Phenotypic, cytogenetic and molecular characterization of a new B-chronic lymphocytic leukaemia (B-CLL) cell line

LeukemiaResearchVol. 11, No. 7, pp. 579-588, 1987.

0145-2126/87$3.00 + .00 © 1987PergamonJournalsLtd.

Printed in Great Britain.

PHENOTYPIC, CYTOGENETIC AND MOLECULAR CHARACTERIZATION OF A NEW B-CHRONIC

LYMPHOCYTIC LEUKAEMIA (B-CLL) CELL LINE FEDERICO CALIGARIS-CAPPIO,* LUCIANA BERGUI,* GIOVANNA REGE-CAMBRIN,* LUISA TESIO,* NICOLA MIGONEt a n d FABIO MALAVASIt *Dipartimento di Scienze Biomediche e Oncologia Umana, Cattedra di Clinica Medica A and tIstituto di Genetica Medica e Centro CNR, Immunogenetica ed Istocompatibilith, University of Turin, Italy

(Received 29 October 1986. Accepted 14 January 1987) Abstract--A lymphoid cell line was established from a patient with B-cell chronic lymphocytic leukaemia (B-CLL) by infecting blood lymphocytes with Epstein-Barr virus (EBV). Immunoglobulin gene rearrangement studies and the presence of a chromosome marker (isochromosome 17q) provided the formal proof that the line has originated from the neoplastic B cells. The morphology and phenotype indicate that the EBV-induced cell line has reached a plasma cell-like stage of differentiation.

Key words: B-chronic lymphocytic leukaemia (B-CLL), EBV.

INTRODUCTION B LYMPHOCYTES are the only cells permissive for Epstein-Barr virus (EBV) infection through a surface receptor which has been characterized as the receptor for the C3d fraction of complement [7, 21, 22]. The ability to be infected by EBV can be traced back to the very early elements of the B-cell lineage [15]. This target cell tropism has been utilized to obtain permanent cell lines from both normal and malignant (Burkitt lymphoma) B cells which have shed considerable light onto the processes of B-cell activation and transformation [30]. However, not all malignant B cells are equally susceptible to EBV transformation. It is generally agreed that the B cells of B-chronic lymphocytic leukaemia (B-CLL) are refractory to EBV-induced in vitro transformation [6, 39, 33, 23] irrespective of the fact that they do express the C3d receptor [34]. Only a few cell lines have been described which fulfill the stringent criteria needed to demonstrate their origin from the neoplastic B cells and not from the nonneoplastic residual B lymphocytes [18, 20, 29]. The purpose of this paper is to present the characterization of a new B-CLL cell line EBV-induced. Abbreviations: EBV, Epstein-Barr virus; B-CLL, B-chronic lymphocytic leukaemia; Ig, immunoglobulin; MoAb, monoclonal antibody; Mrbc, mouse red blood cells; FH, FicollHypaque; EBNA, Epstein-Barr nuclear antigen, PHA, phytohemoagglutinin; PWM, pokeweed mitogen; CD, cluster differentiation; FITC, fluorescein isothiocyanate; TR1TC, tetraethylrhodamine isothiocyanate; IF, immunofluorescence; APAAP, alkaline phosphatase anti alkaline phosphatase. Correspondence to: Dr F. Caligaris-Cappio, Clinica Medica A, Via Genova 3, 10126 Torino, Italy.

Immunoglobulin (Ig) gene rearrangement studies and the detection of a chromosome marker provided the formal proof that the cell line has indeed originated from the transformation of malignant B-CLL cells. PATIENT The patient was a 64-yr old male with a well-documented diagnosis of B-CLL stage II according to Rai's [31] of three-year duration. Few cervical nodes could be appreciated. The spleen was not palpable. Cell morphology revealed small, mature-looking lymphocytes without nucleoli. The phenotypic features were investigated evaluating the expression of surface Ig, the reactivity with a whole range of B cell- and plasma cell-associated monoclonal antibodies (MoAbs) and the presence of receptors for mouse red blood cells (Mrbc; see below). At the time of cell line establishment the patient was untreated and the white cell count was 6 × 104/~tm 3. MATERIALS

AND METHODS

Cell cultures Mononuclear cells obtained from peripheral blood by FicollHypaque (FH) centrifugation were washed twice in RPMI1640 medium supplement with 20% foetal calf serum (FCS) and incubated with the supernatant from the EBV-infected marmoset cell line EB95-8 [27]. Half millilitre of the supernatant was added to 5 x 106 cells resuspended in 2 ml of medium and the cells were seeded in 24-well culture plates (Costar) at 37°C in a humidified atmosphere containing 5% CO2. Cultures were run in Iscove's Modified Dulbecco medium (IMDM, Gibco) supplemented with 20% heat-inactivated FCS and conditioned with 5% supernatant of a vigorously growing 579

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EBV cell line. Either normal B-lymphoblastoid cell lines or the Burkitt lymphoma cell lines Daudi and Raji were used. Clusters of cells became apparent one week after the stimulation. The cultures were maintained in conditions of overcrowding and the growing cells split only when large clumps were clearly visible in the wells. The cells were then carefully divided and expanded in 24-well culture plates (Costar). In order to improve the growth efficiency irradiated murine macrophages obtained from peritoneal washings of female Balb/C mice were sometimes used as supporting feeder. Finally, the growing cells were transferred in 25-cm 2 culture flasks (Costar) and cultured in IMDM medium supplemented with 20% FCS.

Cell line characterization (a) Morphology. The morphology of growing cells was defined on cytocentrifuge smears stained with May-GriinwaldGiemsa (MGG). (b) Assay for Epstein-Barr nuclear antigen (EBNA). An indirect immunofluorescence (IF) assay was performed to determine the presence of EBNA utilizing essentially the method of Reedman and Klein [32]. (c) Tumour origin. Two methodologies were utilized to demonstrate that the growing cells were indeed derived from neoplastic B lymphocytes: Ig gene rearrangement studies and cytogenetic techniques. lg gene rearrangement studies. Ig gene rearrangement was comparatively evaluated on cell line elements and the patient peripheral blood mononuclear cells. High molecular weight DNA extracted from 20 x 10 6 cells, was digested with three restriction endonucleases (SacI, EcoRI and BamHI), size fractionated through 0.65--0.8% agarose gel electrophoresis and hybridized, following Southern blot, to nick-translated (32p) DNA probes, as previously described [10]. DNA configuration at the Ig heavy chain (IgH) locus was analysed using as probe a 3.3-kilobase (1 kb = 1000 base pairs) fragment containing a major portion of the heavy chain "joining" (JH) segments [10]. The k light chain (Igk) locus was investigated with a probe represented by a 2.7-kb EcoRI-segment, carrying the constant portion of the k gene (Ck). The ~. light chain (Ig~) locus was investigated by means of a 1.2-kb BamHI-fragment carrying the Kern-, O~-C ). isotype. The k and ). probes were isolated from the recombinant phages Huk and Hu 5, respectively [17, 18]. A switch probe (Sw), represented by a 2.2-kb SacIfragment, containing most of the u-switch region, was also used in order to obtain a more general characterization of the polymorphisms mapped within the ~t, trl and tr2 switch regions [25]. The C7 loci were analysed by means of an 8.0-kb HindlII segment containing the C71 gene (this probe hybridizes with all five C genes, i.e. the four activity ones and a pseudogene [40, 9, 26]. The heavy chain ~t gene locus was investigated by means of a 1.2-kb EcoRI-fragment carrying almost the entire constant portion of the ~t gene (C~t). Chromosome analysis. Cytogenetic analysis was comparatively performed on cell line growing elements and on the patient's peripheral blood mononuclear cells. Unstimulated cell line elements were investigated following standard procedures. The patient's peripheral blood mononuclear cells were separated on Ficoll-Hypaque (FH) gradient. 50 x 106 ceils were further purified by spontaneous rosette formation with sheep erythrocytes (E-rosette) in order to perform selective cytogenetic analysis on T- and B-cell populations. The cells were pretreated

with neuraminidase (Sigma) and galactose-oxidase (Sigma) to increase the response to mitogens [43]. Three parallel cultures were set up in RPMI 1640 supplemented with 20% FCS: unseparated mononuclear cells, E-rosette forming cells (T lymphocytes) and non-E-rosette forming cells (99% monoclonal B lymphocytes). Unseparated mononuclear cells and E-rosette forming-cell were stimulated with phytohemoagglutinin (PHA, Wellcome) 10 ~tg/ml. Non-E-rosette forming cells were stimulated with pokeweed mitogen (PWM, Gibco) at a dilution 1:100 ml. The cells were harvested after 72-hr culture: colcemid (Gibco) was added 20 min before harvesting. Chromosomes were G-banded using a modification of Seabright's technique [36]. (d) Phenotype. The phenotype of patient's and growing cell line lymphoid cells was investigated on cell suspensions resuspended in phosphate buffered saline (PBS) and cytocentrifuge smears (2.5 × 105cells/slide) air-dried and fixed with cold acetone. The relevant Abs included reagents recognizing the different anti-B cluster differentiation (CD) antigens CD9 (BA2, Hybritech cat. No. KO462), CD10 (J5, Coulter cat. No. 6602143 and RFAL1, Ref. [4]), CD19 (B4, Coulter cat. No. 6602683), CD20 (B1, Coulter cat. No. 66602140), CD21 (RFB6, ref. 4), CD22 (RFB4, ref. 4), CD24 (BA1, Hybritech cat. No. KO452) and the plasma cell related antigen PCA-1 (Coulter, cat. No. CU6602714). The MoAb panel was completed with the following reagents: RFT1 (2) and Leul (Becton-Dickinson, cat. No. 6300) both detecting the CD5 antigen. Anti-HLA-DR monomorphic antigen (Becton-Dickinson, cat. No. 7450). FMC7 (Sera-Lab), which recognizes a subset of peripheral blood B- cells and strongly reacts with the vast majority of cells in B-prolymphocytic leukaemia and hairy cell leukaemia [5]. A10 detects the T10 antigen expressed by normal and neoplastic plasmacells, some lymphohaemopoietic cells and large granular lymphocytes [24]. Leu4 (Becton-Dickinson, cat. No. 7340) which identifies T cells carrying CD3 T-cell specific antigen. MoAbs were either used as culture supernatants in 1:5 final dilution or as purified Ig (2.5-10 Ixg/ ml final dilution). MoAb reactivity was defined in IF experiments and revealed by Goat (G)-anti-Mouse (M)-IgG-fluorescein isothiocyanate (FITC) labelled (Becton-Dickinson, cat. No. 9031) or rat-anti-M-IgM-FITC (Becton-Dickinson, cat. No. 1143). In selected experiments, MoAb reactivity was evaluated with the more sensitive immunohistochemical APAAP technique [28]. The expression of Ig was investigated with polyclonal rabbit (R)-anti human-Ig isotypes M,D,G,A,K, directly conjugated with tetraethylrhodamineisothiocyanate (TRITC; Dakopatts, cat. Nos. R-152, R-148, R-151, R-153, T-154, R-155) used at 1:50 final dilution. All direct and indirect IF staining procedures were performed as detailed by Janossy et al. [19]. The ability to form Mrbc-rosettes was evaluated according to Forbes and Zalewsky [11].

RESULTS The m o r p h o l o g y and clinical course of the patient were typical of B-CLL. As for the p h e n o t y p e (see Table 1), the cells reacted with CD19, 20, 21, 22, 24 and were unreactive with CD9, C D 1 0 and F M C 7 M o A b s . They were also H L A D R + and formed rosettes with Mrbc in an appreciable percentage. Nevertheless, the "classical"

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FIG. 2. Southern blotting hybridization of the JH switch (Sw) region and C;~-containing probes to SacI and EcoRI digested DNAs of patient's fresh neoplastic cells (lane 1), EBVtransformed B-cells from the same patient (lane 2) and of normal, unrearranged DNA, as control (lane C). The length of kb of the hybridizing fragments are reported. The rearranged segments are marked with asterisks; the arrows point to the position where the germ line JH-containing segments should have migrated. The Ig Ix-, o:1- and o:2-switch (s) regions where the polymorphic switch-containing segments map are indicated.

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FIG. 3. Both the patient's monoclonal B cells (A) and the EBV-derived cell line (B) show the same chromosome marker: isochromosome 17. Arrows point to the two homologous chromosomes 17: the normal 17 is on the left, while the iso (17q) is on the right.

21

22

A new B-CLL cell line TABLE 1. PHENOTYPIC ANALYSIS OF EBV-INDUCED CELL LINE AS COMPARED TO THE PATIENT'S

(Pt)

FRESH PERIPHERAL BLOOD B

LYMPHOCYTES

Cell line %

Pt %

<1 >90? <1 80 <1 90 80

>90* 10 48 80 <1 <1 <1 >90 >90 80 80 > 90 <5 <1

Sig cylg Mrbc:~ HLA-DR CD5 CD9 CD10 CD19 CD20 CD21 CD22 CD24 T10 PCA1

* ~t÷, 6+, ~.~ ? ~t+, )t+ (see Fig. IB). $ Mrbc = rosettes with mouse red blood cells.

B-CLL phenotype [3] was not fulfilled, since the slg expression was stronger than expected and, above all, CD5 was never detected even with the sensitive A P A A P technique.

Cell line characterization The cells, cultured as above described, have been kept continuously growing for more than 9 months. The characterization was begun after 6 months of continuous growth in culture. (a) General features. The cells grew in clusters or clumps and were not adherent to the vessel wall. When cytospins were made and stained with MGG, the morphology (Fig. 1A) was typical of large, plasmacytoid elements with a basophilic cytoplasm and one or two nucleoli. The growing cells had a mean doubling time of 4-5 days. The E B N A IF staining was very bright in more than 90% of the cells.

585

which map at known sites within the IgH cluster [25], and a few of them are polymorphic. Thus, by means of this probe, it appears that both o{1- and tr2-switch regions are in the germ-line configuration on both chromosomes, since two relatively common allelic fragments (7.4 and 6.9 kb "alleles" at the Sol1 region and 4.9 and 4.8 kb fragments at the Str2 region, see Fig. 2) are detectable. The presence of only one band (2.7 kb) at the ~t-switch locus, is consistent with an homozygous genotype, although hemizygosity cannot be excluded. The same Sw probe can detect the JH-containing Sad fragments since the 3' ends of these segments overlap with the ~t-switch region (see Fig. 1: Ref. [25]. The appearance of only one arranged JH band (4.4Kb) following hybridization with the Sw probe might suggest that a deletion of a relevant portion of the ~t-switch region had occurred in one chromosome 14, before EBV transformation. The hypothesis that this phenomenon could have been derived from an "aberrant" switch, e.g. to Y3 or Yl, occurring in the non-productive chromosome, cannot be excluded although no rearrangement was observed either probing with a yprobe or with a ~t-probe, following BamHI and SacI respective digestion, (data not shown). The Igk locus was then tested with the CK probe following BamH! digestion (data not shown). The Igk locus was then tested with the Ck probe following BamHI digestion (data not shown). The absence of any detectable fragment both before and after EBV transformation suggests that deletions at both Ck allelic regions have occurred as it has been generally observed in ;~ producing B-cell lines [37]. The Ig;t region was analysed with C~.2 probe after EcoRI digestion (Fig. 2). Two novel fragments which do not correspond to any known polymorphic "allele" yet described (Ref. [41] and personal observations) appear, confirming a rearrangement in at least one IgA carrying chromosome. Chromosome studies (Fig. 3 and Table 2). The karyotype was 46, xy, i(17q) on 100% of cell line metaphases. The cytogenetic investigations of the patient's mononuclear cells demonstrated that in the culture of lymphocytes depleted of E-rosette-forming cells (99% monoclonal B cells) all the metaphases had the same 46, xy,i(17q) karotype. In the unfractionated mononuclear cell culture 48% of the metaphases had a normal karyotype while 52% carried the isochromosome 17q. All the

(b) Demonstration of tumour origin Ig gene rearrangement studies (Fig. 2). The analysis of the Ig-gene configuration confirmed that both the patient's malignant cells and the corresponding EBVtransformed cell line shared identical patterns of DNA rearrangement at the IgH and L loci. The evidence of two novel JH containing fragments and no germ-line band (Fig. 2) suggests that both chromosomes have been involved in V-D-JH rearrangement. To assess in more detail the extent of similarities of the configuration at the IgH locus between the two D N A samples (before and after EBV transformation) the pattern of hybridisation with a ~tSw probe have been compared following SacI digestion (Fig. 2). It has been shown that this probe cross-hybridizes with several germ-line fragments containing ~t-switch homologous sequences most of

TABLE2. KARYOTYPE ANALYSIS OF EBV-INDUCED CELL LINE IN COMPARISON WITH PATIENT (Pt) FRESH UNSEPARATED PERIPHERAL BLOOD LYMPHOCYTES ( P B L ) , SEPARATED B AND T CELLS

Cell line PtPBL + PHA-t Pt B + PWM:~ Pt T + PHA

46, xy

46, xy, i(17q)

0/15" 24/50 0/5 10/10

15/15 26/50 5/5 0/10

* Number of positive mitosis/number of mitosis examined. t PHA = phytohemoagglutinin. $ PWM = pokeweed mitogen.

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F. CALIGARIS.CAPPIOet al.

metaphases were normal in the E-rosette forming cell cutlure. The conclusion is that the patients leukemic cells have the same chromosomal abnormality observed in the cell line obtained with EBV induction. (c) Phenotype. The detailed phenotype characterization of the cell line in comparison with the patient fresh cells is reported in Table 1. The first observation is that in the cell line slg were no longer detectable, while in contrast cy Ig of the ~t/). type were detectable (Fig. 1B). Delta heavy chains could not be observed on the surface neither in the cytoplasm. Further, all the Bcell specific CD antigens were no longer expressed on the surface (neither in the cytoplasm). The T1 antigen (CD5) was not displayed by the transformed cells which had also lost the capacity to form rosettes with Mrbc. H L A - D R in contrast was maintained by the transformed cells which also acquired the T10 antigen. The plasma cell specific antigen PCA1 was expressed very weakly and its reactivity could be well appreciated only with the sensitive A P A A P technique. DISCUSSION The exceeding difficulty of obtaining permanent cell lines EBV-induced from monoclonal B-CLL lymphocytes is well recognized [6, 39, 20, 23, 33]. Frequently, the growing cells derive from the transformation of normal residual B cells. Therefore, we aimed at demonstrating that our EBV-induced cell line was indeed originating from the transformation of the malignant clone. The evidence achieved is two-fold. First, the Ig gene rearrangement which gives the molecular fingerprint of Ig genes, provides clear evidence (Fig. 2) that both the patient's malignant cells and the corresponding EBV-transformed cell line share identical patterns of D N A rearrangement at the JH and C IgH and L loci. Second, both cell populations share the same chromosome marker, the isochromosome 17q (Fig. 3). This cytogenetic abnormality is very unusual in CLL [13] but was described in two other EBV-induced CLL cell lines [42]. Abnormalities of chromosome 17 were also observed in other EBV-induced B-CLL cell lines [29] suggesting the possible interest of exploring the relationship of chromosome 17 and EBV transformability in BCLL cells. The EBV-induced B-CLL cell line obtained has more differentiated characteristics than the patient's cells from which it was established. Both the morphology and the phenotype clearly indicate the features of a plasma cell-like stage of differentiation. The transformed cells have lost slg and strongly express cylg of the p heavy chain type only. Further, they do not express the B-cell associated antigens CD19, 20, 21, 22, 24 as normal B cells do when differentiate to plasma cells [1]. The strongly cyu+, B-cell antigen negative cells are also T10+ and react, albeit weakly, with the MoAb PCA1. Both antigens are positive on normal as well as malignant plasma cells, while not expressed by mature B lymphocytes [1]. The EBV-induced cell line elements differ from normal plasma cells since they retain the Ia positivity [14]. However, this may not be surprising since human myeloma cell lines are very often Ia+ [30].

A point which is worth discussing is the phenotype of the B-CLL cells from which the line is originated. As shown in Table 1, the leukaemic B lymphocytes strongly express monoclonal slg and do not react with CD5 MoAb, thus differing from the "conventional" B-CLL which are almost invariably weakly slg+ and CD5+ [3]. Interestingly, also the EBV-induced CLL cell lines insofar described were derived from "non conventional" CLL cases. The patient's cells were strongly slg positive [42] and a monoclonal serum peak could also be observed [18]. These features are unusual in "conventional" B-CLL [3], and are consistent with the presentation of the rare CD5-cases [35]. Further, we have been unable to obtain EBV-induced lines from 9 patients with "conventional" weakly slg+ and CD5+ B-CLL, but two other strongly slg+, CD5-CLL cases yielded E B N A + cell cultures growing for several weeks and expressing in the cytoplasm the same H and L chain of the leukaemic ceils (manuscript in preparation). These observations point out to the possibility that EBV-transformability is related to the stage of differentiation of malignant B cells. The intensity of slg expression or the presence of an Ig serum peak and the absence of CD5 might characterize a slightly more differentiated malignant B lymphocyte which gives the morphological and clinical picture of CLL and can also be transformed by EBV. This hypothesis is supported by the finding [8] that EBV-induced cell lines can be easily obtained from B-prolymphocytic leukaemia, a strongly slg+, usually [12, 38] CD5-, B-CLL variant. In conclusion, the EBV-induced B-CLL cell line here presented may prove to be a valuable model for understanding the mechanisms which hinder the differentiation of B-CLL cells. Acknowledgements--This work was supported by Progetto Finalizzato Oncologia, C.N.R., Roma, grants No. 84.00482.44 and 84.00581.44 and partly by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.). L.B. and G.R.C. are recipient of a scholarship from the Comitato G. Ghirotti, Torino. The authors are greatly indebted to Prof. M. Siniscalco, New York, U.S.A., for providing the EB95-8 marmoset cell line, to Prof. G. Janossy, London, U.K., for providing the MoAbs of the RF series used in the present study and to Prof. H. Van Den Berghe, Leuven, Belgium, for helpful advice and criticism in analysing the karyotypes.

REFERENCES 1. Anderson K. C., Bates M. P., Slaughenhoupt B. L., Pinkus G. S., Schlossman S. F. & Nadler L. M. (1984) Expression of human B cell-associated antigens on leukemias and lymphomas: a model of B-cell differentiation. Blood 63, 1424. 2. Caligaris-Cappio F., Gobbi M., Bofill M. & Janossy G. (1982) Infrequent normal B lymphocytes express features of B-chronic lymphocytic leukemia. J. exp. Med. 155,623. 3. Caligaris-Cappio F. & Janossy G. (1985) Surface markers in chronic lymphoid leukemias of B-cell type. Sere. Hemat 1, 1.

A new B-CLL cell line 4. Campana D., Janossy G., Bofill M., Trejdosiewicz L., Ma D., Hoffbrand A. V., Mason D. Y., Lebacq A. M. & Forster H. K. (1985) Human B-cell development--I. Phenotypic differences of B lymphocytes in the bone marrow and peripheral lymphoid tissue. J. Immun. 134, 1524. 5. Catovsky D., Cherchi M., Brooks D., Bradley, J. & Zola H. (1981) Heterogeneity of B-cell leukemias demonstrated by the monoclonal antibody FMC7. Blood 58, 406. 6. Chang R. S., Fillingame R. A., Paglieroni T. & Glassy F. J. (1976) A procedure for quantifying susceptibility of human lymphocytes to transformation by Epstein-Barr viruses (39508). Proc. Soc. exp. Biol. Med. 153, 193. 7. Fearon D. T. (1984) Cellular receptors for fragments of the third component of complement. Immun. Today 5, 105. 8. Finerty S., Rickinson A. B., Epstein M. A. & Platt-Mills T. A. E. (1982) Interaction of Epstein-Barr virus with leukaemic B cells in vitro---II. Cell line establishment from prolymphocytic leukaemia and Waidenstrom's macroglobulinaemia. Int. J. Cancer 30, 1. 9. Flanagan S. G. & Rabbits T. H. (1982) Arrangements of human immunoglobulin heavy chain constant region genes implies evolutionary duplication of a segment containing and genes. Nature, Lond. 300, 709. 10. Foa R., Migone N., Saitta M., Fierro M. T., Giubellino M. C., Lusso P., Cordero di Montezemolo L., Miniero R. & Lauria F. (1984) Different stages of B-cell differentiation in non-T acute lymphoblastic leukemia. J. clin. Invest. 74, 1756. 11. Forbes I. J. & Zalewsky P. U. (1976) A subpopulation of human B lymphocytes that rosette with mouse erythrocytes. Clin. exp. Immun. 26, 99 12. Gobbi M., Caligaris-Cappio F. & Janossy G. (1983) Normal equivalent cells of B-cell malignancies: analysis with monoclonal antibodies. Br. J. Haemat. 54, 393. 13. Goh K. O. (1987) Chromosomal abnormalities in chronic lymphocytic leukemia. Canc. Genet. Cytogenet. 16, 103. 14. Halper J., Fu S. M., Wang C. Y., Winchester R. & Kunkel H. G. (1978) Patterns of expression of human "Ia-like" antigens during the terminal stages of B-cell development. J. Immun. 120, 1480. 15. Hansson M., Falk K. & Ernberg I. (1983) Epstein-Barr virus transformation of human pre-B cells. J. exp. Med. 158, 616. 16. Hieter P. A., Maizel J. V. & Leder P. (1982) Evolution of human immunoglobulin KJ region genes. J. biochem. Chem. 257, 1516. 17. Hieter P. A., Korsmeyer S. J., Waldmann T. & Leder P. (1981) Human immunoglobulin K light-chain genes are deleted or rearranged in k.-producing B cells. Nature, Lond. 290, 368. 18. Hurley J. N., Fu S. M., Kunkel H. G., McKenna G. & Scharff M. D. (1978) Lymphoblastoid cell lines from patients with chronic lymphocytic leukemia: identification of tumor origin by idiotypic analysis. Proc. natn. Acad. Sci. U.S.A. 75, 5706. 19. Janossy G., Bollum F. J., Bradstock K. F. & Ashley J. (1980) Cellular phenotypes of normal and leukemic hemopoietic cells determined by analysis with selected antibody combinations. Blood 56, 430. 20. Karande A., Fialkow P. J., Nilsson K., Povey S., Klein G., Najfeld V. & Penfold G. (1980) Establishment of

587

a lymphoid cell line from leukemia cells of a patient with chronic lymphocytic leukemia. Int. J. Cancer 26, 551. 21. Klein G., Yefenof E., Falk K. & Westman A. (1978) Relationship between Epstein-Barr (EBV)-production and the loss of the EBV receptor/complement complex in a series of sublines derived from the same original Burkitt's lymphoma. Int. J. Cancer 21,552. 22. Yefenof E., Klein G., Jondal M. & Oldstone M. B. A. (1976) Surface markers on human B- and T-lymphocytes-IX. Two-color immunofluorescence studies on the association between EBV receptors and complement receptors on the surface of lymphoid cell lines. Int. J. Cancer 17, 693. 23. Levitt M. L., Barry W. E., Helfrich M. K., Kaiser-McCaw Hecht B. & Henderson E. E. (1983) Characterization of Epstein-Barr virus-carrying cell lines established from chronic lymphocytic leukemia. Cancer Res. 43, 1195. 24. Malavasi F., Caligaris-Cappio F., Milanese C., Dellabona P., Richiardi P. & Carbonara A. O. (1984) Characterization of a murine monoclonal antibody specific for human early lymphohemopoietic cells. Hum. Irnmun. 9, 9. 25. Migone N., Feder J., Cann H., vanWest B., Hwang J., Takahashi N., Hanjo T., Piazza A. & Cavalli-Sforza L. L. (1983) Multiple DNA fragment polymorphisms associated with immunoglobulin ~t chain switch-like regions in man. Proc. natn. Acad. Sci. U.S.A. 80, 467. 26. Migone N., Olivero S., deLange G., Delacroix D. L., Boschis D., Altruda F., Silengo L., DeMarchi M. & Carbonara, A. O. (1984) Multiple gene deletions within the human immunoglobulin heavy-chain cluster. Proc. natn. Acad. Sci. U.S.A. 81, 5811. 27. Miller G. & Lipman M. (1973) Release of infectious Epstein-Barr virus by transformed marmoset leukocytes. Proc. natn. Acad. Sci. U.S.A. 70, 190. 28. Moir D. G., Ghosh A. K., Abdulaziz Z., Knight P. M. & Mason D. Y. (1983). Immunoenzymatic staining of haematological samples with monoclonal antibodies. Br. J. Haemat. 55, 395. 29. Najfeld V., Fialkow P. J., Karande A., Nilsson K., Klein G. & Penfold G. (1980) Chromosome analysis of lymphoid cell lines derived from patients with chronic lymphocytic leukemia. Int. J. Cancer 26, 543. 30. Nilsson K. & Klein G. 1982 Phenotypic and cytogenetic characteristics of human B-lymphoid cell lines and their relevance for the etiology of Burkitt's lymphoma. Adv. Cancer Res. 37, 319. 31. Rai K. R., Sawitsky A., Cronkite E. P., Chanana A., Levy R. N. & Pasternak B. S. (1975) Clinical staging of chronic lymphocytic leukemia. Blood 46, 219. 32. Reedman B. M. & Klein G. (1973) Cellular localization of an Epstein-Barr virus (EBV)-associated complementfixing antigen in producer and non-producer lymphoblastoid cell lines. Int. J. Cancer 11, 499. 33. Rickinson A. B., Finerty S. & Epstein M. A. (1982) Interaction of Epstein-Barr virus with leukaemic B cells in vitro-I. Abortive infection and rare cell line establishment from chronic lymphocytic leukaemic cells. Clin. exp. Immun. 50, 347. 34. Ross G. D. (1979) Identification of human lymphocyte subpopulations by surface marker analysis. Blood 53, 799.

588

F. CALIGARIS-CAPPIOet al.

35. Royston I., Majda J., Baird S., Meserve B. & Griffiths J. (1980) Human T cell antigens defined by monoclonal antibodies: the 65,000 dalton antigen of T cells (T65) is also found on chronic lymphocytic leukaemic cells bearing surface immunoglobulins. J. Immun. 1125, 725. 36. Seabright M. (1971) A rapid banding technique for human chromosomes. Lancet ii, 971. 37. Siminovitch K. A., Bakhshi A., Goldman P. & Korsmeyer S. J. (1985) A uniform deleting element mediates the loss of k genes in human B cells. Nature, Lond. 316, 260. 38. Stein H., Lennert K., Feller A. C. & Mason D. Y. (1984) Immunohistological analysis of human lymphoma: correlation of histological and immunological categories. Adv. Cancer Res. 42, 67. 39. Takada K., Yamamoto K. & Osato T. (1980) Analysis of the transformation of human lymphocytes by Epstein-

40.

41.

42.

43.

Barr virus---II. Abortive response of leukemic cells to the transforming virus, lntervirology 13, 223. Takahashi N., Ueda S., Obata M., Nikaido T., Sumiko N. & Honjo T. (1982) Structure of human immunoglobulin gamma genes: implication for evolution of a gene family. Cell 29, 671. Taub R. A., Hollis G. F., Hieter P. A., Korsmeyer S. J., Waldmann T. A. & Leder P. (1983) Variable amplification of immunoglobulin ;t light-chain genes in human populations. Nature, Lond. 304, 172. Vahdati M., Graafiand H. & Emberger J. M. (1983) Isochromosome 17q in cell lines of two cases of B-cell chronic lymphocytic leukemia. Cancer Genet. Cytogenet. 6, 227. Zafar H. N., Pittman S., Cherchi M. & Catovsky D. (1981) Stimulation of chronic lymphatic leukemia cells by pokeweed mitogen after treatment with neuraminidasegalactose oxidase. Clin. exp. Immun. 44, 124.