Int. J . Cancer: 21, 166-170 (1978)
TWO E-ROSETTE-FORMING LYMPHOID CELL LINES Shigeru MORIKAWA l, z, Eiji TATSUMI 3, Mitsuo BABA l , Takayuki HARADAand Kimio YASUHIRA Department of Pathology, Shimane Medical College, Shimane; The First Division of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto 606; and Department of Pathology, Chest Disease Research Institute, Kyoto 606, Japan
Two E-rosette (spontaneous rosette with sheep red blood cells)-forming lymphoid cell lines were established. One (HPB-ALL) was derived from a young male Japanese patient with acute lymphoblastic leukemia (ALL), and the other (HPB-MLT) was from a 62-year-old female Japanese patient with a leukemic T-lymphoid malignancy. Formation of E rosettes, absence of any immunoglobulin determinants, absence of E B N A (Epstein-Barr virus associated nuclear antigen) and very limited stimulating ability in mixed lymphocyte culture, were characteristics mostly identical with those of so far established 1-cell-derived lymphoid cell lines, M O L T , CCRF-CEM, CCRF-HS-B2, RPMI-8402 and JM. Only HPB-MLT, however, has been derived from an aged patient with 1-lymphoid malignancy.
Since Minowada et al. (1972) reported the establishment in vitro of the first lymphoid T-cell line, MOLT, four similar lines, CCRF-CEM (Foley et al., 1965), CCRF-HSB2 (Adams et al., 1968), RPMI-8402 (Minowada and Moore, 1975) and JM (Schneider et al., 1977) have been reported. Before MOLT, the characteristics of lymphoid cell lines were confined to those of B lymphocytes (Nilsson and Pontkn, 1975). The above lymphoid T and other cell lines (Klein et al., 1974; Epstein et al., 1976; Kaplan and Gartner, 1977) have contributed to our knowledge about the characteristics of cultured lymphoid cell lines, and to modern immuno-biological concepts concerning normal and malignant lymphoid cells. We report here two newly established lymphoid cell Iines of a T-cell nature, called HPB-ALL and HPB-MLT. HPB-MLT was derived from the leukemic lymphocytes in an elderly patient with T-lymphoid malignancy in contrast to the so far known lymphoid T-cell lines, including HPB-ALL, which all are derived from young patients. Our results indicate that there are no marked differences between HPB-ALL and HPB-MLT. MATERIAL AND METHODS
Patients The donor patient of HPB-ALL was a 14-yearold boy, who was diagnosed as having ALL and thymoma in November 1973. The leukocyte count in the peripheral blood was 5.6 x 104/,d,and 94% were leukemic cells. In the bone marrow, 86% of the non-erythroid nucleated cells were abnormal blasts. The patient died 7 months after diagnosis. The donor patient of HPB-MLT was a 62-yearold female, who presented in November 1974 with lymphadenopathy in the neck and the axilla,
hepatomegaly and marked splenomegaly. The leukocyte count in the peripheral blood was 9.4 x lo4/ p l , the proportion of lzukemic lymphocytes being 96%. The leukemic cells were pleomorphic. The cell size varied from 8 to 24pm in diameter. The small cells had a round nucleus with well-condensed chromatin and scanty cytoplasm. In the larger cells, a lobated or indented nucleus with less condensed chromatin and a well characterized cytoplasm was evident. E-rosette formation and absence of surface immunoglobulin with direct immunofluorescence indicated that the leukemic cells were of a T-cell nature. Erythrodermia was absent, although dermal alteration in the neck due to leukemic infiltration was present. The non-erythroid nucleated cells in the bone marrow contained 52 % abnormal lymphocytes. The patient died after 5 months. Cell culture Heparinized blood was obtained from the patients before leukemia treatment by venipuncture. Plasma rich in leukemic cells was separated after erythrocyte sedimentation at room temperature for 30 min. The cells were washed twice in medium RPMI1640 (Nissui, Tokyo, Japan) by centrifugation at 200 g for 5 min, and then suspended in the nutrient medium, RPMI-I640 supplemented with 20% heat-inactivated fetal calf serum (FCS) (GIBCO, New York). Cultures were set up in small glass dishes containing 3 ml cell suspension. The initial cell concentration was about 107/ml. All the dishes were incubated at 37" C in an atmosphere of 5 % CO, and 95% air with 100% humidity. Half of the medium was replaced with fresh medium every 2 days. CeN surface immunoglobulin Direct immunofluorescence was carried out according to the method described by Piessens et al. (1973) with slight modifications. Fluoresceinconjugated goat anti-human a, ?, p, x , or 3, gamma globulin (Hyland, California) was diluted five-fold with phosphate-buffered saline (PBS). One-tenth ml of the diluted conjugate and 0.1 ml of PBS containing 5 x106 cells were incubated at 4" C for 60 min. After washing three times with PBS by centrifugation at 200 g for 5 min, the celfs were suspended in 0.1 ml of PBS containing 50% glycerol, and were observed under a fluorescence microscope. Received : September 29, 1977. Present address: Department of Pathology, Chest Disease Research Institute, Kyoto University, Kyoto 606, Japan.
167
LYMPHOID T-CELL LINES
E rosettes mycin-C (80 pg/ml) (Kyowa-Hakko, Tokyo, Japan) E-rosette-forming ability of the cells of HPB- at 37" C for 30 min, and after three washes suspended ALL and HPB-MLT was assayed according to the as stimulators in the culture medium (RPMI-I640 method described by Jondal et al. (1972) with with 10% FCS) at a concentration of 0.2 x 106/ml, modifications. Sheep red blood cells and HPB-ALL 0.5 x 106/ml or 1.0 x 106/mI. For comparison, cells or HPB-MLT cells were suspended in PBS con- from two lymphoblastoid B-cell lines were treated taining 50% FCS at a concentration of 1O8/ml and prepared in the same way, and used as stimuand 5 x 10s/ml, respectively. One-tenth ml of each lators. They were established in our laboratory suspension was mixed and incubated at 37" C for from the peripheral blood lymphocytes of two 10 min, then centrifuged at 1 O O g and left at room norinal individuals (Nilsson, 1971), designated temperature or 4" C. Rosetting cells were counted OKA and NAI, respectively, have y , ,u, x and A immediately after centrifugation, and after 1 h or chains on the surface, express EBNA, do not form 8 h incubation under either of the temperature E rosettes, but form EAC rosettes. One-half ml of stimulator cell suspension and 0.5 ml of responder conditions. cell suspension were mixed in a glass tube and EAC rosettes (rosettes wirh SRBC coated with incubated for 6 % days in an atmosphere of 5 % CO, and 95% air with 100% humidity. For the 19s antibody and complement) The 19s fraction of rabbit serum raised against last 12 h, 0.1 ml of RPMI-1640 containing 1 pCi SRBC was obtained from commercially available tritiated thymidine (specific radioactivity 5 Ci/mmole) hemolysin (Toshiba, Tokyo, Japan) by Sephadex was added. Tubes were prepared in triplicate for G-200 (Pharmacia, Uppsala, Sweden) gel filtration each cell combination. Incorporation of tritiated in PBS, and was diluted with PBS at twice the thymidine into acid-precipitable fraction was maximal dilution required to show agglutination. measured by liquid scintillation counting. One-tenth ml of SRBC in PBS (108/mI) and RESULTS 0.1 ml of the diluted rabbit 19s anti-SRBC serum were mixed and incubated at 37" C for General characteristics 30 min. The SRBC coated with 19s antibody The behavior of the leukemic cells in culture (EA.19S) was washed three times with PBS, and 0.1 ml of EA.19S (lOs/ml) and 0.1 ml of 10-fold until establishment was quite similar in both HPBdiluted AKR mouse serum, used as complement, ALL and HPB-MLT. From the initiation of culture were mixed and incubated at 37" C for 15 min. the cells showed slow but definite multiplication. The EAC formed was washed three times with About 1 month later the original cultures were PBS, and then adjusted to a cell concentration of divided into subcultures. Four months after the 108/ml. One-tenth ml of EAC and 0.1 ml of cultured start of culture the proliferation increased. The cells (5 x 106/ml in PBS) were mixed and incubated cells grew in a single-cell suspension, forming for 60 min at 37" C. Immediately after incubation, only loose clumps. This tendency to proliferate was evident in both cases, and the two T-cell rosetting cells were counted. As a control, EA.19S lines could be readily distinguished from normal rosettes and E rosettes were assayed after 60 min B-lymphocyte-derived lines by phase-contrast microincubation at 37" C in PBS. scopy. EBNA (Epstein-Barr virus-associated nuclear antigen) EBNA was assayed by anticomplement immunofluorescence according to the method of Reedman and Klein (1973) with slight modifications. Cells on slides were fixed in acetone at room temperature for 10 min, and incubated for 50 min at 37" C with 10-fold-diluted fresh serum from a healthy adult who had a moderate titer of anti-VCA (viral capsid antigen) of Epstein-Barr virus. After washing with PBS, fluorescein-conjugated goat anti-human BIC/BIA globulin diluted 1 :I0 (Hyland, California) was layered on the cells. After the incubation at 37" C for 40 min, the smeared cells were washed with PBS, mounted in PBS containing 50% glycerol, and observed under a fluorescence microscope. Mixed lymphocyte reaction ( M LR ) Mononuclear cells were separated from heparinized peripheral venous blood of two healthy individuals by Ficoll Hypaque-Conray discontinuous gradient (Boyum, 1968), washed three times with RPMI-1640, and finally suspended in RPMI-1640 with 10% FCS, and used as responders. The HPBALL and HPB-MLT cells were treated with mito-
Morphology The cells were round and small (9-12 pm in diameter), and resembled lymphocytes. The cytoplasm was scanty and slightly stained in the periphery (May-Griinwald-Giemsa). The nuclei had dense or fine chromatin, sometimes an indentation and one nucleolus or two. HPB-ALL and HPB-MLT could not be distinguished morphologically.
TABLE I FREQUENCY OF E-ROSETTE-FORMING CELLS Incubation conditions Cell line
22°C
HPB-ALL HPB-MLT
'
lh
Oh'
'
8h 40 c
22" C
4" c
24% 5 5 % 78% 63% 88% 21 % 52% 78% 73% 84%
' Cells with at least three SRBC were considered rosetting cells. Over 300 cells were counted. The sum of the cells with three or four SRBC was less than 4%. - 'Immediately after centrifugation rosetting cells were counted.
168
MORIKAWA ET AL. TABLE I1
FREQUENCY OF EAC-ROSETTE-FORMING CELLS '
HPB-ALL HPB-MLT
EAC
EA. 19s
E'
48 % 40 %
15%
11%
10%
9%
' Cells with at least three SRBC were considered rosetlinp cells. Over 300 SRBC were counted. - "on-treated SRBC and cells of the T-cell lines were mixed in PBS, and after 1 h incubation at 37" C rosetting cells were counted.
Surjace immunoglobulin
The HPB-ALL and HPB-MLT cells did not stain with goat anti-human y, p , a, x or 1. For comparison, the OKA and NAI cells stained readily, as expected, under the same conditions.
E rosettes The proportion of E-rosette-forming cells tested under different conditions is shown in Table I. A significant proportion of the cells formed E rosettes immediately after 100 g centrifugation following 10 min incubation at 37" C. After a 1-h incubation, a larger proportion of the cells formed E rosettes at room temperature as well as at 4" C. After incubation for 8 h, the percentage of E rosettes increased at 4" C.
EAC rosettes Under the test conditions used by us, the proportion of EAC-rosette-forming cells was nearly 50% (Table 11). Under the same conditions the frequency of EA.19S rosettes and E rosettes was about 10%. This was interpretated as meaning that the cells did not actually form EA.19S rosettes and that the true frequencies of EAC rosettes formed by HPB-ALL and HPB-MLT were 36.0% and 30.8 %, respectively.
EBNA More than 95% of Raji, OKA and NAI cells had a nucleus with a definite mottled fluorescence. In contrast, HPB-ALL and HPB-MLT cells showed no nuclear fluorescence. M i x e d lymphocyte reaction ( M L R )
Incorporation of tritiated thymidine into the acid precipitable fraction was calculated as counts per minute (cpm) (Table 111). The stimulation index (SI) was defined as the ratio of cpm obtained from the mixed culture divided by the sum of the cpm from the responder cells and the mitomycin-Ctreated stimulator cells, each cultured independently. SI of OKA or NAI lymphoblastoid B-cell lines ranged from 5.1 to 17.5, and a sufficient stimulating effect was observed even when the stimulator cell population contained 0.1 x lo6 cells. In contrast, SI of HPB-ALL and HPB-MLT ranged from 0.9
TABLE 111 STIMULATORY CAPACITY OF HPB-ALL A N D HPB-MLT IN MLR Incorporation of tritiated thymidine ( c p n i f s ~ ) Stimulating cell
Number o f stimulating cells
HPB-ALL
0.5 x 106 0.25 x lo8 0.1 x 106 0.5 X lo8 0.25 x lo6 0.1 x 1 0 8
Donor Of
Responding cell alone
N-1'
48294466
N-2'
68504569
0.5 x 106 0.25 x lo6 0.1 x 108 0.5 x 106 0.25 x lo6 0.1 x 1 0 8
N-1
48294466
N-2
68504 569
0.5 x lo8 0.1 x 106 0.5 x lo8 0.1 x 108
N- 1
5495 4521
N-2
6261 1501
0.5 x lo6 0.1 x 1 0 6
N- 1
5495f521
0.5 x lo6 0.1 x 106
N-2
6261 f501
Stimulating cell alone
SI (fSD)
MLR
63&11 564~12 50k-13 63111 56k 12 50113
5802j~586 6261 f421 50361490 8971 A606 8822 1 6 13 601 3 *779
98A14 741t12 63413 98 5 14 74k12 63&13
63 17 h821 69241759 5004 161 5 8249 1 9 0 6 621 8 f997 75821 504
'
1.2t0.2 1.3 1 0 . 2 1.010.2 1.3f0.1 1.3k0.1 0.9 &O. 1
~_________
HPB-MLT
~
~
OKA
NAI
@.
1.3+0.2 1.4*0.2 1.010.2 1.2f0.2 0.910.2 1.1 +0.1 ~
_
_
143 13 1 891.19 174&31 94&22
7874713995 40061k348.5 85069*3423 3232912579
17.51 1.8 7.211.5 13.1 + l . l 5.110.5
143k31 89f19 174&31 94f22
7735915241 38871 k3527 8826014219 32538*2422
13.611.5 7.0*0.9 13.7 t1.2 5.110.6
_
The number of responding cells was always 0.5 x 10 - * SI = stimulation index. - a N-I and N-2 are the two donors of respondel lymphocytes. - 'OKA and NAI are lymphoblastoid B-cell lines established in our laboratory.
_
_
~
169
LYMPHOID T-CELL LINES
to 1.4, and the cells were considered to be nonstimulating in comparison with the lymphoblastoid B-cell lines. DISCUSSION
.
A slow continuous proliferation for a few months from initiation of culture was observed when MOLT (Minowada et af., 1972), CCRF-CEM (Foley et al., 1965), and our cell lines were established. Formation of a single-cell suspension was described with MOLT (Minowada et al., 1972). Both HPB-ALL and HPB-MLT formed E rosettes, which are regarded as a marker for human T cells (Lay et al., 1971; Jondal et al., 1972). Except for CCRF-CEM (West and Herberman, 1974), all known T-cell lines form E rosettes. Complement receptors were found on a significant proportion of both HPB-ALL and HPB-MLT cells, but consistent results were not obtained with other T-cell lines. CCRF-HSB2 and CCRF-CEM have no complement receptors, whereas MOLT was determined to have a dual T- and B-cell nature (West and Herberman, 1974) because of the presence of complement receptors. The implication of complement receptors in some of the T-cell lines remained to be defined. Immunoglobulin determinants were not detected on the cell surface of HPB-ALL or HPB-MLT with direct immunofluorescence, as is the case in other T-cell lines. Detection of an immunoglobulin determinant with other methods may be possible in some of the T-cell lines, since this has been done with a murine T lymphoma cell line (Haustein et al., 1974). Since EBNA-positive lymphoid cell lines are derived from either Burkitt lymphoma or normal lymphocytes (Huber et al., 1976), and since so far known T-cell lines lack EBNA (Minowada and Moore, 1975; Pauly et al., 1975; Kaplan et al., 1974), the absence of EBNA appears to be a prerequisite for proving T-cell derivation. This condition was fulfilled by HPB-ALL and HPB-MLT. HPB-ALL and HPB-MLT, like the other T-cell lines, showed very limited stimulating ability in MLR (Han and Minowada, 1973; Pauly et al., 1975; Royston et al., 1974a, b). This can be at-
tributed to the T-cell origen of these cells because several experiments indicate that in man and mouse alloantigens which concern stimulation in MLR are preferentially, if not exclusively, expressed on B cells (Lohrman et af., 1974; Lozner et af., 1974; Simpson, 1975; Plate and McKenzie, 1973). Furthermore, Cresswell and Geier (1975) succeeded in immunochemical purification of the alloantigens which are selectively expressed on lymphoblastoid B-cell lines and effect stimulation in MLR. There are, however, some contradictory reports (Chess et al., 1974; Sonde1 et al., 1975). Callewaert et al. (1975), using CCRF-HSB2, demonstrated that this T - B disparity is only quantitative. The possibility cannot be ruled out that this limited stimulation in MLR may be due to changes accompanying establishment in culture or to malignant transformation and/or humoral factor($ synthesized by the cultured cells, suppressing the function of responder cells. Lastly, it should be noted that HPB-MLT is apparently the first T-cell line derived from an elderly patient. All other cell lines were obtained from young patients. The donor patient of MOLT was the oldest (19 years). T-lymphoid malignancies in elderly patients form an emerging clinical entity (Yodoi et al., 1974; Sumiya et al., 1973; Brouet et al., 1975), apparently constituting a continuous disease spectrum with mycosis fungoides and Skzary syndrome (Lutzner et al., 1975), and having no consistent counterpart among so far used clinical terms describing lymphoid malignancies. The clinical picture of the donor patient of HPB-MLT simulated prolymphocytic leukemia (Catovsky et af., 1973; Galton et al., 1974). In any case, since T-lymphoid malignancies in young and elderly patients are clinically distinct, and since they may represent different T-cell subsets, some difference between HPB-MLT and the other T-cell lines may be discovered, although at this time these lines are considered to be similar. ACKNOWLEDGEMENT
Our thanks are due to Ms. M. Ohara for assistance with the manuscript .
DEUX LIGNEES DE CELLULES LYMPHOfDES FO R M A N T DES ROSETTES E Deux IignQs de cellules lymphordes formant des rosettes E (rosettes spontandes avec des erythrocytes de mouton) ont ete &&lies. L’une (HPB-ALL) provenait d’un jeune Japonais atteint de leuc6mie lymphoblastique aigue (ALL) et l’autre (HPB-MLT) d’une Japonaise d e 62 ans atteinte d’une n b p lasie lympho’ide A cellules IeucQmiques T. La formation d e rosettes E, l’absence de determinants immunoglobuliniques, I’absence d’EBNA (antigene nuclkaire associe au virus d’Epstein-Barr) et une capacit6 de stimulation tres limit& en culture mixte de lymphocytes sont des caractkristiques pour la plupart identiques B celles des ligntes lymphoides derivks de cellules T Btablies jusqu’ici: MOLT, CCRF-CEM, CCRF-HS-B2, RPMI-8402 et JM. Toutefois, la IignBe HPB-MLT est la seule qui provienne d’une malade agee atteinte d’une affection lympholde de type T. REFERENCES
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TWO E-ROSETTE-FORMING LYMPHOID CELL LINES Shigeru MORIKAWA l, z, Eiji TATSUMI 3, Mitsuo BABA l , Takayuki HARADAand Kimio YASUHIRA Department of Pathology, Shimane Medical College, Shimane; The First Division of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto 606; and Department of Pathology, Chest Disease Research Institute, Kyoto 606, Japan
Two E-rosette (spontaneous rosette with sheep red blood cells)-forming lymphoid cell lines were established. One (HPB-ALL) was derived from a young male Japanese patient with acute lymphoblastic leukemia (ALL), and the other (HPB-MLT) was from a 62-year-old female Japanese patient with a leukemic T-lymphoid malignancy. Formation of E rosettes, absence of any immunoglobulin determinants, absence of E B N A (Epstein-Barr virus associated nuclear antigen) and very limited stimulating ability in mixed lymphocyte culture, were characteristics mostly identical with those of so far established 1-cell-derived lymphoid cell lines, M O L T , CCRF-CEM, CCRF-HS-B2, RPMI-8402 and JM. Only HPB-MLT, however, has been derived from an aged patient with 1-lymphoid malignancy.
Since Minowada et al. (1972) reported the establishment in vitro of the first lymphoid T-cell line, MOLT, four similar lines, CCRF-CEM (Foley et al., 1965), CCRF-HSB2 (Adams et al., 1968), RPMI-8402 (Minowada and Moore, 1975) and JM (Schneider et al., 1977) have been reported. Before MOLT, the characteristics of lymphoid cell lines were confined to those of B lymphocytes (Nilsson and Pontkn, 1975). The above lymphoid T and other cell lines (Klein et al., 1974; Epstein et al., 1976; Kaplan and Gartner, 1977) have contributed to our knowledge about the characteristics of cultured lymphoid cell lines, and to modern immuno-biological concepts concerning normal and malignant lymphoid cells. We report here two newly established lymphoid cell Iines of a T-cell nature, called HPB-ALL and HPB-MLT. HPB-MLT was derived from the leukemic lymphocytes in an elderly patient with T-lymphoid malignancy in contrast to the so far known lymphoid T-cell lines, including HPB-ALL, which all are derived from young patients. Our results indicate that there are no marked differences between HPB-ALL and HPB-MLT. MATERIAL AND METHODS
Patients The donor patient of HPB-ALL was a 14-yearold boy, who was diagnosed as having ALL and thymoma in November 1973. The leukocyte count in the peripheral blood was 5.6 x 104/,d,and 94% were leukemic cells. In the bone marrow, 86% of the non-erythroid nucleated cells were abnormal blasts. The patient died 7 months after diagnosis. The donor patient of HPB-MLT was a 62-yearold female, who presented in November 1974 with lymphadenopathy in the neck and the axilla,
hepatomegaly and marked splenomegaly. The leukocyte count in the peripheral blood was 9.4 x lo4/ p l , the proportion of lzukemic lymphocytes being 96%. The leukemic cells were pleomorphic. The cell size varied from 8 to 24pm in diameter. The small cells had a round nucleus with well-condensed chromatin and scanty cytoplasm. In the larger cells, a lobated or indented nucleus with less condensed chromatin and a well characterized cytoplasm was evident. E-rosette formation and absence of surface immunoglobulin with direct immunofluorescence indicated that the leukemic cells were of a T-cell nature. Erythrodermia was absent, although dermal alteration in the neck due to leukemic infiltration was present. The non-erythroid nucleated cells in the bone marrow contained 52 % abnormal lymphocytes. The patient died after 5 months. Cell culture Heparinized blood was obtained from the patients before leukemia treatment by venipuncture. Plasma rich in leukemic cells was separated after erythrocyte sedimentation at room temperature for 30 min. The cells were washed twice in medium RPMI1640 (Nissui, Tokyo, Japan) by centrifugation at 200 g for 5 min, and then suspended in the nutrient medium, RPMI-I640 supplemented with 20% heat-inactivated fetal calf serum (FCS) (GIBCO, New York). Cultures were set up in small glass dishes containing 3 ml cell suspension. The initial cell concentration was about 107/ml. All the dishes were incubated at 37" C in an atmosphere of 5 % CO, and 95% air with 100% humidity. Half of the medium was replaced with fresh medium every 2 days. CeN surface immunoglobulin Direct immunofluorescence was carried out according to the method described by Piessens et al. (1973) with slight modifications. Fluoresceinconjugated goat anti-human a, ?, p, x , or 3, gamma globulin (Hyland, California) was diluted five-fold with phosphate-buffered saline (PBS). One-tenth ml of the diluted conjugate and 0.1 ml of PBS containing 5 x106 cells were incubated at 4" C for 60 min. After washing three times with PBS by centrifugation at 200 g for 5 min, the celfs were suspended in 0.1 ml of PBS containing 50% glycerol, and were observed under a fluorescence microscope. Received : September 29, 1977. Present address: Department of Pathology, Chest Disease Research Institute, Kyoto University, Kyoto 606, Japan.
167
LYMPHOID T-CELL LINES
E rosettes mycin-C (80 pg/ml) (Kyowa-Hakko, Tokyo, Japan) E-rosette-forming ability of the cells of HPB- at 37" C for 30 min, and after three washes suspended ALL and HPB-MLT was assayed according to the as stimulators in the culture medium (RPMI-I640 method described by Jondal et al. (1972) with with 10% FCS) at a concentration of 0.2 x 106/ml, modifications. Sheep red blood cells and HPB-ALL 0.5 x 106/ml or 1.0 x 106/mI. For comparison, cells or HPB-MLT cells were suspended in PBS con- from two lymphoblastoid B-cell lines were treated taining 50% FCS at a concentration of 1O8/ml and prepared in the same way, and used as stimuand 5 x 10s/ml, respectively. One-tenth ml of each lators. They were established in our laboratory suspension was mixed and incubated at 37" C for from the peripheral blood lymphocytes of two 10 min, then centrifuged at 1 O O g and left at room norinal individuals (Nilsson, 1971), designated temperature or 4" C. Rosetting cells were counted OKA and NAI, respectively, have y , ,u, x and A immediately after centrifugation, and after 1 h or chains on the surface, express EBNA, do not form 8 h incubation under either of the temperature E rosettes, but form EAC rosettes. One-half ml of stimulator cell suspension and 0.5 ml of responder conditions. cell suspension were mixed in a glass tube and EAC rosettes (rosettes wirh SRBC coated with incubated for 6 % days in an atmosphere of 5 % CO, and 95% air with 100% humidity. For the 19s antibody and complement) The 19s fraction of rabbit serum raised against last 12 h, 0.1 ml of RPMI-1640 containing 1 pCi SRBC was obtained from commercially available tritiated thymidine (specific radioactivity 5 Ci/mmole) hemolysin (Toshiba, Tokyo, Japan) by Sephadex was added. Tubes were prepared in triplicate for G-200 (Pharmacia, Uppsala, Sweden) gel filtration each cell combination. Incorporation of tritiated in PBS, and was diluted with PBS at twice the thymidine into acid-precipitable fraction was maximal dilution required to show agglutination. measured by liquid scintillation counting. One-tenth ml of SRBC in PBS (108/mI) and RESULTS 0.1 ml of the diluted rabbit 19s anti-SRBC serum were mixed and incubated at 37" C for General characteristics 30 min. The SRBC coated with 19s antibody The behavior of the leukemic cells in culture (EA.19S) was washed three times with PBS, and 0.1 ml of EA.19S (lOs/ml) and 0.1 ml of 10-fold until establishment was quite similar in both HPBdiluted AKR mouse serum, used as complement, ALL and HPB-MLT. From the initiation of culture were mixed and incubated at 37" C for 15 min. the cells showed slow but definite multiplication. The EAC formed was washed three times with About 1 month later the original cultures were PBS, and then adjusted to a cell concentration of divided into subcultures. Four months after the 108/ml. One-tenth ml of EAC and 0.1 ml of cultured start of culture the proliferation increased. The cells (5 x 106/ml in PBS) were mixed and incubated cells grew in a single-cell suspension, forming for 60 min at 37" C. Immediately after incubation, only loose clumps. This tendency to proliferate was evident in both cases, and the two T-cell rosetting cells were counted. As a control, EA.19S lines could be readily distinguished from normal rosettes and E rosettes were assayed after 60 min B-lymphocyte-derived lines by phase-contrast microincubation at 37" C in PBS. scopy. EBNA (Epstein-Barr virus-associated nuclear antigen) EBNA was assayed by anticomplement immunofluorescence according to the method of Reedman and Klein (1973) with slight modifications. Cells on slides were fixed in acetone at room temperature for 10 min, and incubated for 50 min at 37" C with 10-fold-diluted fresh serum from a healthy adult who had a moderate titer of anti-VCA (viral capsid antigen) of Epstein-Barr virus. After washing with PBS, fluorescein-conjugated goat anti-human BIC/BIA globulin diluted 1 :I0 (Hyland, California) was layered on the cells. After the incubation at 37" C for 40 min, the smeared cells were washed with PBS, mounted in PBS containing 50% glycerol, and observed under a fluorescence microscope. Mixed lymphocyte reaction ( M LR ) Mononuclear cells were separated from heparinized peripheral venous blood of two healthy individuals by Ficoll Hypaque-Conray discontinuous gradient (Boyum, 1968), washed three times with RPMI-1640, and finally suspended in RPMI-1640 with 10% FCS, and used as responders. The HPBALL and HPB-MLT cells were treated with mito-
Morphology The cells were round and small (9-12 pm in diameter), and resembled lymphocytes. The cytoplasm was scanty and slightly stained in the periphery (May-Griinwald-Giemsa). The nuclei had dense or fine chromatin, sometimes an indentation and one nucleolus or two. HPB-ALL and HPB-MLT could not be distinguished morphologically.
TABLE I FREQUENCY OF E-ROSETTE-FORMING CELLS Incubation conditions Cell line
22°C
HPB-ALL HPB-MLT
'
lh
Oh'
'
8h 40 c
22" C
4" c
24% 5 5 % 78% 63% 88% 21 % 52% 78% 73% 84%
' Cells with at least three SRBC were considered rosetting cells. Over 300 cells were counted. The sum of the cells with three or four SRBC was less than 4%. - 'Immediately after centrifugation rosetting cells were counted.
168
MORIKAWA ET AL. TABLE I1
FREQUENCY OF EAC-ROSETTE-FORMING CELLS '
HPB-ALL HPB-MLT
EAC
EA. 19s
E'
48 % 40 %
15%
11%
10%
9%
' Cells with at least three SRBC were considered rosetlinp cells. Over 300 SRBC were counted. - "on-treated SRBC and cells of the T-cell lines were mixed in PBS, and after 1 h incubation at 37" C rosetting cells were counted.
Surjace immunoglobulin
The HPB-ALL and HPB-MLT cells did not stain with goat anti-human y, p , a, x or 1. For comparison, the OKA and NAI cells stained readily, as expected, under the same conditions.
E rosettes The proportion of E-rosette-forming cells tested under different conditions is shown in Table I. A significant proportion of the cells formed E rosettes immediately after 100 g centrifugation following 10 min incubation at 37" C. After a 1-h incubation, a larger proportion of the cells formed E rosettes at room temperature as well as at 4" C. After incubation for 8 h, the percentage of E rosettes increased at 4" C.
EAC rosettes Under the test conditions used by us, the proportion of EAC-rosette-forming cells was nearly 50% (Table 11). Under the same conditions the frequency of EA.19S rosettes and E rosettes was about 10%. This was interpretated as meaning that the cells did not actually form EA.19S rosettes and that the true frequencies of EAC rosettes formed by HPB-ALL and HPB-MLT were 36.0% and 30.8 %, respectively.
EBNA More than 95% of Raji, OKA and NAI cells had a nucleus with a definite mottled fluorescence. In contrast, HPB-ALL and HPB-MLT cells showed no nuclear fluorescence. M i x e d lymphocyte reaction ( M L R )
Incorporation of tritiated thymidine into the acid precipitable fraction was calculated as counts per minute (cpm) (Table 111). The stimulation index (SI) was defined as the ratio of cpm obtained from the mixed culture divided by the sum of the cpm from the responder cells and the mitomycin-Ctreated stimulator cells, each cultured independently. SI of OKA or NAI lymphoblastoid B-cell lines ranged from 5.1 to 17.5, and a sufficient stimulating effect was observed even when the stimulator cell population contained 0.1 x lo6 cells. In contrast, SI of HPB-ALL and HPB-MLT ranged from 0.9
TABLE 111 STIMULATORY CAPACITY OF HPB-ALL A N D HPB-MLT IN MLR Incorporation of tritiated thymidine ( c p n i f s ~ ) Stimulating cell
Number o f stimulating cells
HPB-ALL
0.5 x 106 0.25 x lo8 0.1 x 106 0.5 X lo8 0.25 x lo6 0.1 x 1 0 8
Donor Of
Responding cell alone
N-1'
48294466
N-2'
68504569
0.5 x 106 0.25 x lo6 0.1 x 108 0.5 x 106 0.25 x lo6 0.1 x 1 0 8
N-1
48294466
N-2
68504 569
0.5 x lo8 0.1 x 106 0.5 x lo8 0.1 x 108
N- 1
5495 4521
N-2
6261 1501
0.5 x lo6 0.1 x 1 0 6
N- 1
5495f521
0.5 x lo6 0.1 x 106
N-2
6261 f501
Stimulating cell alone
SI (fSD)
MLR
63&11 564~12 50k-13 63111 56k 12 50113
5802j~586 6261 f421 50361490 8971 A606 8822 1 6 13 601 3 *779
98A14 741t12 63413 98 5 14 74k12 63&13
63 17 h821 69241759 5004 161 5 8249 1 9 0 6 621 8 f997 75821 504
'
1.2t0.2 1.3 1 0 . 2 1.010.2 1.3f0.1 1.3k0.1 0.9 &O. 1
~_________
HPB-MLT
~
~
OKA
NAI
@.
1.3+0.2 1.4*0.2 1.010.2 1.2f0.2 0.910.2 1.1 +0.1 ~
_
_
143 13 1 891.19 174&31 94&22
7874713995 40061k348.5 85069*3423 3232912579
17.51 1.8 7.211.5 13.1 + l . l 5.110.5
143k31 89f19 174&31 94f22
7735915241 38871 k3527 8826014219 32538*2422
13.611.5 7.0*0.9 13.7 t1.2 5.110.6
_
The number of responding cells was always 0.5 x 10 - * SI = stimulation index. - a N-I and N-2 are the two donors of respondel lymphocytes. - 'OKA and NAI are lymphoblastoid B-cell lines established in our laboratory.
_
_
~
169
LYMPHOID T-CELL LINES
to 1.4, and the cells were considered to be nonstimulating in comparison with the lymphoblastoid B-cell lines. DISCUSSION
.
A slow continuous proliferation for a few months from initiation of culture was observed when MOLT (Minowada et af., 1972), CCRF-CEM (Foley et al., 1965), and our cell lines were established. Formation of a single-cell suspension was described with MOLT (Minowada et al., 1972). Both HPB-ALL and HPB-MLT formed E rosettes, which are regarded as a marker for human T cells (Lay et al., 1971; Jondal et al., 1972). Except for CCRF-CEM (West and Herberman, 1974), all known T-cell lines form E rosettes. Complement receptors were found on a significant proportion of both HPB-ALL and HPB-MLT cells, but consistent results were not obtained with other T-cell lines. CCRF-HSB2 and CCRF-CEM have no complement receptors, whereas MOLT was determined to have a dual T- and B-cell nature (West and Herberman, 1974) because of the presence of complement receptors. The implication of complement receptors in some of the T-cell lines remained to be defined. Immunoglobulin determinants were not detected on the cell surface of HPB-ALL or HPB-MLT with direct immunofluorescence, as is the case in other T-cell lines. Detection of an immunoglobulin determinant with other methods may be possible in some of the T-cell lines, since this has been done with a murine T lymphoma cell line (Haustein et al., 1974). Since EBNA-positive lymphoid cell lines are derived from either Burkitt lymphoma or normal lymphocytes (Huber et al., 1976), and since so far known T-cell lines lack EBNA (Minowada and Moore, 1975; Pauly et al., 1975; Kaplan et al., 1974), the absence of EBNA appears to be a prerequisite for proving T-cell derivation. This condition was fulfilled by HPB-ALL and HPB-MLT. HPB-ALL and HPB-MLT, like the other T-cell lines, showed very limited stimulating ability in MLR (Han and Minowada, 1973; Pauly et al., 1975; Royston et al., 1974a, b). This can be at-
tributed to the T-cell origen of these cells because several experiments indicate that in man and mouse alloantigens which concern stimulation in MLR are preferentially, if not exclusively, expressed on B cells (Lohrman et af., 1974; Lozner et af., 1974; Simpson, 1975; Plate and McKenzie, 1973). Furthermore, Cresswell and Geier (1975) succeeded in immunochemical purification of the alloantigens which are selectively expressed on lymphoblastoid B-cell lines and effect stimulation in MLR. There are, however, some contradictory reports (Chess et al., 1974; Sonde1 et al., 1975). Callewaert et al. (1975), using CCRF-HSB2, demonstrated that this T - B disparity is only quantitative. The possibility cannot be ruled out that this limited stimulation in MLR may be due to changes accompanying establishment in culture or to malignant transformation and/or humoral factor($ synthesized by the cultured cells, suppressing the function of responder cells. Lastly, it should be noted that HPB-MLT is apparently the first T-cell line derived from an elderly patient. All other cell lines were obtained from young patients. The donor patient of MOLT was the oldest (19 years). T-lymphoid malignancies in elderly patients form an emerging clinical entity (Yodoi et al., 1974; Sumiya et al., 1973; Brouet et al., 1975), apparently constituting a continuous disease spectrum with mycosis fungoides and Skzary syndrome (Lutzner et al., 1975), and having no consistent counterpart among so far used clinical terms describing lymphoid malignancies. The clinical picture of the donor patient of HPB-MLT simulated prolymphocytic leukemia (Catovsky et af., 1973; Galton et al., 1974). In any case, since T-lymphoid malignancies in young and elderly patients are clinically distinct, and since they may represent different T-cell subsets, some difference between HPB-MLT and the other T-cell lines may be discovered, although at this time these lines are considered to be similar. ACKNOWLEDGEMENT
Our thanks are due to Ms. M. Ohara for assistance with the manuscript .
DEUX LIGNEES DE CELLULES LYMPHOfDES FO R M A N T DES ROSETTES E Deux IignQs de cellules lymphordes formant des rosettes E (rosettes spontandes avec des erythrocytes de mouton) ont ete &&lies. L’une (HPB-ALL) provenait d’un jeune Japonais atteint de leuc6mie lymphoblastique aigue (ALL) et l’autre (HPB-MLT) d’une Japonaise d e 62 ans atteinte d’une n b p lasie lympho’ide A cellules IeucQmiques T. La formation d e rosettes E, l’absence de determinants immunoglobuliniques, I’absence d’EBNA (antigene nuclkaire associe au virus d’Epstein-Barr) et une capacit6 de stimulation tres limit& en culture mixte de lymphocytes sont des caractkristiques pour la plupart identiques B celles des ligntes lymphoides derivks de cellules T Btablies jusqu’ici: MOLT, CCRF-CEM, CCRF-HS-B2, RPMI-8402 et JM. Toutefois, la IignBe HPB-MLT est la seule qui provienne d’une malade agee atteinte d’une affection lympholde de type T. REFERENCES
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