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secondary AML-derived T lymphoid cell line K2-MDS
Leukemia Research 24 (1999) 103 – 108 www.elsevier.com/locate/leukres
Establishment of a myelodysplastic syndrome (MDS)/secondary AML-derived T lymphoid cell line K2-MDS Mitsuhiro Matsuda a,*, Yasuhiro Maeda a, Yoshiyasu Sumimoto a, Hiroyuki Nawata a, Tetsuaki Sano a, Masaki Higashishiba a, Hisae Haga b, Yoichi Tatsumi a, Fusanari Horiuchi a, Kiyohiro Irimajiri b, Akihisa Kanamaru a a
Third Department of Internal Medicine, School of Medicine, Kinki Uni6ersity, 377 -2 Ohno-Higashi, Osaka-Sayama, Osaka 589 -8511, Japan b Faculty of Pharmaceutical Science, Kinki Uni6ersity, Osaka, Japan Received 24 May 1999; accepted 24 August 1999
Abstract We have established a T lymphoid cell line, K2-MDS, from the peripheral blood mononuclear cells (PBMC) of a patient with acute myeloblastic leukemia (AML) transformed myelodysplastic syndrome (MDS). K2-MDS cells are positive for the expression of CD4, CD5, CD13, CD25, CD71, CD95, HLA-DR and cytoplasmic CD3. Southern blotting analysis shows T cell receptor (TCR) b chain genes rearrangements, whereas immunoglobulin heavy chain (IgH) genes are not rearranged. Further, the patient PBMC contains TCR b chain genes rearrangements in the same manner as K2-MDS cells. The data indicate that K2-MDS is a T lymphoid cell line derived from a myelodysplastic clone in the patient PBMC. This new MDS-derived cell line K2-MDS may be a useful in vitro model for studies on the pathogenetic mechanisms leading to MDS. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Myelodysplastic syndrome/secondary AML; T lymphoid cell line; Myelodysplastic T lymphoid clone; T cell receptor genes rearrangements; Southern blotting analysis; DNA from peripheral blood slides
1. Introduction Myelodysplastic syndromes (MDS) are characterized by the ineffective proliferation or production of cells with abnormalities of maturation and function leading to peripheral blood cytopenia and a preleukemic state [1]. The pathogenesis of MDS is supposed to be a multistep process involving two or more genetic alterations which cause clonal proliferation of an abnormal stem cell [2]. There is a good evidence indicating that these conditions arise from clonal expansion of the progeny of an aberrant pluripotential stem cell [3–5]. Abbre6iations: AML, acute myeloblastic leukemia; HPRT, hypoxanthine phosphoribosyl-transferase; IgH, immunoglobulin heavy chain; MDS, myelodysplastic syndrome; MoAb, monoclonal antibody; MPO, myeloperoxidase; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PGK, phosphoglycerate kinase; RFLP, restriction fragment length polymorphisms; SDS, sodium dodecyl sulphate; TCR, T cell receptor. * Corresponding author. Tel.: + 81-723-660221, ext. 3128; fax: + 81-723-683732.
Cytogenetic involvement of myeloid and erythroid cell lineages has been shown in MDS. Some reports have shown peripheral blood lymphopenia and functional abnormalities of B and T cells [6,7]. Some B cell lines were established from bone marrow or peripheral blood of MDS or de novo acute myeloblastic leukemia (AML)/secondary AML after MDS, suggesting the derivation of B lymphocytes from the MDS or AML stem cell [8–11]. As for T cells involvement in MDS, using the restriction fragment length polymorphisms (RFLP) of the X-chromosome phosphoglycerate kinase (PGK) and hypoxanthine phosphoribosyl-transferase (HPRT) genes, Karasawa et al. have showed the presence of MDS clone originated from a progenitor cell to differentiate into T cells [12]. However, there has been no appropriate model for study about T lymphoid clone in MDS. In this report, we describe a unique T lymphoid cell line (K2-MDS) derived from peripheral blood of MDS and discuss the possible involvement of T lymphoid clone in MDS.
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2. Materials and methods
2.1. Origin of the cell line A 62-year-old male was admitted to our department because of anemia. The bone marrow specimen revealed a hypocellular with trilineage dysplasia, diagnosed as MDS (FAB subtype, refractory anemia with excess blasts). The patient had been supported mainly with red blood cell transfusions, but 14 months later white blood cells in peripheral blood increased up to 15.0 × 109/l with 10% myeloblasts, 60% monocytes and 17% eosinophiles including blastic cells. Bone marrow examination showed normocelluar with 30.1% myeloblast and promyelocytes, 11.5% monocytes and 8.4% eosinophiles (1.9% myelocytes, 2.5% metamyelocytes, 2.8% band formed and 1.2% segmented eosinophiles). These data made us diagnose as a overt leukemia (FAB subtype, M4Eo). Thereafter, he was refractory to therapy with cytarabine and died of progressive leukemia. The immunophenotypic profile of peripheral blood mononuclear cells (PBMC) at overt leukemia is shown in Table 1. Serum antibody for HTLV-I was negative. PBMC at overt leukemia were prepared by the standard method and grown in RPMI 1640 (GIBCO, Grand Island, NY, USA) with 10% fetal calf serum without any stimulation. The cultures were fed weekly by replacing half of the medium.
2.2. Morphology K2-MDS cells were studied after staining with Wright and Giemsa solutions.
2.3. Immunophenotyping The cells were immunophenotyped with a flow cytometry (FACS Calibur, Becton Dickinson Immunocytometry Systems, CA, USA) using monoclonal antibodies (MoAbs) directed against a number of lineage-associated and -independent antigens (Table 1). Some specific MoAbs were purchased from Becton Dickinson Immunocytometry Systems, DAKO (Glostrup, Denmark) and Immunotech (Marseilles, France). 32.2 (MoAb for CD64) and IV.3 (MoAb for CD32) were kindly provided by Dr Alan D. Schreiber of University of Pennsylvania (Philadelphia, PA, USA) and H107 (MoAb for CD23) was from Dr J. Yodoi of Kyoto University (Kyoto, Japan). The cells were washed twice with PBS and then incubated with MoAbs at 4°C for 30 min. Following incubation with the MoAbs and subsequent washing steps, cells were stained with FITC-conjugated goat anti-mouse F(ab’)2 IgG (Tago, Burlingame, CA, USA) at 4°C for 30 min. In every determination, a negative control of irrelevant
mouse MoAb of the same isotype as the MoAb under investigation was included (IgG1, IgG2, IgM controls). Addition of propidium iodide (10 mg/ml; Sigma) allowed gating out of dead cells. In cases of cytoplasmic staining for cytoplasmic CD3 or myeloperoxidase (MPO), 70% ethanol solution was used for cell fixation.
2.4. Cytogenetic analysis Cytogenetic analysis was performed on bone marrow cells of the patient at overt leukemia and K2-MDS cells. Chromosome preparations were made by SRL, Inc (Tokyo, Japan) by routine methods [13].
2.5. Preparation of genomic DNA Genomic DNA extraction from K2-MDS cells was carried out by the conventional method. Briefly, the cells were washed out with ice-cold phosphate-buffered Table 1 Immunophenotypic profile on the peripheral blood mononuclear cells (PBMC) of the patient at overt leukemia and on K2-MDS cells. Cells were analyzed with a flow cytometry (FACS Calibur) and the numbers in this table represent the percentages of the positive cells detected by the respective antibodya Antigens (CD)
Patient (%)
K2-MDS (%)
B cell: CD10 CD19 CD20 CD23
ND 0 0 ND
0 0 0 0
2 1 ND ND ND ND ND ND
0 45 99 0 0 0 0 99
Progenitor cell/activation: CD25 CD33 CD34 Class 1 Class II Class III CD71 CD95 HLA-DR
0 98 ND ND 27 ND ND ND
99 1 0 0 0 94 40 99
Myelomonocytic: CD13 CD14 CD16 CD32 CD64 MPO
78.9 ND 14 62 52 ND
78 0 0 0 0 0
T-cell: CD3 CD4 CD5 CD7 CD8 TCRa/b TCRg/d Cytoplasmic CD3
a
CD, cluster differentiation; MPO, myeloperoxide; ND, not done.
M. Matsuda et al. / Leukemia Research 24 (2000) 103–108
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Fig. 1. Morphological study of K2-MDS cells ( ×132). Staining K2-MDS cells with Wright and Giemsa solutions shows single and round cells in suspension, partly in clumps. Most are mononucleated cells with one to three nucleoli, abundant cytoplasm with vacuolization. Some cells are mitotic.
saline (PBS) and resuspended in 0.5 ml digestion buffer (100 mM NaCl, 10 mM Tris – HCl [pH 8.0], 25 mM EDTA [pH 8.0], 0.5% SDS, 0.1 mg/ml proteinase K), followed by incubation with shaking at 50°C for 12 h. Genomic DNA was isolated by ethanol precipitation after phenol/chloroform extraction. DNA was also extracted from stored peripheral blood slides as previously reported [14,15]. Briefly, the smears were scraped off the glass slide with a stainless steel razor blade and powdered, and the material was transferred into a 1.5 ml Eppendorf tube. The material was first lysed in a 1.5 ml of distilled water and in 500 ml of a nonionic detergent (Nonidet P 40, 0.1% solution, lysis time 10 min) and the nuclear pellets were incubated in a 200 ml lysis buffer (10 mM Tris–HCl [pH 8.0], 10 mM NaCl, 10 mM EDTA), with sodium dodecyl sulphate (SDS; 2%) and proteinase K (2 mg/ml) at 37°C for 12 h. DNA was extracted with phenol and chloroform. DNA was precipitated from the aqueous phase with sodium chloride and ethanol. After incubation at –80°C for 20 min, centrifugation was carried out at 15 000 rpm in a microcentrifuge. After the DNA pellets were dried, they were redissolved in 20 ml distilled water.
2.6. Analysis of receptor genes rearrangements by Southern blotting DNA extracted from K2-MDS cells and stored peripheral blood slides was accessed for receptor genes rearrangements by SRL, Inc. T cell receptor (TCR) b
chain and immunoglobulin heavy chain (IgH) genes rearrangements were studied by using primers for TCR b chain (Cb1, Jb1 and Jb2) and IgH (JH and Cm) prepared by SRL, Inc.
2.7. Effect of cytokines and inducers The proliferative response of K2-MDS cells to various cytokines and inducers was examined. After 1× 105/ml K2-MDS cells were incubated with IL-2 (used at 10 IU/ml), IL-3 (50 ng/ml), GM-CSF (5 ng/ml), G-CSF (50 ng/ml), PMA (10 ng/ml), PHA (10 mg/ml) and Con-A (10 mg/ml) for 24 or 48 h, the proliferation was assessed by 3H-thymidine uptake assay as described previously [11]. Cytokines (IL-2, IL-3, GM-CSF and G-CSF) were purchased from Boehringer Mannheim, Germany and inducers (PMA, PHA and Con-A) were from Sigma.
3. Results
3.1. Establishment of the cell line During the first 8 weeks of patient PBMC suspension culture, the number of the cells increased slowly. After the eighth week, however, cells proliferated rapidly with a doubling time of 24–30 h, giving rise to a continuous cell line K2-MDS. K2-MDS cells have grown without any growth factor over 24 months or over 200 passages.
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3.2. Morphological and immunological characterization Fig. 1 represents K2-MDS cells stained with Wright and Giemsa solutions. K2-MDS cells are single and round cells in suspension, partly in clumps. Most are mononucleated cells with one to three nucleoli, abundant cytoplasm with vacuolization. Some cells are mitotic. Table 1 shows the immunological findings on PBMC of the patient at overt leukemia and on K2MDS cells. The data demonstrate that K2-MDS cells express T lymphoid marker (CD4, CD5, CD25, CD71, CD95, HLA-DR and cytoplasmic CD3), whereas PBMC of the patient expresses myelomonocytic markers (CD13, CD16, CD32, CD33, CD34 and CD64). Every histogram of flow cytometer on K2-MDS cells shows a single peak (data not shown). After 12 or 24 month culture, the same results were obtained (data not shown), suggesting the stability of the cell surface expression over long culture.
from individual clones. All K2-MDS clones indicate pseudotetraploid described above, suggesting that K2MDS cells are clonal.
3.4. Analysis of receptor genes rearrangements DNA extracted from K2-MDS cells was examined for TCR b chain (Cb1, Jb1 and Jb2) and IgH (JH and Cm) genes rearrangements by Southern blotting. TCR b chain genes rearrangements (Cb1, Jb1 and Jb2) were observed (Fig. 2), whereas no rearrangement was detected in IgH genes (data not shown). Next, we investigated whether TCR b chain genes in the patient PBMC were rearranged or not. We extracted DNA from stored peripheral blood slides of the patient at overt leukemia. Southern blotting analysis shows that TCR b chain genes (Cb1) in the patient PBMC are rearranged as well as K2-MDS cells (Fig. 3).
3.5. Effect of cytokines and inducers 3.3. Cytogenetic analysis Cytogenetic investigations of bone marrow cells of the patient at overt leukemia showed 46, XY, + der (1;7)(q10;p10), -7. Of 20 mitoses of K2-MDS cells analyzed, more than 18 (90%) had pseudotetraploid with the following karyotypes: 78-81, XXY, + add (1) (p 11), + 2, + add (3) (q 27-29), +4, + add (5) (q35), + 7, +add (8) (q 24), add (11) (p1), add (12) (q 15-22), del (13) (q 32), add (14) (p 11), -16, add (17) (p 11), add (19) (p 13), +add (19), +20, indicating the discrepancy of the karyotype between the patient cells and K2-MDS cells. Further we performed limiting dilution method to isolate an individual clone. After culture for three weeks, we have obtained several clones derived
In the 3H-thymidine uptake assay, none of the cytokines and inducers used (IL-2, IL-3, GM-CSF, GCSF, PMA, PHA and Con-A) had significant proliferation-stimulating or -inhibiting effects on K2MDS cells for 24 or 48 h (data not shown).
4. Discussion The presence of T lymphoid surface markers and the monoclonal rearrangements of T cell receptor genes by Southern blotting analysis clearly show the T lymphoid origin of K2-MDS cells. Southern blotting indicates T cell receptor b chain genes rearrangements on both
Fig. 2. T cell receptor (TCR) b chain genes rearrangements in K2-MDS cells. DNA extracted from K2-MDS cells was digested with EcoR V (E), BamH I (B) or Hind III (H). The Jb1, Jb2 and Cb1 probes show the presence of rearranged TCR b chain genes (arrows) in K2-MDS cells. Human placentas were used as a control.
M. Matsuda et al. / Leukemia Research 24 (2000) 103–108
Fig. 3. T cell receptor (TCR) b chain (Cb1) genes rearrangements in the patient PBMC. DNA from the patient PBMC was digested with EcoR V (E), BamH I (B). The Cb1 probes show the presence of rearranged TCR b chain genes (arrows) in the patient PBMC. Human placentas were used as a control.
K2-MDS cells and the patient PBMC. K2-MDS cells have grown for more than 24 months since the establishment in 1997. Also these cells proliferate independently of any growth factor, indicating that the cells are immortal. PCR analysis using primers designed to amplify Bam W region of the EBV genome shows that the transformation of EBV is not associated in this cell line (data not shown). As discussed previously, a clonal population is best identified by unique mutational events, such as a distinctive chromosomal abnormality or discrete gene rearrangements [16]. K2-MDS cells have manifestated a highly complex, near triploid karyotype. Such a cytogenetic picture is more generally typical of late passage AML cell line or solid tumor cell line. All K2-MDS clones obtained by limiting dilution method indicate the chromosomal abnormality (data not shown), suggesting that K2-MDS cells are clonal. However, the discrepancy of the karyotype between the patient’s cells and K2-MDS cells may be conflicting with the notion that this cell line originated from the myelodysplastic clone. Therefore, we examined TCR b chain genes rearrangements by Southern blotting using DNA from stored peripheral blood slides of the patient. We have examined only TCR b chain (Cb1) genes rearrangement because of the limited amount of DNA obtained from the patient PBMC. The data demonstrate that TCR b
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chain (Cb1) genes in the patient PBMC have two rearrangement bands (Fig. 3), one of which is identical to K2-MDS cells (Fig. 2 and Fig. 3), suggesting that this cell line is derived from a myelodysplastic T lymphoid clone in the patient PBMC. Steube et al. reported previously that the failure of MDS cells to respond ex vivo might be a failure of the culture system or lack of any proliferative responsiveness of the MDS cells [11]. Interestingly, we have established a T lymphiod cell line, not myeloid or other lineages-associated cell line without any stimulation. Every histogram of flow cytometer on K2-MDS cells shows a single peak and the same surface markers were expressed after 24 month culture, suggesting that K2MDS cells have the capacity to expand monoclonally over long culture. The mechanism of CD13 expression on K2-MDS cells remains unclear. They express neither MPO nor other myeloid markers such as CD33 or CD34. Furthermore, the treatment with some hematopoietic factors (IL-3, G-CSF and GM-CSF) did not affect the proliferation of K2-MDS cells. As assessed by the morphological evaluation, none of these cytokines affected the differentiation of K2-MDS cells (data not shown). These results make it difficult to conclude that K2-MDS cells have also myeloid features and that K2-MDS cells are unique progenitor clone with intermediate differentiation towards both the myeloid and lymphoid lineages. In this study the proliferative response of K2-MDS cells to the cytokines and inducers used was not observed. It is to be clarified whether other cyokines affect the proliferation or the differentiation of K2-MDS cells for the further study about a T lymphoid feature. Further studies on the biological and molecular features of K2-MDS cells could characterize the differentiating mechanisms of the myelodysplastic progenitors toward T lymphoid lineages in MDS. In addition, this unique cell line may be a useful tool for studying the hematopoietic differentiation or commitment process from the multipotent stem cells.
Acknowledgements We thank Dr Tatsumi Ohashi of SRL. Inc. for helpful discussion and to S. Yoshida, M. Nakagawa and S. Nagayama for excellent technical assistance. M. Matsuda contributed to the concept and design, analysis of the data, drafted and revised the paper. Y. Maeda contributed to the concept and design, analysis and interpretation of the data and provided critical comments for the revision. Y. Sumimoto, H. Nawata, T. Sano and M. Higashishiba provided technical support. H. Haga provided study materials and technical support. Y. Tatsumi, F. Horiuchi, K. Irimajiri assembled
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the data. A. Kanamaru obtained the funding and gave final approval.
References [1] Jacobs A, Clark RE. Pathogenesis and clinical variations in the myelodysplastic syndromes. Clin Haematol 1986;5:925. [2] Raskind WH, Tirumali N, Jacobson R, Singer J, Fialkow PJ. Evidence for a multistep pathogenesis of a myelodysplastic syndrome. Blood 1984;63:1318. [3] Prchal JY, Throckmorton DW, Carol AJ, Fuson EW, Gams RA, Prchal JF. A common progenitor for human myeloid and lymphoid cells. Nature 1987;274:590. [4] Janssen JWG, Buschle M, Layton M, Drexler HG, Lyons J, Van den Berghe H, et al. Clonal analysis of myelodysplastic syndromes: evidence of multiple stem cell origin. Blood 1989;73:248. [5] Tefferi A, Thibodeau SN, Soldberg LA. Clonal studies in the myelodysplastic syndrome using X-linked restriction fragment length polymorphisms. Blood 1990;75:1770. [6] Bynoe AG, Scott CS, Ford P, Roberts BE. Decreased T helper cells in the myelodysplastic syndrome. Blood 1983;61:449. [7] Anderson RW, Volsky DJ, Greenberg B. Lymphocyte abnormalities in preleukaemia. I. Leuk Res 1983;7:389. [8] Bergamaschi G, Stella CC, Cattoretti G, Invernizzi R, Maserati E, Nalli G, et al. Establishment and characterization of a B-cell line derived from a patient with a myelodysplastic syndrome which expresses myelomonocytic and lymphoid markers. Br J Haematol 1991;78:167.
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[9] Lau RC, Squire J, Brisson L, Kamel-Reid S, Grunberger T, Dube´ I, et al. Lymphoid blast crisis of B-lineage phenotype with monosomy 7 in a patient with juvenile chronic myelogenous leukemia (jCML). Leukemia 1994;8:903. [10] Attias D, Grunberger T, Vanek W, Estrov Z, Cohen A, Lau R, et al. B-lineage lymphoid blast crisis in juvenile chronic myelogenous leukemia. II. Interleukin-1-mediated autocrine growth regulation of the lymphoblasts. Leukemia 1995;9:884. [11] Steube KG, Gignac SM, Hu ZB, Teepe D, Harms D, Kabisch H, et al. In vitro culture studies of childhood myelodysplastic syndrome: establishment of the cell line MUTZ-1. Leuk Lymphoma 1997;25:345. [12] Karasawa M, Tsukamoto N, Morita K, Maehara T, Okamoto K, Sakai H, et al. Clonality of T-lymphocytes in myelodysplastic syndromes and chronic myeloproliferative disorders, Myelodysplastic Syndromes: Advances in Research and Treatment 1995. p. 121. [13] Seabright M. A rapid banding technique for human chromosomes. Lancet 1971;2:971. [14] Fey MF, Pilkington SP, Summers C, Wainscoat JS. Molecular diagnosis of haematological disorders using DNA from stored bone marrow slides. Br J Haematol 1987;67:489. [15] Maeda Y, Horiuchi F, Morita S, Matsuda M, Shirakawa C, Masaki H, et al. Detection of minimal residual disease using clonospecific primers for CDRIII in patients with acute B lymphocytic leukemia with or without Philadelphia chromosome: possibility of clinical application as a tool for improving prognosis. Exp Hematol 1994;22:881. [16] Luzzatto L. Leukaemia is a genetic disorder of somatic cells. Haematologica (Pavia) 1990;75:105.
Establishment of a myelodysplastic syndrome (MDS)/secondary AML-derived T lymphoid cell line K2-MDS Mitsuhiro Matsuda a,*, Yasuhiro Maeda a, Yoshiyasu Sumimoto a, Hiroyuki Nawata a, Tetsuaki Sano a, Masaki Higashishiba a, Hisae Haga b, Yoichi Tatsumi a, Fusanari Horiuchi a, Kiyohiro Irimajiri b, Akihisa Kanamaru a a
Third Department of Internal Medicine, School of Medicine, Kinki Uni6ersity, 377 -2 Ohno-Higashi, Osaka-Sayama, Osaka 589 -8511, Japan b Faculty of Pharmaceutical Science, Kinki Uni6ersity, Osaka, Japan Received 24 May 1999; accepted 24 August 1999
Abstract We have established a T lymphoid cell line, K2-MDS, from the peripheral blood mononuclear cells (PBMC) of a patient with acute myeloblastic leukemia (AML) transformed myelodysplastic syndrome (MDS). K2-MDS cells are positive for the expression of CD4, CD5, CD13, CD25, CD71, CD95, HLA-DR and cytoplasmic CD3. Southern blotting analysis shows T cell receptor (TCR) b chain genes rearrangements, whereas immunoglobulin heavy chain (IgH) genes are not rearranged. Further, the patient PBMC contains TCR b chain genes rearrangements in the same manner as K2-MDS cells. The data indicate that K2-MDS is a T lymphoid cell line derived from a myelodysplastic clone in the patient PBMC. This new MDS-derived cell line K2-MDS may be a useful in vitro model for studies on the pathogenetic mechanisms leading to MDS. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Myelodysplastic syndrome/secondary AML; T lymphoid cell line; Myelodysplastic T lymphoid clone; T cell receptor genes rearrangements; Southern blotting analysis; DNA from peripheral blood slides
1. Introduction Myelodysplastic syndromes (MDS) are characterized by the ineffective proliferation or production of cells with abnormalities of maturation and function leading to peripheral blood cytopenia and a preleukemic state [1]. The pathogenesis of MDS is supposed to be a multistep process involving two or more genetic alterations which cause clonal proliferation of an abnormal stem cell [2]. There is a good evidence indicating that these conditions arise from clonal expansion of the progeny of an aberrant pluripotential stem cell [3–5]. Abbre6iations: AML, acute myeloblastic leukemia; HPRT, hypoxanthine phosphoribosyl-transferase; IgH, immunoglobulin heavy chain; MDS, myelodysplastic syndrome; MoAb, monoclonal antibody; MPO, myeloperoxidase; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PGK, phosphoglycerate kinase; RFLP, restriction fragment length polymorphisms; SDS, sodium dodecyl sulphate; TCR, T cell receptor. * Corresponding author. Tel.: + 81-723-660221, ext. 3128; fax: + 81-723-683732.
Cytogenetic involvement of myeloid and erythroid cell lineages has been shown in MDS. Some reports have shown peripheral blood lymphopenia and functional abnormalities of B and T cells [6,7]. Some B cell lines were established from bone marrow or peripheral blood of MDS or de novo acute myeloblastic leukemia (AML)/secondary AML after MDS, suggesting the derivation of B lymphocytes from the MDS or AML stem cell [8–11]. As for T cells involvement in MDS, using the restriction fragment length polymorphisms (RFLP) of the X-chromosome phosphoglycerate kinase (PGK) and hypoxanthine phosphoribosyl-transferase (HPRT) genes, Karasawa et al. have showed the presence of MDS clone originated from a progenitor cell to differentiate into T cells [12]. However, there has been no appropriate model for study about T lymphoid clone in MDS. In this report, we describe a unique T lymphoid cell line (K2-MDS) derived from peripheral blood of MDS and discuss the possible involvement of T lymphoid clone in MDS.
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2. Materials and methods
2.1. Origin of the cell line A 62-year-old male was admitted to our department because of anemia. The bone marrow specimen revealed a hypocellular with trilineage dysplasia, diagnosed as MDS (FAB subtype, refractory anemia with excess blasts). The patient had been supported mainly with red blood cell transfusions, but 14 months later white blood cells in peripheral blood increased up to 15.0 × 109/l with 10% myeloblasts, 60% monocytes and 17% eosinophiles including blastic cells. Bone marrow examination showed normocelluar with 30.1% myeloblast and promyelocytes, 11.5% monocytes and 8.4% eosinophiles (1.9% myelocytes, 2.5% metamyelocytes, 2.8% band formed and 1.2% segmented eosinophiles). These data made us diagnose as a overt leukemia (FAB subtype, M4Eo). Thereafter, he was refractory to therapy with cytarabine and died of progressive leukemia. The immunophenotypic profile of peripheral blood mononuclear cells (PBMC) at overt leukemia is shown in Table 1. Serum antibody for HTLV-I was negative. PBMC at overt leukemia were prepared by the standard method and grown in RPMI 1640 (GIBCO, Grand Island, NY, USA) with 10% fetal calf serum without any stimulation. The cultures were fed weekly by replacing half of the medium.
2.2. Morphology K2-MDS cells were studied after staining with Wright and Giemsa solutions.
2.3. Immunophenotyping The cells were immunophenotyped with a flow cytometry (FACS Calibur, Becton Dickinson Immunocytometry Systems, CA, USA) using monoclonal antibodies (MoAbs) directed against a number of lineage-associated and -independent antigens (Table 1). Some specific MoAbs were purchased from Becton Dickinson Immunocytometry Systems, DAKO (Glostrup, Denmark) and Immunotech (Marseilles, France). 32.2 (MoAb for CD64) and IV.3 (MoAb for CD32) were kindly provided by Dr Alan D. Schreiber of University of Pennsylvania (Philadelphia, PA, USA) and H107 (MoAb for CD23) was from Dr J. Yodoi of Kyoto University (Kyoto, Japan). The cells were washed twice with PBS and then incubated with MoAbs at 4°C for 30 min. Following incubation with the MoAbs and subsequent washing steps, cells were stained with FITC-conjugated goat anti-mouse F(ab’)2 IgG (Tago, Burlingame, CA, USA) at 4°C for 30 min. In every determination, a negative control of irrelevant
mouse MoAb of the same isotype as the MoAb under investigation was included (IgG1, IgG2, IgM controls). Addition of propidium iodide (10 mg/ml; Sigma) allowed gating out of dead cells. In cases of cytoplasmic staining for cytoplasmic CD3 or myeloperoxidase (MPO), 70% ethanol solution was used for cell fixation.
2.4. Cytogenetic analysis Cytogenetic analysis was performed on bone marrow cells of the patient at overt leukemia and K2-MDS cells. Chromosome preparations were made by SRL, Inc (Tokyo, Japan) by routine methods [13].
2.5. Preparation of genomic DNA Genomic DNA extraction from K2-MDS cells was carried out by the conventional method. Briefly, the cells were washed out with ice-cold phosphate-buffered Table 1 Immunophenotypic profile on the peripheral blood mononuclear cells (PBMC) of the patient at overt leukemia and on K2-MDS cells. Cells were analyzed with a flow cytometry (FACS Calibur) and the numbers in this table represent the percentages of the positive cells detected by the respective antibodya Antigens (CD)
Patient (%)
K2-MDS (%)
B cell: CD10 CD19 CD20 CD23
ND 0 0 ND
0 0 0 0
2 1 ND ND ND ND ND ND
0 45 99 0 0 0 0 99
Progenitor cell/activation: CD25 CD33 CD34 Class 1 Class II Class III CD71 CD95 HLA-DR
0 98 ND ND 27 ND ND ND
99 1 0 0 0 94 40 99
Myelomonocytic: CD13 CD14 CD16 CD32 CD64 MPO
78.9 ND 14 62 52 ND
78 0 0 0 0 0
T-cell: CD3 CD4 CD5 CD7 CD8 TCRa/b TCRg/d Cytoplasmic CD3
a
CD, cluster differentiation; MPO, myeloperoxide; ND, not done.
M. Matsuda et al. / Leukemia Research 24 (2000) 103–108
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Fig. 1. Morphological study of K2-MDS cells ( ×132). Staining K2-MDS cells with Wright and Giemsa solutions shows single and round cells in suspension, partly in clumps. Most are mononucleated cells with one to three nucleoli, abundant cytoplasm with vacuolization. Some cells are mitotic.
saline (PBS) and resuspended in 0.5 ml digestion buffer (100 mM NaCl, 10 mM Tris – HCl [pH 8.0], 25 mM EDTA [pH 8.0], 0.5% SDS, 0.1 mg/ml proteinase K), followed by incubation with shaking at 50°C for 12 h. Genomic DNA was isolated by ethanol precipitation after phenol/chloroform extraction. DNA was also extracted from stored peripheral blood slides as previously reported [14,15]. Briefly, the smears were scraped off the glass slide with a stainless steel razor blade and powdered, and the material was transferred into a 1.5 ml Eppendorf tube. The material was first lysed in a 1.5 ml of distilled water and in 500 ml of a nonionic detergent (Nonidet P 40, 0.1% solution, lysis time 10 min) and the nuclear pellets were incubated in a 200 ml lysis buffer (10 mM Tris–HCl [pH 8.0], 10 mM NaCl, 10 mM EDTA), with sodium dodecyl sulphate (SDS; 2%) and proteinase K (2 mg/ml) at 37°C for 12 h. DNA was extracted with phenol and chloroform. DNA was precipitated from the aqueous phase with sodium chloride and ethanol. After incubation at –80°C for 20 min, centrifugation was carried out at 15 000 rpm in a microcentrifuge. After the DNA pellets were dried, they were redissolved in 20 ml distilled water.
2.6. Analysis of receptor genes rearrangements by Southern blotting DNA extracted from K2-MDS cells and stored peripheral blood slides was accessed for receptor genes rearrangements by SRL, Inc. T cell receptor (TCR) b
chain and immunoglobulin heavy chain (IgH) genes rearrangements were studied by using primers for TCR b chain (Cb1, Jb1 and Jb2) and IgH (JH and Cm) prepared by SRL, Inc.
2.7. Effect of cytokines and inducers The proliferative response of K2-MDS cells to various cytokines and inducers was examined. After 1× 105/ml K2-MDS cells were incubated with IL-2 (used at 10 IU/ml), IL-3 (50 ng/ml), GM-CSF (5 ng/ml), G-CSF (50 ng/ml), PMA (10 ng/ml), PHA (10 mg/ml) and Con-A (10 mg/ml) for 24 or 48 h, the proliferation was assessed by 3H-thymidine uptake assay as described previously [11]. Cytokines (IL-2, IL-3, GM-CSF and G-CSF) were purchased from Boehringer Mannheim, Germany and inducers (PMA, PHA and Con-A) were from Sigma.
3. Results
3.1. Establishment of the cell line During the first 8 weeks of patient PBMC suspension culture, the number of the cells increased slowly. After the eighth week, however, cells proliferated rapidly with a doubling time of 24–30 h, giving rise to a continuous cell line K2-MDS. K2-MDS cells have grown without any growth factor over 24 months or over 200 passages.
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3.2. Morphological and immunological characterization Fig. 1 represents K2-MDS cells stained with Wright and Giemsa solutions. K2-MDS cells are single and round cells in suspension, partly in clumps. Most are mononucleated cells with one to three nucleoli, abundant cytoplasm with vacuolization. Some cells are mitotic. Table 1 shows the immunological findings on PBMC of the patient at overt leukemia and on K2MDS cells. The data demonstrate that K2-MDS cells express T lymphoid marker (CD4, CD5, CD25, CD71, CD95, HLA-DR and cytoplasmic CD3), whereas PBMC of the patient expresses myelomonocytic markers (CD13, CD16, CD32, CD33, CD34 and CD64). Every histogram of flow cytometer on K2-MDS cells shows a single peak (data not shown). After 12 or 24 month culture, the same results were obtained (data not shown), suggesting the stability of the cell surface expression over long culture.
from individual clones. All K2-MDS clones indicate pseudotetraploid described above, suggesting that K2MDS cells are clonal.
3.4. Analysis of receptor genes rearrangements DNA extracted from K2-MDS cells was examined for TCR b chain (Cb1, Jb1 and Jb2) and IgH (JH and Cm) genes rearrangements by Southern blotting. TCR b chain genes rearrangements (Cb1, Jb1 and Jb2) were observed (Fig. 2), whereas no rearrangement was detected in IgH genes (data not shown). Next, we investigated whether TCR b chain genes in the patient PBMC were rearranged or not. We extracted DNA from stored peripheral blood slides of the patient at overt leukemia. Southern blotting analysis shows that TCR b chain genes (Cb1) in the patient PBMC are rearranged as well as K2-MDS cells (Fig. 3).
3.5. Effect of cytokines and inducers 3.3. Cytogenetic analysis Cytogenetic investigations of bone marrow cells of the patient at overt leukemia showed 46, XY, + der (1;7)(q10;p10), -7. Of 20 mitoses of K2-MDS cells analyzed, more than 18 (90%) had pseudotetraploid with the following karyotypes: 78-81, XXY, + add (1) (p 11), + 2, + add (3) (q 27-29), +4, + add (5) (q35), + 7, +add (8) (q 24), add (11) (p1), add (12) (q 15-22), del (13) (q 32), add (14) (p 11), -16, add (17) (p 11), add (19) (p 13), +add (19), +20, indicating the discrepancy of the karyotype between the patient cells and K2-MDS cells. Further we performed limiting dilution method to isolate an individual clone. After culture for three weeks, we have obtained several clones derived
In the 3H-thymidine uptake assay, none of the cytokines and inducers used (IL-2, IL-3, GM-CSF, GCSF, PMA, PHA and Con-A) had significant proliferation-stimulating or -inhibiting effects on K2MDS cells for 24 or 48 h (data not shown).
4. Discussion The presence of T lymphoid surface markers and the monoclonal rearrangements of T cell receptor genes by Southern blotting analysis clearly show the T lymphoid origin of K2-MDS cells. Southern blotting indicates T cell receptor b chain genes rearrangements on both
Fig. 2. T cell receptor (TCR) b chain genes rearrangements in K2-MDS cells. DNA extracted from K2-MDS cells was digested with EcoR V (E), BamH I (B) or Hind III (H). The Jb1, Jb2 and Cb1 probes show the presence of rearranged TCR b chain genes (arrows) in K2-MDS cells. Human placentas were used as a control.
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Fig. 3. T cell receptor (TCR) b chain (Cb1) genes rearrangements in the patient PBMC. DNA from the patient PBMC was digested with EcoR V (E), BamH I (B). The Cb1 probes show the presence of rearranged TCR b chain genes (arrows) in the patient PBMC. Human placentas were used as a control.
K2-MDS cells and the patient PBMC. K2-MDS cells have grown for more than 24 months since the establishment in 1997. Also these cells proliferate independently of any growth factor, indicating that the cells are immortal. PCR analysis using primers designed to amplify Bam W region of the EBV genome shows that the transformation of EBV is not associated in this cell line (data not shown). As discussed previously, a clonal population is best identified by unique mutational events, such as a distinctive chromosomal abnormality or discrete gene rearrangements [16]. K2-MDS cells have manifestated a highly complex, near triploid karyotype. Such a cytogenetic picture is more generally typical of late passage AML cell line or solid tumor cell line. All K2-MDS clones obtained by limiting dilution method indicate the chromosomal abnormality (data not shown), suggesting that K2-MDS cells are clonal. However, the discrepancy of the karyotype between the patient’s cells and K2-MDS cells may be conflicting with the notion that this cell line originated from the myelodysplastic clone. Therefore, we examined TCR b chain genes rearrangements by Southern blotting using DNA from stored peripheral blood slides of the patient. We have examined only TCR b chain (Cb1) genes rearrangement because of the limited amount of DNA obtained from the patient PBMC. The data demonstrate that TCR b
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chain (Cb1) genes in the patient PBMC have two rearrangement bands (Fig. 3), one of which is identical to K2-MDS cells (Fig. 2 and Fig. 3), suggesting that this cell line is derived from a myelodysplastic T lymphoid clone in the patient PBMC. Steube et al. reported previously that the failure of MDS cells to respond ex vivo might be a failure of the culture system or lack of any proliferative responsiveness of the MDS cells [11]. Interestingly, we have established a T lymphiod cell line, not myeloid or other lineages-associated cell line without any stimulation. Every histogram of flow cytometer on K2-MDS cells shows a single peak and the same surface markers were expressed after 24 month culture, suggesting that K2MDS cells have the capacity to expand monoclonally over long culture. The mechanism of CD13 expression on K2-MDS cells remains unclear. They express neither MPO nor other myeloid markers such as CD33 or CD34. Furthermore, the treatment with some hematopoietic factors (IL-3, G-CSF and GM-CSF) did not affect the proliferation of K2-MDS cells. As assessed by the morphological evaluation, none of these cytokines affected the differentiation of K2-MDS cells (data not shown). These results make it difficult to conclude that K2-MDS cells have also myeloid features and that K2-MDS cells are unique progenitor clone with intermediate differentiation towards both the myeloid and lymphoid lineages. In this study the proliferative response of K2-MDS cells to the cytokines and inducers used was not observed. It is to be clarified whether other cyokines affect the proliferation or the differentiation of K2-MDS cells for the further study about a T lymphoid feature. Further studies on the biological and molecular features of K2-MDS cells could characterize the differentiating mechanisms of the myelodysplastic progenitors toward T lymphoid lineages in MDS. In addition, this unique cell line may be a useful tool for studying the hematopoietic differentiation or commitment process from the multipotent stem cells.
Acknowledgements We thank Dr Tatsumi Ohashi of SRL. Inc. for helpful discussion and to S. Yoshida, M. Nakagawa and S. Nagayama for excellent technical assistance. M. Matsuda contributed to the concept and design, analysis of the data, drafted and revised the paper. Y. Maeda contributed to the concept and design, analysis and interpretation of the data and provided critical comments for the revision. Y. Sumimoto, H. Nawata, T. Sano and M. Higashishiba provided technical support. H. Haga provided study materials and technical support. Y. Tatsumi, F. Horiuchi, K. Irimajiri assembled
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the data. A. Kanamaru obtained the funding and gave final approval.
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