rubenzer G, Holler C, Hofstaetter E, Stoecher M, Egle A, et al. Expression levels of CD38 in T cells predict course of disease in male patients with B-chronic lymphocytic leukemia. Blood 2006; 108 :2950-2956.  Back to cited text no. 9
    
10.Giannopoulos K, Schmitt M, Kowal M, Wlasiuk P, Bojarska-Junak A, Chen J, et al. Characterization of regulatory T cells in patients with B-cell chronic lymphocytic leukemia. Oncol Rep 2008; 20 :677-682.  Back to cited text no. 10
    
11.Beyer M, Schultzze JL. Regulatory T cells in cancer. Blood 2006; 108 :804-811.  Back to cited text no. 11
    
12.Biancotto A, Dagur P, Fuchs J, Wiestner A, Bagwell B, McCoy JP Jr. Phenotypic complexity of T regulatory subsets in patients with B-chronic lymphocytic leukemia. Mod Pathol 2012; 25 :246-259.  Back to cited text no. 12
    
13.Jak M, Mous R, Remmerswaal EB, Spijker R, Jaspers A, Yagüe A, et al. Enhanced formation and survival of CD4 + CD25hi Foxp3 + T-cells in chronic lymphocytic leukemia. Leuk Lymphoma 2009; 50 :788-801.  Back to cited text no. 13
    
14.Dasgupta A, Mahapatra M, Saxena R. Flow cytometric immunophenotyping of regulatory T cells in chronic lymphocytic leukemia: comparative assessment of various markers and use of novel antibody panel with CD127 as alternative to transcription factor FoxP3. Leuk Lymphoma 2013; 54 :778-789.  Back to cited text no. 14
    
15.D'Arena G, Laurenti L, Minervini MM, Deaglio S, Bonello L, De Martino L, et al. Regulatory T-cell number is increased in chronic lymphocytic leukemia patients and correlates with progressive disease. Leuk Res 2011; 35 : 363-368.  Back to cited text no. 15
    
16.D'Arena G, Simeon V, D' Auria F, Statulo T, Di Sanzo P, De Martino L, et al. Regulatory T cells in chronic lymphocytic leukemia: actor or innocent bystander?. Am J Blood Res 2013; 3 :52-57.  Back to cited text no. 16
    
17.Jadidi-Niaragh F, Ghalamfarsa G, Yousefi M, Tabrizi M, Shokri F. Regulatory T cells in chronic lymphocytic leukemia: implication for immunotherapeutic interventions. Tumor Biol 2013; 34 :2031-2039.  Back to cited text no. 17
    
18.Lad D, Varma S, Varma N, Sachdeva M, Bose P, Malhotra P. Regulatory T cells in B-cell chronic lymphocytic leukemia: their role in disease progression and autoimmune cytopenias. Leuk Lymphoma 2013; 54 :1012-1019.  Back to cited text no. 18
    



 
 
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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