|Year : 2012 | Volume
| Issue : 2 | Page : 111-115
Study of CD200 in chronic lymphocytic leukemia
Ahmad Baraka1, Hatem M. Salem2
1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||18-Feb-2012|
|Date of Acceptance||10-Mar-2012|
|Date of Web Publication||23-Jun-2014|
Clinical Pathology Department, Faculty of Medicine, Zagazig University, Zagazig
Source of Support: None, Conflict of Interest: None
CD200 is a transmembrane protein expressed on multiple cell types (e.g. thymocytes, activated T cells, B cells, dendritic cells, and endothelial cells). It regulates antitumor immunity and its overexpression is associated with poor prognosis in chronic lymphocytic leukemia (CLL). A soluble variant of CD200 (sCD200) is detectable in human serum, and an elevated serum level was shown in patients with CLL.
The aim of the study is to investigate the relationship between the levels of CD200 and clinical staging in patients with CLL.
Subjects and methods
The study included 50 patients, divided into two groups. The patient group included 30 de-novo patients with CLL (18 men and 12 women), whose age ranged from 49 to 70 years with a mean±SD of 57.82±4.94 years. They were classified according to the modified Rai staging system into three classes: (a) low-risk patients, which included seven cases (stage 0); (b) intermediate-risk patients, which included eight cases (stages I and II); and (c) high-risk patients, which included 15 cases (stages III and IV). The control group included 20 individuals (14 men and six women), whose age ranged from 50 to 72 years with a mean±SD of 58.54±5.1 years. CD200 levels were measured by flow cytometry.
Higher levels of CD200 were observed in the patient group compared with the control group. In addition, there was a significant increase of CD200 expression in the intermediate group of patients compared with low-risk patients, and a significant increase in the CD200 level in the high-risk group compared with intermediate-risk patients.
A positive correlation between the level of CD200 expression and clinical staging system of CLL was observed in our study; thus, measurement of CD200 may have a prognostic role in patients with CLL. In addition, it can be used as a follow-up marker for clinical response to treatment in CLL. CD200 blockade by immunotherapeutic agents may represent a novel approach for clinical treatment of CLL and a number of B-cell-derived neoplasms.
Keywords: CD200, chronic lymphocytic leukemia, flow cytometry, prognosis
|How to cite this article:|
Baraka A, Salem HM. Study of CD200 in chronic lymphocytic leukemia. Egypt J Haematol 2012;37:111-5
| Introduction|| |
Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative malignancy characterized by progressive lymphocytosis caused by the clonal accumulation of CD5+/CD19+ B cells in peripheral blood, bone marrow, and lymphoid organs 1. The clinical staging systems developed by Rai and Binet have been widely utilized and accepted as prognostic tools with limitations for predicting early-stage disease progression 2,3. Patients are classified into low, intermediate, and high-risk groups on the basis of presence of lymphadenopathy, splenomegaly, anemia, and thrombocytopenia 4.
Despite advances in the ability to diagnose high-risk patients during earlier stages of the disease, CLL remains incurable. Chemotherapeutic approaches have largely failed to result in major advances, but with the development of therapeutic monoclonal antibodies, a novel form of targeted cancer therapy has emerged either as monotherapy or in combination with chemotherapy 5.
CD200 is a type 1 transmembrane protein, related to the B7 family of costimulatory receptors, with two extracellular domains, a single transmembrane region, and a cytoplasmic tail with no known signaling motif. CD200 is expressed by thymocytes, activated T cells, B cells, dendritic cells, and endothelial cells 6.
The expression of the receptor for CD200 (CD200R1) is restricted to the monocyte/macrophage lineage and certain populations of T cells 7. Interaction of CD200 with its receptor imparts an immunosuppressive signal leading to inhibition of macrophages 8, induction of regulatory T cells 9, switching of cytokine profiles from Th1 to Th2 10, and inhibition of tumor-specific T-cell immunity 11.
CD200 is also involved in the immunosuppression in B-cell neoplasias. CLL cells express CD200, which leads to the inhibition of the Th1 response in mixed lymphocyte reactions 6. Kretz Rommel et al. 12 demonstrated that CD200 expression in Burkitt’s lymphoma cell lines prevents the rejection of tumor cells by human peripheral blood monocytes. Suppression of patients T cells is a key event in CLL pathogenesis and is mediated by direct cell–cell contact of malignant CLL cells with T cells 13. One critical molecule in this context is the immune-modulatory molecule CD200, which was shown to be upregulated in CLL cells and is sufficient to downregulate a Th1 immune response, including the production of cytokines such as IL-2 and IFN-γ 14. Furthermore, CD200 has been implicated in the induction of regulatory T cells, which are thought to hamper tumor-specific effector T-cell immunity 9. The immunosuppressive impact is underscored by the correlation between CD200 expression and poor prognosis in multiple myeloma and acute myeloid leukemia 15.
Aim of this work
We aimed to investigate the relationship between the levels of CD200 on CLL cells and clinical staging in patients with CLL.
| Subjects and methods|| |
This study was carried out in Clinical Pathology and Internal Medicine Departments of the Zagazig University Hospital. It included 50 individuals, divided into two groups. The patient group included 30 de-novo patients with CLL (18 men and 12 women), whose age ranged from 49 to 70 years with a mean±SD of 57.82±4.94 years. The control group included 20 individuals (14 men and six women), whose age ranged from 50 to 72 years with a mean±SD of 58.54±5.1 years. The diagnosis of CLL was based on full clinical history, lymphocyte morphology, and immunophenotyping. CLL patients were classified into three groups according to the modified Rai staging system: (a) low-risk patients, which included seven cases with lymphocytosis only in the peripheral blood and bone marrow (stage 0); (b) intermediate-risk patients, which included eight cases with lymphocytosis and lymphadenopathy (stage I) and/or hepatosplenomegaly (stage II); and (c) high-risk patients, which included 15 cases with lymphocytosis together with anemia (HB<11g/dl; stage III) and/or thrombocytopenia (platelets count<100×103/μl; stage IV) 1–16.
After informed consent was obtained, all the participants were subjected to the following: routine laboratory investigations including estimation of complete blood count and levels of liver enzymes, urea, and creatinine; analysis of peripheral blood film to confirm diagnosis; and flow cytometry by lymphoma panel (CD19, CD20, CD23, CD5, CD79b, FMC7, and SmIgM).
Measurement of CD200 by flow cytometry
CD200 is measured using R&D kits containing fluorescein isothiocyanate (FITC) labeled monoclonal anti-human CD200 antibodies. These are designated to quantitatively determine the percentage of cells bearing CD200 within a population and qualitatively determine the density of CD200 on cell surfaces.
Principle of the test
Monoclonal antibody reagents are added to the sample. The fluorochrome-labeled antibodies bind specifically to the surface antigens of the leukocytes. An aliquot of the stained blood sample is introduced into the flow cytometer and passed in a narrow stream through the path of a laser beam. The cells that are stained by the fluorochrome FITC are exited at 488 nm, and when exposed to the laser beam, they emit light at 580 nm, which is detected by special filters. Data are collected and processed by the software of the flow cytometer. The cells also interact with the laser beam by scattering the light. The forward scattered light provides a measure that correlates well with cell size, whereas the side scattered light is an indicator of cellular granulations.
Peripheral blood was collected in sterile ethylenediaminetetraacetic acid vacutainer tubes. Cells were washed three times in an isotonic phosphate buffer to remove any contaminated serum components, followed by centrifugation at 500g for 5 min. A volume of 50 μl of packed cells was transferred into a 5 ml tube for staining with monoclonal antibodies.
Cells Fc-blocked by treatment with 1 μg of human IgG/105 cells for 15 min at room temperature before staining; the excess blocking IgG should not be washed from this reaction. Transfer 25 μl of the Fc-blocked cells (1×105 cells) into a 5 ml tube, add 10 μl of CD200 FITC reagent, and incubate for 30 min at 2–8°C. Remove unreacted CD200 reagent by washing the cells twice in 4 ml of PBS. Finally, resuspend the cells in 200–400 μl of PBS for analysis by flow cytometry. As a control for analysis, cells in a separate tube should be treated with FITC-labeled mouse IgG antibodies.
Collected data are organized, tabulated, and statistically analyzed using the SPSS software computer package (version 13; SPSS Inc., Chicago, Illinois, USA) running on an IBM compatible computer. For qualitative data, the frequency and percentage distribution were calculated, and for comparison between groups the χ2-test was used. For quantitative data, the mean, SD, and sometimes minimum and maximum were calculated, and for comparison between two groups Student’s t-test was used. The correlation between two variables (bivariate correlation) was estimated by calculating Pearson’s correlation coefficient r; the mark ‘−’ means inverse correlation, whereas no mark before a value indicates positive (proportional) correlation. The correlation was considered to be mild when r was less than 0.3, moderate when r ranged from 0.3 to 0.7, and powerful when r was greater than 0.7. For interpretation of results P less than 0.05 was considered significant.
For estimation of the cut-off value, the following equation was used: cut-off value=[(mean control+2 SD)/highest positive level]×100.
| Results|| |
Fifty individuals were included in this study, 30 patients with CLL and 20 controls. In the present study, there was a statistically significant increase in the number of white blood cells (WBCs) and percentage of lymphocytes in the patient group (28.07±21.19×103/μl and 81.31±8.24%, respectively) compared with the control group (7.41±1.47×103/μl and 29.32±5.57%, respectively). As regards the red blood cell (RBC) count and hemoglobin concentration, there was a statistically significant decrease in the patient group compared with the control group (3.16±2.06×106, 5.34±0.45×106/μl and 9.42±3.07, 12.60±0.84 g/dl, respectively). There was a statistically significant decrease in platelet count in the patient group (171.15±123.80×103/μl) compared with the control group (257.62±47.32×103/μl) [Table 1].
|Table 1: Comparison between the patient and control groups as regards (white blood cells, red blood cells, platelets) counts, hemoglobin level and lymphocytes percentage|
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As regards CD200 percentage expression, there was a statistically significant increase in CD200 in the patient group compared with the control group (73.83±18.19 and 11.17±7.51%, respectively; [Table 2]. CD200 was higher in the intermediate-risk group (61.71±6.74%) compared with the low-risk group (46.69±6.52%), and a significant increase was observed in the high-risk group (86.92±6.27%) compared with intermediate-risk patients [Table 3]. A positive correlation of CD200 with WBC count and lymphocyte percentage, and a negative correlation with RBC count and platelets count is shown in [Table 4] and [Figure 1], [Figure 2], [Figure 3] and [Figure 4]. CD200 levels measured by flow cytometry in the three stages of CLL and in controls are shown in [Figure 5], [Figure 6], [Figure 7] and [Figure 8]. These results showed a positive correlation between clinical staging of the disease and CD200 expression.
|Figure 1: Positive correlation between CD200 and white blood cell count.|
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|Figure 5: CD200 was expressed on 7.40% of chronic lymphocytic leukemia cells (control individual).|
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|Figure 6: CD200 was expressed on 42.15% of chronic lymphocytic leukemia cells (low-risk patient).|
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|Figure 7: CD200 was expressed on 60.57% of chronic lymphocytic leukemia cells (intermediate-risk patient).|
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|Figure 8: CD200 was expressed on 94.70% of chronic lymphocytic leukemia cells (high-risk patient).|
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|Table 2: Comparison between patient and control groups as regards CD200 expression|
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|Table 3: Relationship between clinical staging of chronic lymphocytic leukemia and CD200 expression|
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|Table 4: Correlation between CD200 and white blood cells, lymphocytes, red blood cells and platelets|
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| Discussion|| |
CLL is an indolent B-cell non-Hodgkin’s lymphoma, characterized by the monoclonal expansion of B lymphocytes in the peripheral blood, bone marrow, and lymphoid organs. The disease has a typical clinical presentation, but the clinical course is variable with survival times ranging from 3 to 20 years 17.
CD200 is transmembrane protein broadly expressed on a variety of cell types, which delivers immunoregulatory signals through binding to receptors (CD200Rs) expressed on monocytes/myeloid cells and T lymphocytes. Signals delivered through the CD200 : CD200R axis have been shown to play an important role in the regulation of antitumor immunity, and overexpression of CD200 has been reported in a number of malignancies, including CLL, as well as on cancer stem cells 6.
CD200 expression was significantly higher in the patient group compared with the control group. These results are in concordance with those of Moreaux et al. 11, who reported that the majority of normal tissues were negative for CD200 expression. CD200 was expressed at low levels by CD34 hematopoietic progenitors, dendritic cells, and peripheral B cells. They also found a significant overexpression of CD200 in CLL compared with normal B cells. In addition, Deaglio et al. 18 reported that the level of CD200 in CLL patients was 92.4±13%, which is in accordance with that reported in the present study, although it is higher.
Our results showed a significant increase in CD200 expression in the high-risk group of patients compared with intermediate-risk or low-risk groups. In addition, a statistically significant increase in CD200 expression in the intermediate-risk group of patients compared with the low-risk group and a positive correlation between clinical staging and CD200 level were observed. These results were in agreement with those of Wong et al. 19, who found that CD200 levels correlate with the Rai disease stage and lymphocyte-doubling time. They concluded that CD200 levels in serum may provide prognostic and/or diagnostic information in CLL and may contribute to the efficacy of treatments targeting cell-surface CD200.
In our results there was mild positive correlation between WBC count and CD200, whereas there was a strong positive correlation between lymphocyte percentage and CD200 expression. In contrast, there was a strong negative correlation between RBC count and CD200 expression.
CD200 seems to be a very good candidate target for immunotherapy for a number of hematological disorders including CLL 20. Moreaux et al. 11 demonstrated that CD200 mRNA is overexpressed in at least eight cancers compared with their normal counterparts and, within a given tumor category, is associated with a bad prognosis. Given the immunosuppressive role of CD200, overexpression of CD200 in cancer tissues may facilitate an immune escape of cancer cells and provide a mechanism whereby they are able to avoid detection by the immune system and remain as residual disease.
As indicated above, antibodies to CD200 can abrogate its immunosuppressive effect, and in particular, restore tumor immune control in murine models. Treatment of CD200-expressing tumors with anti-CD200 antibodies inhibits tumor growth, indicating the potential for anti-CD200 therapy as a promising approach for CLL.
| Conclusion|| |
CD200 levels on CLL cells correlate with the clinical staging system of CLL; higher levels were reported with high-risk patients, indicating poor prognosis. Measurement of CD200 may have a prognostic role in patients with CLL; it can be used as a follow-up marker for clinical response to treatment. CD200 blockade by immunotherapeutic agents may represent a novel approach for clinical treatment of CLL and a number of B-cell-derived neoplasms.
| References|| |
|1.||Van Bockstaele F, Verhasselt B, Philippé J. Prognostic markers in chronic lymphocytic leukemia: a comprehensive review. Blood Rev. 2009;23:25–47 |
|2.||Rai KR, Sawitsky A, Cronkite EP. Clinical staging of chronic lymphocytic leukemia. Blood. 1975;46:219–234 |
|3.||Binet JL, Leporrier M, Dighiero G. A clinical staging system for chronic lymphocytic leukemia. Prognostic significance. Cancer. 1977;40:855–864 |
|4.||Nabhan C, Shanafelt TD, Kay NE. Controversies in the front-line management of chronic lymphocytic leukemia. Leuk Res. 2008;32:679–688 |
|5.||McWhirter JR, Kretz Rommel A, Saven A, Maruyama T, Potter KM, Mockridge CI. Antibodies selected from combinatorial libraries block a tumor antigen that plays a key role in immunomodulation. Proc Natl Acad Sci USA. 2006;103:1041–1046 |
|6.||Barclay AN, Wright GJ, Brooke G, Brown MH. CD200 and membrane protein interactions in the control of myeloid cells. Trends Immunol. 2002;23:285–290 |
|7.||Wright GJ, Cherwinski H, Foster Cuevas M, Brooke G, Puklavec MJ. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol. 2003;171:3034–3046 |
|8.||Jenmalm MC, Cherwinski H, Bowman EP, Phillips JH, Sedgwick JD. Regulation of myeloid cell function through the CD200 receptor. J Immunol. 2006;176:191–199 |
|9.||Gorczynski RM. CD200 and its receptors as targets for immunoregulation. Curr Opin Investig Drugs. 2005;6:483–488 |
|10.||Gorczynski RM, Cohen Z, Fu XM, Lei J. Anti-rat OX-2 blocks increased small intestinal transplant survival after portal vein immunization. Transplant Proc. 1999;31:577 |
|11.||Moreaux J, Veyrune JL, Reme T, De Vos J, Klein B. CD200: a putative therapeutic target in cancer. Biochem Biophys Res Commun. 2008;366:117–122 |
|12.||Kretz Rommel A, Qin F, Dakappagari N, Ravey EP, McWhirter J, Oltean D. CD200 expression on tumor cells suppresses antitumor immunity: new approaches to cancer immunotherapy. J Immunol. 2007;178:5595–5605 |
|13.||Görgün G, Holderried TAW, Zahrieh D, Neuberg D, Gribben JG. Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells. J Clin Invest. 2005;115:1797–1805 |
|14.||Kretz Rommel A, Bowdish KS. Rationale for anti-CD200 immunotherapy in B-CLL and other hematologic malignancies: new concepts in blocking immune suppression. Expert Opin Biol Ther. 2008;8:5–15 |
|15.||Tonks A, Hills R, White P, Rosie B, Mills KI, Burnett AK, et al. CD200 as a prognostic factor in acute myeloid leukaemia. Leukemia. 2007;21:566–568 |
|16.||Rai KR, Han T. Prognostic factors and clinical staging in chronic lymphocytic leukemia. Hematol Oncol Clin North Am. 1990;4:447–456 |
|17.||Binet JL, Caligaris Cappio F, Catovsky D, Cheson B, Davis T, Dighiero G. Perspectives on the use of new diagnostic tools in the treatment of chronic lymphocytic leukemia. Blood. 2006;107:859–861 |
|18.||Deaglio S, Vaisitti T, Bergui L, Bonello L, Horenstein AL, Tamagnone L, et al. CD38 and CD100 lead a network of surface receptors relaying positive signals for B-CLL growth and survival. Blood. 2005;105:3042–3050 |
|19.||Wong K, Shaha S, Spaner D. Potential role for serum soluble CD200 in human chronic lymphocytic leukemia. Leuk Res. 2009;33(Suppl 1):S144–S145 |
|20.||Pallasch CP, Ulbrich S, Brinker R, Hallek M, Uger RA, Wendtner CM. Disruption of T cell suppression in chronic lymphocytic leukemia by CD200 blockade. Leuk Res. 2009;33:460–464 |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4]