107 @ 1990 The Japanese Society of Pathology
Expression of Leukocyte Common Antigen (CD45) on Various Human Leukemia/Lymphoma Cell Lines
Akinobu Nakano1s2,Takayuki Harada2, Shigeru Morikawa2, and Yuzuru Katol
CD45 antigen (leukocyte common antigen), a unique and ubiquitous membrane glycoprotein with a molecular mass of about 200 kDa, is expressed on almost all hematopoietic cells except for mature erythrocytes. However, the biological function of this glycoprotein still remains to be resolved. In order to clarify the role of CD45 antigen in hematopoietic cell differentiation and function, its expression on human leukemia/lymphoma cell lines was studied by membrane immunofluorescence. Thirty-eight established cell lines were analyzed using T29/33, a monoclonal antibody (MoAb) that recognizes the common epitopes of this glycoprotein molecule. Conventional cell marker studies were also carried out on these cell lines to compare their CD45 expression. It was shown that CD45 expression varies among B-lineage cells depending on cell differentiation, in contrast to its stable expression on leukemic T cell ( 6 / 6 , positive) and myeloid (5/5, positive) lineage cell lines. On the other hand, only two out of six histiomonocytoid lineage cell lines were positive. Human T cell leukemia/lymphoma virus type I (HTLV-I)-associated T cell lines derived from peripheral blood leukocytes of patients with adult T cell leukernia/lymphoma (ATL/L) in Japan did not express CD45 on their cell surface. Taken together, these observations suggest that CD45 has a functional role i n hematopoietic cell activation and differentiation. Acta Pathol Jpn 40: 107-115, 1990. Key words : Leukocyte common antigen, Human,
Leukemia/Lymphoma, Cell line
Received June 29, 1989. Accepted for publication October 5, 1989. 'First Division of Internal Medicine, Department of Medicine, *The Department of Pathology 1st Unit, Shimane Medical University, Izumo. Mailing address: Akinobu Nakano, M.D. (+RE{#),The Department of Pathology 1st Unit, Shimane Medical University, 89-1 Enya-cho, Izumo, Shimane 693, Japan.
INTRODUCTION Human leukocyte common antigen (CD45; L-CA in the rat, T200 or Ly-5 in the mouse) is a family of highmolecular-mass (170-240 kDa) glycoproteins, and is expressed exclusively on cells of hematopoietic lineage, excluding mature erythroid cells (1-3). This unique molecule is thought to be implicated in a number of immunological processes, including NK- (4-6) and CTLinduced cytolysis (7,8), induction of both suppressor Tcell activity (9) and cytotoxic T cell activity (lo), B cell differentiation and function (11, 12), and regulation of IL-2 receptor expression (13). CD45 (L-CA) shows heterogeneity in its molecular size, antigenicity and glycosylation and its different forms are unique to different lymphoid types (1, 14, 15). More recently, analysis of cloned cDNA has revealed that human CD45 consists of a single integral-membrane polypeptide chain with a large intracytoplasmic domain (16, 17). The molecular heterogeneity of CD45 is suggested to be based on varying mRNA splicing (9,18-20) and abundant glycosylation sites displayed on the molecule (7, 18). Hematopoietic cell type-specific differences in the size of CD45 mRNA have been detected in the rat (19), mouse (21) and human (17). However, the relationship between the molecular heterogeneity of CD45 and its functional aspects in hematopoietic cell differentiation as well as activation of these cells is still obscure. In order to estimate the expression of CD45 on hematopoietic cells at various stages of differentiation, 38 human leukemia/malignant lymphoma cell lines of defined cellular lineage in hematopoiesis were examined with a monoclonal antibody (MoAb) T29/33 (22) by membrane immunofluorescence assay. Conventional cell marker and surface marker studies were also carried out on these cell lines.
108
Expression of th CD45 Antigen in Cell Lines (Nakano et a/.)
MATERIALS AND METHODS Cell lines Table 1 shows a list of the 38 hematopoietic cell lines used. Cell lines nos. 2, 5, 19, 22-27, and 34-38 were established and analyzed immunologically in our laboratory (23-26). The other 2 3 cell lines were kindly pro-
vided by Dr. J. Minowada (Fujisaki Cancer Cell Laboratory, Okayama, Japan) and their characteristics have been described elsewhere (27, 28). These cell lines were divided into 3 types of hematopoietic lineage based on membrane marker criteria, histochemical properties and biological activities. 1 ) Lymphoid-lineage
cell lines : These cell lines
Table 1. Reactivity of T29/33 Monoclonal Antibody (Anti-CD45) with Human Hematopoietic Cell Lines
Cell line
Origin Cell type
No.
Designation
Diagnosis
Source
$1 n2 e3 $4 n5 ii6 $7 $8 ti9 G10 411 112 413 114 a15 d16 dl7 $18 it19 820 421 422 #23 d24 1125 d26 $27 #28 #29 #30 531 k32 c33 Y34 Y35 it36 #37 r38
REH HCF-MLp-N NALM-16 NALM-6 HPB-Null HPE MLp K BALM-1 BALM 2 Daudi Rail P3HR-1 B46M DND-39 ARH-77 U 266 RPMI-8226 TALL 1 MOLT-4 HPB-ALL CCRF CEM CCRF-HSB-2 HPB-MLT HPB CTL I HPB-ATL-T HPB-ATL-2 HPB-ATL-0 HLN-ATL-0 NALM 1 K562 KG-1 ML 3 HL-60 u937 HPL-Hod-l HPL-Hod-2 HLN-Ret- 1 HLN-Ret 2 HPE-Ret-3
ALL ML (UD) ALL ALL ALL ML (UD) ALL ALL Burkitt's Burkitt's Burkitt's Burkitt's Burkitt's MM MM MM ALL ALL ALL ALL ALL LML
PBL CSF PBL PBL PBL PE PBL PBL LT LT LT LT LT PBL PBL PBL BM PBL PBL PBL PBL PBL PBL PBL PBL PBL LN PBL PE BM PBL PBL PL PL PL LN LN PE
s. s
ATL/L ATL/L ATL/L ATL/L CML-BC CML AML AML APL ML (DH) ML ML (Hod) ML (Rs) ML (Rs) ML (Rs)
non T, non B non T, non B non T, non B pre-B pre-B B-Blast [ A , p ] B-Blast [ x , 8, p ] B-Blast [ x , 8, p ] B-Blast [ x , 8, p ] B-Blast [ p ] B-Blast [ p ] B-Blast [ x , 8, p ] B-Blast AF [ x , AF [ A , €1 AF [ A 1 T-Blast T-Blast T-Blast T-Blast T-Blast T-Blast T>' T" T\' T" T" MY MY (Er) MY MY MY Mon Hist Hist Hist Hist Hist
I.
Year established 1977 1987 1977 1979 1977 1979 1976 1976 1967 1964 1967 1969 1978 1968 1966 1977 1971 1973 1964 1966 1974 1981 1983 1983 1986 1985 1975 1975 1978 1982 1977 1975 1972 1974 1972 1974 1979
CD45
% positive 87 95
Fluorescence intensitv
+-+
0 0 17 78 100 61 82 88 75 74 99 13.4 5.7 0 97 81 86 96 92 85 10 0 0 0 91 36 62 100 65 92 91
+/*-it
+-8 +-it +-it
+-+ f-+ i-+ +-it
f-+
+-+ -
+-it
i-+ +-it
f-+ +-it +-it
f- t
+-it
+- i + f-+ +-it
f-+
0 0 0 0 91.8
f-it
ALL, acute lymphocytic leukemia ; ML (UD), malignant lymphoma, undefined ; Burkitt's, Burkitt's lymphoma ; MM, multiple myeloma ; LML, leukemic malignant lymphoma (T) ; S.S, SBzary's syndrome; ATL/L, adult T-cell leukemia/lymphoma ; CML, chronic myelogenic leukemia-blastic crisis ; AML, acute myelogenic leukemia ; APL, acute promyelogenic leukemia ; ML (DH), malignant diffuse histiocytic lymphoma; ML (Hod), Hodgkin's disease; ML (Rs), reticulum cell sarcoma. PBL, peripheral blood leukocyte; CSF, cerebrospinal fluid; PE, peritoneal effusion; LT, lymph node tumor; BM, bone marrow cell; LN, lymph node; PL, pleural effusion. AF, antibody-forming cell ; My, myeloid ; Er, erythloid ; Mon, monocytoid ; Hist, histiocytoid; T", HTLV-I-producing cell, [ 1 : immunoglobulin light and/or heavy chain produced.
109
Acta Pathologica Japonica 40 ( 2 ) : 1990
consisted of 3 subsets. First, non-T-, non-B-(null) cell lines, which do not express any specified T-, B- cell markers and show high terminal deoxytransferase activity and common ALL antigen (27) (#1-3). Second, Bcell lines (#4-16) including leukemic pre-B cells (NALM6, HPB-Null)and antibody-forming cells derived from myeloma patients (#14-16). Among them, cell lines nos. 9 - 1 3 were derived from African Burkitt's lymphoma cells (27). And last, the T-cell line group consisted of 2 subgroups, T-blast cell lines (#17-22) derived from T cell leukemia, and T cell lines which harbor the human T lymphotropic virus type I (HTLV-I) genome and produce C-type retrovirus (#23-27).
2) Myeloid-lineage cell lines : The K 5 6 2 and NALM-I lines were Ph' chromosome-positive CML-derived cells (29, 30), the latter cell line originating from cells of a lymphoid-type blast crisis of CML. The other 3 lines were derived from AML. 3) Monohistiocyte-lineage cell lines : Five out of 6 cell lines (#34-38) were established from patients with malignant lymphoma diagnosed as Hodgkin's disease or reticulum cell sarcoma in our laboratory (26). U937 is a phagocytic cell line established from a patient with malignant diffuse histiocytic lymphoma (31). All cell lines were maintained in RPMI-1640 medium supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (FCS) (KC Biologicals, Lenexa, USA) and kanamycin ( 6 0 ~ g / m l ) at 37°C in a 5% C0,/95% air atmosphere. All cultures used were free from bacterial or mycoplasma infection. Monoclona I a n t i bodies T29/33 was purchased from Hybritech Inc. (San Diego, USA). This MoAb was obtained by fusion of spleen cells from BALB/c mice immunized against the human cell line CCRF-CEM with a non-producer mouse myeloma cell line(1). T29/33 was able to react with human T200 glycoprotein from various hematopoietic cell sources (22). B A - l (CD24) and BA-2 (CD9) were also purchased from Hybritech. HB-5(CD21) was kindly provided by Dr. M. Cooper (Cellular Immunology Unit of the Tumor Institute, University of Alabama, Birmingham, AL USA).
Membrane immunofluorescence Cell-surface markers were examined by indirect membrane immunofluorescence. Cells were incubated with mouse MoAbs for 30 min a t 4°C in suspension. After
being washed twice with Eagle's minimum essential medium (MEM) (Nissui, Tokyo), they were stained with FITC-conjugated F(ab), fragment of goat anti-mouse IgG from Cappel (Malvern, PA, USA) for 30 min at 4°C. lmmunofluorescent samples were examined using a Zeiss epifluorescence microscope or by a FACSTAR fluorescence-activated cell sorter (Becton Dickinson FAGS Systems, USA) to measure the relative fluorescence intensities of individual cells. Data obtained from the FACSTAR were analyzed using an attached HewlettPackard computer. Cytoplasmic immunoglobulin For detection of cytoplasmic immunoglobulin, direct immunofluorescence staining was performed. Cells were smeared and fixed with acetone for 5 min, washed with phosphate-buffered saline (PBS), and then incubated with fluorescein-conjugated goat anti-human immunoglobulins (MBL, Japan) for 4 5 min. Specifically positive cells were observed and photographed by epifl uo rescence microscopy. Western blot analysis for CD45 antigen Molecules recognized by MoAb T29/33 were analyzed by the Western blotting method. After being washed with PBS, 5 x 1 0 7 - 1 x108 cells were lysed with 1 ml of extraction buffer (0.5% Nonidet P-40 in 0.02 M Tris-0.3 M NaCI, 1.0 m M CaCI,-0.5 mM MgCI,-2 m M EDTA10% glycerol-2.0 mM phenylmethylsulfonyl fluoride, pH 9.0). Insoluble material was removed by centrifugation for 2 0 min at 15,000 rpm. Electrophoresis was performed using a 7.5% acrylamide gel under reducing conditions (SDS-PAGE) and transferred elect rophoretically to a nitrocellulose membrane. The membrane was then incubated with MoAb T29/33 (diluted 1 : 250) overnight and incubated with Western blotting-grade affinity-purified goat anti-mouse IgG (H L) horseradish peroxidase conjugate (Bio-Rad Laboratories, Richmond, USA) (diluted 1 : 2,000 or 1 : 3,000). As the substrate for the peroxidase reaction, 3, 3'-diaminobenzidine tetrahydrochloride was used.
+
RESULTS 1. Detection o f CD45 antigen in various hematopoietic cell lines The reactivity of each cell line with MoAb T29/33 was expressed as the percentage of fluorescence-positive cells by indirect immunofluorescence, and the intensity of specific fluorescence of each smear was given an arbi-
110
Expression of th CD45 Antigen in Cell Lines (Nakano et a/.)
trary value ranging from negative staining (-) to strongly brilliant staining ( + ) ; (+) was used to denote weakly but significantly stained cells. Cell lines showing variable staining results were examined repeatedly and subjected to cytofluorometric analysis by FACSTAR. More than 90% of peripheral blood leukocytes from healthy donors had been shown to express the CD45 antigen in a control study. With regard to the lymphoid-lineage cell lines, two out of three null cell lines (REH and MLp-N) were T29/33positive and one (NALM-16) was completely negative. A variety of reactivity with T29/33 was evident for Blineage cell lines, and low expression of CD45 antigen was observed in the pre-B cell lines (especially NALM-6) and in the antibody-forming cell lines originating from myeloma cells (especially RPMI-8226). On the other hand, B-blast cell lines including Burkitt's cell lines expressed the CD45 antigen strongly. All cells of T-blastic lineage expressed the CD45 antigen constantly and strong Iy. Interestingly, however, HTLV-I-positive T-cell lines derived from peripheral blood leukocytes of ATL patients lacked expression of CD45. An ATL cell line (HLN-ATL0) established from lymph node cells showed reactivity with T29/33, in spite of a lack of CD45 expression on cells derived from PBL (HPB-ATL-0) of the same patient. All myeloid-lineage cell lines expressed the CD45 antigen constantly, even though the intensity of the expression varied. A relatively small population in the NALM-1 cell line was positive for T29/33. Variable reactivity with MoAb T29/33 was also o b served in the group of cell lines of mono-histiocyte lineage, in contrast t o all of the granulocytic cell lines (KG1, ML-3 and HL-60) expressing the CD45 antigen. Two out of 6 cell lines (U937 and HPE-Ret-3) showed a high percentage of antigen expression and the other cell lines were completely unreactive with MoAb T29/33.
2. Western blotting analysis of CD45 antigen in lymphoid cell lines positive a n d negative for membrane fluorescence Western blotting analysis was carried out on eight lymphoblastoid cell lines and three ATL-derived T-cell lines; two of them derived from peripheral blood leukocytes were negative for CD45 membrane fluorescence. Figure 2 shows a typical result of CD45-positive bands on the blotted gels of lymphoblastoid cell lines using MoAb T29/33. The distribution of isoforms of CD45 antigen in fluorescence-positive cell lines is illustrated schematically in Fig. 1. Variable levels of production of CD45-isoform antigens were observed in T-blastic line-
HTLV I
p roduc i i g
HPB-ATL-2 HPBATL-0 HLNATL-0
NOT DETECTEO
PEL PEL LN
Figure 1. Schema of CD45 on lymphoid-lineage cell line detected by Western blotting analysis. Solid rectangles ; definitive blotted bands, Rectangles with dots ; weakly blotted bands.
MW
205
-
11697.466-
1
2
3
4
5
6
7
8
Figure 2. Western blotting analysis of CD45 antigen. Lane I ; HPB-MLT, Lane 2 ; TALL-1 Lane 3 ; HPB-ALL, Lane 4 ; HSB2, Lane 5 ; P3HR-1, Lane 6 : Raji, Lane 7 ; BALM-1, and Lane 8 ; Daudi.
age cells even though they expressed the CD45 antigen equally on their cell surface. HPB-ALL produces 150-, 1 7 0 - and 200-kDa isoforms, HSB-2 and HPB-MLT produce 170-, 200- and 220-kDa isoforrns, and TALL1 contains larger molecular isoforms of 2 2 0 and 2 4 0 kDa. CD45-isoform patterns were rather restricted among four B-blastoid cell lines. BALM-1 had two isoforms of 2 0 0 and 2 2 0 kDa. Additional blotting bands in the gel were observed with Daudi, Raji and P3HR--l cells, which were Burkitt's lymphoma cell lines harboring the EBV genome. As for ATL-cell lines, membrane CD45-positive HLN-ATL-0 cells formed a 170-kDa precipitating band in the gel. HPB-ATL-0 cells, which were negative for membrane CD45 antigen, formed a weak 170-kDa isoform band by Western blotting analysis. ATL-2 cells did not show any detectable band in this study.
111
Acta Pathologica Japonica 40 (2) : 1990
Figure 3. Cytoplasmic immunoglobulins of plasmacytoma cell lines, ARH-77 (a) and RPMI-8226 (b) are shown by direct immunofluorescence (objective).
3' Relationship
maturation and differentiation to CD45 expression in B-lineage cell h e s Of
Variable reactivity to T29/33 was evident for B-lineage cell lines in which restricted isoform patterns were observed by immunoblotting analysis. Expression of some antigens and markers related to B-cell differTable 2.
entiation and/or maturation was analyzed in B-lineage cell lines and compared with CD45 expression (Table 3). Pre-B cell lines, thought to be immature cells, expressed CD45 antigen only to a limited grade and extent, in contrast to B-blastic cell lines. In the latter group of cell lines, both CD45 antigen and surface immunoglobulins were expressed constantly, whereas reactivities to
Expression of CD45 on Fresh Leukemia/Lymphoma Cells from Various Origins in Primary Culture
Primary culture No. (Cell line)
Patient Age
Clinical diagnosis AML
Cell source
CuIture duration
% positive
PBL
83.4 97.1 96.3 91.2 33.7 97.3 > 99.0 97.3 85.5 95.0 83.2 > 99.0 > 99.0 > 99.0 94.4 > 99.0 > 99.0 > 99.0 95.1 82.7 75.6 >99.0 > 99.0 55.8
1 HPB-Nod.
68
Sex M
2 HPB-Wat.
55
M
AML
PEL
3 HPB-Ued.
62
F
ALL
PEL
HPB-Tom.
35
M
ALL
PEL
5 HLN-Kan. 6 HLN-Wak. 7 HPB-Wak.
61 50 50
M F F
ML ML ML
LN LN PEL
8 HPL-Kaw.
85
M
ML
PL
9 HCF-Nis.
49
M
ML
CSF
60 42
M M
ML ATL/L
PEL PEL
61
F
ATL/L
PEL
70
F
s.s
PBL
Expression of Leukocyte Common Antigen (CD45) on Various Human Leukemia/Lymphoma Cell Lines
Akinobu Nakano1s2,Takayuki Harada2, Shigeru Morikawa2, and Yuzuru Katol
CD45 antigen (leukocyte common antigen), a unique and ubiquitous membrane glycoprotein with a molecular mass of about 200 kDa, is expressed on almost all hematopoietic cells except for mature erythrocytes. However, the biological function of this glycoprotein still remains to be resolved. In order to clarify the role of CD45 antigen in hematopoietic cell differentiation and function, its expression on human leukemia/lymphoma cell lines was studied by membrane immunofluorescence. Thirty-eight established cell lines were analyzed using T29/33, a monoclonal antibody (MoAb) that recognizes the common epitopes of this glycoprotein molecule. Conventional cell marker studies were also carried out on these cell lines to compare their CD45 expression. It was shown that CD45 expression varies among B-lineage cells depending on cell differentiation, in contrast to its stable expression on leukemic T cell ( 6 / 6 , positive) and myeloid (5/5, positive) lineage cell lines. On the other hand, only two out of six histiomonocytoid lineage cell lines were positive. Human T cell leukemia/lymphoma virus type I (HTLV-I)-associated T cell lines derived from peripheral blood leukocytes of patients with adult T cell leukernia/lymphoma (ATL/L) in Japan did not express CD45 on their cell surface. Taken together, these observations suggest that CD45 has a functional role i n hematopoietic cell activation and differentiation. Acta Pathol Jpn 40: 107-115, 1990. Key words : Leukocyte common antigen, Human,
Leukemia/Lymphoma, Cell line
Received June 29, 1989. Accepted for publication October 5, 1989. 'First Division of Internal Medicine, Department of Medicine, *The Department of Pathology 1st Unit, Shimane Medical University, Izumo. Mailing address: Akinobu Nakano, M.D. (+RE{#),The Department of Pathology 1st Unit, Shimane Medical University, 89-1 Enya-cho, Izumo, Shimane 693, Japan.
INTRODUCTION Human leukocyte common antigen (CD45; L-CA in the rat, T200 or Ly-5 in the mouse) is a family of highmolecular-mass (170-240 kDa) glycoproteins, and is expressed exclusively on cells of hematopoietic lineage, excluding mature erythroid cells (1-3). This unique molecule is thought to be implicated in a number of immunological processes, including NK- (4-6) and CTLinduced cytolysis (7,8), induction of both suppressor Tcell activity (9) and cytotoxic T cell activity (lo), B cell differentiation and function (11, 12), and regulation of IL-2 receptor expression (13). CD45 (L-CA) shows heterogeneity in its molecular size, antigenicity and glycosylation and its different forms are unique to different lymphoid types (1, 14, 15). More recently, analysis of cloned cDNA has revealed that human CD45 consists of a single integral-membrane polypeptide chain with a large intracytoplasmic domain (16, 17). The molecular heterogeneity of CD45 is suggested to be based on varying mRNA splicing (9,18-20) and abundant glycosylation sites displayed on the molecule (7, 18). Hematopoietic cell type-specific differences in the size of CD45 mRNA have been detected in the rat (19), mouse (21) and human (17). However, the relationship between the molecular heterogeneity of CD45 and its functional aspects in hematopoietic cell differentiation as well as activation of these cells is still obscure. In order to estimate the expression of CD45 on hematopoietic cells at various stages of differentiation, 38 human leukemia/malignant lymphoma cell lines of defined cellular lineage in hematopoiesis were examined with a monoclonal antibody (MoAb) T29/33 (22) by membrane immunofluorescence assay. Conventional cell marker and surface marker studies were also carried out on these cell lines.
108
Expression of th CD45 Antigen in Cell Lines (Nakano et a/.)
MATERIALS AND METHODS Cell lines Table 1 shows a list of the 38 hematopoietic cell lines used. Cell lines nos. 2, 5, 19, 22-27, and 34-38 were established and analyzed immunologically in our laboratory (23-26). The other 2 3 cell lines were kindly pro-
vided by Dr. J. Minowada (Fujisaki Cancer Cell Laboratory, Okayama, Japan) and their characteristics have been described elsewhere (27, 28). These cell lines were divided into 3 types of hematopoietic lineage based on membrane marker criteria, histochemical properties and biological activities. 1 ) Lymphoid-lineage
cell lines : These cell lines
Table 1. Reactivity of T29/33 Monoclonal Antibody (Anti-CD45) with Human Hematopoietic Cell Lines
Cell line
Origin Cell type
No.
Designation
Diagnosis
Source
$1 n2 e3 $4 n5 ii6 $7 $8 ti9 G10 411 112 413 114 a15 d16 dl7 $18 it19 820 421 422 #23 d24 1125 d26 $27 #28 #29 #30 531 k32 c33 Y34 Y35 it36 #37 r38
REH HCF-MLp-N NALM-16 NALM-6 HPB-Null HPE MLp K BALM-1 BALM 2 Daudi Rail P3HR-1 B46M DND-39 ARH-77 U 266 RPMI-8226 TALL 1 MOLT-4 HPB-ALL CCRF CEM CCRF-HSB-2 HPB-MLT HPB CTL I HPB-ATL-T HPB-ATL-2 HPB-ATL-0 HLN-ATL-0 NALM 1 K562 KG-1 ML 3 HL-60 u937 HPL-Hod-l HPL-Hod-2 HLN-Ret- 1 HLN-Ret 2 HPE-Ret-3
ALL ML (UD) ALL ALL ALL ML (UD) ALL ALL Burkitt's Burkitt's Burkitt's Burkitt's Burkitt's MM MM MM ALL ALL ALL ALL ALL LML
PBL CSF PBL PBL PBL PE PBL PBL LT LT LT LT LT PBL PBL PBL BM PBL PBL PBL PBL PBL PBL PBL PBL PBL LN PBL PE BM PBL PBL PL PL PL LN LN PE
s. s
ATL/L ATL/L ATL/L ATL/L CML-BC CML AML AML APL ML (DH) ML ML (Hod) ML (Rs) ML (Rs) ML (Rs)
non T, non B non T, non B non T, non B pre-B pre-B B-Blast [ A , p ] B-Blast [ x , 8, p ] B-Blast [ x , 8, p ] B-Blast [ x , 8, p ] B-Blast [ p ] B-Blast [ p ] B-Blast [ x , 8, p ] B-Blast AF [ x , AF [ A , €1 AF [ A 1 T-Blast T-Blast T-Blast T-Blast T-Blast T-Blast T>' T" T\' T" T" MY MY (Er) MY MY MY Mon Hist Hist Hist Hist Hist
I.
Year established 1977 1987 1977 1979 1977 1979 1976 1976 1967 1964 1967 1969 1978 1968 1966 1977 1971 1973 1964 1966 1974 1981 1983 1983 1986 1985 1975 1975 1978 1982 1977 1975 1972 1974 1972 1974 1979
CD45
% positive 87 95
Fluorescence intensitv
+-+
0 0 17 78 100 61 82 88 75 74 99 13.4 5.7 0 97 81 86 96 92 85 10 0 0 0 91 36 62 100 65 92 91
+/*-it
+-8 +-it +-it
+-+ f-+ i-+ +-it
f-+
+-+ -
+-it
i-+ +-it
f-+ +-it +-it
f- t
+-it
+- i + f-+ +-it
f-+
0 0 0 0 91.8
f-it
ALL, acute lymphocytic leukemia ; ML (UD), malignant lymphoma, undefined ; Burkitt's, Burkitt's lymphoma ; MM, multiple myeloma ; LML, leukemic malignant lymphoma (T) ; S.S, SBzary's syndrome; ATL/L, adult T-cell leukemia/lymphoma ; CML, chronic myelogenic leukemia-blastic crisis ; AML, acute myelogenic leukemia ; APL, acute promyelogenic leukemia ; ML (DH), malignant diffuse histiocytic lymphoma; ML (Hod), Hodgkin's disease; ML (Rs), reticulum cell sarcoma. PBL, peripheral blood leukocyte; CSF, cerebrospinal fluid; PE, peritoneal effusion; LT, lymph node tumor; BM, bone marrow cell; LN, lymph node; PL, pleural effusion. AF, antibody-forming cell ; My, myeloid ; Er, erythloid ; Mon, monocytoid ; Hist, histiocytoid; T", HTLV-I-producing cell, [ 1 : immunoglobulin light and/or heavy chain produced.
109
Acta Pathologica Japonica 40 ( 2 ) : 1990
consisted of 3 subsets. First, non-T-, non-B-(null) cell lines, which do not express any specified T-, B- cell markers and show high terminal deoxytransferase activity and common ALL antigen (27) (#1-3). Second, Bcell lines (#4-16) including leukemic pre-B cells (NALM6, HPB-Null)and antibody-forming cells derived from myeloma patients (#14-16). Among them, cell lines nos. 9 - 1 3 were derived from African Burkitt's lymphoma cells (27). And last, the T-cell line group consisted of 2 subgroups, T-blast cell lines (#17-22) derived from T cell leukemia, and T cell lines which harbor the human T lymphotropic virus type I (HTLV-I) genome and produce C-type retrovirus (#23-27).
2) Myeloid-lineage cell lines : The K 5 6 2 and NALM-I lines were Ph' chromosome-positive CML-derived cells (29, 30), the latter cell line originating from cells of a lymphoid-type blast crisis of CML. The other 3 lines were derived from AML. 3) Monohistiocyte-lineage cell lines : Five out of 6 cell lines (#34-38) were established from patients with malignant lymphoma diagnosed as Hodgkin's disease or reticulum cell sarcoma in our laboratory (26). U937 is a phagocytic cell line established from a patient with malignant diffuse histiocytic lymphoma (31). All cell lines were maintained in RPMI-1640 medium supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (FCS) (KC Biologicals, Lenexa, USA) and kanamycin ( 6 0 ~ g / m l ) at 37°C in a 5% C0,/95% air atmosphere. All cultures used were free from bacterial or mycoplasma infection. Monoclona I a n t i bodies T29/33 was purchased from Hybritech Inc. (San Diego, USA). This MoAb was obtained by fusion of spleen cells from BALB/c mice immunized against the human cell line CCRF-CEM with a non-producer mouse myeloma cell line(1). T29/33 was able to react with human T200 glycoprotein from various hematopoietic cell sources (22). B A - l (CD24) and BA-2 (CD9) were also purchased from Hybritech. HB-5(CD21) was kindly provided by Dr. M. Cooper (Cellular Immunology Unit of the Tumor Institute, University of Alabama, Birmingham, AL USA).
Membrane immunofluorescence Cell-surface markers were examined by indirect membrane immunofluorescence. Cells were incubated with mouse MoAbs for 30 min a t 4°C in suspension. After
being washed twice with Eagle's minimum essential medium (MEM) (Nissui, Tokyo), they were stained with FITC-conjugated F(ab), fragment of goat anti-mouse IgG from Cappel (Malvern, PA, USA) for 30 min at 4°C. lmmunofluorescent samples were examined using a Zeiss epifluorescence microscope or by a FACSTAR fluorescence-activated cell sorter (Becton Dickinson FAGS Systems, USA) to measure the relative fluorescence intensities of individual cells. Data obtained from the FACSTAR were analyzed using an attached HewlettPackard computer. Cytoplasmic immunoglobulin For detection of cytoplasmic immunoglobulin, direct immunofluorescence staining was performed. Cells were smeared and fixed with acetone for 5 min, washed with phosphate-buffered saline (PBS), and then incubated with fluorescein-conjugated goat anti-human immunoglobulins (MBL, Japan) for 4 5 min. Specifically positive cells were observed and photographed by epifl uo rescence microscopy. Western blot analysis for CD45 antigen Molecules recognized by MoAb T29/33 were analyzed by the Western blotting method. After being washed with PBS, 5 x 1 0 7 - 1 x108 cells were lysed with 1 ml of extraction buffer (0.5% Nonidet P-40 in 0.02 M Tris-0.3 M NaCI, 1.0 m M CaCI,-0.5 mM MgCI,-2 m M EDTA10% glycerol-2.0 mM phenylmethylsulfonyl fluoride, pH 9.0). Insoluble material was removed by centrifugation for 2 0 min at 15,000 rpm. Electrophoresis was performed using a 7.5% acrylamide gel under reducing conditions (SDS-PAGE) and transferred elect rophoretically to a nitrocellulose membrane. The membrane was then incubated with MoAb T29/33 (diluted 1 : 250) overnight and incubated with Western blotting-grade affinity-purified goat anti-mouse IgG (H L) horseradish peroxidase conjugate (Bio-Rad Laboratories, Richmond, USA) (diluted 1 : 2,000 or 1 : 3,000). As the substrate for the peroxidase reaction, 3, 3'-diaminobenzidine tetrahydrochloride was used.
+
RESULTS 1. Detection o f CD45 antigen in various hematopoietic cell lines The reactivity of each cell line with MoAb T29/33 was expressed as the percentage of fluorescence-positive cells by indirect immunofluorescence, and the intensity of specific fluorescence of each smear was given an arbi-
110
Expression of th CD45 Antigen in Cell Lines (Nakano et a/.)
trary value ranging from negative staining (-) to strongly brilliant staining ( + ) ; (+) was used to denote weakly but significantly stained cells. Cell lines showing variable staining results were examined repeatedly and subjected to cytofluorometric analysis by FACSTAR. More than 90% of peripheral blood leukocytes from healthy donors had been shown to express the CD45 antigen in a control study. With regard to the lymphoid-lineage cell lines, two out of three null cell lines (REH and MLp-N) were T29/33positive and one (NALM-16) was completely negative. A variety of reactivity with T29/33 was evident for Blineage cell lines, and low expression of CD45 antigen was observed in the pre-B cell lines (especially NALM-6) and in the antibody-forming cell lines originating from myeloma cells (especially RPMI-8226). On the other hand, B-blast cell lines including Burkitt's cell lines expressed the CD45 antigen strongly. All cells of T-blastic lineage expressed the CD45 antigen constantly and strong Iy. Interestingly, however, HTLV-I-positive T-cell lines derived from peripheral blood leukocytes of ATL patients lacked expression of CD45. An ATL cell line (HLN-ATL0) established from lymph node cells showed reactivity with T29/33, in spite of a lack of CD45 expression on cells derived from PBL (HPB-ATL-0) of the same patient. All myeloid-lineage cell lines expressed the CD45 antigen constantly, even though the intensity of the expression varied. A relatively small population in the NALM-1 cell line was positive for T29/33. Variable reactivity with MoAb T29/33 was also o b served in the group of cell lines of mono-histiocyte lineage, in contrast t o all of the granulocytic cell lines (KG1, ML-3 and HL-60) expressing the CD45 antigen. Two out of 6 cell lines (U937 and HPE-Ret-3) showed a high percentage of antigen expression and the other cell lines were completely unreactive with MoAb T29/33.
2. Western blotting analysis of CD45 antigen in lymphoid cell lines positive a n d negative for membrane fluorescence Western blotting analysis was carried out on eight lymphoblastoid cell lines and three ATL-derived T-cell lines; two of them derived from peripheral blood leukocytes were negative for CD45 membrane fluorescence. Figure 2 shows a typical result of CD45-positive bands on the blotted gels of lymphoblastoid cell lines using MoAb T29/33. The distribution of isoforms of CD45 antigen in fluorescence-positive cell lines is illustrated schematically in Fig. 1. Variable levels of production of CD45-isoform antigens were observed in T-blastic line-
HTLV I
p roduc i i g
HPB-ATL-2 HPBATL-0 HLNATL-0
NOT DETECTEO
PEL PEL LN
Figure 1. Schema of CD45 on lymphoid-lineage cell line detected by Western blotting analysis. Solid rectangles ; definitive blotted bands, Rectangles with dots ; weakly blotted bands.
MW
205
-
11697.466-
1
2
3
4
5
6
7
8
Figure 2. Western blotting analysis of CD45 antigen. Lane I ; HPB-MLT, Lane 2 ; TALL-1 Lane 3 ; HPB-ALL, Lane 4 ; HSB2, Lane 5 ; P3HR-1, Lane 6 : Raji, Lane 7 ; BALM-1, and Lane 8 ; Daudi.
age cells even though they expressed the CD45 antigen equally on their cell surface. HPB-ALL produces 150-, 1 7 0 - and 200-kDa isoforms, HSB-2 and HPB-MLT produce 170-, 200- and 220-kDa isoforrns, and TALL1 contains larger molecular isoforms of 2 2 0 and 2 4 0 kDa. CD45-isoform patterns were rather restricted among four B-blastoid cell lines. BALM-1 had two isoforms of 2 0 0 and 2 2 0 kDa. Additional blotting bands in the gel were observed with Daudi, Raji and P3HR--l cells, which were Burkitt's lymphoma cell lines harboring the EBV genome. As for ATL-cell lines, membrane CD45-positive HLN-ATL-0 cells formed a 170-kDa precipitating band in the gel. HPB-ATL-0 cells, which were negative for membrane CD45 antigen, formed a weak 170-kDa isoform band by Western blotting analysis. ATL-2 cells did not show any detectable band in this study.
111
Acta Pathologica Japonica 40 (2) : 1990
Figure 3. Cytoplasmic immunoglobulins of plasmacytoma cell lines, ARH-77 (a) and RPMI-8226 (b) are shown by direct immunofluorescence (objective).
3' Relationship
maturation and differentiation to CD45 expression in B-lineage cell h e s Of
Variable reactivity to T29/33 was evident for B-lineage cell lines in which restricted isoform patterns were observed by immunoblotting analysis. Expression of some antigens and markers related to B-cell differTable 2.
entiation and/or maturation was analyzed in B-lineage cell lines and compared with CD45 expression (Table 3). Pre-B cell lines, thought to be immature cells, expressed CD45 antigen only to a limited grade and extent, in contrast to B-blastic cell lines. In the latter group of cell lines, both CD45 antigen and surface immunoglobulins were expressed constantly, whereas reactivities to
Expression of CD45 on Fresh Leukemia/Lymphoma Cells from Various Origins in Primary Culture
Primary culture No. (Cell line)
Patient Age
Clinical diagnosis AML
Cell source
CuIture duration
% positive
PBL
83.4 97.1 96.3 91.2 33.7 97.3 > 99.0 97.3 85.5 95.0 83.2 > 99.0 > 99.0 > 99.0 94.4 > 99.0 > 99.0 > 99.0 95.1 82.7 75.6 >99.0 > 99.0 55.8
1 HPB-Nod.
68
Sex M
2 HPB-Wat.
55
M
AML
PEL
3 HPB-Ued.
62
F
ALL
PEL
HPB-Tom.
35
M
ALL
PEL
5 HLN-Kan. 6 HLN-Wak. 7 HPB-Wak.
61 50 50
M F F
ML ML ML
LN LN PEL
8 HPL-Kaw.
85
M
ML
PL
9 HCF-Nis.
49
M
ML
CSF
60 42
M M
ML ATL/L
PEL PEL
61
F
ATL/L
PEL
70
F
s.s
PBL
