Leukemia Research 37 (2013) 1616–1621
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CD200 expression in patients with Multiple Myeloma: Another piece of the puzzle Concetta Conticello a , Raffaella Giuffrida b , Nunziatina Parrinello c , Simona Buccheri b , Luana Adamo b , Maria Rita Sciuto b , Cristina Colarossi a , Eleonora Aiello a , Annalisa Chiarenza c , Alessandra Romano c , Edvige Salomone c , Massimo Gulisano b , Rosario Giustolisi a , Francesco Di Raimondo c,∗ a
Department of Experimental Oncology, Mediterranean Institute of Oncology (IOM), Viagrande, Catania, Italy IOM (Mediterranean Institute of Oncology) Ricerca, Viagrande, Catania, Italy c Department of Biomedical Sciences, Hematology Section, University of Catania, Catania, Italy b
a r t i c l e
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Article history: Received 30 May 2012 Accepted 5 August 2013 Available online 23 September 2013 Keywords: CD200 Multiple Myeloma ERK pathway MEK inhibitor T cells Immunoregulation
a b s t r a c t CD200 is a relatively ubiquitously expressed molecule that plays a role in cancer immune evasion through interaction with its receptors. High expression levels of CD200 have been described in different human malignancies. For example, CD200 has been shown to be targeted after RAS/RAF/MEK/ERK activation in melanoma. Here we present the analysis of CD200 expression in human Multiple Myeloma (MM) samples. We found that CD200-positive cells express ERK and p-ERK. Moreover, UO126, a MEK inhibitor, reduces CD200 expression. Furthermore, we observe that CD200-positive cells show reduced immunogenicity compared to normal lymphocytes and that such immunogenicity increases when UO126 is used. We therefore hypothesize that CD200 expression in MM could suppress antitumor response and that antiCD200 treatment might be therapeutically beneficial in CD200-expressing tumors. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Immune evasion in cancer plays a pivotal role in the failure of natural host antitumor immune response. Molecular signaling pathways activated in the tumor environment help to create an immunosuppressive network whereby the cancer escapes detection [1]. It has recently been hypothesized that CD200 may play an important role in the tumour’s ability to evade immune detection [2,3]. CD200 (initially described as the Ox-2 tumor antigen) is a transmembrane glycoprotein that has been studied as a therapeutic target for its role in immunoregulation and tolerance. CD200 is a highly conserved member of the Ig superfamily. It is expressed on a variety of cell types, including myeloid cells, endothelium, ovarian cells, placental trophoblasts, and neurons. Interaction of CD200 with its receptor (CD200R), an inhibitory receptor mainly expressed on myeloid/monocyte lineage cells,
Abbreviations: MM, Multiple Myeloma; ISS, International Staging System; MLR, Mixed Lymphocyte Reaction; BM, bone marrow; PC, plasma cell. ∗ Corresponding author at: Department of Biomedical Sciences, Hematology Section, University of Catania, Via Citelli 7, 95100 Catania, Italy. Tel.: +39 095 7435911; fax: +39 095 7435913. E-mail address: [email protected] (F. Di Raimondo). 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.08.006
leads to the suppression of T-cells mediated immune responses [4]. Analysis of gene expression data shows that CD200 is significantly overexpressed in many human cancers, both of hematopoietic and non-hematopoietic origin [5]. CD200 has been described in melanoma as a downstream target of RAS/RAF/MEK/ERK activation [6]. Moreover, higher expression of CD200 has been reported in malignancies of the B cell lineage [7–9] and in acute myeloid leukemia [10]. In particular, Moreaux and co-workers [7] showed a correlation between CD200 mRNA expression and reduced eventfree survival [(14 months vs. 24 months) in Multiple Myeloma (MM)], independently from known adverse MM prognostic factors (high ISS score, beta-2 microglobulin). In addition to its function as a negative prognostic factor, Oltenau and co-workers [8] found a correlation between CD200 expression levels and age ≥65 years, low lactate dehydrogenase, and low hemoglobin level. Here we confirm the findings that CD200 protein is expressed in human myeloma samples at higher levels. We show that plasma cells isolated from MM samples expressed ERK, which is partially phosphorylated (p-ERK). When the ERK pathway is inhibited by UO126 expression of CD200 is reduced. In addition, we observe that CD200 expression is immunologically significant, as MM cell lines with endogenous levels of CD200 exhibit a weak activation of T responder cells in Mixed Lymphocyte Reaction (MLR). This low immunogenicity is strongly reversed by UO126 (ERK inhibitor).
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Thus, we hypothesize that expression of CD200 in MM may suppress antitumor response and that anti-CD200 treatment might be therapeutically beneficial for the treatment of CD200-expressing tumors. 2. Materials and methods 2.1. Primary cells/bone marrow cultures 51 BM biopsies and 7 marrow aspirates were obtained as part of routine examination from patients with MM who gave informed consent and were managed at the Division of Hematology, Ospedale Ferrarotto, University of Catania. All samples were selected for plasma cells (PC) infiltration between 5% and 100%. BM biopsies were fixed with 10% buffered formalin or fixed and decalcified in Lowry’s solution. BM aspirates were collected in tubes containing EDTA and cells were isolated by Ficoll Hypaque (Cedarlane labs, Ontario, Canada) density gradient centrifugation. Cells were maintained in RPMI 1640 medium (Gibco, Invitrogen) supplemented with 2 mM l-glutamine and 100 U/ml pennicillin–streptomycin and 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA). Cells were kept in a 5% CO2 atmosphere at a density of 5 × 105 cells/ml.
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antigen on responder cells and for the percentage of CD4-T responder cells, both used as an indicator for response to stimulation. 2.7. ERK inhibition Briefly, three MM cell lines (KMS18, KMS27 and ARH77) that showed different pERK levels were treated for 24 h with 30 mol/l U0126 (Promega, Madison, WI), a MEK1/2-specific inhibitor to block ERK1/2 activation, with parallel control cultures treated with carrier (DMSO) and flow cytometric analyses were done as previously described. 2.8. Statistical analysis A two-tailed paired t test was used to analyze the statistical significance of the results. Values of p < 0.05 was considered statistically significant. One asterisk indicates p < 0.05, two asterisks p < 0.005. Data are presented as mean values ± SD of the mean.
3. Results
2.2. Cell lines
3.1. Samples
The human MM cell lines KMS27, KMS18 and ARH77 – all plasmocytomas of B-cell origin – were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were maintained in RPMI 1640 medium (Gibco, Invitrogen) supplemented with 2 mM l-glutamine and 100 U/ml penicillin–streptomycin and 10% FBS (Invitrogen) and were kept in a 5% CO2 atmosphere at a density of 5 × 105 cells/ml.
A total of 58 samples from patients with MM [48 samples from patients at diagnosis, 4 samples from resistant/refractory MM patients, 4 samples from MM patients evolved from previously monoclonal gammopathy of undetermined significance (MGUS) and 2 MGUS samples] were examined. The percentage of PCs in the collected samples was variable, ranging from 5% to 100%. The characteristics of the Patients such as cytogenetic features, PCs percentage and prior therapies are detailed in Table 1A and B.
2.3. Immunostaining procedure Immunohistochemical staining was performed on 4 m thick formalin-fixed paraffin-embedded BM biopsies. After deparaffination–hydration, sections were permeabilized with PBS containing 0.4% Triton X-100 for 30 min and blocked with PBS containing 4% BSA for 30 min. The samples were incubated, overnight at 4 ◦ C, with the following primary antibodies: goat anti-human CD200 (1:100; R&D Systems, Minneapolis, MN) and mouse anti-human ERK (1:300; Cell Signaling, Danvers, MA). The following day, after washing, slides were incubated with LSAB+ kit (Dako Corp., Carpintera, CA) at room temperature. Staining was detected using diaminobenzedine (DAB) as chromogen. Sections were counterstained with hematoxylin. CD-138 staining was performed with automated immunostainer Bench Mark XT (Roche Ventana, Tucson, AZ). Omission of the primary antibodies was used as negative control. Bone marrow biopsies by B-cell chronic lymphocytic leukemia were used as positive control for CD200, showing a diffuse membranous localization in neoplastic cells as previously described [11]. The percentage of positive cells for CD200 was assessed as follows: (+) 1–25% of plasma cells (CD138+) with weak staining; (++) 25–50% of plasma cells (CD138+) with moderate staining; (+++) >50% of plasma cells (CD138+) with strong staining (Fig. 1A). 2.4. Flow cytometry 105 BM primary cells and cell lines were washed with cold PBS containing 1% BSA and incubated for 20 min at 4 ◦ C with fluorochrome-conjugated mAbs: anti-CD 25 FITC (clone B1.49.9), anti-CD45 ECD (J.33), anti-CD4 PE, (13 B8.2), anti-CD138 PC5 (BA38), anti-CD38 FITC (T16), (all from Beckman Coulter). Isotype control monoclonal antibody was used as negative control. Labeled cells, incubated with each conjugated Ab or corresponding isotype control (1:100), were washed with PBS and analyzed by flow cytometry using EPICS-XL 4-color flow cytometer (Beckman Coulter). For CD200 evaluation, after washing, cells incubated with mouse anti-human CD200 (1:50, BD Pharmingen, San Jose, CA), were incubated for 40 min at 4 ◦ C with phycoerythrin-conjugated anti-mouse secondary antibody (1:100, Jackson Laboratories, West Grove, PA) and then evaluated for fluorescence intensity. 2.5. Western blotting Western Blot analysis was carried out on proteins isolated from cell lysates of the three cell lines as previously described [12]. Rabbit anti-human Erk (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal anti-human p-Erk (1:1000, Santa Cruz) primary antibodies were used. 2.6. Mixed Lymphocyte Reaction Mixed Lymphocyte Reactions (MLR) were set up in 12-well plates using 1 × 106 T responder cells. T cells were enriched by incubating the cells for 1 h in tissue culture flasks and taking the non-adherent cell fraction. Stimulator MM cell lines (250,000 per reaction) were added to the MLRs at the start of the experiment, and cells were collected after 24 h and analyzed by flow cytometer for the expression of CD25
3.2. CD200 protein is expressed in MM cells We have analyzed CD200 protein expression in 58 human Multiple Myeloma samples, including 51 BM biopsies and 7 bone marrow aspirates from patients at different stages of disease (Table 1A and B), as well as in three myeloma cell lines (KMS27, KMS18, ARH77). Through immunohistochemical analysis performed on BM biopsies, we found that 80% of the samples bearing more than 50% of CD-138 positive plasma cells showed a diffuse membrane localization of CD200, with a variable staining intensity ranging from weak (+) to strong (+++) (Table 1A, Fig. 1A). While all primary tumor cells analyzed in this study by flow cytometry were essentially positive for CD200, as compared to the respective isotype controls, the percentage of CD200 expression varied widely (Fig. 1B). Furthermore, we found different percentages of cells positive for CD200 in the three myeloma cell lines by flow cytometric immunophenotyping (Fig. 1C). Heterogeneity in the CD200 cell surface expression level of the MM samples was not correlated with any of the various clinical parameters of the patients from whom MM cells were derived. 3.3. Erk pathway is active in CD200 positive cells In order to study whether CD200 protein expression is regulated by ERK activation, we evaluated the activation of the ERK pathway by immunohistochemistry on the CD-200 positive BM biopsies from human MM samples (Fig. 2A), and Western Blot analysis on the three MM cell lines (Fig. 2B). Results obtained on the cell lines have shown that KMS27 and KMS18, respectively presenting 77.3% and 11.8% of CD200 positivity by flow cytometric analysis, expressed Erk and its activated form (p-Erk). On the other hand, ARH77 cell line, which resulted negative for CD200 expression (1.7%), showed only a slight band for ERK while the phosphorylated form was completely absent (Fig. 2B). In order to assess directly the role of ERK expression in CD200positive cells, we exposed MM cell lines to UO126, a potent and specific MEK1/2 pharmacologic inhibitor. Treatment with U0126 for 24 h strongly abolished the expression of CD200 (Fig. 2C), thus
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Fig. 1. CD200 expression on CD-138 positive MM samples. (A) CD-200 immunohistochemical analysis on a CD-138 positive sample from a MM bone marrow biopsy. (a and c) CD-138 and (b and d) CD-200 immuno-staining performed on serial sections of a MM bone marrow biopsy. (e–g) Examples of CD200 immunostainings with variable intensity: weak (+), moderate (++) and strong (+++). (a and b) Magnification 20×; (c–g) magnification (40×). In brown CD-138 and CD-200 staining is shown, nuclei are counter-stained in blue with hematoxylin. These are representative images taken from samples n. 41 (for a, b, c, d and g), n. 20 (for e) and n. 35 (for f) of immunostainings performed on the 51 bone marrow MM biopsies, reported in Table 1A. (B) Flow cytometric analysis of CD200 expression on MM primary cells. MM primary cells were analyzed for surface expression of CD-200 by flow cytometry as described in Section 2. Data are expressed as % of FACS staining. These data represent results from 7 independent experiments. (C) Flow cytometric analysis of CD-200 expression on MM cell lines. Three MM cell lines, KMS-18, KMS-27 and ARH-77 MM, were analyzed for surface expression of CD-200 by flow cytometry as described in Section 2. These data represent results from 3 independent experiments. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)
suggesting that the ERK pathway plays a pivotal role in the expression of cellular CD200 antigen. 3.4. MM CD200 expressing cells present a reduced immunogenicity in Mixed Lymphocyte Reaction cultures The majority of MM cells express CD200 (Table 1), the CD200:CD200R axis may be an important pathway involved in suppression of T cell activity by MM cells. Thus, to investigate the functional importance of CD200 expression on myeloma cells with regards to immune tolerance, we initially determined (i) the expression of surface activation marker CD25 on alloreactive T cells and (ii) the percentage of CD4-T cells during MLR by adding MM cell lines with varying CD200 expression levels to MLR. Moreover, since UO126 is an ERK inhibitor and it is able to reduce CD200 expression, we have explored the effects of UO126 treatment on CD200 expressing MM cell lines. KMS27 and KMS18 cells were used in this functional study as stimulators while CD4T cells from healthy blood donors were used as responder cells. The study was designed to investigate the functional consequences of the presence of CD200 expression on tumor cell-induced immunity. An inhibitory effect on T cell activation was observed when MM cell lines KMS27 and KMS18, which harbor intermediatehigh CD200 and p-ERK expression, were added to MLRs. This is evidenced by the low levels of CD25 antigen expression and by
the low percentage of CD4-T cells. Therefore, KMS18 and KMS27 MM cell lines, which are poorly immunogenic when used alone, acquired immunogenicity after treatment with UO126, through an increase both in the expression of CD25 (about threefold) and in the percentage of CD4-T cells. In contrast, antigen expression and T cells percentage were augmented when MLRs were performed in the presence of ARH77 MM cells, which harbor very low CD200 expression and lack p-ERK (Fig. 3). We therefore come to the conclusion that CD200 expression is correlated with a reduced immunogenicity of MM cells in MLR cultures. 4. Discussion Tumors use a number of strategies to hinder the function of critical cells of the immune system and thus evade host immunity. Several studies provide evidences that CD200 is a strong inhibitor of the antitumor immune response [3,5,13,16–18]. Indeed, CD200 has been found to be expressed on multiple human cancers, both of haematopoietic and non-haematopoietic origin [6–10,14,17]. CD200 exerts its effects through interaction with its receptor, CD200R. Since CD200R is expressed by macrophages, dendritic cells, and specific T cells such as follicular Th cells, tumors expressing CD200 can negatively influence the immune system through multiple pathways. The presence of CD200 on the surface of a cancer cell may thus represents a mechanism for reinforcing
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Table 1 The characteristics of the samples. Samples (BM biopsy)
Immunoglubulin heavy chain type
Durie–Salmon stage
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
IgG IgG IgA IgG IgA IgA IgG IgG IgA IgG IgG Light chain only, Bence Jones protein IgG IgA IgA IgG IgA IIgG IIgD IgG IgG IgG IgG IgA IgGK IgGK IgGK IgGK IgA IgA IgG IgA IgG IgA IgG IgG IgA IgA IgG Lambda Chain IgA IgG IgA IgG IgG IgG IgG IgA IgG IgG IgG
II III III III III III III III III III III III III III III III III III III III III III III III III III II III II III III II III II II II II IIIb II II II II III MGUS IIa Ia Ia IIa MGUS IIa Ia
% PCs 50 50 50 50 50 78 60 50 75 70 65 60 70 90 80 90 90 90 60 80 80 90 50 50 50 70 90 50 50 60 60 100 60 80 90 60 60 70 50 50 100 50 100 10–20 20 20 10 10 5 10 40
CD 200 score
Cytogenetics
Status of disease
++ − − + ++ + + + + + + ++ − + + + + − − + +++ + ++ +++ − ++ ++ − +++ +++ ++ +++ +++ +++ ++ +++ +++ +++ +++ − +++ ++ +++ − ++ − − − − − +++
Nd Nd Nd Nd del 13 Nd Nd Nd del 13 del13q del 13 Nd del13q Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd del13q Nd Normal Normal del 13q Failed t (11,14), del 13q Failed Nd Normal t (11,14) Normal Nd Failed Nd t (11,14) Normal Nd Normal Normal Failed t (11,14) Nd Failed Nd
Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Resistant Newly diagnosed Relapsed Newly diagnosed Newly diagnosed Relapsed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Relapsed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Progression from MGUS Newly diagnosed Progression from MGUS Progression from MGUS Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Progression from MGUS
Samples (BM cells)
Immunoglubulin heavy chain type
Durie–salmon stage
% PCs
Cytogenetics
Status of disease
1 2 3 4 5 6 7
IgG IgG IgA IgA IgG IgG IgG
III III III III III II III
50 50 60 50 75 15 95
Nd Nd Abnormal T(4,14) 46,XY Nd 46, XY
Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed
Relapsed patients, relapsed to the first line of chemotherapy including thalidomide and desametazone. Relapsed/resistant, resistance to 2 or more line of chemotherapy including VAD regimen and/or thalidomide and desametazone and/or MP regimen. Nd, not determined.
suppression of immune reactivity to self-antigens [13] and functional blockade of this immunomodulatory molecule might provide a promising strategy to enhance immunogenicity of human cancer cells. MM cells were previously reported to express CD200 in 78% of examined cases by gene expression profiling, in 86% of cases by flow cytometric immunophenotyping analysis, with variable fluorescence intensity [7], and in about 70% of cases by immunostaining analysis [8,9,14].
Here we confirm by immunostaining and flow cytometric analysis that CD200 protein is expressed in about 80% of MM population, without any correlations with disease markers, and in two out of three myeloma cell lines. Furthermore, we show that Erk pathway is active in CD200-positive MM cells and that expression of CD200 protein on MM cells is regulated by ERK. It is noteworthy that the mitogen-activated protein kinase (MEK)/extracellular signalregulated kinase (ERK) signal transduction pathway significantly contributes to the pathogenesis of MM, by supporting MM-cell
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Fig. 2. Erk pathway is active in CD200 positive samples. (A) Immunohistochemical analysis for Erk and p-Erk on a CD-200 positive MM bone marrow sample with high levels of CD-138 positive PCs. (a and d) CD-138, (b and e) CD200, and (c and f) Erk staining on serial sections of a MM bone marrow biopsy. (a–c) Magnification 20×; (d–f) magnifications (40×). In brown CD-138, CD-200 and Erk staining is shown, nuclei are counter-stained in blue with hematoxylin. This is a representative image taken from sample n. 21 of immunostainings performed on 51 bone marrow samples reported on Table 1A. (B) Western blot analysis for Erk and P-Erk performed on the three MM cell lines (KMS-18, KMS-27 e ARH77). Actin is used as loading control. (C) Effects of 24 h treatment with UO126 on CD200 expression in human MM cell lines. KMS27 and KMS18 cell lines untreated (−) or treated with U0126 or carrier (DMSO) alone for 24 h. Cells were then analyzed by flow cytometer for CD200 expression. Data are expressed as % of FACS staining. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)
%CD25 FACS staining
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Fig. 3. MM CD200 expressing cells have a reduced immunogenicity in MLRs that is reversed by ERK inhibition. We performed an allogenic MLR to determine the immunogenicity of CD200 expressing cells. Responder T cells and stimulator MM cells (KMS18, KMS27 and ARH77) were cultured together and treated with U0126 for 24 h. Cells were then stained for (A) CD25 and (B) CD4 expression and analyzed by flow cytometry.
growth and survival, angiogenesis and cell-adhesion phenomena, thus conferring growth and drug resistance in the BM milieu [15]. Moreover, we show that the expression of CD200 is reduced on CD200-positive MM cell lines by blocking Erk pathway with U0126, a MEK1/2-specific inhibitor able to block ERK1/2 activation. The observation that CD200 is a downstream target of RAS/RAF/MEK/ERK activation has also been described by Petermann and co-workers for melanoma where CD200 expression is immunologically significant, as endogenous levels of CD200 in melanoma cell lines attenuate the ability of dendritic cells (DCs) to activate T cells in MLR [6]. Finally, we show that endogenous expression of CD200 in MM cell lines is immunologically significant, as CD200 expressing MM cells exhibit a weak activation of T responder cells in MLR. This effect is largely abolished by using UO126. To investigate the hypothesis that CD200 expression in MM cells is one of the contributors for their poor immunogenicity, similarly to what it has already been described for other B malignant cells [16,17], 2 MM cell lines, which constitutively express CD200 at levels that resemble those expressed by primary MM cells were used as targets in MLR. A CD200–negative cell line, ARH77, was used as control. In the MLR with the CD200-positive cell lines the value of CD25 expression and of the percentage of CD4-T cells were augmented greater than threefold by the MEK inhibitor UO126 that is able to decrease CD200 expression, suggesting that CD200 reduction may affect anti-tumor immunity. A similar effect has been described in previous works on the expression of CD200 in CLL cells and in a subset of solid tumors, particularly melanoma. We have extended these results to MM. In the above mentioned studies the immunosuppressive effect due to the interaction between CD200 and CD200R, present on myeloid cells and specific T-cell subsets, can be abolished by antiCD200 monoclonal antibodies [18], as well as by shRNA-mediated knockdown of CD200 [6]. These studies suggest that anti-CD200
C. Conticello et al. / Leukemia Research 37 (2013) 1616–1621
treatment might be therapeutically beneficial for treating CD200expressing cancers. While we cannot demonstrate it, we hypothesize that CD200 expression may suppress antitumor response even in MM patients. We believe that these findings may have important implications for the development of anti-myeloma therapies that target directly the CD200–CD200R interaction.
[4] [5] [6]
[7]
Conflict of interest [8]
All authors have no conflict of interest to report.
[9]
Acknowledgements [10]
None. No funding to declare. Contributions: C.C. and R.G. equally contributed to this work. C.C. provided the conception and design of the study, acquisition of data, analysis and interpretation of data. R.G. supplied the design of study, analysis and interpretation of data, drafting the article, revised it critically for important intellectual content; N.P., S.B., L.A., M.R.S. and C.C. performed the experiments, supplied the acquisition of data, drafting of manuscript; E.A. performed the experiments; A.C., A.R. and E.S. collected the samples and supplied the acquisition of data; M.G. and R.G. were responsible for the article critically for important intellectual content; and F.D.R. provided conception and design of the study, revised the article critically for important intellectual content and gave final approval of the version to be submitted.
[11]
[12]
[13] [14]
[15]
[16]
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Leukemia Research journal homepage: www.elsevier.com/locate/leukres
CD200 expression in patients with Multiple Myeloma: Another piece of the puzzle Concetta Conticello a , Raffaella Giuffrida b , Nunziatina Parrinello c , Simona Buccheri b , Luana Adamo b , Maria Rita Sciuto b , Cristina Colarossi a , Eleonora Aiello a , Annalisa Chiarenza c , Alessandra Romano c , Edvige Salomone c , Massimo Gulisano b , Rosario Giustolisi a , Francesco Di Raimondo c,∗ a
Department of Experimental Oncology, Mediterranean Institute of Oncology (IOM), Viagrande, Catania, Italy IOM (Mediterranean Institute of Oncology) Ricerca, Viagrande, Catania, Italy c Department of Biomedical Sciences, Hematology Section, University of Catania, Catania, Italy b
a r t i c l e
i n f o
Article history: Received 30 May 2012 Accepted 5 August 2013 Available online 23 September 2013 Keywords: CD200 Multiple Myeloma ERK pathway MEK inhibitor T cells Immunoregulation
a b s t r a c t CD200 is a relatively ubiquitously expressed molecule that plays a role in cancer immune evasion through interaction with its receptors. High expression levels of CD200 have been described in different human malignancies. For example, CD200 has been shown to be targeted after RAS/RAF/MEK/ERK activation in melanoma. Here we present the analysis of CD200 expression in human Multiple Myeloma (MM) samples. We found that CD200-positive cells express ERK and p-ERK. Moreover, UO126, a MEK inhibitor, reduces CD200 expression. Furthermore, we observe that CD200-positive cells show reduced immunogenicity compared to normal lymphocytes and that such immunogenicity increases when UO126 is used. We therefore hypothesize that CD200 expression in MM could suppress antitumor response and that antiCD200 treatment might be therapeutically beneficial in CD200-expressing tumors. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Immune evasion in cancer plays a pivotal role in the failure of natural host antitumor immune response. Molecular signaling pathways activated in the tumor environment help to create an immunosuppressive network whereby the cancer escapes detection [1]. It has recently been hypothesized that CD200 may play an important role in the tumour’s ability to evade immune detection [2,3]. CD200 (initially described as the Ox-2 tumor antigen) is a transmembrane glycoprotein that has been studied as a therapeutic target for its role in immunoregulation and tolerance. CD200 is a highly conserved member of the Ig superfamily. It is expressed on a variety of cell types, including myeloid cells, endothelium, ovarian cells, placental trophoblasts, and neurons. Interaction of CD200 with its receptor (CD200R), an inhibitory receptor mainly expressed on myeloid/monocyte lineage cells,
Abbreviations: MM, Multiple Myeloma; ISS, International Staging System; MLR, Mixed Lymphocyte Reaction; BM, bone marrow; PC, plasma cell. ∗ Corresponding author at: Department of Biomedical Sciences, Hematology Section, University of Catania, Via Citelli 7, 95100 Catania, Italy. Tel.: +39 095 7435911; fax: +39 095 7435913. E-mail address: [email protected] (F. Di Raimondo). 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.08.006
leads to the suppression of T-cells mediated immune responses [4]. Analysis of gene expression data shows that CD200 is significantly overexpressed in many human cancers, both of hematopoietic and non-hematopoietic origin [5]. CD200 has been described in melanoma as a downstream target of RAS/RAF/MEK/ERK activation [6]. Moreover, higher expression of CD200 has been reported in malignancies of the B cell lineage [7–9] and in acute myeloid leukemia [10]. In particular, Moreaux and co-workers [7] showed a correlation between CD200 mRNA expression and reduced eventfree survival [(14 months vs. 24 months) in Multiple Myeloma (MM)], independently from known adverse MM prognostic factors (high ISS score, beta-2 microglobulin). In addition to its function as a negative prognostic factor, Oltenau and co-workers [8] found a correlation between CD200 expression levels and age ≥65 years, low lactate dehydrogenase, and low hemoglobin level. Here we confirm the findings that CD200 protein is expressed in human myeloma samples at higher levels. We show that plasma cells isolated from MM samples expressed ERK, which is partially phosphorylated (p-ERK). When the ERK pathway is inhibited by UO126 expression of CD200 is reduced. In addition, we observe that CD200 expression is immunologically significant, as MM cell lines with endogenous levels of CD200 exhibit a weak activation of T responder cells in Mixed Lymphocyte Reaction (MLR). This low immunogenicity is strongly reversed by UO126 (ERK inhibitor).
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Thus, we hypothesize that expression of CD200 in MM may suppress antitumor response and that anti-CD200 treatment might be therapeutically beneficial for the treatment of CD200-expressing tumors. 2. Materials and methods 2.1. Primary cells/bone marrow cultures 51 BM biopsies and 7 marrow aspirates were obtained as part of routine examination from patients with MM who gave informed consent and were managed at the Division of Hematology, Ospedale Ferrarotto, University of Catania. All samples were selected for plasma cells (PC) infiltration between 5% and 100%. BM biopsies were fixed with 10% buffered formalin or fixed and decalcified in Lowry’s solution. BM aspirates were collected in tubes containing EDTA and cells were isolated by Ficoll Hypaque (Cedarlane labs, Ontario, Canada) density gradient centrifugation. Cells were maintained in RPMI 1640 medium (Gibco, Invitrogen) supplemented with 2 mM l-glutamine and 100 U/ml pennicillin–streptomycin and 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA). Cells were kept in a 5% CO2 atmosphere at a density of 5 × 105 cells/ml.
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antigen on responder cells and for the percentage of CD4-T responder cells, both used as an indicator for response to stimulation. 2.7. ERK inhibition Briefly, three MM cell lines (KMS18, KMS27 and ARH77) that showed different pERK levels were treated for 24 h with 30 mol/l U0126 (Promega, Madison, WI), a MEK1/2-specific inhibitor to block ERK1/2 activation, with parallel control cultures treated with carrier (DMSO) and flow cytometric analyses were done as previously described. 2.8. Statistical analysis A two-tailed paired t test was used to analyze the statistical significance of the results. Values of p < 0.05 was considered statistically significant. One asterisk indicates p < 0.05, two asterisks p < 0.005. Data are presented as mean values ± SD of the mean.
3. Results
2.2. Cell lines
3.1. Samples
The human MM cell lines KMS27, KMS18 and ARH77 – all plasmocytomas of B-cell origin – were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were maintained in RPMI 1640 medium (Gibco, Invitrogen) supplemented with 2 mM l-glutamine and 100 U/ml penicillin–streptomycin and 10% FBS (Invitrogen) and were kept in a 5% CO2 atmosphere at a density of 5 × 105 cells/ml.
A total of 58 samples from patients with MM [48 samples from patients at diagnosis, 4 samples from resistant/refractory MM patients, 4 samples from MM patients evolved from previously monoclonal gammopathy of undetermined significance (MGUS) and 2 MGUS samples] were examined. The percentage of PCs in the collected samples was variable, ranging from 5% to 100%. The characteristics of the Patients such as cytogenetic features, PCs percentage and prior therapies are detailed in Table 1A and B.
2.3. Immunostaining procedure Immunohistochemical staining was performed on 4 m thick formalin-fixed paraffin-embedded BM biopsies. After deparaffination–hydration, sections were permeabilized with PBS containing 0.4% Triton X-100 for 30 min and blocked with PBS containing 4% BSA for 30 min. The samples were incubated, overnight at 4 ◦ C, with the following primary antibodies: goat anti-human CD200 (1:100; R&D Systems, Minneapolis, MN) and mouse anti-human ERK (1:300; Cell Signaling, Danvers, MA). The following day, after washing, slides were incubated with LSAB+ kit (Dako Corp., Carpintera, CA) at room temperature. Staining was detected using diaminobenzedine (DAB) as chromogen. Sections were counterstained with hematoxylin. CD-138 staining was performed with automated immunostainer Bench Mark XT (Roche Ventana, Tucson, AZ). Omission of the primary antibodies was used as negative control. Bone marrow biopsies by B-cell chronic lymphocytic leukemia were used as positive control for CD200, showing a diffuse membranous localization in neoplastic cells as previously described [11]. The percentage of positive cells for CD200 was assessed as follows: (+) 1–25% of plasma cells (CD138+) with weak staining; (++) 25–50% of plasma cells (CD138+) with moderate staining; (+++) >50% of plasma cells (CD138+) with strong staining (Fig. 1A). 2.4. Flow cytometry 105 BM primary cells and cell lines were washed with cold PBS containing 1% BSA and incubated for 20 min at 4 ◦ C with fluorochrome-conjugated mAbs: anti-CD 25 FITC (clone B1.49.9), anti-CD45 ECD (J.33), anti-CD4 PE, (13 B8.2), anti-CD138 PC5 (BA38), anti-CD38 FITC (T16), (all from Beckman Coulter). Isotype control monoclonal antibody was used as negative control. Labeled cells, incubated with each conjugated Ab or corresponding isotype control (1:100), were washed with PBS and analyzed by flow cytometry using EPICS-XL 4-color flow cytometer (Beckman Coulter). For CD200 evaluation, after washing, cells incubated with mouse anti-human CD200 (1:50, BD Pharmingen, San Jose, CA), were incubated for 40 min at 4 ◦ C with phycoerythrin-conjugated anti-mouse secondary antibody (1:100, Jackson Laboratories, West Grove, PA) and then evaluated for fluorescence intensity. 2.5. Western blotting Western Blot analysis was carried out on proteins isolated from cell lysates of the three cell lines as previously described [12]. Rabbit anti-human Erk (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal anti-human p-Erk (1:1000, Santa Cruz) primary antibodies were used. 2.6. Mixed Lymphocyte Reaction Mixed Lymphocyte Reactions (MLR) were set up in 12-well plates using 1 × 106 T responder cells. T cells were enriched by incubating the cells for 1 h in tissue culture flasks and taking the non-adherent cell fraction. Stimulator MM cell lines (250,000 per reaction) were added to the MLRs at the start of the experiment, and cells were collected after 24 h and analyzed by flow cytometer for the expression of CD25
3.2. CD200 protein is expressed in MM cells We have analyzed CD200 protein expression in 58 human Multiple Myeloma samples, including 51 BM biopsies and 7 bone marrow aspirates from patients at different stages of disease (Table 1A and B), as well as in three myeloma cell lines (KMS27, KMS18, ARH77). Through immunohistochemical analysis performed on BM biopsies, we found that 80% of the samples bearing more than 50% of CD-138 positive plasma cells showed a diffuse membrane localization of CD200, with a variable staining intensity ranging from weak (+) to strong (+++) (Table 1A, Fig. 1A). While all primary tumor cells analyzed in this study by flow cytometry were essentially positive for CD200, as compared to the respective isotype controls, the percentage of CD200 expression varied widely (Fig. 1B). Furthermore, we found different percentages of cells positive for CD200 in the three myeloma cell lines by flow cytometric immunophenotyping (Fig. 1C). Heterogeneity in the CD200 cell surface expression level of the MM samples was not correlated with any of the various clinical parameters of the patients from whom MM cells were derived. 3.3. Erk pathway is active in CD200 positive cells In order to study whether CD200 protein expression is regulated by ERK activation, we evaluated the activation of the ERK pathway by immunohistochemistry on the CD-200 positive BM biopsies from human MM samples (Fig. 2A), and Western Blot analysis on the three MM cell lines (Fig. 2B). Results obtained on the cell lines have shown that KMS27 and KMS18, respectively presenting 77.3% and 11.8% of CD200 positivity by flow cytometric analysis, expressed Erk and its activated form (p-Erk). On the other hand, ARH77 cell line, which resulted negative for CD200 expression (1.7%), showed only a slight band for ERK while the phosphorylated form was completely absent (Fig. 2B). In order to assess directly the role of ERK expression in CD200positive cells, we exposed MM cell lines to UO126, a potent and specific MEK1/2 pharmacologic inhibitor. Treatment with U0126 for 24 h strongly abolished the expression of CD200 (Fig. 2C), thus
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Fig. 1. CD200 expression on CD-138 positive MM samples. (A) CD-200 immunohistochemical analysis on a CD-138 positive sample from a MM bone marrow biopsy. (a and c) CD-138 and (b and d) CD-200 immuno-staining performed on serial sections of a MM bone marrow biopsy. (e–g) Examples of CD200 immunostainings with variable intensity: weak (+), moderate (++) and strong (+++). (a and b) Magnification 20×; (c–g) magnification (40×). In brown CD-138 and CD-200 staining is shown, nuclei are counter-stained in blue with hematoxylin. These are representative images taken from samples n. 41 (for a, b, c, d and g), n. 20 (for e) and n. 35 (for f) of immunostainings performed on the 51 bone marrow MM biopsies, reported in Table 1A. (B) Flow cytometric analysis of CD200 expression on MM primary cells. MM primary cells were analyzed for surface expression of CD-200 by flow cytometry as described in Section 2. Data are expressed as % of FACS staining. These data represent results from 7 independent experiments. (C) Flow cytometric analysis of CD-200 expression on MM cell lines. Three MM cell lines, KMS-18, KMS-27 and ARH-77 MM, were analyzed for surface expression of CD-200 by flow cytometry as described in Section 2. These data represent results from 3 independent experiments. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)
suggesting that the ERK pathway plays a pivotal role in the expression of cellular CD200 antigen. 3.4. MM CD200 expressing cells present a reduced immunogenicity in Mixed Lymphocyte Reaction cultures The majority of MM cells express CD200 (Table 1), the CD200:CD200R axis may be an important pathway involved in suppression of T cell activity by MM cells. Thus, to investigate the functional importance of CD200 expression on myeloma cells with regards to immune tolerance, we initially determined (i) the expression of surface activation marker CD25 on alloreactive T cells and (ii) the percentage of CD4-T cells during MLR by adding MM cell lines with varying CD200 expression levels to MLR. Moreover, since UO126 is an ERK inhibitor and it is able to reduce CD200 expression, we have explored the effects of UO126 treatment on CD200 expressing MM cell lines. KMS27 and KMS18 cells were used in this functional study as stimulators while CD4T cells from healthy blood donors were used as responder cells. The study was designed to investigate the functional consequences of the presence of CD200 expression on tumor cell-induced immunity. An inhibitory effect on T cell activation was observed when MM cell lines KMS27 and KMS18, which harbor intermediatehigh CD200 and p-ERK expression, were added to MLRs. This is evidenced by the low levels of CD25 antigen expression and by
the low percentage of CD4-T cells. Therefore, KMS18 and KMS27 MM cell lines, which are poorly immunogenic when used alone, acquired immunogenicity after treatment with UO126, through an increase both in the expression of CD25 (about threefold) and in the percentage of CD4-T cells. In contrast, antigen expression and T cells percentage were augmented when MLRs were performed in the presence of ARH77 MM cells, which harbor very low CD200 expression and lack p-ERK (Fig. 3). We therefore come to the conclusion that CD200 expression is correlated with a reduced immunogenicity of MM cells in MLR cultures. 4. Discussion Tumors use a number of strategies to hinder the function of critical cells of the immune system and thus evade host immunity. Several studies provide evidences that CD200 is a strong inhibitor of the antitumor immune response [3,5,13,16–18]. Indeed, CD200 has been found to be expressed on multiple human cancers, both of haematopoietic and non-haematopoietic origin [6–10,14,17]. CD200 exerts its effects through interaction with its receptor, CD200R. Since CD200R is expressed by macrophages, dendritic cells, and specific T cells such as follicular Th cells, tumors expressing CD200 can negatively influence the immune system through multiple pathways. The presence of CD200 on the surface of a cancer cell may thus represents a mechanism for reinforcing
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Table 1 The characteristics of the samples. Samples (BM biopsy)
Immunoglubulin heavy chain type
Durie–Salmon stage
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
IgG IgG IgA IgG IgA IgA IgG IgG IgA IgG IgG Light chain only, Bence Jones protein IgG IgA IgA IgG IgA IIgG IIgD IgG IgG IgG IgG IgA IgGK IgGK IgGK IgGK IgA IgA IgG IgA IgG IgA IgG IgG IgA IgA IgG Lambda Chain IgA IgG IgA IgG IgG IgG IgG IgA IgG IgG IgG
II III III III III III III III III III III III III III III III III III III III III III III III III III II III II III III II III II II II II IIIb II II II II III MGUS IIa Ia Ia IIa MGUS IIa Ia
% PCs 50 50 50 50 50 78 60 50 75 70 65 60 70 90 80 90 90 90 60 80 80 90 50 50 50 70 90 50 50 60 60 100 60 80 90 60 60 70 50 50 100 50 100 10–20 20 20 10 10 5 10 40
CD 200 score
Cytogenetics
Status of disease
++ − − + ++ + + + + + + ++ − + + + + − − + +++ + ++ +++ − ++ ++ − +++ +++ ++ +++ +++ +++ ++ +++ +++ +++ +++ − +++ ++ +++ − ++ − − − − − +++
Nd Nd Nd Nd del 13 Nd Nd Nd del 13 del13q del 13 Nd del13q Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd del13q Nd Normal Normal del 13q Failed t (11,14), del 13q Failed Nd Normal t (11,14) Normal Nd Failed Nd t (11,14) Normal Nd Normal Normal Failed t (11,14) Nd Failed Nd
Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Resistant Newly diagnosed Relapsed Newly diagnosed Newly diagnosed Relapsed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Relapsed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Progression from MGUS Newly diagnosed Progression from MGUS Progression from MGUS Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Progression from MGUS
Samples (BM cells)
Immunoglubulin heavy chain type
Durie–salmon stage
% PCs
Cytogenetics
Status of disease
1 2 3 4 5 6 7
IgG IgG IgA IgA IgG IgG IgG
III III III III III II III
50 50 60 50 75 15 95
Nd Nd Abnormal T(4,14) 46,XY Nd 46, XY
Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed Newly diagnosed
Relapsed patients, relapsed to the first line of chemotherapy including thalidomide and desametazone. Relapsed/resistant, resistance to 2 or more line of chemotherapy including VAD regimen and/or thalidomide and desametazone and/or MP regimen. Nd, not determined.
suppression of immune reactivity to self-antigens [13] and functional blockade of this immunomodulatory molecule might provide a promising strategy to enhance immunogenicity of human cancer cells. MM cells were previously reported to express CD200 in 78% of examined cases by gene expression profiling, in 86% of cases by flow cytometric immunophenotyping analysis, with variable fluorescence intensity [7], and in about 70% of cases by immunostaining analysis [8,9,14].
Here we confirm by immunostaining and flow cytometric analysis that CD200 protein is expressed in about 80% of MM population, without any correlations with disease markers, and in two out of three myeloma cell lines. Furthermore, we show that Erk pathway is active in CD200-positive MM cells and that expression of CD200 protein on MM cells is regulated by ERK. It is noteworthy that the mitogen-activated protein kinase (MEK)/extracellular signalregulated kinase (ERK) signal transduction pathway significantly contributes to the pathogenesis of MM, by supporting MM-cell
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Fig. 2. Erk pathway is active in CD200 positive samples. (A) Immunohistochemical analysis for Erk and p-Erk on a CD-200 positive MM bone marrow sample with high levels of CD-138 positive PCs. (a and d) CD-138, (b and e) CD200, and (c and f) Erk staining on serial sections of a MM bone marrow biopsy. (a–c) Magnification 20×; (d–f) magnifications (40×). In brown CD-138, CD-200 and Erk staining is shown, nuclei are counter-stained in blue with hematoxylin. This is a representative image taken from sample n. 21 of immunostainings performed on 51 bone marrow samples reported on Table 1A. (B) Western blot analysis for Erk and P-Erk performed on the three MM cell lines (KMS-18, KMS-27 e ARH77). Actin is used as loading control. (C) Effects of 24 h treatment with UO126 on CD200 expression in human MM cell lines. KMS27 and KMS18 cell lines untreated (−) or treated with U0126 or carrier (DMSO) alone for 24 h. Cells were then analyzed by flow cytometer for CD200 expression. Data are expressed as % of FACS staining. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)
%CD25 FACS staining
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Fig. 3. MM CD200 expressing cells have a reduced immunogenicity in MLRs that is reversed by ERK inhibition. We performed an allogenic MLR to determine the immunogenicity of CD200 expressing cells. Responder T cells and stimulator MM cells (KMS18, KMS27 and ARH77) were cultured together and treated with U0126 for 24 h. Cells were then stained for (A) CD25 and (B) CD4 expression and analyzed by flow cytometry.
growth and survival, angiogenesis and cell-adhesion phenomena, thus conferring growth and drug resistance in the BM milieu [15]. Moreover, we show that the expression of CD200 is reduced on CD200-positive MM cell lines by blocking Erk pathway with U0126, a MEK1/2-specific inhibitor able to block ERK1/2 activation. The observation that CD200 is a downstream target of RAS/RAF/MEK/ERK activation has also been described by Petermann and co-workers for melanoma where CD200 expression is immunologically significant, as endogenous levels of CD200 in melanoma cell lines attenuate the ability of dendritic cells (DCs) to activate T cells in MLR [6]. Finally, we show that endogenous expression of CD200 in MM cell lines is immunologically significant, as CD200 expressing MM cells exhibit a weak activation of T responder cells in MLR. This effect is largely abolished by using UO126. To investigate the hypothesis that CD200 expression in MM cells is one of the contributors for their poor immunogenicity, similarly to what it has already been described for other B malignant cells [16,17], 2 MM cell lines, which constitutively express CD200 at levels that resemble those expressed by primary MM cells were used as targets in MLR. A CD200–negative cell line, ARH77, was used as control. In the MLR with the CD200-positive cell lines the value of CD25 expression and of the percentage of CD4-T cells were augmented greater than threefold by the MEK inhibitor UO126 that is able to decrease CD200 expression, suggesting that CD200 reduction may affect anti-tumor immunity. A similar effect has been described in previous works on the expression of CD200 in CLL cells and in a subset of solid tumors, particularly melanoma. We have extended these results to MM. In the above mentioned studies the immunosuppressive effect due to the interaction between CD200 and CD200R, present on myeloid cells and specific T-cell subsets, can be abolished by antiCD200 monoclonal antibodies [18], as well as by shRNA-mediated knockdown of CD200 [6]. These studies suggest that anti-CD200
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treatment might be therapeutically beneficial for treating CD200expressing cancers. While we cannot demonstrate it, we hypothesize that CD200 expression may suppress antitumor response even in MM patients. We believe that these findings may have important implications for the development of anti-myeloma therapies that target directly the CD200–CD200R interaction.
[4] [5] [6]
[7]
Conflict of interest [8]
All authors have no conflict of interest to report.
[9]
Acknowledgements [10]
None. No funding to declare. Contributions: C.C. and R.G. equally contributed to this work. C.C. provided the conception and design of the study, acquisition of data, analysis and interpretation of data. R.G. supplied the design of study, analysis and interpretation of data, drafting the article, revised it critically for important intellectual content; N.P., S.B., L.A., M.R.S. and C.C. performed the experiments, supplied the acquisition of data, drafting of manuscript; E.A. performed the experiments; A.C., A.R. and E.S. collected the samples and supplied the acquisition of data; M.G. and R.G. were responsible for the article critically for important intellectual content; and F.D.R. provided conception and design of the study, revised the article critically for important intellectual content and gave final approval of the version to be submitted.
[11]
[12]
[13] [14]
[15]
[16]
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