A Non·T, Non·B Human Leukemia Cell Line (NALM·1): Establishment of the Cell Line and Presence of Leukemia·Associated Antigens1. 2 J. Minowada,3 T. Tsubota,3 M. F. Greaves,4 and T. R. Walters 5 .
Since our previous report of the establishment of leukemic T -cell lines MOLT and RPMI 8402 (1, 2) and the suggestion by Wilson and Nossal (3) that CLL might be a leukemia of the B-cells and ALL might be a T-cell cancer, numerous studies at classifying human lymphoid leukemias have been reported [(4-14), reviewed in (15)]. It has been established that the great majority of CLL are monoclonal B-cell leukemias (4, 5, 7). ALL, however, is more heterogeneous, and there is as yet no general agreement on its classification or subgrouping. Many investigators have reported that cells from 10-20% of patients with ALL have a T-cell membrane phenotype (6, 7, 10). Rare B-cell-like cases of ALL have been reported (7), but most cases of ALL are generally negative in reactivity with T- and B-cell markers and are referred to as non-T, non-B ALL (or "null" cell ALL). This group is also characterized by the presence of a leukemia-specific antigen (16). More recently, this common form of ALL has also been found to express some more membrane antigens in common with both T-cells (13, 17, 18) and B-cells (19-21) as well as to possess the thymic enzyme terminal deoxynucleotidyl transferase (22, 23). The "B-cell" antigens are, in fact, widely expressed in all non-T acute and chronic leukemias and provide no evidence of a B-cell origin of acute leukemias (20, 21). Our analyses suggest that ALL might primarily involve cells with a variable degree of T-cell differentiation as reflected in the expression of either full or partial T-cell phenotype (17, 18). We report here that cells from line NALM-1, established from a case of Phi-positive CML in blastic crisis, have many characteristics that differ from previously studied cells of human T- and B-cell lines (2)_ Evidence is presented that the NALM-1 line originated from a "lymphoid" cell type of the blastic crisis of CML (24). A specific non-T, non-B ALL antigen known to be present in null cell ALL and "lymphoid" blastic crisis of CML (16, 24) was found on the NALM-1 cells.
ABBREVIATIONS USED: CLL = chronic lymphocytic leukemia(s); ALL = acute lymphoblastic leukemia(s); CML = chronic myelocytic leukemia; SRBC = sheep red blood cells; PBS = phosphate-buffered saline; AET = 2-amino-ethylisothiouronium bromide; EAC = erythrocyte-antibodycomplement; SmIg = surface immunoglobulins; FITC = fluoresceinisothiocyanate; EBV = Epstein-Barr virus; VCA = Epstein-Barr virus capsid antigen; EBNA = Epstein-Barr virus-associated nuclear antigen; BLAA = blast leukemia-associated antigen; nT = normal T-cell antigen; HTLA = human thymus-leukemia antigen; nB = normal B-cell antigen; AML = acute myeloid leukemia.
Received November 11.1976; accepted January 25.1977. Supported by Public Health Service research grants CA14413 and CA17609 from the National Cancer Institute. 3 Cell Culture Laboratory. Department of Immunology and Immunochemistry Research. Roswell Park Memorial Institute. New York State Department of Health. 666 Elm St.. Buffalo. N.Y. 14263_ 4 Imperial Cancer Research Fund. Tumour Immunology Unit. Zoology Department. University College. London. England. 5 Department of Pediatrics. New Jersey Medical School. Newark, N.J. 07103. 6 We thank Dr. L. F. Sinks. Dr. S. Nakazawa. and Dr. G_ Janossy for helpful discussion. and Mrs_ I. Rakowski for cytochemical analysis. We acknowledge Dr. Paul I. Teraski for his HLA typing of the cell lines and Mr. W. R. Oleszko for EBV determination. I
2
MATERIALS AND METHODS Cell culture. - The donor patient was a 3-year-old girl who had been treated for CML since June 1975 and was 83
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presenting, at the time of cell culture, a feature of ALL on the basis of clinical, hematologic, and cytochemical findings. On December 6, 1975, her peripheral white blood cell count was 10,000/mm3 with 85% leukemic blasts. A portion of her fresh leukemic cells, which consisted of about 80% of mononuclear cells isolated by Hypaque-Ficoll gradient centrifugation, was used for setting up a culture in a Falcon plastic flask (#3024; 250 cm2) with 25 ml of nutrient medium RPMI-1640 supplemented with 10% (vol/vol) heat-inactivated fetal calf serum and antibiotics (penicillin, 100 D/ml; streptomycin, 50 J,lg/ml). The initial cell density was approximately 5-10 X 10 6 cells/ml. The culture was incubated at 37' C under a 5% CO 2 atmosphere in a humidified incubator. The culture was fed once a week by addition of about 5-7 ml of fresh medium. By the end of the first month in culture, the cells remained viable, though an active proliferation was not apparent. During the second month of incubation, it became apparent that the cells were proliferating slowly_ In the third month of incubation, the proliferation of cultured cells became steady, and subcultures of NALM-l were established. Surface marker assays. - Rosette formation with SRBC was used as a T-cell surface marker. The NALM-l cells were washed once with PBS and suspended in saline at a density of 1 X 107 cells/ ml. Two-tenths ml of the cell suspension was mixed with 0.2 ml of either untreated SRBC suspension (1 % suspension in PBS by volume) or AET-treated SRBC (25), and then centrifuged at 200 X g at room temperature for 3 minutes. The cell pellets were incubated undisturbed for
ABSTRACT-A permanent hematopoietic cell line, designated NALM·1, was established from the peripheral blood of a patient who was in blastic crisis of Ph'-positive chronic myleocytic leukemia_ By means of a panel of specific xenoantisera, the NALM-1 cells were found to express a specific antigen of acute lymphoblastic leukemia and blast leukemia-associated antigen_ The cells exhibited no cell-surface receptors for sheep erythrocytes, IgG, or complement; neither cell-surface immunoglobulins nor cytoplasmic immunoglobulin were observed. Furthermore, normal T-cell or B-cell antigens, detectable by the antisera used in this study, were not found in the NALM-1 line_-J Natl Cancer Inst 59: 83-87,1977_
VOL. 59. NO.1. JULY 1977
6
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MINOWADA, TSUBOTA, GREAVES, AND WALTERS
TABLE
RESULTS Growth Characteristics The NALM-l cells grow in suspension at a much slower rate in the nutrient medium RPMI-1640 supplemented with 10% (vol/vol) heat-inactivated fetal calf serum than do the cells of most previously established lymphoid cell lines. Under 5% CO 2 in a 37" C humidified atmosphere, the cells showed a doubling time of 3-5 days. In a closed culture bottle their growth rate was much reduced. The NALM-l cells could tolerate much longer intervals without being fed (5 - 7 days) than the usual interval of 2-3 days for the cells of most other lymphoid cell lines. As shown in text-figure 1, the NALM-l cells exhibited two unique growth characteristics:
l. -General specificity xenoantisera
Cell specificity (No. tested) Antiserum (immunogen)
Absorbed with cells of:
Rabbit-72 (E-ALL)b RBC, bone marrow, tonsil, AML, . thymocytes Rabbit-52 (MOLT)d RBC,B-LCL RBC,B-LCL,N-PBLf Rabbit-57 (B411-4)" RBC,T-LCL RBC,T-LCL,PHA-BL i RBC,T-LCL,N-PBL
Antigens detecting
ALL C nT, HTLA HTLA nB, BLAAh nB BLAA
Normal cells Leukemic cells Celliines a Refer· Thymus T-cells B-cells E+-ALL E--ALL SmIg+-CLL E+-CLL T-LCL B-LCL ences (16) (30) (30) (4) (41) (44) (1) (4) (30)
+ +
+ + +
+ +
+ + +
+ + + +
+ +
(15,16,26) (1,17,27)'
+
(1,17,27)'
+ +
a T-LCL == T-cell lymphoid cell lines MOLT, RPMI 8402, CCRF-HBS-2, and CCRF-CEM. B-LCL == B-celllymphoid cell lines that are common Ig+-B-celllymphoid cell lines. Thirty cell lines were randomly chosen out of our 108 cell lines. bE = SRBC-T-cell rosettes. C Non-T, non-B ALL antigens. d MOLT == A leukemia T-cell line (MOLT-2) that has HLA (AI, AI0) (BWI7, B18) without detectable "B-cell" antigens . • Tsubota T, Minowada J, Nakazawa S, et al: Submitted for publication. f N-PBL == Pooled normal peripheral blood lymphocytes from 40 blood donors, collected by leukophoresis. "B411-4 == A normal Ig+-B-celliine that has HLA (A3, AW23) (B7, -) and extra "B-cell" antigens. h BLAA are
Since our previous report of the establishment of leukemic T -cell lines MOLT and RPMI 8402 (1, 2) and the suggestion by Wilson and Nossal (3) that CLL might be a leukemia of the B-cells and ALL might be a T-cell cancer, numerous studies at classifying human lymphoid leukemias have been reported [(4-14), reviewed in (15)]. It has been established that the great majority of CLL are monoclonal B-cell leukemias (4, 5, 7). ALL, however, is more heterogeneous, and there is as yet no general agreement on its classification or subgrouping. Many investigators have reported that cells from 10-20% of patients with ALL have a T-cell membrane phenotype (6, 7, 10). Rare B-cell-like cases of ALL have been reported (7), but most cases of ALL are generally negative in reactivity with T- and B-cell markers and are referred to as non-T, non-B ALL (or "null" cell ALL). This group is also characterized by the presence of a leukemia-specific antigen (16). More recently, this common form of ALL has also been found to express some more membrane antigens in common with both T-cells (13, 17, 18) and B-cells (19-21) as well as to possess the thymic enzyme terminal deoxynucleotidyl transferase (22, 23). The "B-cell" antigens are, in fact, widely expressed in all non-T acute and chronic leukemias and provide no evidence of a B-cell origin of acute leukemias (20, 21). Our analyses suggest that ALL might primarily involve cells with a variable degree of T-cell differentiation as reflected in the expression of either full or partial T-cell phenotype (17, 18). We report here that cells from line NALM-1, established from a case of Phi-positive CML in blastic crisis, have many characteristics that differ from previously studied cells of human T- and B-cell lines (2)_ Evidence is presented that the NALM-1 line originated from a "lymphoid" cell type of the blastic crisis of CML (24). A specific non-T, non-B ALL antigen known to be present in null cell ALL and "lymphoid" blastic crisis of CML (16, 24) was found on the NALM-1 cells.
ABBREVIATIONS USED: CLL = chronic lymphocytic leukemia(s); ALL = acute lymphoblastic leukemia(s); CML = chronic myelocytic leukemia; SRBC = sheep red blood cells; PBS = phosphate-buffered saline; AET = 2-amino-ethylisothiouronium bromide; EAC = erythrocyte-antibodycomplement; SmIg = surface immunoglobulins; FITC = fluoresceinisothiocyanate; EBV = Epstein-Barr virus; VCA = Epstein-Barr virus capsid antigen; EBNA = Epstein-Barr virus-associated nuclear antigen; BLAA = blast leukemia-associated antigen; nT = normal T-cell antigen; HTLA = human thymus-leukemia antigen; nB = normal B-cell antigen; AML = acute myeloid leukemia.
Received November 11.1976; accepted January 25.1977. Supported by Public Health Service research grants CA14413 and CA17609 from the National Cancer Institute. 3 Cell Culture Laboratory. Department of Immunology and Immunochemistry Research. Roswell Park Memorial Institute. New York State Department of Health. 666 Elm St.. Buffalo. N.Y. 14263_ 4 Imperial Cancer Research Fund. Tumour Immunology Unit. Zoology Department. University College. London. England. 5 Department of Pediatrics. New Jersey Medical School. Newark, N.J. 07103. 6 We thank Dr. L. F. Sinks. Dr. S. Nakazawa. and Dr. G_ Janossy for helpful discussion. and Mrs_ I. Rakowski for cytochemical analysis. We acknowledge Dr. Paul I. Teraski for his HLA typing of the cell lines and Mr. W. R. Oleszko for EBV determination. I
2
MATERIALS AND METHODS Cell culture. - The donor patient was a 3-year-old girl who had been treated for CML since June 1975 and was 83
J NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at University of Sussex on October 8, 2012
presenting, at the time of cell culture, a feature of ALL on the basis of clinical, hematologic, and cytochemical findings. On December 6, 1975, her peripheral white blood cell count was 10,000/mm3 with 85% leukemic blasts. A portion of her fresh leukemic cells, which consisted of about 80% of mononuclear cells isolated by Hypaque-Ficoll gradient centrifugation, was used for setting up a culture in a Falcon plastic flask (#3024; 250 cm2) with 25 ml of nutrient medium RPMI-1640 supplemented with 10% (vol/vol) heat-inactivated fetal calf serum and antibiotics (penicillin, 100 D/ml; streptomycin, 50 J,lg/ml). The initial cell density was approximately 5-10 X 10 6 cells/ml. The culture was incubated at 37' C under a 5% CO 2 atmosphere in a humidified incubator. The culture was fed once a week by addition of about 5-7 ml of fresh medium. By the end of the first month in culture, the cells remained viable, though an active proliferation was not apparent. During the second month of incubation, it became apparent that the cells were proliferating slowly_ In the third month of incubation, the proliferation of cultured cells became steady, and subcultures of NALM-l were established. Surface marker assays. - Rosette formation with SRBC was used as a T-cell surface marker. The NALM-l cells were washed once with PBS and suspended in saline at a density of 1 X 107 cells/ ml. Two-tenths ml of the cell suspension was mixed with 0.2 ml of either untreated SRBC suspension (1 % suspension in PBS by volume) or AET-treated SRBC (25), and then centrifuged at 200 X g at room temperature for 3 minutes. The cell pellets were incubated undisturbed for
ABSTRACT-A permanent hematopoietic cell line, designated NALM·1, was established from the peripheral blood of a patient who was in blastic crisis of Ph'-positive chronic myleocytic leukemia_ By means of a panel of specific xenoantisera, the NALM-1 cells were found to express a specific antigen of acute lymphoblastic leukemia and blast leukemia-associated antigen_ The cells exhibited no cell-surface receptors for sheep erythrocytes, IgG, or complement; neither cell-surface immunoglobulins nor cytoplasmic immunoglobulin were observed. Furthermore, normal T-cell or B-cell antigens, detectable by the antisera used in this study, were not found in the NALM-1 line_-J Natl Cancer Inst 59: 83-87,1977_
VOL. 59. NO.1. JULY 1977
6
84
MINOWADA, TSUBOTA, GREAVES, AND WALTERS
TABLE
RESULTS Growth Characteristics The NALM-l cells grow in suspension at a much slower rate in the nutrient medium RPMI-1640 supplemented with 10% (vol/vol) heat-inactivated fetal calf serum than do the cells of most previously established lymphoid cell lines. Under 5% CO 2 in a 37" C humidified atmosphere, the cells showed a doubling time of 3-5 days. In a closed culture bottle their growth rate was much reduced. The NALM-l cells could tolerate much longer intervals without being fed (5 - 7 days) than the usual interval of 2-3 days for the cells of most other lymphoid cell lines. As shown in text-figure 1, the NALM-l cells exhibited two unique growth characteristics:
l. -General specificity xenoantisera
Cell specificity (No. tested) Antiserum (immunogen)
Absorbed with cells of:
Rabbit-72 (E-ALL)b RBC, bone marrow, tonsil, AML, . thymocytes Rabbit-52 (MOLT)d RBC,B-LCL RBC,B-LCL,N-PBLf Rabbit-57 (B411-4)" RBC,T-LCL RBC,T-LCL,PHA-BL i RBC,T-LCL,N-PBL
Antigens detecting
ALL C nT, HTLA HTLA nB, BLAAh nB BLAA
Normal cells Leukemic cells Celliines a Refer· Thymus T-cells B-cells E+-ALL E--ALL SmIg+-CLL E+-CLL T-LCL B-LCL ences (16) (30) (30) (4) (41) (44) (1) (4) (30)
+ +
+ + +
+ +
+ + +
+ + + +
+ +
(15,16,26) (1,17,27)'
+
(1,17,27)'
+ +
a T-LCL == T-cell lymphoid cell lines MOLT, RPMI 8402, CCRF-HBS-2, and CCRF-CEM. B-LCL == B-celllymphoid cell lines that are common Ig+-B-celllymphoid cell lines. Thirty cell lines were randomly chosen out of our 108 cell lines. bE = SRBC-T-cell rosettes. C Non-T, non-B ALL antigens. d MOLT == A leukemia T-cell line (MOLT-2) that has HLA (AI, AI0) (BWI7, B18) without detectable "B-cell" antigens . • Tsubota T, Minowada J, Nakazawa S, et al: Submitted for publication. f N-PBL == Pooled normal peripheral blood lymphocytes from 40 blood donors, collected by leukophoresis. "B411-4 == A normal Ig+-B-celliine that has HLA (A3, AW23) (B7, -) and extra "B-cell" antigens. h BLAA are
