Proc. Natl. Acad. Sci. USA Vol. 75, No. 11, pp. 5706-5710, November 1978 Medical Sciences
Lymphoblastoid cell lines from patients with chronic lymphocytic leukemia: Identification of tumor origin by idiotypic analysis (B lymphocyte/cell cloning/Epstein-Baff virus/tumor-specific antigen)
JOHN N. HURLEY*, SHU MAN Fu*, HENRY G. KUNKEL*, GILLIES MCKENNAt, AND MATTHEW D. SCHARFFt
* The Rockefeller University, New York, New York 10021; and t Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Contributed by Henry G. Kunkel, August 28,1978
Multiple lymphoblastoid cell lines have been ABSTRACT derived from two patients with chronic lymphocytic leukemia with an associated monoclonal immunoglobulin (Ig) band. Idiotypic antisera raised against the monoclonal serum Ig bands were shown to be specific for the membrane Ig of the patients' leukemic cells. The idiotypic determinants in these patients thereby constitute tumor-specific antigens. Surface and intracellular immunofluorescence studies utilizing these idiotypic antisera were used to identify the cell lines of leukemic origin. These studies showed that certain cell lines from each patient were derived from the leukemic cells while other cell lines were derived from residual normal B lymphocytes. The leukemic cell lines were variable and contained different percentages of lymphoid cells with the idiotype specific membrane Ig and, in addition, different percentages of plasma cells with intracellular Ig of the same specificity. Specific Ig synthesis was also demonstrated by hemagglutination-inhibition analysis of cell line supernatants. Aside from Ig specificity, no differences have been found between the leukemic cell lines and those derived from normal cells. One of the leukemic cell lines was cloned in soft agarose. All the clones were shown to be of leukemic origin.
Studies of surface markers and chromosomal analyses indicate that cell lines obtained from patients with various types of leukemia are rarely derived from the leukemic cells (1-5). The majority of the cell lines are lymphoblastoid lines with B lymphocyte characteristics and are derived from residual normal B lymphocytes in the patient's blood (6). In the case of chronic lymphocytic leukemia (CLL), a number of reports have appeared implying the establishment of continuous lines of leukemic origin (7, 8). However, in such cases it is extremely difficult to document the leukemic origin of the transformed line because both the original tumor cells and cells from the line have B cell characteristics. In some cases of CLL, a monoclonal immunoglobulin (Ig) band is found in the serum in addition to marked lymphocytosis (9). In previous studies, it was documented that idiotypic antisera to these Ig bands stained the membranes of the majority of the leukemic lymphocytes and stained the cytoplasm of the cells responsible for the Ig band (10). In the present study numerous continuous cell lines were obtained from these patients. Analyses with the idiotypic antisera have demonstrated that some of these lines were derived from the same cell lineage as the leukemic lymphocytes bearing similar idiotypic determinants. These lines represent proven success in establishing cell lines of CLL origin. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
MATERIALS AND METHODS Patient Identification, Antisera, Immunofluorescence, and Cell Preparation. Patient Se is a 62-year-old woman who has had CLL for 3 years and who has a K IgM band in the serum. On initial clinical presentation, the leukocyte count (WBC) was 50,000/mm3, the serum monoclonal macroglobulin was present at a concentration of 4 mg/ml, and the patient had marked splenomegaly. Patient Ei is an 87-year-old woman with CLL and a K IgM monoclonal band in the serum. Patient Ei has been described in detail previously (10). Antisera were prepared in New Zealand Red rabbits against the serum 1gM components from these patients after purification by Pevikon block electrophoresis. Rhodamine-conjugated F(ab')2 fragments specific for y,, , a, K, X, IgM protein Se (IgM Se), and IgM protein Ei (IgM Ei) were prepared and characterized by methods previously described (11). Unconjugated idiotypic antisera specific for IgM Ei and for IgM Se were also prepared. These antisera were absorbed with pooled normal human serum, pooled IgG, and monoclonal IgM proteins isolated from other patients, and were shown to be highly specific for the protein used in the immunization both by hemagglutination inhibition and by fluorescence. Idiotypic antiserum to IgM Se did not react with normal B cells or with cells from patient Ei by fluorescence; the same individual specificity was true for idiotypic antiserum to IgM Ei. Hemagglutination and hemagglutination inhibition were carried out as described (12). Mononuclear cell preparations from peripheral blood were isolated by Ficoll/Hypaque gradient centrifugation. Patient Se underwent theraputic splenectomy. A portion of the spleen was teased with a sterile needle and forceps in RPMI 1640 medium with L-glutamine (Microbiological Associates, Bethesda, MD). Larger pieces of tissue were allowed to settle. Mononuclear spleen cells from this supernatant cell suspension were then isolated by the Ficoll/Hypaque gradient technique. Surface and intracellular immunofluorescent staining for Ig and idiotypic determinants was performed as described previously (10). Rosette formation with sheep erythrocytes was performed as described (13). Peroxidase staining was determined by a modification of the technique of Kaplow (14) utilizing 3,3'dimethoxybenzidine (Sigma). The presence of Epstein-Barr virus nuclear antigen (EBNA) was tested by the method of Reedman and Klein (15). Cell Cultures and Initiation of Cell Lines. The culture medium used was RPMI 1640 with L-glutamine supplemented Abbreviations: CLL, chronic lymphocytic leukemia; WBCl4eukocyte
count; EBV, Epstein-Barr virus; EBNA, EBV nuclear antigen. 5706
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Table 1. Immunofluorescent analysis of peripheral blood and spleen mononuclear cells from patient Se with antisera to different Igs % positive with antiserum to Sample Id* IgM IgD IgG IgA K A Peripheral blood Surface Ig October 1976t 13 19 12 0 0 16 2 34 March 1978t 44 ND 0 0 33 2 Intracellular Ig March 1978 0.2 0.2 0 0 0 0.2 0 Spleen 12 Intracellular Ig 12 0 0 0 15 0 ND, not determined. * Antiserum specific for idiotypic determinants of the IgM K paraprotein from patient Se. t Sixty-five percent of the peripheral mononuclear cells formed sheep erythrocyte rosettes. At this time the WBC was 7000/mm3. Forty-two percent of the peripheral mononuclear cells formed sheep erythrocyte rosettes while 17% were peroxidase positive. At this time the WBC was 12,000/mm3.
with 2 mM additional glutamine, penicillin at 100 units/ml, streptomycin at 100 ,g/ml (Grand Island Biological), and 10% fetal bovine serum (lot P60512, Reheis Chemical Company, Phoenix, AZ). The source of Epstein-Barr virus (EBV) was culture medium supernatants from marmoset cell line B95-8 (16). This cell line was obtained from Pfizer, through the National Cancer Institute, Bethesda, MD. Four-day culture supernatants from the B95-8 cell line were filtered through a 0.8-aum filter (Nalge, Rochester, NY) and used without freezing or storage. Cell lines were initiated in 16-mm tissue culture plates (FB-16-24-TC, Linbro Scientific, Hamden, CT) by mixing 1 ml of culture medium containing 2 X 106 mononuclear cells with 1 ml of filtered B95-8 culture supernatant. Culture plates were kept in a humidified incubator at 370C with a 5% CO2 in air atmosphere. Cultures were fed twice weekly. After 2-4 weeks, cells reached a sufficient density to be transferred to larger tissue culture flasks (Falcon). After a cell line was established, the cultures were split 3:1 every third day and fed with fresh culture medium. Cell Cloning in Soft Agarose. Cloning was carried out according to the method described by Coffino et al. (17), using the culture medium described above. By this method, the human lymphoblastoid cell line 8866 was found to clone at an efficiency of approximately 10%.
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RESULTS Establishment of Cell Lines. A large number of continuous cell lines were derived from the peripheral blood mononuclear cell population from patients Se and Ei with monoclonal IgM
bands in the serum. EBV-containing supernatants from cell line B95-8 were utilized in this earlier work. Cell lines were obtained from these patients at a frequency similar to normal individuals. Cell lines were also derived from the spleen mononuclear cells of patient Se in a similar manner. Two cell lines from the peripheral blood of patient Ei were derived without the addition of the EBV-containing supernatants. The frequency of derivation of these spontaneous lines was much lower than the frequency found for the EBV-infected cultures. Characterization of Anti-Idiotypic Antisera. As detailed in the Materials and Methods section, anti-idiotypic antisera were obtained against the isolated IgM band proteins of patients Se and Ei. These antisera confirmed the previous observations on patient Ei that the same idiotypic determinants were present on the band serum Ig as were present on the membrane of the respective leukemic cells and in the cytoplasm of the circulating plasma cells (10). Table 1 shows data on patient Se regarding this point. Although cell lines were derived from patient Se at a time when the WBC was 50,000/mm3, characterization of the patient's peripheral blood mononuclear cells with antiidiotypic antiserum was performed after the patient had been treated extensively and the WBC had been reduced significantly. At a time when the WBC was 7,000/mm3, surface staining of the peripheral mononuclear cells of this patient showed that 13% of these cells were stained with the anti-idiotypic reagent, while an anti-IgM reagent stained 19% of the cells and an anti-IgD reagent stained 12%. This shows that, even after extensive treatment, the majority of this patient's Igbearing lymphocytes had surface ,u, 6, and K chains present and shared the idiotypic determinants of the patient's IgM K serum band. A later study at a time when the patient's WBC had increased to 12,000/mm3 showed an increase in the idiotypebearing lymphocytes to 34%. Isolated T lymphocytes from this patient did not stain for idiotypic determinants with this reagent. Intracellular staining of these cells showed the presence of 0.2% circulating plasma cells that contained IgM with the idiotype of the serum band. Intracellular staining of the mononuclear cells of the spleen of patient Se showed 12% plasma cells that contained IgM that shared the idiotype of the serum band. The idiotypic antiserum specific for the serum band from patient Se, therefore, stained the surface of the leukemic lymphocytes and the cytoplasm of the plasma cells of this patient that are responsible for the serum Ig band and was specific for this patient's leukemic cell lineage.
Table 2. Percentage of cells in different Se and Ei cell lines showing membrane Ig and cytoplasmic Ig as detected by fluorescence with various Ig antisera* Surface Ig, % Intracellular Ig, % Line Se Idt A Ei Id IgM IgD K Se Idt Ei Idt IgG IgM IgD IgG SeD 67 0 72 37 0 67 0 24 0 29 0 0 SeAl 0 0 80 22 0 33 0 0 0 43 0 0 Se21 0 0 96 0 0 39 0 0 0 42 0 0 SeA2 0 0 0 0 0 0 0 0 0 0 0 28 Ei26 0 75 80 0 0 15 0 0 46 51 0 0 Ei39 0 4 4 0 0 2 0 0 27 22 0 0 Ei81 0 0 0 0 0 0 0 0 34 35 0 0 Ei 151 0 0 0 0 0 0 0 0 0 0 0 27 * No cell lines were stained with an anti-IgA reagent. t Antiserum specific for the idiotypic determinants of the IgM K paraprotein from patient Se. t Antiserum specific for the idiotypic determinants of the IgM K paraprotein from patient Ei.
K
X
30 40 38 20 49 26 24 0
0 0 0 0 0 0 0 25
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FIG. 1. Surface staining on living cells and intracellular immunofluorescent staining on a fixed preparation of cell line SeD with an anti-IgM Se idiotypic reagent. (X 1800.) (a) Phase-contrast micrograph of surface staining preparation; (b) same field under fluorescent microscopy showing four positive cells of differing intensities. (c) Phase-contrast micrograph of intracellular staining preparation; (d) same field under fluorescent microscopy, showing three positive cells.
Cell Line Igs and Their Idiotypes. Immunofluorescent staining with rhodamine-conjugated F(ab')2 fragments of the
anti-idiotypic sera characterized above was used to characterize the surface and intracellular Igs of the cell lines derived from these patients. Results for representative cell lines are shown in Table 2. Of five cell lines derived from the spleen and two lines derived from the peripheral blood of patient Se, only cell line SeD, which was derived from spleen, was found to stain with the anti-IgM Se idiotypic reagent. In surface staining, the idiotypic antiserum stained 67% of the cells of line SeD, while 24% were stained intracellularly. The intensity of both the surface and intracellular staining varied considerably from cell to cell. This variation can be noted in Fig. 1. Twelve of 17 cell lines derived from the peripheral blood of patient Ei were found to stain for intracellular IgM sharing the idiotype of the patient's band. In cell line Ei 26, 75% of the cells stained for surface Ig with the IgM Ei idiotype while only 4% did so in the case of Ei 39. None of the cell lines from patient Ei had detectable surface IgD. Some of the cell lines that were positive for production of the idiotypic determinants by intracellular staining did not have surface Ig detectable by surface staining. Ei 81 is an example of this type of cell line. Supernatant culture medium from each cell line was tested for the presence of the idiotypic determinants of each of these patient's serum bands by the hemagglutination-inhibition system. Results for various cell line supernatants are shown in Table 3 for the IgM Se idiotypic system. Only supernatants from cell line SeD and its clones (to be described later) were found to inhibit this system. In a hemagglutination-inhibition system specific for IgM, it was found that SeD, SeAl, Se 21, and Ei 26
secreted equivalent amounts of IgM into the culture medium. Thus, only cell line SeD and its clones secreted an Ig that shared the idiotypic determinants of IgM Se. The leukemic origin of cell line SeD is, therefore, demonstrated both by fluorescence and by hemagglutination inhibition. A hemagglutinationinhibition assay was also performed for the idiotypic determinants of IgM Ei In all cases, culture supernatants from cell lines positive by intracellular staining for a cytoplasmic Ig sharing the patient's idiotypic determinants were found to inhibit the hemagglutination-inhibition system specific for that patient's idiotypic determinants. All culture supernatants from cell lines negative for an idiotypic determinant by intracellular staining were negative in the corresponding hemagglutination-inhibiTable 3. Hemagglutination-inhibition assay of 3-day culture supernatants from various cell lines for the detection of the idiotypic determinants of IgM Se Dilution of cell line supernatant Cell line 0 1:2 1:4 1:8 1:16 1:32 1:64 SeD (parental) SeD.2 (clone) SeD.9 (clone) SeAl Se2l
SeA2 Ei26
+ + +
+ + +
+
+
-
-
+
+
-
-
-
-
+ + + + +
+ + + + +
+ + + + +
+ + + +
+ + + + + + +
Erythrocyte coat, IgM Se. Antiserum made against IgM Se, absorbed with pooled normal human sera. Control endpoint inhibition with IgM Se was 1 ,4g/ml. A + indicates hemagglutination.
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Table 4. Characterization of clones of cell line SeD by immunofluorescence*
Surface Ig, % Intracellular Ig, % Line Idt IgM K Idt IgD IgM IgD SeD (parental) 67 72 37 67 24 29 0 SeD.2 (clone) 75 91 26 66 41 51 0 SeD.4 (clone) 68 96 22 87 35 42 0 SeD.6 (clone) 58 79 *6 78 30 31 0 SeD.9 (clone) 65 99 32 56 38 36 0 * No cell lines were stained with anti-IgA, IgG, or X reagents. t Antiserum specific for the idiotypic determinants of the IgM K paraprotein from patient Se.
tion assay. Thus, fluorescence and hemagglutination inhibition confirm each other in the identification of cell lines of leukemic origin.
Several cell lines from patient Ei, when studied by hemagglutination inhibition and by intracellular staining within 2 months of initiation of the cell lines, were found to be mixtures of cells positive for idiotypic determinants, IgM, and IgG. Over a period of 6 months, changes in the hemagglutination-inhibition results and staining patterns were noted. In some of the cases, Ig detected in the supernatants no longer had the IgM Ei idiotypic determinants. Thus, presumably nonleukemic B cells in these lines became the dominant cells. In other cases, for example Ei 26, the leukemic cells continued to dominate in the mass culture. Leukemic Lines without Addition of EBV. The two cell lines from patient Ei derived without addition of the EBVcontaining supernatant from cell line B95-8 were both found to be of leukemic origin. Ei 39 in Table 2 is one of these lines. Studies on cell line Ei 39, however, showed that the cells were infected with EBV because the nuclei were EBNA positive. Clones of Cell Line SeD. After approximately 6 months of continuous culture, cell line SeD was cloned in soft agarose. The cloning efficiency was found to be approximately 0.1%. Seven clones of this cell line were characterized both by immunofluorescent staining and by hemagglutination inhibition. The immunofluorescent staining results for representative clones are shown in Table 4. In general, higher percentages of the cells were positive for surface IgM in the cloned lines than in the parental line. In addition, the anti-A antiserum stained more cells than the anti-idiotypic and the anti-K antisera. The latter difference might be due to the different strengths of the antisera. Supernatant culture fluids from all the clones were found to inhibit the IgM Se idiotypic hemagglutination-inhibition system (Table 3). No qualitative differences between clones or between the parental line and any of the clones were found. Attempts to clone certain idiotype-positive cell lines from patient Ei were not successful. DISCUSSION In the present study, anti-idiotypic antisera proved powerful tools for the identification of the nature of various cell lines derived from two patients with CLL. Many lines were derived that showed membrane and cytoplasmic Ig that bore no relation to the Ig of the leukemic cells. In other instances, related Ig would be detected early after initiation of the lines but would then disappear with the appearance of an unrelated Ig. However, in a number of instances (1 for Se and 12 for Ei), long-term lines were obtained that clearly made the same Ig as the leukemic cell as identified by the anti-idiotypic antisera. Subelones were obtained from cell line SeD that clearly showed similar continuous production of Ig of the same idiotype. These findings point out the need to clone cell lines to isolate the cell population of interest and to ensure its stability. The possibility of spontaneous mutation in culture exists. However, during
K
30 35 36 27 30
study of cell line SeD and its clones over a period of over 12 months, no changes in the expression of the idiotypic markers were observed. It is, therefore, unlikely that mutations were the cause of the changes seen in Ig production in some Ei cell lines. The surface staining of lines SeD and Ei 26 varied in intensity. This varied staining has also been observed in vio (10). It was documented in the peripheral blood of patient Ei that the varied staining is due to different amounts of Ig on the cell surface (10). Immunofluorescence with double fluorochromes also demonstrated that cells that stained brightly for intracellular IgM in line SeD had no detectable surface Ig (data not shown). It seems probable that the varied intensity of surface staining is related to the cell cycle in view of previous observations with other lymphoblastoid cell lines (18-20). Some of the Ei lines such as Ei 81 had no detectable surface IgM, while a certain percentage of the cells stained intracellularly for IgM Ei This differs from Ei 26 and Ei 39, which had a variable percentage of surface staining. In the previous studies (10), the majority of the Ei leukemic B lymphocytes bore surface IgD. None of the Ei lines studied in the present investigation contained cells with surface IgD. Whether these differences reflect transformation of cells at different stages of differentiation remains to be answered. All lines from patient Se were derived from cultures infected with exogenous EBV. Two cell lines from patient Ei were derived without addition of exogenous EBV. Both spontaneous lines from patient Ei were positive for the IgM Ei idiotype. Most lymphoblastoid lines derived without addition of exogenous EBV have been found to be positive for EBNA and are thought to be infected by EBV from cells carrying the EBV genome from a previous exposure to this virus (21). Evidence has been obtained that the CLL lines derived in the present study are EBNA positive. Both patients Ei and Se differed from the majority of patients with CLL in that a monoclonal IgM band was found in the serum in addition to marked lymphocytosis. Idiotypic antisera to the serum IgM band proved that the serum IgM band, the membrane IgM on the leukemic lymphocytes, and the intracellular IgM in the plasma cells were idiotypically identical. Evidence was obtained (10) that this Ig band reflected differentiation of a few of the leukemic lymphocytes to IgM-secreting plasma cells. Recently, this differentiation has been markedly accelerated in vitro with various stimulating materials (22). The question of whether this differentiation capability played a role in the successful development of long term lymphoblastoid lines remains unclear. Attempts have also been made to obtain long term lymphoblastoid lines from CLL patients without related monoclonal bands. In these cases, lines are difficult to obtain. A few lines from these patients have been derived; their relationship to the patient's leukemic cells is not yet known. Idiotypic antisera are difficult to obtain (23) for these more common types of CLL, and this hampers a definitive answer to the question of the origin of these cell lines.
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We thank Dr. B. Hampar and Dr. E. Tan for supplying the reagents used in the EBNA determinations and Dr. S. Harris for providing patient material. The excellent assistance of Ms. R. Brooks is gratefully appreciated. S.M.F. is a scholar of the Leukemia Society of America.
G.M. is a medical scientist trainee supported by Grant 5T5 GM1674 from the National Institute of General Medical Sciences. This investigation was supported by U.S. Public Health Service Grants RR-102 and AI-10811 and a grant from the National Leukemia Association. 1. Epstein, A. L. & Kaplan, H. S. (1974) Cancer 34, 1851-1872. 2. Kaplan, J., Shope, T. C. & Peterson, W. D. (1974) J. Exp. Med. 139, 1070-1076. 3. Nilsson, K. & Sundstrom, C. (1974) Int. J. Cancer 13, 808823. 4. Lozzio, C. B. & Lozzio, B. B. (1975) Blood 45,321-334. 5. Rosenfeld, C., Goutner, A., Choquet, C., Venvat, A. M., Kayibanda, B., Pico, J. L. & Greaves, M. F. (1977) Nature (London) 267,841-843. 6. Belpomme, D., Minowada, J. & Moore, G. E. (1972) Cancer 30, 282-287. 7. Sawitsky, B., Douglas, S. D., Lipton, R. & Sawitsky, A. (1974) Acta Haematol. 52, 83-94. 8. Saxon, A., Stevens, R. H., Quan, S. G. & Golde, D. W. (1978) J.
Immunol. 120, 777-782.
9. Azar, H. A., Hill, W. T. & Osserman, E. F. (1957) Am. J. Med. 23,239-249.
Proc. Natl. Acad. Sci. -USA 75 (1978) 10. Fu, S. M., Winchester, R. J., Feizi, T., Walzer, P. D. & Kunkel, H. G. (1974) Proc. Natl. Acad. Sci. USA 71, 4487-4490. 11. Fu, S. M., Winchester, R. J. & Kunkel, H.G. (1975)J. Immunol. 114,250-252. 12. Natvig, J. B. & Kunkel, H. G. (1967) Nature (London) 215, 68-69. 13. Hoffman, T. & Kunkel, H. G. (1976) in In Vitro Methods in Cell-Mediated and Tumor Immunity, eds. Bloom, B. R. & David, J. R. (Academic, New York), pp. 71-81. 14. Kaplow, L. S. (1965) Blood 26,215-219. 15. Reedman, B. M. & Klein, G. (1973) Int. J. Cancer 11, 499520. 16. Miller, G. & Lipman, M. (1973) Proc. Natl. Acad. Sci. USA 70, 190-194. 17. Coffino, P., Baumal, R., Laskov, R. & Scharff, M. D. (1972) J.
Cell. Physiol. 79,429-440.
18. Buell, D. N. & Fahey, J. L. (1969) Science 164, 1524-1525. 19. Takahashi, M., Yagi, Y., Moore, G. E. & Pressman, D. (1969) J. Immunol. 103, 834-843. 20. Kwok, S. Y. & Litwin, S. D. (1976) Cell. Immunol. 25, 256265. 21. Nilsson, K. & Ponten, J. (1975) Int. J. Cancer 15,321-341. 22. Fu, S. M., Chiorazzi, N., Kunkel, H. G. & Halper, J. P. (1978) Clin. Res. 26, 514A. 23. Hough, D. W., Eady, R. P., Hamblin, T. J., Stevenson, F. K. & Stevenson, G. T. (1976) J. Exp. Med. 144, 960-969.
Lymphoblastoid cell lines from patients with chronic lymphocytic leukemia: Identification of tumor origin by idiotypic analysis (B lymphocyte/cell cloning/Epstein-Baff virus/tumor-specific antigen)
JOHN N. HURLEY*, SHU MAN Fu*, HENRY G. KUNKEL*, GILLIES MCKENNAt, AND MATTHEW D. SCHARFFt
* The Rockefeller University, New York, New York 10021; and t Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Contributed by Henry G. Kunkel, August 28,1978
Multiple lymphoblastoid cell lines have been ABSTRACT derived from two patients with chronic lymphocytic leukemia with an associated monoclonal immunoglobulin (Ig) band. Idiotypic antisera raised against the monoclonal serum Ig bands were shown to be specific for the membrane Ig of the patients' leukemic cells. The idiotypic determinants in these patients thereby constitute tumor-specific antigens. Surface and intracellular immunofluorescence studies utilizing these idiotypic antisera were used to identify the cell lines of leukemic origin. These studies showed that certain cell lines from each patient were derived from the leukemic cells while other cell lines were derived from residual normal B lymphocytes. The leukemic cell lines were variable and contained different percentages of lymphoid cells with the idiotype specific membrane Ig and, in addition, different percentages of plasma cells with intracellular Ig of the same specificity. Specific Ig synthesis was also demonstrated by hemagglutination-inhibition analysis of cell line supernatants. Aside from Ig specificity, no differences have been found between the leukemic cell lines and those derived from normal cells. One of the leukemic cell lines was cloned in soft agarose. All the clones were shown to be of leukemic origin.
Studies of surface markers and chromosomal analyses indicate that cell lines obtained from patients with various types of leukemia are rarely derived from the leukemic cells (1-5). The majority of the cell lines are lymphoblastoid lines with B lymphocyte characteristics and are derived from residual normal B lymphocytes in the patient's blood (6). In the case of chronic lymphocytic leukemia (CLL), a number of reports have appeared implying the establishment of continuous lines of leukemic origin (7, 8). However, in such cases it is extremely difficult to document the leukemic origin of the transformed line because both the original tumor cells and cells from the line have B cell characteristics. In some cases of CLL, a monoclonal immunoglobulin (Ig) band is found in the serum in addition to marked lymphocytosis (9). In previous studies, it was documented that idiotypic antisera to these Ig bands stained the membranes of the majority of the leukemic lymphocytes and stained the cytoplasm of the cells responsible for the Ig band (10). In the present study numerous continuous cell lines were obtained from these patients. Analyses with the idiotypic antisera have demonstrated that some of these lines were derived from the same cell lineage as the leukemic lymphocytes bearing similar idiotypic determinants. These lines represent proven success in establishing cell lines of CLL origin. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
MATERIALS AND METHODS Patient Identification, Antisera, Immunofluorescence, and Cell Preparation. Patient Se is a 62-year-old woman who has had CLL for 3 years and who has a K IgM band in the serum. On initial clinical presentation, the leukocyte count (WBC) was 50,000/mm3, the serum monoclonal macroglobulin was present at a concentration of 4 mg/ml, and the patient had marked splenomegaly. Patient Ei is an 87-year-old woman with CLL and a K IgM monoclonal band in the serum. Patient Ei has been described in detail previously (10). Antisera were prepared in New Zealand Red rabbits against the serum 1gM components from these patients after purification by Pevikon block electrophoresis. Rhodamine-conjugated F(ab')2 fragments specific for y,, , a, K, X, IgM protein Se (IgM Se), and IgM protein Ei (IgM Ei) were prepared and characterized by methods previously described (11). Unconjugated idiotypic antisera specific for IgM Ei and for IgM Se were also prepared. These antisera were absorbed with pooled normal human serum, pooled IgG, and monoclonal IgM proteins isolated from other patients, and were shown to be highly specific for the protein used in the immunization both by hemagglutination inhibition and by fluorescence. Idiotypic antiserum to IgM Se did not react with normal B cells or with cells from patient Ei by fluorescence; the same individual specificity was true for idiotypic antiserum to IgM Ei. Hemagglutination and hemagglutination inhibition were carried out as described (12). Mononuclear cell preparations from peripheral blood were isolated by Ficoll/Hypaque gradient centrifugation. Patient Se underwent theraputic splenectomy. A portion of the spleen was teased with a sterile needle and forceps in RPMI 1640 medium with L-glutamine (Microbiological Associates, Bethesda, MD). Larger pieces of tissue were allowed to settle. Mononuclear spleen cells from this supernatant cell suspension were then isolated by the Ficoll/Hypaque gradient technique. Surface and intracellular immunofluorescent staining for Ig and idiotypic determinants was performed as described previously (10). Rosette formation with sheep erythrocytes was performed as described (13). Peroxidase staining was determined by a modification of the technique of Kaplow (14) utilizing 3,3'dimethoxybenzidine (Sigma). The presence of Epstein-Barr virus nuclear antigen (EBNA) was tested by the method of Reedman and Klein (15). Cell Cultures and Initiation of Cell Lines. The culture medium used was RPMI 1640 with L-glutamine supplemented Abbreviations: CLL, chronic lymphocytic leukemia; WBCl4eukocyte
count; EBV, Epstein-Barr virus; EBNA, EBV nuclear antigen. 5706
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Table 1. Immunofluorescent analysis of peripheral blood and spleen mononuclear cells from patient Se with antisera to different Igs % positive with antiserum to Sample Id* IgM IgD IgG IgA K A Peripheral blood Surface Ig October 1976t 13 19 12 0 0 16 2 34 March 1978t 44 ND 0 0 33 2 Intracellular Ig March 1978 0.2 0.2 0 0 0 0.2 0 Spleen 12 Intracellular Ig 12 0 0 0 15 0 ND, not determined. * Antiserum specific for idiotypic determinants of the IgM K paraprotein from patient Se. t Sixty-five percent of the peripheral mononuclear cells formed sheep erythrocyte rosettes. At this time the WBC was 7000/mm3. Forty-two percent of the peripheral mononuclear cells formed sheep erythrocyte rosettes while 17% were peroxidase positive. At this time the WBC was 12,000/mm3.
with 2 mM additional glutamine, penicillin at 100 units/ml, streptomycin at 100 ,g/ml (Grand Island Biological), and 10% fetal bovine serum (lot P60512, Reheis Chemical Company, Phoenix, AZ). The source of Epstein-Barr virus (EBV) was culture medium supernatants from marmoset cell line B95-8 (16). This cell line was obtained from Pfizer, through the National Cancer Institute, Bethesda, MD. Four-day culture supernatants from the B95-8 cell line were filtered through a 0.8-aum filter (Nalge, Rochester, NY) and used without freezing or storage. Cell lines were initiated in 16-mm tissue culture plates (FB-16-24-TC, Linbro Scientific, Hamden, CT) by mixing 1 ml of culture medium containing 2 X 106 mononuclear cells with 1 ml of filtered B95-8 culture supernatant. Culture plates were kept in a humidified incubator at 370C with a 5% CO2 in air atmosphere. Cultures were fed twice weekly. After 2-4 weeks, cells reached a sufficient density to be transferred to larger tissue culture flasks (Falcon). After a cell line was established, the cultures were split 3:1 every third day and fed with fresh culture medium. Cell Cloning in Soft Agarose. Cloning was carried out according to the method described by Coffino et al. (17), using the culture medium described above. By this method, the human lymphoblastoid cell line 8866 was found to clone at an efficiency of approximately 10%.
Proc. Natl. Acad. Sci. USA 75 (1978)
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RESULTS Establishment of Cell Lines. A large number of continuous cell lines were derived from the peripheral blood mononuclear cell population from patients Se and Ei with monoclonal IgM
bands in the serum. EBV-containing supernatants from cell line B95-8 were utilized in this earlier work. Cell lines were obtained from these patients at a frequency similar to normal individuals. Cell lines were also derived from the spleen mononuclear cells of patient Se in a similar manner. Two cell lines from the peripheral blood of patient Ei were derived without the addition of the EBV-containing supernatants. The frequency of derivation of these spontaneous lines was much lower than the frequency found for the EBV-infected cultures. Characterization of Anti-Idiotypic Antisera. As detailed in the Materials and Methods section, anti-idiotypic antisera were obtained against the isolated IgM band proteins of patients Se and Ei. These antisera confirmed the previous observations on patient Ei that the same idiotypic determinants were present on the band serum Ig as were present on the membrane of the respective leukemic cells and in the cytoplasm of the circulating plasma cells (10). Table 1 shows data on patient Se regarding this point. Although cell lines were derived from patient Se at a time when the WBC was 50,000/mm3, characterization of the patient's peripheral blood mononuclear cells with antiidiotypic antiserum was performed after the patient had been treated extensively and the WBC had been reduced significantly. At a time when the WBC was 7,000/mm3, surface staining of the peripheral mononuclear cells of this patient showed that 13% of these cells were stained with the anti-idiotypic reagent, while an anti-IgM reagent stained 19% of the cells and an anti-IgD reagent stained 12%. This shows that, even after extensive treatment, the majority of this patient's Igbearing lymphocytes had surface ,u, 6, and K chains present and shared the idiotypic determinants of the patient's IgM K serum band. A later study at a time when the patient's WBC had increased to 12,000/mm3 showed an increase in the idiotypebearing lymphocytes to 34%. Isolated T lymphocytes from this patient did not stain for idiotypic determinants with this reagent. Intracellular staining of these cells showed the presence of 0.2% circulating plasma cells that contained IgM with the idiotype of the serum band. Intracellular staining of the mononuclear cells of the spleen of patient Se showed 12% plasma cells that contained IgM that shared the idiotype of the serum band. The idiotypic antiserum specific for the serum band from patient Se, therefore, stained the surface of the leukemic lymphocytes and the cytoplasm of the plasma cells of this patient that are responsible for the serum Ig band and was specific for this patient's leukemic cell lineage.
Table 2. Percentage of cells in different Se and Ei cell lines showing membrane Ig and cytoplasmic Ig as detected by fluorescence with various Ig antisera* Surface Ig, % Intracellular Ig, % Line Se Idt A Ei Id IgM IgD K Se Idt Ei Idt IgG IgM IgD IgG SeD 67 0 72 37 0 67 0 24 0 29 0 0 SeAl 0 0 80 22 0 33 0 0 0 43 0 0 Se21 0 0 96 0 0 39 0 0 0 42 0 0 SeA2 0 0 0 0 0 0 0 0 0 0 0 28 Ei26 0 75 80 0 0 15 0 0 46 51 0 0 Ei39 0 4 4 0 0 2 0 0 27 22 0 0 Ei81 0 0 0 0 0 0 0 0 34 35 0 0 Ei 151 0 0 0 0 0 0 0 0 0 0 0 27 * No cell lines were stained with an anti-IgA reagent. t Antiserum specific for the idiotypic determinants of the IgM K paraprotein from patient Se. t Antiserum specific for the idiotypic determinants of the IgM K paraprotein from patient Ei.
K
X
30 40 38 20 49 26 24 0
0 0 0 0 0 0 0 25
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Proc. Natl. Acad. Sci. USA 75 (1978)
FIG. 1. Surface staining on living cells and intracellular immunofluorescent staining on a fixed preparation of cell line SeD with an anti-IgM Se idiotypic reagent. (X 1800.) (a) Phase-contrast micrograph of surface staining preparation; (b) same field under fluorescent microscopy showing four positive cells of differing intensities. (c) Phase-contrast micrograph of intracellular staining preparation; (d) same field under fluorescent microscopy, showing three positive cells.
Cell Line Igs and Their Idiotypes. Immunofluorescent staining with rhodamine-conjugated F(ab')2 fragments of the
anti-idiotypic sera characterized above was used to characterize the surface and intracellular Igs of the cell lines derived from these patients. Results for representative cell lines are shown in Table 2. Of five cell lines derived from the spleen and two lines derived from the peripheral blood of patient Se, only cell line SeD, which was derived from spleen, was found to stain with the anti-IgM Se idiotypic reagent. In surface staining, the idiotypic antiserum stained 67% of the cells of line SeD, while 24% were stained intracellularly. The intensity of both the surface and intracellular staining varied considerably from cell to cell. This variation can be noted in Fig. 1. Twelve of 17 cell lines derived from the peripheral blood of patient Ei were found to stain for intracellular IgM sharing the idiotype of the patient's band. In cell line Ei 26, 75% of the cells stained for surface Ig with the IgM Ei idiotype while only 4% did so in the case of Ei 39. None of the cell lines from patient Ei had detectable surface IgD. Some of the cell lines that were positive for production of the idiotypic determinants by intracellular staining did not have surface Ig detectable by surface staining. Ei 81 is an example of this type of cell line. Supernatant culture medium from each cell line was tested for the presence of the idiotypic determinants of each of these patient's serum bands by the hemagglutination-inhibition system. Results for various cell line supernatants are shown in Table 3 for the IgM Se idiotypic system. Only supernatants from cell line SeD and its clones (to be described later) were found to inhibit this system. In a hemagglutination-inhibition system specific for IgM, it was found that SeD, SeAl, Se 21, and Ei 26
secreted equivalent amounts of IgM into the culture medium. Thus, only cell line SeD and its clones secreted an Ig that shared the idiotypic determinants of IgM Se. The leukemic origin of cell line SeD is, therefore, demonstrated both by fluorescence and by hemagglutination inhibition. A hemagglutinationinhibition assay was also performed for the idiotypic determinants of IgM Ei In all cases, culture supernatants from cell lines positive by intracellular staining for a cytoplasmic Ig sharing the patient's idiotypic determinants were found to inhibit the hemagglutination-inhibition system specific for that patient's idiotypic determinants. All culture supernatants from cell lines negative for an idiotypic determinant by intracellular staining were negative in the corresponding hemagglutination-inhibiTable 3. Hemagglutination-inhibition assay of 3-day culture supernatants from various cell lines for the detection of the idiotypic determinants of IgM Se Dilution of cell line supernatant Cell line 0 1:2 1:4 1:8 1:16 1:32 1:64 SeD (parental) SeD.2 (clone) SeD.9 (clone) SeAl Se2l
SeA2 Ei26
+ + +
+ + +
+
+
-
-
+
+
-
-
-
-
+ + + + +
+ + + + +
+ + + + +
+ + + +
+ + + + + + +
Erythrocyte coat, IgM Se. Antiserum made against IgM Se, absorbed with pooled normal human sera. Control endpoint inhibition with IgM Se was 1 ,4g/ml. A + indicates hemagglutination.
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Hurley et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
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Table 4. Characterization of clones of cell line SeD by immunofluorescence*
Surface Ig, % Intracellular Ig, % Line Idt IgM K Idt IgD IgM IgD SeD (parental) 67 72 37 67 24 29 0 SeD.2 (clone) 75 91 26 66 41 51 0 SeD.4 (clone) 68 96 22 87 35 42 0 SeD.6 (clone) 58 79 *6 78 30 31 0 SeD.9 (clone) 65 99 32 56 38 36 0 * No cell lines were stained with anti-IgA, IgG, or X reagents. t Antiserum specific for the idiotypic determinants of the IgM K paraprotein from patient Se.
tion assay. Thus, fluorescence and hemagglutination inhibition confirm each other in the identification of cell lines of leukemic origin.
Several cell lines from patient Ei, when studied by hemagglutination inhibition and by intracellular staining within 2 months of initiation of the cell lines, were found to be mixtures of cells positive for idiotypic determinants, IgM, and IgG. Over a period of 6 months, changes in the hemagglutination-inhibition results and staining patterns were noted. In some of the cases, Ig detected in the supernatants no longer had the IgM Ei idiotypic determinants. Thus, presumably nonleukemic B cells in these lines became the dominant cells. In other cases, for example Ei 26, the leukemic cells continued to dominate in the mass culture. Leukemic Lines without Addition of EBV. The two cell lines from patient Ei derived without addition of the EBVcontaining supernatant from cell line B95-8 were both found to be of leukemic origin. Ei 39 in Table 2 is one of these lines. Studies on cell line Ei 39, however, showed that the cells were infected with EBV because the nuclei were EBNA positive. Clones of Cell Line SeD. After approximately 6 months of continuous culture, cell line SeD was cloned in soft agarose. The cloning efficiency was found to be approximately 0.1%. Seven clones of this cell line were characterized both by immunofluorescent staining and by hemagglutination inhibition. The immunofluorescent staining results for representative clones are shown in Table 4. In general, higher percentages of the cells were positive for surface IgM in the cloned lines than in the parental line. In addition, the anti-A antiserum stained more cells than the anti-idiotypic and the anti-K antisera. The latter difference might be due to the different strengths of the antisera. Supernatant culture fluids from all the clones were found to inhibit the IgM Se idiotypic hemagglutination-inhibition system (Table 3). No qualitative differences between clones or between the parental line and any of the clones were found. Attempts to clone certain idiotype-positive cell lines from patient Ei were not successful. DISCUSSION In the present study, anti-idiotypic antisera proved powerful tools for the identification of the nature of various cell lines derived from two patients with CLL. Many lines were derived that showed membrane and cytoplasmic Ig that bore no relation to the Ig of the leukemic cells. In other instances, related Ig would be detected early after initiation of the lines but would then disappear with the appearance of an unrelated Ig. However, in a number of instances (1 for Se and 12 for Ei), long-term lines were obtained that clearly made the same Ig as the leukemic cell as identified by the anti-idiotypic antisera. Subelones were obtained from cell line SeD that clearly showed similar continuous production of Ig of the same idiotype. These findings point out the need to clone cell lines to isolate the cell population of interest and to ensure its stability. The possibility of spontaneous mutation in culture exists. However, during
K
30 35 36 27 30
study of cell line SeD and its clones over a period of over 12 months, no changes in the expression of the idiotypic markers were observed. It is, therefore, unlikely that mutations were the cause of the changes seen in Ig production in some Ei cell lines. The surface staining of lines SeD and Ei 26 varied in intensity. This varied staining has also been observed in vio (10). It was documented in the peripheral blood of patient Ei that the varied staining is due to different amounts of Ig on the cell surface (10). Immunofluorescence with double fluorochromes also demonstrated that cells that stained brightly for intracellular IgM in line SeD had no detectable surface Ig (data not shown). It seems probable that the varied intensity of surface staining is related to the cell cycle in view of previous observations with other lymphoblastoid cell lines (18-20). Some of the Ei lines such as Ei 81 had no detectable surface IgM, while a certain percentage of the cells stained intracellularly for IgM Ei This differs from Ei 26 and Ei 39, which had a variable percentage of surface staining. In the previous studies (10), the majority of the Ei leukemic B lymphocytes bore surface IgD. None of the Ei lines studied in the present investigation contained cells with surface IgD. Whether these differences reflect transformation of cells at different stages of differentiation remains to be answered. All lines from patient Se were derived from cultures infected with exogenous EBV. Two cell lines from patient Ei were derived without addition of exogenous EBV. Both spontaneous lines from patient Ei were positive for the IgM Ei idiotype. Most lymphoblastoid lines derived without addition of exogenous EBV have been found to be positive for EBNA and are thought to be infected by EBV from cells carrying the EBV genome from a previous exposure to this virus (21). Evidence has been obtained that the CLL lines derived in the present study are EBNA positive. Both patients Ei and Se differed from the majority of patients with CLL in that a monoclonal IgM band was found in the serum in addition to marked lymphocytosis. Idiotypic antisera to the serum IgM band proved that the serum IgM band, the membrane IgM on the leukemic lymphocytes, and the intracellular IgM in the plasma cells were idiotypically identical. Evidence was obtained (10) that this Ig band reflected differentiation of a few of the leukemic lymphocytes to IgM-secreting plasma cells. Recently, this differentiation has been markedly accelerated in vitro with various stimulating materials (22). The question of whether this differentiation capability played a role in the successful development of long term lymphoblastoid lines remains unclear. Attempts have also been made to obtain long term lymphoblastoid lines from CLL patients without related monoclonal bands. In these cases, lines are difficult to obtain. A few lines from these patients have been derived; their relationship to the patient's leukemic cells is not yet known. Idiotypic antisera are difficult to obtain (23) for these more common types of CLL, and this hampers a definitive answer to the question of the origin of these cell lines.
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We thank Dr. B. Hampar and Dr. E. Tan for supplying the reagents used in the EBNA determinations and Dr. S. Harris for providing patient material. The excellent assistance of Ms. R. Brooks is gratefully appreciated. S.M.F. is a scholar of the Leukemia Society of America.
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Proc. Natl. Acad. Sci. -USA 75 (1978) 10. Fu, S. M., Winchester, R. J., Feizi, T., Walzer, P. D. & Kunkel, H. G. (1974) Proc. Natl. Acad. Sci. USA 71, 4487-4490. 11. Fu, S. M., Winchester, R. J. & Kunkel, H.G. (1975)J. Immunol. 114,250-252. 12. Natvig, J. B. & Kunkel, H. G. (1967) Nature (London) 215, 68-69. 13. Hoffman, T. & Kunkel, H. G. (1976) in In Vitro Methods in Cell-Mediated and Tumor Immunity, eds. Bloom, B. R. & David, J. R. (Academic, New York), pp. 71-81. 14. Kaplow, L. S. (1965) Blood 26,215-219. 15. Reedman, B. M. & Klein, G. (1973) Int. J. Cancer 11, 499520. 16. Miller, G. & Lipman, M. (1973) Proc. Natl. Acad. Sci. USA 70, 190-194. 17. Coffino, P., Baumal, R., Laskov, R. & Scharff, M. D. (1972) J.
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18. Buell, D. N. & Fahey, J. L. (1969) Science 164, 1524-1525. 19. Takahashi, M., Yagi, Y., Moore, G. E. & Pressman, D. (1969) J. Immunol. 103, 834-843. 20. Kwok, S. Y. & Litwin, S. D. (1976) Cell. Immunol. 25, 256265. 21. Nilsson, K. & Ponten, J. (1975) Int. J. Cancer 15,321-341. 22. Fu, S. M., Chiorazzi, N., Kunkel, H. G. & Halper, J. P. (1978) Clin. Res. 26, 514A. 23. Hough, D. W., Eady, R. P., Hamblin, T. J., Stevenson, F. K. & Stevenson, G. T. (1976) J. Exp. Med. 144, 960-969.
