Clin. exp. Immunol. (1976) 25, 367-376.

Surface receptors on human haematopoietic cell lines CH. HUBER,* C. SUNDSTROM, K. NILSSON & H. WIGZELLDepartmentofImmunologyandDepartment ofPathology and Wallenberg Laboratory, University of Uppsala, Uppsala, Sweden (Received 15 March 1976)

SUMMARY

The expression of complement receptors, of Fc receptors, of SRBC receptors and of S-Ig was investigated on human haematopoietic cell lines of proved malignant derivation. According to their origin and to a panel of phenotypic markers these lines have been classified into lymphoma lines, myeloma lines and leukaemia lines. Results were compared with those obtained on non-malignant EBV carrying lymphoblastoid cell lines (LCL). Among the lymphoid cell lines the LCL showed a common pattern of B-lymphocyte surface markers, i.e. surface immunoglobulins, C3 receptors but low density of Fc receptors. The nonBurkitt lymphoma lines bore in varying degree these B-lymphocyte markers. The lines U-698 M and DG-75 were exceptional in having only surface immunoglobulin. The Burkitt lymphoma lines had all B-lymphocyte markers. The myeloma lines differed from the lymphoid lines in lacking C3 and Fc receptors and showed only trace amounts of surface immunoglobulins. In contrast to lymphoid and myeloma lines, the leukaemia lines were completely lacking surface immunoglobulins, but showed C3 and Fc receptors in variable densities. One line, the ALL derived line MOLT-3 showed the capacity to spontaneous rosette formation with SRBC. The findings that LCL presented a homogeneous pattern of B-lymphocyte surface markers may be of value in order to discriminate between these lines and lines derived from haematopoietic malignancies other than Burkitt lymphomas. INTRODUCTION Several experimental approaches have been used to characterize normal and neoplastic human haematopoietic cell lines, e.g. morphology (Moore, Kitamura & Toshima, 1968; Nilsson & Ponten, 1975b), immunoglobulin production (Tanigaki et al., 1966; Finegold, Fahey & Granger, 1967; Nilsson, 197 la), agglutination with Concanavalin A (Glimelius, Nilsson & Ponten, 1975), presence of Epstein-Barr virus (EBV) related antigens (Nilsson et al., 1971; Klein et al., 1974; Klein, 1973), fl2-microglobulin production (Nilsson, Evrin & Welsh, 1974a), chromosome analyses (Jarvis et al., 1974; Zech et al., 1976) and stainability with fluorescent anti-actin antibodies (Fagraeus et al., 1975). The recent development of immunological techniques to distinguish between T- and B-lymphocyte surface markers (Jondal, Wigzell & Aiuti, 1973b) offers a new possibility for comparative studies of normal and malignant haematopoietic cell populations. Shevach et al. (1972, 1973), Jondal et al. (1973b) and Theofilopoulos, Dixon & Bokisch (1974) studied a group of established lymphoid cell lines for the presence of surface located immunoglobulin (S-Ig), for C3 and Fc receptors and for spontaneous rosette formation with SRBC. Evidence was presented that the majority of these lines were derived from Blymphocyte precursors, while a small minority was suggested to be of T-lymphocyte origin. Till now no attempts were made to correlate the expression of surface markers to the normal or neoplastic state of these cell lines. Present address: Medizinische Universitatsklinik, Innsbruck, Austria. Correspondence: Dr C. Sundstrum, Wallenberg Laboratory, P.O. Box 562, S-751 22 Uppsala, Sweden. *

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Ch. Huber et al.

In the present study we investigated S-Ig, C3, Fc and SRBC receptors on a battery of haematopoietic cell lines. These lines were classified according to their origin, karyotype, morphology, association with EBV, presence of contractile proteins and production of immunoglobulin and f32-microglobulin as lymphoblastoid cell lines (LCL) of possible non-neoplastic origin and lymphoma, leukaemia and myeloma lines of malignant derivation (Nilsson et al., 1975b). The aim of the study was to answer the following questions. (1) Do normal and neoplastic cells differ in their surface receptor pattern? (2) Can quantitative characterization by lymphocyte surface markers help in the classification of haematopoietic cell lines?

MATERIALS AND METHODS Cell line. Ten lymphoma lines, five LCL, two myeloma lines and three leukaemia lines were studied. Cell lines with the prefix U- in Table 1 were established in this laboratory with the Spongostan grid culture technique as described (Nilsson, 1971a). The origin of the other lines employed as well as selected references are found in Table 1. Cell lines were maintained in 50 ml Erlenmeyer flasks containing approximately 20 ml of Ham's F-10 tissue culture medium (Gibco, New York, USA) supplemented with 10% postnatal calf serum and antibiotics (100 i.u./ml penicillin; 50,pg/ml streptomycin; 1P25 pg/ml amphotericin B). The medium was changed twice a week. For surface-receptor studies cells were harvested from logarithmically growing stock cultures and washed three times in PBS or VBSS. Cell concentration was adjusted to approx. 4 x 106 cells/ml. Preparation of indicator particles (EAC, EA7S, ZyC, EN) for rosette assays. C3 receptors were demonstrated using EACand C-coated zymosan beads (Sigma Chem., St Louis, Missouri). Both methods have been previously described (Huber et al., 1971; Huber & Wigzell, 1975). Briefly, to prepare EAC a 5% suspension of SRBC in VBSS or PBS was sensitized with an equal volume of 19S rabbit anti-Forsmann antibody (Haemoderivate, Wien, Austria; 10,000 H.U./ml) in a final dilution of 1:700 in VBSS for 20 min at 0°C (EA,9s). After three washes 1 ml 5% EA19s in VBSS was incubated with 0 1 ml fresh CBA mouse serum for 30 min at 37°C (EAC). After three washes EAC were resuspended to a final concentration of 0 5% in VBSS and were used within 4 days. Zymosan (Zy) beads activated by boiling (Huber & Wigzell, 1975) were suspended in VBSS to a concentration of 1 mg/ml. 1 ml Zy suspension was incubated with 0 05 ml CBA-serum or 0 5 ml fresh human serum for 30 min at 37°C (ZyMC and ZyHC). After three washes the beads were resuspended in 1 ml VBSS and used within 4 days. For preparation of EA7s 5% SRBC in PBS were sensitized with a 7S rabbit anti-Forssman serum in a final dilution of 1:600 in PBS for 30 min at 0°C. After three washes EA7S were resuspended to a 0.5% suspension in PBS and used within 4 days. EN were prepared by incubating 5% SRBC suspension in PBS with 50 U neuraminidase (Behringwerke, Marburg, Germany) for 30 min at 37°C. EN were washed three times and resuspended to a 0 5% suspension which was used within 4 days. Rosette assays for detection ofC3 and Fc receptors. Twenty-five microlitres cell suspension and 25,u1 indicator particles were mixed in round-bottomed micro titre plates (system Cook, Greiner, Nurtingen, Germany) and were centrifuged in a Wifug X3 (Wifug, Stockholm, Sweden) for 5 min at approximately 90 g. EAC and EA7S were incubated at 37°C, ZyC at 0°C for 30 min. EA,9s, Zy and Zy coated with Zy-absorbed sera (4 mg Zy/ml serum) served as specificity controls. The cell pellets were gently resuspended in 0 5% trypan blue in PBS and mounted on slides. Two hundred viable cells were investigated at x 600 magnification. Cells binding three or more SRBC and two or more Zy beads were considered to be positive. The majority of lines were tested repeatedly and the percentage of rosette-forming cells was expressed as the mean of several independent experiments. Rosette assay for detection of spontaneous rosette forming cells. Twenty-five microlitres cell suspension was mixed with 25 ,1 EN, incubated at 37°C for 15 min, spun down at 90 g for 7 min, and then incubated for 60 min at 0°C. The further procedure was essentially the same as described above. Preparation of soluble immune complexes. Human-pooled IgG, obtained from Cohn fraction II (Behriglobulin, Behringwerke, Marburg, Germany) was further purified by chromatography on DEAE-SH-cellulose (Serva, Heidelberg, Germany) using 0-015 M phosphate solution (pH 8 0) as elution buffer. After dialysis against PBS the protein concentration was adjusted to 10 mg/ml by vacuum filtration. Heat aggregation was performed by heating to 63'C for 20 min. Aggregated IgG (AIgG) was obtained from the void of an Sepharose-6B column (Pharmacia, Uppsala, Sweden). MIgG was obtained from the 7 S peak of a sucrose gradient (10-35% sucrose in PBS) (Merck, Darmstadt, Germany) after 16 hr centrifugation with a SW40 rotor (Beckman Institute, USA) at 37,000 rev./min at 4°C. After extensive dialysis MIgG was adjusted to 10 mg/ml in PBS. Quantitative precipitin curves were constructed for BSA-7S rabbit anti-BSA complexes. Increasing amounts of BSA (Schwarzmann, New York, USA) were incubated with constant dilutions of the antiserum at 4°C overnight. The precipitates were washed afterwards three times and the amount of precipitate bound IgG was assessed by means of the protein A assay using '25I-labelled protein A from Staphylococcus aureus (strain Cowan I) (Dorval, Welsh & Wigzell, 1974). Soluble BSA-

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Surface receptors on haematopoietic cell lines TABLE 1. Haematopoietic cell lines used for surface marker studies

Origin

Designation U-61M U-255Bm U-303L U-704L

Type of lymphoid cell line*

EBV

References

Hodgkin's disease Myeloma (bone marrow) Healthy donor (lymph node)

LCL LCL

+ +

LCL

+

Hodkin's disease

LCL

+

Ponten (1967); Nilsson et al. (1971) Nilsson (1971a, b); Nilsson et al. (1970, 1971) Nilsson (1971a) Nilsson et al. (1971) Sundstrom (to be published)

LCL

+

Sundstrom (to be published)

LCL

+

Yata & Klein (1969)

Lymphoma

-

Lymphoma

-

(lymph node) U-718L

Maku U-698M U-715M U-937

S 95

DG-75 Daudi

Raji Namalva IV P3HRIK U-266B1 RPMI 8226

K562 MOLT-3 MOLT-A

Hodkin's disease

(lymph node) Burkitt's lymphoma (tumour tissue) Lymphosarcoma (tonsil) Lymphosarcoma (lymph node) Reticulum cell sarcoma (pleural effusion) Hodkin's disease (pleural effusion) Undifferentiated lymphoma (pleural effusion) Burkitt's lymphoma (tumour tissue) Burkitt's lymphoma (tumour tissue) Burkitt's lymphoma (lymphoma tissue) Burkitt's lymphoma (tumour tissue) Myeloma (peripheral blood) Myeloma (peripheral blood) Chronic myeloid leukaemia Acute lymphatic leukaemia (peripheral blood) Acute lymphatic leukaemia (peripheral blood) *

Lymphoma

Nilsson & Sundstrom (1974b); Klein et al. (1974) Nilsson & Sundstrum (1974b) Klein et al. (1974) Sundstrom & Nilsson (1976)

+

Nilsson, Ghetie & Sjuquist (1975a);

Lymphoma

-

Hausen et al. (1972) Ben-Bassat et al. (1976)

Lymphoma

+

Klein et al., (1968)

Lymphoma

+

Pulvertaft (1965)

Lymphoma

+

Reedman & Klein (1973)

Lymphoma

+

Hinuma & Grace (1967)

Myeloma

-

Myeloma

-

Leukaemia Leukaemia

-

Lozzio & Lozzio (1975) Minowada, Ohnuma & Moore (1972)

Leukaemia

-

Minowada et al. (1972)

Lymphoma

zur

-

Nilsson et al., (1970) Nilsson (1971b) Matsuoka et al. (1967)

According to the classification proposed by Nilsson & Ponten (1975b).

anti-BSA complexes used were prepared at seven times the antigen excess with respect to the point of equivalency. The concentration of BSA was adjusted to 100 pg/ml. Purified human IgG myeloma proteins were a gift of Dr J. Natvig, Oslo, Norway. Binding of soluble immune complexes to established lines. 5 x 105 cells in 100pul PBS were incubated with 10,pl AIgG, or BSAanti BSA for 30 min at 0C. After four washes in PBS cells were resuspended in 100,ul PBS and again incubated for 30 min at OC with 125I-labelled protein A (5-0-10-Ox 104 ct/min, specific activity see Dorval et al. (1974)). After three washes cell-bound radioactivity was assessed in a gamma counter GC 30 (Intertechnique, France) or by means of autoradiography. [3H]Thymidine labelling. Approximately I x 106 cells were incubated with 1 pCi [3H]thymidine (Radiochemical Centre, Amersham, Bucks) (specific activity 5000 mCi/mM) in 1 ml medium for 60 min at 370C. Cells were pelleted and smeared on gelatinized glass microslides. The percentage of cells in DNA synthesis was then evaluated by means of autoradiography. Autoradiography. Autoradiograms from 125I1 or 3H-labelled cell suspensions were prepared using the dipping film technique with Ilford C5 emulsion (Ilford, Essex). Details of the technique and of the evaluation of the exposed autoradiograms have been described (Huber, Michlmayr & Huber, 1974a). The dipped slides were usually exposed for 1 week.

Ch. Huber et al.

370

Demonstration of surface immunoglobulins. Surface located immunoglobulin was demonstrated by direct and indirect immunofluorescence. Cells (2 x 105) were washed three times and resuspended in 50,ul PBS. In the direct test they were then mixed with 25 Jl of fluorescein isothiocynate (FITC) conjugated polyvalent rabbit anti-human immunoglobulin (FITCraIg) (Dakopatts. Copenhagen) diluted 1:5 in PBS and incubated for 30 min at 40C. In the indirect technique the first incubation with raIg for 30 min at +44C was followed by an additional incubation with FITC-conjugated swine antirabbit immunoglobulin (30 min, +40C). After three final washings in PBS cells were resuspended in PBS-glycerol (1:1). A drop of this suspension was placed on a slide under a coverslip. Slides were examined without delay in a Leitz UV microscope equipped with a Ploem vertical illumination system.

RESULTS C3 receptors

All LCL and the majority of the lymphoma lines formed

rosettes

with EAC and ZyC (Table 2)

TABLE 2. Surface markers in human haematopoietic cell lines

Surface markers

ZyMC ZyHC

EAC

(% rosettes)

Cell line

Lymphoblastoid cell lines U-61M U-255Bm U-303L U-704 U-718 Maku Non-Burkitt lymphoma lines U-698 U-715 U-937 S 95 DG-75 Burkitt lymphoma lines Daudi

Raji Namalva IV P3 HRIK Myeloma lines U-266 RPMI8226 Leukaemia lines K 562 MOLT-3 MOLT-4 Normal blood lymphocytes§

Spontaneous rosette formation BSA-aBSA with SRBC AIgG EN (autoradiography)*

Fc receptor

C3 receptor

EA7S (% rosettes)

n.d. ++ n.d. ++ n.d. n.d.

0 0 0 0 0 0

+ + + + + +

+ +++ n.d.

0 0 0 0 0

+ +

+4+

+++ ++ n.d.

0 0 0 0

+ + + +

0 0

n.d.

+ n.d.

0 0

+ +

62 19 3 17-3

++++ n.d. + 29-3

+++ n.d. +

7 64 0 66-0

-

(±75)

(±45)

94 20 70 93 59 77

82 n.d. 93 32 73 72

78 34 51 32 10 67

0 4 0 1 0 5

+++

0 61 29 93 0

0 11 11 68 0

0 6 11 73 0

1 83 31 2 0

++++ n.d. ++++ +++

14 95 53 0

50 93 38 0

39 75 26 0

0 3 3 15

+4+-+

+±+

+++ ++

0 0

0 0

0 0

21 28 42 24-0

13 n.d. 41 26-7

15 1 11 20-0

(±78) (±67) (±53)

Ig

++

n.d. ++ + n.d.

++t n.d.

n.d. = Not determined. * Data are given in arbitrary scoring units and described by Huber et al. (1974b) t only line strongly binding 125I-SpA even in the absence of IgG I secretory Ig detectable by weak fluorescence during its transition over the cell surface. § for details see references Huber et al, 1975; Huber et al. (1974b).

(±20)

+ +

-

Surface receptors on haematopoietic cell lines

371

In contrast the two myeloma lines were completely negative. C3 receptors were also demonstrable on the K562 cell line, derived from a patient with chronic myeloid leukemia, and on the acute lymphatic leukaemia line MOLT-4. The specificity of EAC rosette formation of MOLT-3 cells remained questionable because of the capacity to bind EA19s and because of the lack of reactivity with ZyC. If the same C source was used, both methods for demonstration of C3 receptor mostly gained similar results. ZyMC, however, usually gave higher percentages of rosettes and a stronger reaction than ZyHC, which might suggest that this reagent can bind to both, C3b as well to C3d receptors. Fc receptors Fc receptor sites as demonstrated by rosette formation with EA7S were found only in one LCL, two lymphoma lines and the leukaemia line K562 (Table 2). Again both myeloma lines were completely negative. All lines tested with this method, however, bound AIgG and BSA complexes in variable amounts (Table 2). A weak binding of soluble immune complexes was in particular observed on the C3 receptor negative lymphoma line U-698 M, and on the T-cell leukaemia line MOLT-4, whereas both myeloma lines were almost completely negative. TABLE 3. Capacities of various IgG subclasses to bind to Fc receptors

IgGi

IgG2 IgG3 IgG4 (ct/min x 103/2 x 105 cells)t

Raji

19-8

U-715 M K 562

MOLT-A

22-9 45-2 9-2

Human mononuclear blood cells

18-2

14-6 12-8 32-7 7-3 10-5

Cell source

*

36-4 28-3 36-8 18-1 24-3

15-7 14-4 8-9 4-7 95

IgG1-4* 19.5 17-3 26-5 7-2 16-4

Human polyclonal IgG, obtained from a normal serum pool.

t 2 x 105 Cells were incubated with 2 5 pg 125I-labelled IgG (specific activities 1-3,pCi/pg protein) for 30 min at 37°C in the presence of 0-01% sodium azide. After four washes cellbound radioactivity was determined using a gammacounter. Counts given in the table are corrected for differences in the specific activities.

In a series of experiments, which will be reported elsewhere in detail (Huber & Wigzell, to be published), we studied the subclass specificity of Fc receptors on several lines. Table 3 shows one representative experiment, in which the binding capacities of human myeloma proteins IgG1, 2, 3 and 4 were compared with that of polyclonal IgG, obtained from a serum pool. As demonstrated all cell types tested showed a preferential binding of IgGl and IgG3.

Spontaneous rosette formation As demonstrated in Table 2, LCL, lymphoma or myeloma lines were incapable to form spontaneous rosettes with neuraminidase-treated SRBC. The lymphoid leukaemia line MOLT-3 was the only line forming high percentages of spontaneous rosettes. Surprisingly enough, the myeloid leukaemia line K562 showed a low percentage of spontaneous rosettes after prolonged time of incubation (Table 2). Surface immunoglobulins All LCL and lymphoma lines expressed surface immunoglobulins. Leukaemia lines had no detectable surface immunoglobulin while a faint membrane fluorescence was detected on the myeloma cells (Table 2).

Influence ofgrowth phase on the expression of C3 and Fc receptors During repeated testings of our lines with rosette markers considerable variations were observed, B

Ch. Huber et al.

372

TABLE 4. Influence of growth phase on the expression of C3 and Fc-receptors Days of culture

Cellline K 562

Raji

[3H]-TdR LI(%)* EA7s rosettes (%) [3H]-TdR LI (%) EAC rosettes (%) ZyMC rosettes (%)

1

3

5

7

38 54 23 83 75

18 49 n.d. n.d. n.d.

10 45 12 73 63

n.d. n.d. 1 71 72

n.d. = not determined. * [3H]Thymidine labelling index.

which were not easily explainable on the basis of the variation of these methods. In order to exclude an influence of cell cycle phase on the expression of these receptors the percentages of rosette-forming cells was compared with the numbers of DNA-synthesizing cells during a culture period of several days. As shown in Table 4 no obvious correlation between these two parameters was observed.

DISCUSSION We have studied the presence of C3, Fc, SRBC receptors and of S-Ig on several neoplastic haematopoietic lines. Results were compared with those obtained on EBV-carrying LCLs of non-malignant origin. The lymphoma lines were found to bear, in varying degrees, the surface receptor pattern of mature B lymphocytes: C3 and Fc receptors and S-Ig. In agreement with other reports the presence of Fc receptors was rarely demonstrable by rosette formation technique using EA7S (Shevach et al., 1973), but was detectable in autoradiograms by specific binding of soluble-immune complexes. The lines U-698 M DG-75 and P3HRIK differed from the other lymphoma lines in lacking detectable C3 receptors. U-698 M also showed an unusually low density of Fc receptors. However, the demonstration of S-Ig presents a strong argument in favour of a B-lymphatic origin also of these three lines. As reported previously (Huber et al., 1974a; Huber, Dworzak, Fink, Michlmayr, Braunsteiner & Huber, 1974b) cells from eighteen non-Hodgkin-lymphoma lymph nodes expressed the same pattern of B lymphocyte receptors as the present lymphoma lines, indicating that studies on established tumour cell lines can be representative of malignant lymphoid cells in vivo. With one exception, the line U-61M, LCLs showed a common pattern of B lymphocyte surface markers (S-Ig, C3 and a low density of Fc receptors) confirming earlier reports by other authors (Jondal et al., 1973b; Shevach et al., 1972; Shevach et al., 1973; Theofilopoulos et al., 1974; Florentin, Bruley & Belpomme, 1975; Pauly, Minowada, Ham & Moore, 1975). The U-61 M is exceptional in expressing a higher density of Fc receptor. The line in aneuploid (Ponten, 1967) and thus represents an example of an LCL which has been secondarily altered during prolonged cultivation in vitro (Nilsson et al., 1975b). Other phenotypic characteristics of U-61 M such as growth properties, Con A agglutinability (Glimelius, Nilsson & Ponten, 1975) and f2-microglobulin production (Nilsson et al., 1974a) have also changed during the long-term culture. The two myeloma cell lines (U266 B1 and RPMI 8226) constituted a distinct group with virtually no detectable receptors of either C3 or Fc type and by low density of S-Ig. Finally, the leukaemia lines did not match in their receptor markers with any of the above B-cell lines. The Ph 1 chromosome-positive myeloid leukaemia line K562 expressed extensively high values of Fc receptors, sizeable C3 receptor density, lacked S-Ig and could be shown to bind a low but significant number of SRBC. MOLT-3 and MOLT-4 sublines derived from the same acute lymphocytic leukaemia have been reported to be of T-cell origin (Minowada, Ohnuma & Moore, 1972; Pauly et al., 1975; Jondal, Holm & Wigzell, 1972). MOLT-3 cells showed spontaneous rosette formation with SRBC,

Surface receptors on haemaiopoietic cell lines

373 whereas MOLT-4 has lost this capacity during repeated subcultivation (Jondal & Klein, 1973a; West & Herberman, 1974). Both sublines, however, show a high percentage of complement receptors with Fc receptors in lower density. The latter finding is supported by other reports indicating the presence of Fc receptors on activated T cells (van Boxel & Rosenstreich, 1974; Anderson & Grey, 1974) and on murine leukaemia lines of T-cell origin (Anderson & Grey, 1974). We thus would conclude that by extending the present analysis to still larger numbers of normal or malignant cells, subgroups of these lines may be characterized according to their surface receptor pattern. This might lead to a more discriminatory diagnosis of the various malignant disorders affecting the myeloid and lymphoid systems. Moreover, the use of highly defined, monoclonal cell lines expressing certain receptors in a comparatively 'pure' form may greatly facilitate the isolation and biochemical characterization of receptor structures. To prove that a cell line obtained from explanted tumour tissue is in fact representative of the tumour cell population is a particularly important task in the case of haematopoietic malignancies because of the common presence of LCL precursors as contaminants in biopsies from EBV-infected patients (Nilsson, 1971b; Pauly et al., 1975). Such LCL precursors will often, due to a selective growth advantage, overgrow the tumour cells and establish in vitro as an indefinitely proliferating LCL. Since no single specific phenotypic marker for LCL or the different types of malignant haematopoietic cells has been detected, various morphological and functional parameters have been employed to differentiate between LCL and tumour cell lines (Nilsson & Ponten, 1975b; Belpomme, Minowada & Moore, 1972; Lazarus, Barell, Oppenheim & Krishan, 1974; Royston et al., 1974). TABLE 5. Surface receptor pattern in different types of haematopoietic cell lines

Type of cell line LCL Non-Burkitt lymphoma lines Burkitt lymphoma lines Myeloma lines

C

Fc

+ + + -

SRBC

S-Ig

(±)

-

+

+

-

+ +

-

(+)

(+) -

The most important parameters seem to be: (1) Clonality; as revealed by isoenzyme analysis or pattern of Ig synthesis, fresh LCLs are polyclonal while tumour lines can (at least shortly after establishment) be expected to be monoclonal (Fialkow, Klein, Gartler & Clifford, 1970; Fialkow et al., 1973; Bechet et al., 1974; Glade & Chessin, 1968; van Furth et al., 1972). (2) Karyotype; LCLs, analysed shortly after establishment, are normal diploid while all carefully examined malignant cell populations are aneuploid (Jarvis et al., 1974; Zech et al., 1976). Other parameters can be employed in doubtful cases. From the present study it seems clear that characterization of a haematopoietic cell line by B- and T-cell surface markers represents an additional possibility to differentiate between a non-neoplastic LCL and a tumour cell line. The pattern of receptors in LCLs (C3, Fc receptors and S-Ig) was not found in nonBurkitt lymphomas, myelomas and leukaemias (Table 5). However, in the group of Burkitt lymphomas some lines expressed receptors of the same type and in comparable amounts as in LCLs. We therefore conclude that at least in cases of haematopoietic malignancies other than Burkitt lymphomas, surface receptor characterization is valid. This does not exclude, of course, that a malignant lymphoid line of non-Burkitt lymphoma origin may appear with a C3 and Fc receptor pattern identical to that of an LCL. These markers will therefore not have the same high priority as S-Ig studies, which can decide clonality, and chromosome analyses. Restriction of Ig synthesis to one class of heavy and one type of light chain is observed in malignant lymphoproliferative disorders, as shown by studies on biopsy and blood cells (Huber et al., 1974a; Grey, Rabellino & Pirofsky, 1971; Aisenberg & Bloch, 1972; Fr0land & Natvig, 1973; Seligmann, Preud'

374

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Homme & Bronet, 1973) and in established cell lines (Klein et al., 1968). This finding, together with data from chromosome and isoenzyme analyses (Fialkow et al., 1973; Bechet et al., 1974), prove the monoclonal nature of these malignancies. Whether a similar restriction in expression of different Fc and C3 receptors occurs in a monoclonal cell population has not been extensively investigated. Human peripheral blood lymphocytes bear at least two distinct C3 receptor sites: one for C3b and one for C3d. In chronic lymphocytic leukaemia (CLL) C3 receptors were found to be restricted the C3d specificity (Ross, Polley, Rabellino & Grey, 1973). We have applied two different methods for detection of C3 receptors, the rosette formation with EAC-MC, which detects C3b and C3d (Ross et al., 1973) and the ZyC technique which seems to preferentially detect the C3b receptor (Huber & Wigzell, 1975). Since the majority of our lymphoma lines and the LCLs showed a comparable reactivity with EAC as well as with ZyC a restriction of C3 receptors to C3d receptors seems to be rare. This conclusion is also supported by another study in which a defined system for detection of C3b and C3d receptors has been used (Theofilopoulos et al., 1974). On normal human mononuclear blood cells restriction of Fc receptors to the IgG subclasses 1 and 3 has been observed by several groups using different experimental systems (Huber & Fudenberg, 1968; Fr0land, Natvig & Michaelsen, 1974). We therefore studied the subclass specificity of this receptor on several cell lines. Preferential binding of labelled IgCl and IgG3 was found on both normal mononuclear blood cells and on lymphoma and leukaemia lines indicating a similar Fc-receptor specificity. This work was supported by Swedish Cancer Society (55-B75-1 IXA) and NIH Contract no. NO1-CB-43883. The technical assistance of Mrs Agneta Snellman and secretarial work by Mrs Kerstin Lindberg is gratefully acknowledged. We thank Dr J. Natvig, Oslo, for providing human purified myeloma proteins. The work was undertaken during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer.

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Surface receptors on human haematopoietic cell lines.

Clin. exp. Immunol. (1976) 25, 367-376. Surface receptors on human haematopoietic cell lines CH. HUBER,* C. SUNDSTROM, K. NILSSON & H. WIGZELLDepartm...
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