Acta haemat. 57: 257-265 (1977)
Preparation, Purification and in vitro Properties of a Serum against Human Lymphoblastic Leukemia-Associated Antigens F. P andolfi, G. L uzi, M. C. T ozzi and F. A iuti Department of Clinic.al Medicine III and Department of Pediatrics II, University of Rome, Rome
Key Words. Acute lymphoblastic leukemia • Antileukemic sera • Cytotoxicity • Leukemia antigens • Tumor-associated antigens Abstract. A serum against human lymphoblastic leukemia cells was obtained in oculating rabbits. We studied the specificity of the serum before and after particular absorptions in various clinical conditions. The serum was cytotoxic against many cases of acute lymphoblastic leukemia, against continuously cultured Burkitt’s lym phoma cells and against chronic myelogenous leukemia in blast crisis. On the con trary, the serum was not cytotoxic against lymphocytes of normal donors, acute lymphoblastic leukemia in remission and other lymphoproliférative disorders.
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Lymphoproliférative diseases and, particularly, acute lymphoblastic leukemia (ALL) were recently studied in order to identify particular anti genic modifications in neoplastic cells. This kind of immunological study of tracing cellular properties in tumors could produce useful diagnostic, prognostic and therapeutic results. Specific antigens in leukemias were studied by various authors using specific antisera [8, 10-12]. The greatest difficulty in this kind of study is the preparation of specific antisera; it is clear that an inoculated animal produces antibodies against nonspecific and assumed associated leukemic antigens. For their inoculations some authors used animals with tolerance towards human leukocyte antigen (HLA), identical normal human cells [4], or extracts of membranes of continuous cultured cells which might have cross-reacting antigens with leukemic cells [5, 7,10].
P andolfi/L uzi/T ozzi/A iuti
258
Table /. Clinical and immunological data of the 2 patients with ALL whose cells were used for immunization Case No
Sex
1 2
F F
Age years
WBC//d
5 2
6,000 6,800
Blasts %
Rosettes, % E EAC
Smlg
90 88
12 7
ND 0 0 0
21 13
G A M
SmIg=Surface membrane immunoglobulins: IgG, IgA and IgM; N D = n o t done or not available.
We studied the problem from two points of view: (1) antiserum specif icity at various dilutions, and (2) specificity of antisera following a partic ular absorption scheme. Material and Methods
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Serum anti-ALL. Leukemic lymphoblasts used for inoculation were obtained from the peripheral blood of 2 patients with ALL in acute phase whose diagnosis had been confirmed by usual clinical and laboratory methods. Pertinent data con cerning these 2 patients are reported in table I. The cells were obtained from patients before therapy. Lymphoid cells were iso lated by centrifugation on Ficoll-Isopaque gradient, washed twice and suspended in T.C. 199 (Wellcome, England). Cells were stored at -80 °C until inoculation. The suspension of cells was injected intravenously in a rabbit at a dosage of 2X 107 cells. This was followed by two subsequent inoculations of 107 cells each performed at 14 and 21 days, respectively. The two inoculations were made with subcutaneous injec tions in different places, and an equal volume of Freund’s complete adjuvant (Difco, Detroit, Mich.) was added to the cell suspension. After a period of 7 days we performed the first bleeding, and electrophoresis proved an increase of /-globulins. At this time we sacrificed the rabbit. The serum obtained was incubated at 57 °C for 30 min and stored at -20 °C. Serum anti-ALL(a). We absorbed the antiserum anti-ALL with an equal volume of packed cells (red blood cells A, B, Rh positive) for 20 min at 20 °C and for 1 h at + 4 °C. Then we absorbed this antiserum in the same manner with a pool of nor mal lymphocytes (cells/serum ratio 1:2) and with the lymphocytes of the parents whose sons had given us the lymphoblastic cells for inoculation (2X108 lymphocytes/ml). Finally, for absorption we used the lymphocytes of the same patients during the remission phase, obtained after antileukemic therapy. 2X108 lymphocytes isolat ed from peripheral blood were used for 1 ml of antiserum. The absorptions were performed until the serum no longer reacted with these cells as described in table V.
Serum against Human Leukemic Cells
259
Table II. Values of Cl with anti-ALL serum in 10 normal donors and in 10 patients with various diseases Case No.
Serum dilutions 1/8 1/32
1/256
Normal donors 1 2 3 4 5 6 7 8 9 10
0.03 0.22 0.03 0.90 0.83 0.86 0.14 0.15 0.27 0.34
0.02 0.19 0.06 0.30 0.08 0.07 0.06 0.16 0.03 0.09
0.01 0.15 0.01 0.06 0.00 0.00 0.01 0.09 0.00 0.02
0.77 0.18 0.59 0.88 0.89 0.67 0.96 0.64 0.20 0.12
0.17 0.17 0.08 0.06 0.33 0.33 0.30 0.34 0.12 0.03
0.06 0.18 0.06 0.06 0.20 0.18 0.16 0.33 0.04 0.00
Allergy to cow’s milk Asthma Acute bronchopneumonia Macroglobulinemia Encephalomyelitis Carcinoma of the breast Chronic mucocutaneous candidiasis T cell defect T cell defect CVH
Serum anti-ALL(a) (i). The serum obtained as described for serum anti-ALL(a) was absorbed with lymphoblasts of a different patient with ALL in acute phase (packed cells/serum ratio 1:1). Cytotoxicity test. The sera obtained were tested in vitro with cells of normal do nors, patients with ALL and their relatives, patients with various diseases and with continuous cultured cells. We used the cytotoxicity technique in presence of complement as we described in another work [13]. The titre of cytotoxic activity was determined using code sam ples. Dilutions were made within T.C.199. Target cells were used at a final concen tration of 4X 106/ml, using rabbit normal serum as the source of complement. Incu bation time of cells, antisera and complement was 40 min at 37 °C. Death cells were envisaged using uptake of trypan blue. The results are expressed with the cytotoxic index (Cl) [2] and are calculated for significant antiserum dilutions.
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Various diseases 11 12 13 14 25 16 17 18 19 10
260
P andolfi/L uzi/T ozzi/A iuti
Table III. Values of Cl with anti-ALL serum, and clinical and immunological data in 11 children with ALL in acute phase without therapy Case No.
WBC///1
Blasts %
Rosettes, % E
EAC
Serum Cl dilutions 1/8
1 2 3 4
5 6 7 8 9 10 11
60,000 ND 12,000 2,800l
110,000 ND 10,000 80,000 150,000 9,800 4,0001
90 73 62 0l
51 ND 40 90 97 60 0‘
Comments
1/32 1/256
2 4 10 9 15 23 591 131 92 02
1.00 0.92 0.54 0.79 0.69 0.55 0.85 0.71 0.59
33 14 8 ND 14 ND 2 4 80 2 10 4 681 8l
0.94 0.82 0.40 0.92 0.79 0.39 0.88 0.64 0.30 0.99 0.87 0.68 0,98 0.82 0.06 0.64 0.33 0.09 0.611 0.231 0.041
0.972 0.832 0.422
Diagnosis on bone marrow steroid therapy
T cell leukemia diagnosis on bone marrow
1 Peripheral blood. 2 Bone marrow.
The techniques of E rosettes (marker of T lymphocytes), and EAC rosettes (lym phocytes with receptor for C3) and surface membrane immunoglobulins (Smlg), were performed as indicated in a recent work concerning standardization of immu nological techniques [3]. Cases. Both in peripheral blood and in bone marrow, lymphocytes were isolated on Ficoll-Isopaque gradient, washed twice and resuspended in T.C.199. The diagnosis had been confirmed by usual clinical and laboratory methods.
Results
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Table II shows the data of Cl with serum anti-ALL in 10 normal do nors and 10 patients with different diseases, all studied as controls. At a dilution of 1:32, the serum gives an average Cl of 0.15 (range 0.02-0.34; SD 0.10). The same antiserum was tested in 11 cases of ALL in acute phase and before antileukemic therapy (table III). At the same dilution of 1:32, in 9
Serum against Human Leukemic Cells
261
Table IV. Values of Cl with anti-ALL serum in 10 relatives of patients with ALL (No. 1-10), in 5 patients with ALL in CR (No. 11-15), in 6 patients with other lymphoproliférative disorders (No. 16-21) and in continuous cultured cell lines (No. 22-23) Case No.
Serum dilutions 1/8 f/32
1/256
2 3 4 5 6 7 8 9 10
Mother of case 9 (table III) Father of case 9 (table III) Brother of case 9 (table III) Father of case 11 (table IV) Mother of case 11 (table IV) Mother of case 12 (table IV) Father of case 12 (table IV) Mother of case 13 (table IV) Mother of case 2 (table I) Father of case 2 (table I)
0.45 0.12 0.48 0.25 0.35 0.22 0.72 0.00 0.98 0.97
0.12 0.23 0.07 0.20 0.16 0.00 0.02 0.00 0.62 0.54
0.05 0.06 0.06 0.04 0.14 0.00 0.00 0.00 0.18 0.00
11 12 13 14 15
ALL ALL ALL ALL ALL
0.20 0.94 0.43 0.99 0.40
0.18 0.16 0.05 0.81 0.11
0.05 0.05 0.00 0.08 0.04
16 17 18 19 20 21 22 23
Acute myelomonocytic leukemia Acute myeloblastic leukemia CML CML in BC Chronic lymphocytic leukemia Lymphoma Cultured RAJI cell line Cultured cell line from infectious mononucleosis
0.98 0.23
0.20 0.19 0.78 0.68 0.00 0.07 0.96 0.24
0.12 0.19 0.28 0.34 0.00 0.00 0.98 0.01
1
in in in in in
CR CR CR CR (case 2, table I) CR (case 1, table I)
1.00
1.00 0.97 0.18 1.00 0.32
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patients we obtained an average Cl of 0.78 (range 0.64-0.92; SD 0.09). We regard subjects as positive when CT is >0.45 at 1:32 dilution and we have positive controls (table II): 0/20, and positive patients with ALL (table III): 9/11 (p
Preparation, Purification and in vitro Properties of a Serum against Human Lymphoblastic Leukemia-Associated Antigens F. P andolfi, G. L uzi, M. C. T ozzi and F. A iuti Department of Clinic.al Medicine III and Department of Pediatrics II, University of Rome, Rome
Key Words. Acute lymphoblastic leukemia • Antileukemic sera • Cytotoxicity • Leukemia antigens • Tumor-associated antigens Abstract. A serum against human lymphoblastic leukemia cells was obtained in oculating rabbits. We studied the specificity of the serum before and after particular absorptions in various clinical conditions. The serum was cytotoxic against many cases of acute lymphoblastic leukemia, against continuously cultured Burkitt’s lym phoma cells and against chronic myelogenous leukemia in blast crisis. On the con trary, the serum was not cytotoxic against lymphocytes of normal donors, acute lymphoblastic leukemia in remission and other lymphoproliférative disorders.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 10:09:23 PM
Lymphoproliférative diseases and, particularly, acute lymphoblastic leukemia (ALL) were recently studied in order to identify particular anti genic modifications in neoplastic cells. This kind of immunological study of tracing cellular properties in tumors could produce useful diagnostic, prognostic and therapeutic results. Specific antigens in leukemias were studied by various authors using specific antisera [8, 10-12]. The greatest difficulty in this kind of study is the preparation of specific antisera; it is clear that an inoculated animal produces antibodies against nonspecific and assumed associated leukemic antigens. For their inoculations some authors used animals with tolerance towards human leukocyte antigen (HLA), identical normal human cells [4], or extracts of membranes of continuous cultured cells which might have cross-reacting antigens with leukemic cells [5, 7,10].
P andolfi/L uzi/T ozzi/A iuti
258
Table /. Clinical and immunological data of the 2 patients with ALL whose cells were used for immunization Case No
Sex
1 2
F F
Age years
WBC//d
5 2
6,000 6,800
Blasts %
Rosettes, % E EAC
Smlg
90 88
12 7
ND 0 0 0
21 13
G A M
SmIg=Surface membrane immunoglobulins: IgG, IgA and IgM; N D = n o t done or not available.
We studied the problem from two points of view: (1) antiserum specif icity at various dilutions, and (2) specificity of antisera following a partic ular absorption scheme. Material and Methods
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 10:09:23 PM
Serum anti-ALL. Leukemic lymphoblasts used for inoculation were obtained from the peripheral blood of 2 patients with ALL in acute phase whose diagnosis had been confirmed by usual clinical and laboratory methods. Pertinent data con cerning these 2 patients are reported in table I. The cells were obtained from patients before therapy. Lymphoid cells were iso lated by centrifugation on Ficoll-Isopaque gradient, washed twice and suspended in T.C. 199 (Wellcome, England). Cells were stored at -80 °C until inoculation. The suspension of cells was injected intravenously in a rabbit at a dosage of 2X 107 cells. This was followed by two subsequent inoculations of 107 cells each performed at 14 and 21 days, respectively. The two inoculations were made with subcutaneous injec tions in different places, and an equal volume of Freund’s complete adjuvant (Difco, Detroit, Mich.) was added to the cell suspension. After a period of 7 days we performed the first bleeding, and electrophoresis proved an increase of /-globulins. At this time we sacrificed the rabbit. The serum obtained was incubated at 57 °C for 30 min and stored at -20 °C. Serum anti-ALL(a). We absorbed the antiserum anti-ALL with an equal volume of packed cells (red blood cells A, B, Rh positive) for 20 min at 20 °C and for 1 h at + 4 °C. Then we absorbed this antiserum in the same manner with a pool of nor mal lymphocytes (cells/serum ratio 1:2) and with the lymphocytes of the parents whose sons had given us the lymphoblastic cells for inoculation (2X108 lymphocytes/ml). Finally, for absorption we used the lymphocytes of the same patients during the remission phase, obtained after antileukemic therapy. 2X108 lymphocytes isolat ed from peripheral blood were used for 1 ml of antiserum. The absorptions were performed until the serum no longer reacted with these cells as described in table V.
Serum against Human Leukemic Cells
259
Table II. Values of Cl with anti-ALL serum in 10 normal donors and in 10 patients with various diseases Case No.
Serum dilutions 1/8 1/32
1/256
Normal donors 1 2 3 4 5 6 7 8 9 10
0.03 0.22 0.03 0.90 0.83 0.86 0.14 0.15 0.27 0.34
0.02 0.19 0.06 0.30 0.08 0.07 0.06 0.16 0.03 0.09
0.01 0.15 0.01 0.06 0.00 0.00 0.01 0.09 0.00 0.02
0.77 0.18 0.59 0.88 0.89 0.67 0.96 0.64 0.20 0.12
0.17 0.17 0.08 0.06 0.33 0.33 0.30 0.34 0.12 0.03
0.06 0.18 0.06 0.06 0.20 0.18 0.16 0.33 0.04 0.00
Allergy to cow’s milk Asthma Acute bronchopneumonia Macroglobulinemia Encephalomyelitis Carcinoma of the breast Chronic mucocutaneous candidiasis T cell defect T cell defect CVH
Serum anti-ALL(a) (i). The serum obtained as described for serum anti-ALL(a) was absorbed with lymphoblasts of a different patient with ALL in acute phase (packed cells/serum ratio 1:1). Cytotoxicity test. The sera obtained were tested in vitro with cells of normal do nors, patients with ALL and their relatives, patients with various diseases and with continuous cultured cells. We used the cytotoxicity technique in presence of complement as we described in another work [13]. The titre of cytotoxic activity was determined using code sam ples. Dilutions were made within T.C.199. Target cells were used at a final concen tration of 4X 106/ml, using rabbit normal serum as the source of complement. Incu bation time of cells, antisera and complement was 40 min at 37 °C. Death cells were envisaged using uptake of trypan blue. The results are expressed with the cytotoxic index (Cl) [2] and are calculated for significant antiserum dilutions.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 10:09:23 PM
Various diseases 11 12 13 14 25 16 17 18 19 10
260
P andolfi/L uzi/T ozzi/A iuti
Table III. Values of Cl with anti-ALL serum, and clinical and immunological data in 11 children with ALL in acute phase without therapy Case No.
WBC///1
Blasts %
Rosettes, % E
EAC
Serum Cl dilutions 1/8
1 2 3 4
5 6 7 8 9 10 11
60,000 ND 12,000 2,800l
110,000 ND 10,000 80,000 150,000 9,800 4,0001
90 73 62 0l
51 ND 40 90 97 60 0‘
Comments
1/32 1/256
2 4 10 9 15 23 591 131 92 02
1.00 0.92 0.54 0.79 0.69 0.55 0.85 0.71 0.59
33 14 8 ND 14 ND 2 4 80 2 10 4 681 8l
0.94 0.82 0.40 0.92 0.79 0.39 0.88 0.64 0.30 0.99 0.87 0.68 0,98 0.82 0.06 0.64 0.33 0.09 0.611 0.231 0.041
0.972 0.832 0.422
Diagnosis on bone marrow steroid therapy
T cell leukemia diagnosis on bone marrow
1 Peripheral blood. 2 Bone marrow.
The techniques of E rosettes (marker of T lymphocytes), and EAC rosettes (lym phocytes with receptor for C3) and surface membrane immunoglobulins (Smlg), were performed as indicated in a recent work concerning standardization of immu nological techniques [3]. Cases. Both in peripheral blood and in bone marrow, lymphocytes were isolated on Ficoll-Isopaque gradient, washed twice and resuspended in T.C.199. The diagnosis had been confirmed by usual clinical and laboratory methods.
Results
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Table II shows the data of Cl with serum anti-ALL in 10 normal do nors and 10 patients with different diseases, all studied as controls. At a dilution of 1:32, the serum gives an average Cl of 0.15 (range 0.02-0.34; SD 0.10). The same antiserum was tested in 11 cases of ALL in acute phase and before antileukemic therapy (table III). At the same dilution of 1:32, in 9
Serum against Human Leukemic Cells
261
Table IV. Values of Cl with anti-ALL serum in 10 relatives of patients with ALL (No. 1-10), in 5 patients with ALL in CR (No. 11-15), in 6 patients with other lymphoproliférative disorders (No. 16-21) and in continuous cultured cell lines (No. 22-23) Case No.
Serum dilutions 1/8 f/32
1/256
2 3 4 5 6 7 8 9 10
Mother of case 9 (table III) Father of case 9 (table III) Brother of case 9 (table III) Father of case 11 (table IV) Mother of case 11 (table IV) Mother of case 12 (table IV) Father of case 12 (table IV) Mother of case 13 (table IV) Mother of case 2 (table I) Father of case 2 (table I)
0.45 0.12 0.48 0.25 0.35 0.22 0.72 0.00 0.98 0.97
0.12 0.23 0.07 0.20 0.16 0.00 0.02 0.00 0.62 0.54
0.05 0.06 0.06 0.04 0.14 0.00 0.00 0.00 0.18 0.00
11 12 13 14 15
ALL ALL ALL ALL ALL
0.20 0.94 0.43 0.99 0.40
0.18 0.16 0.05 0.81 0.11
0.05 0.05 0.00 0.08 0.04
16 17 18 19 20 21 22 23
Acute myelomonocytic leukemia Acute myeloblastic leukemia CML CML in BC Chronic lymphocytic leukemia Lymphoma Cultured RAJI cell line Cultured cell line from infectious mononucleosis
0.98 0.23
0.20 0.19 0.78 0.68 0.00 0.07 0.96 0.24
0.12 0.19 0.28 0.34 0.00 0.00 0.98 0.01
1
in in in in in
CR CR CR CR (case 2, table I) CR (case 1, table I)
1.00
1.00 0.97 0.18 1.00 0.32
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patients we obtained an average Cl of 0.78 (range 0.64-0.92; SD 0.09). We regard subjects as positive when CT is >0.45 at 1:32 dilution and we have positive controls (table II): 0/20, and positive patients with ALL (table III): 9/11 (p
