Scand. J. Immunol. 10, 153 159. 1979
Mouse Erythrocyte Rosette Formation by Human Lymphoid Cell Lines J. BERTOGLIO, B. LALDJIM & J. F. Laboraioirc d'fmmunologie et de Canccrologie Exp^rimentale, INSERM FRA 24. Centre Lton B^rard. Lyon, France
Bertoglio. J.. Laldjim, B. A Dori. J.F. Mouse Frythrocyle Rosette Formalion by Human Lymphoid Cell Lines. .Stand J. tmmiinfl. 10. !5.l 159. 1^79. Lymphoid cell lines have been studied with respect to mouse erythrocyte rosctic formation, a marker for B cells. Celts from six B-cclt lines out of fifteen tested were able lo form mouse er>throc>te rosettes. When this capacity was studied after neuraminidase pretreatment of the cells, il defined three groups of B tymphoid cell line* according to their reactivity. However, these groups could not he correlated toany of the other H-cell markers, and especially mouse erythrocyte rosette formalion appeared unrelated to ihe class of the immitnoglobulin present on the cell surface. Non B-cell lines were never found to form such rosettes. The capacity of B-cell tines to form mouse erythrocyte roscUes was further demonstrated to be relatively insensitive to trypsin treatment, and results are presented suggesting some degree of cell cycle dependency of the expression ol' this murlter. J. Berioglio. Lahorotoire J'tmmunotogle ft df Candroh^ie Expfrimentate, INSERM FRA 24, Centre Leon B^rard. 2S rue iMcnnev. Mi7i Lvon Cfdvx 2. France.
It has been recently demonstrated that rosette formation wi(h mouse erythrocytes is a marker for hutiian B-type lymphocytes [II. 25]. Previous reports from this laboratory [4] and from other investigators [10] would suggest that only a subpopulation of normal B lymphocytes possesses this membrane property. Furthermore, among B-type malignant haemopathies, only cells from typical chronic lymphatic leukaemia have been reported to form mouse erythrocyte rosettes [9. 26]. and this property has been considered useful in making a dilferential diagnosis between chronic lymphatic leukaemia and lymphoma [3. 5]. As an approach to further define subpopulations of normal B cells, we have investigated tiiouse erythrocytes (MRBO rosette formation by human lymphoid cell lines (LCD. The vast majority of long-term cell lines derived from peripheral blood of cither normal donors or patients with various lymphoproHferative disorders share characteristics with B lymphocytes [13. Ift.24]. They usually e.\hibit one or more normal B-cell markers, which have already 0300-9475/79/0800-0153 S02,00
been used in attempts to characterise homogeneous populations of human cell lines [27]; these litics are assumed to represent the in vitro counterpart of the normal B-cell subsets they originated from, or various steps in the differentiation process [20]. The results presented below indicate that some human lymphoid B-celt lines tnay form MRBC rosettes and that this property may help in subciassifying them in three groups. Furihermore. it appeared that MRBC rosette formation is not associated with membrane expression of any given class of imtnunoglobulin and tnay depend on the cell cycle phases. MATERIAL AND METHODS Lymphoid tell lines Eighteen lymphoid cell lines established cither from normal donors or from paiients with various malignant haemopathies have been used iTable I). The wdlcharactcri/ed Molt-4. Raji, P3J and Dnudi lines were obtained from G. de Th6 at the I ARC. Lyon: the K562-4 line, initially established hy B. Lo7.7io. und the RI'MI 74S1. MIO and 8.122 hncs were obtained by
r 1979 Blackwell Scientific Publications
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154
J. Hertoiilio, B. Laidjim A J. F. Dore
courlcsv ol" N. Carpentier, Geneve; ihe LHN-1.^. Pon. Del and Reh-6 lines were obtained rVom ICIG in Villcjuif; and Cot-3, Boe and PTL line.s were established in this laboratory. C20I and C2O2 were established Ironi normal cord blood lymphocyies by lipstein-Barr virus^(lW5 strain) infeciion ;ind were u gifl from C. Desgranges (lARC. Lyon), and ihc KM.3 line was obtained Ironi U. Schneider (hrlangcn). Suspension cultures ol" ihese cell lines were propagated in cotnpiele culture medium consisting of RPMI IA40 wiih 10-15",. hcai-inactivmed Tetal calf serum. 50|xgslreptomycin/inl. and KKt units penicillin ml. Membrune marker determination was routinely performed 11 h after ihe last suhi.ijlttjre. For MRBC rosette formation studies according to the cell growth, cells were seeded al 10" cells ml after having been maintained for 24 h at siituration density. 4 • II)" cells were then harvested every 24 h and processed tor roseite formalion. Cell viability was assessed by trypan blue exclusion. Enzymatic treatment of cells Seuraminiduse treatment. 4 • 10" washed cells were treated for 30 min al .17 C with either 2.5 or 5 units'K>^ cells of Vihrio cholera neuraminidase (Bcrhingwerke). The cells were then washed thrice in Hanks' balanced salt solution (HBSS) and processed for roscilc formation. Trypsin treatment of cells. ATlcr ihree washes in serum-free medium, 20 •: 10" cultured cells were incubaled for 5-10 min at 37 C with O.OI", lo O.T^ Irypsin solution (Gibco), then washed thrice in cold complete medium, adjusted to 4 ' 10" cells/ml. and processed for rosette formation. Membrane tnarkers Detection of C3 receptor by EAC rosette formation. EAC complex formation was performed by incubating a0.5''o sheep erythrocyte (SRHC) suspension for 30 min at 37 C in an equal volume of a subiigglutinaling dilution of rabbit anti-shccp erythrocyte IgM puritied by chroma to graphy on a Sephadex G-200 column. After three wiisbcs. sheep erylhrocyle-IgM complexes were adjusted to 5 • 10" ml and further incubated tbr 30 min at 37 C with fresh C57Bt6 scrum diluted 1:5 in barbital fVeronal*) bulTer (0.005 M. pH 7..1). After three more wa-shes. EAC complexes were adjusted to KP/ml. stored at 4 C. and used within a week. EAC rosette formalion was performed bj incubating 10" cells in 50 \i\ of HBSS with 50 (il of t A C complex suspension for .10 min at M C. After proper dilution, the percentage of rosette-forming cells was counted in an hiicmocyiomcier. Each lest was done in triplicate atui controls were performed using unsensili^^cd SRBC and SKBC" IgM complexes. ImmtinofluoreMituf detection of surface immiinogli'htdins. 10" cells were pelleted in 3 ml plastic test lubes and resuspended in 50 jil of a proper dilution of tiuorescein isothiocyanate (KITC)-lnbelled globuhn fraction from monospeciflc goat antisera to heavy or light chain (Meloy Laboratories Inc.). After 30 min incubation on ice. three washes were performed in a large excess of cold HBSS. The cells were suspended in biilVercd gl>ccrol, layered on microscope slides, and the percentage iif positive cells counted by using a Leitz onhoplan microscope.
Formation of mouse frythrofvte rti.\pttes. The technique originally described by Stathopoutos |26I for demonstration of mouse crythrocyie rosetlcs was jpplicd. Briefly, to 25O|j.l of a cell suspension (4 •• lO'/ml in HBSSi 50 [xl of MRBC-ahsorbed tetal calf serum and 250 i.tt of a !"„ suspension in HHSS of freshly drawn C57B16 mouse erylhrocytes were added. The tni.ntiire was incubated for 10 min al 37"C. centrifugcd 5 min at 400 g. and further incubated for I h at 4 C Each test was done in triplicate and the percentage of cells forming rosettes with mouse crythrocytes was then counted in a haemocytomctcr after gentle resuspension. Any cell bearing three or more erylhrocytes bound to its membrane was recorded as M R BC-rosette positive.
RESULTS \fou.\i' erythrocyte rosette formation by human lympliohla.stoid cell titles Results of ihe mouse eryilirocyic rosette formation test, together with those of the standard tests for B-cell markers from eighteen LCL are presetited in Table 1. It lirst appeared that both the Molt-4 line, which is thought to be of T-cell origin [2. 21], and the K562 line of myeloid origin, which laeks any of the T or B usual markers 115. 19]. did not form rosettes with mouse erythrocytes. Nine other cell lines, whose B nature cottid be assessed with regard to other markers, did not form MRBC rosettes. Only cells frotn six out of fifteen B-cell lines under study appeared lo form a significant percentage of rosettes with MRBC. In none of these lines could more than 50% cells be demonstrated to form roseltes with the technique used. It is noteworthy that not only cells from lines exhibiting membrane ii-ehain showed this property, so thai among these six mouse erythrocyte roselting lines all types of" heavy and light chains were represented. For each considered line, the percetitage of rosetting cells was relatively reproducible provided the cells were harvested at a constant time after they have been subcultured. Furthermore MRBC rosette-forming capacity appeared to have good stability in long-term culture, since two batches of the normal-derived LHN-t.l line, frozen at a 3 year interval, formed the same percentage of MRBC roseltes when thawed on the same day and further cultured for 2 weeks before being tested.
Mouse Erythrocyte Rosettes on Lymphoid Lines
155
TABLK I. MRBC rosctie formation by LCL
Cell line
Origin*
LHN-13 C-201 C-202 KM-3 REH PTL MOl.T-4 RI»Mi-MIO R I'M 1-8.122 COT-3 BOE K 562-4 RPMI-7481 DEL PON P?J DALDI RAJI
PBL CBL CBL c-ALL c-ALL c-ALL T-ALl. AML AML AML AMML CML CML LS LS BL BL BL
r-AC-RKCJ M-RFC§
Membrune imniunoglobulint Y
a
0 0 0 0
80
0
0
0
0
0
0
0
(1
22 17 0 0 60 0 20 0 0 0 0 30 0 0 0 100
0 0
1)
I)
hO
0
0
(1
60 0 0 7 30 0 0
0
0
11
0
0 0 0 0 0
K
>.
0 — 8 0 0 0 0
0 0 0 0 0 — 0 0 0
70 — 0 0 0 80 0 80 0 0 50 0 30 -_ 70 0 100
0
0
0 — — 0 0
60 0 0
0
80 0 0 0 0 — 0 0 0 5
38 61 49 0 0 57 65 85 90
9 0 0 0 0 16 0 0 22
68
43
80 0 37 0 45 0 27 75
I« 0 0 0 0 0 0 50
• T or t - A L L acute lymphoblastic leukaemia wiih T or non-T. non-U menibnine phcnoiypc; CML=chronic myelogenous leukaemia; BL - Burkitl's lymphomii: LS lymphosarcoma^ A M L acute myeloblastic leukaemia: A M M L acute myelomonocytic leukaemia; CBL-EBV-transformed cord blood lymphocytes; PBL peripheral blood lymphocylcs from a normal donor. t Percentage of cells reactive wiih monospecific antisera. J Percentage of cells forming rosettes with FAC complexes. § Percentage of cells forming rosettes with mouse erytbrocytcs.
Effect of enzymatic treatment of lymphoid cell lines on rosette formation To attain further insight Into the nature of MRBC binding of LCL. we invesligated the effects of trypsin and neuraminidase treatment of
the cells
an
MRBC
rosette
formation.
Trypsin treatment of three MRBC rosetting lines showed that this membrane property is quite resistant to such an enzyme treattnent (Table II). None of the enzyme concentrations
used (0.01-0.1°-;;) was able to destroy the capacity v>\' Raji cells to form MKBC rosettes. Furthermore, the trypsin concentrations needed to completely inhibit MRBC rosette formation by Col-3 and PTL litie were much more important than those usually ellicicnt in oiher types of T- or B-cell markers; Peliegrino ci al. [22], for instance, have reported that rosette formation with monkey erythrocytes could be inhibited by 0.01";, trypsin concentration, and we have found that this concentration was also
TABLE (I. The effect of trypsin treatment of lymphoid cell lines on the percenta^ of rosette-forming
Trypsin concentration ("„ of stock solution) Cell line RAJI COT-3 PTL
0
0.01
55 54
40 33
17
17
Mean of two experiments.
0.02
0.05
0.1
44 20 —
40 4
42 0 —
8
156
J. Berfoglio, B. Laidjim A J. F. Dore
sufficienl to abolish EAC rosette formation by the LCL under study (dala not sliovvn). Almost all types of rosettes that have been described between human lytnphocytes and erythrocytes from various animals have been reported to be modified by neuraminidase treatment of either erythrocytes or lymphocytes [8]. Lytiiphocyte pretreatment wiih neuraminidase has been described to increase dramatically the percentage of MRBC rosetteforming lymphocytes In peripheral blood [10, 23J. In this study neuraminidase treatment of cells from cultured cell lities generated various types of results aecording to the capacity of the untreated cells from the lines to spontaneously form MRBC rosettes. Three groups of B-LCL could be identified by this procedure (Table 111). Group 1 is composed of LCLs whose cells formed spontaneous MRBC rosettes. In this group neuraminidase treatment had no significant elfect or slightly increased the percentage of rosetting cells, Group If is composed of LCLs whose cells formed MRBC rosettes only when pretreated with neuraminidase. and
group III contains the fines whose ceffs could not be demonstrated to form such rosettes even after neuraminidase treatment. No correlation seemed to appear between Ifiese three groups atid the expression of other B-cell markers. f--vidence that neuraminidase treatment did not induce rosette formation tfirough nonspecific interactions could be drawn frotn the observations that neitfier the T-type Moft-4 nor the non-lymphoid K562 could be indueed to form MRBC rosettes; furthermore, we verified that this treatment did not enable B-celi lines to form rosettes with sheep erythrocytes. Effect of cell growth on MRliC rosette fortnation hy LCL The expression of most of tlie membrane properties of euliured cells has been reported to depetid on the cell cycle [7, 14. 17. 18]. This phenomenon could account for ihe rather broad variations in the ability to bind MRBC of cells from some of the lines under study, when repeatedly tested at difl"erent times. As
Ml. The effect of neuraminidase treatment of lymphoid cell lines on MRBC rosette formation
Units of 111
Cell line Group 1 COT-3 RAJI PTL BOF
RPM 1-8322 1-HN-I.T Group II P3-J DAUDI REH
C-201 C-202 DEL Group III RPMI-7481 RPMI-641()
2.5
43 SO 16 16 22 9
56 4S 29 18 52 17
51 47 35
I9.S 39.3 28 12 27 8
11 38 33 32 29 13
1 0 0 0
1 0 1 0
0
0 0
0 t)
0 0 0 0 0 0
PON
0
KM-3
0
Non-B LCL K 562-4 MOlT-4
ISC 10" cells
0
0 0
0
5
34 56 32
Mottse Erythrocyte Rosettes on Lymplwid Lines
157
-40
-30
-20
-40
-30
-20
24 TIME AFTER
48 72 SUBCULTURE
96
FIG. I. Relationship heiween mouse cryihrocytc (MRBCt rosettes (•) nnti growth ol' cells in culture (0). (.A) Cells from PTL line; (U) cells from Col-3 line.
iin approach to study such variaiions of the expression of MRBC roseltc formation, we studied the percentage of rosette-forming cells according to the culture conditions for two ditTercnt LCL. In six ditTcreni experiments using Ihe PTL line, the percentage of rosetteforming cells appeared to significantly decrease within 24 li after Ihe culture had Ix'en split from saturation densily down to 10" cells.nil. As the cells progressively multiplied, ihc percentage of roscite-form ing cells increased and eventually reached its starting value (Fig. lA). In conirast. no variation could be demonstrated under the same conditions using Col-3 tine (Fig. lBj.
DISCUSSION Several reports have now established thai MRBC rosette formation is characteristic of a subpopulation of normal B-cells [4, II, 25]. and it first did not appear unlikely that MRBC rosetting lymphocytes were those that exhibited membrane immunoglobulin |i-chain [10]. The study of patients with imtiiunodeliciencies has conftrnied these results, and the study of both inali^nanl haemopathic!: and fetal liuman liver
has suggested that MRBC rosette formation may occur at some given stage of maturation [6. 12]. In the present study we investigated the formation of MRBC rosettes by hutnan lymphoid cell lines. The results from both the T-typc Moli-4 line and from the non-lympholdderivod K562 line are in agreement wiih ihe B nature of MRBC rosetting capacity. However, even though all of the MRBC rosette-forming lines are B-derived, not all the B-derived lines may form rosettes with MRBC. Furthermore, live non-rosetting lines have been shown to possess membrane ^-chains, and among the MRBC rosette-form ing lines, two had no delectable or very few membrane immunoglobulins. whilst all types of light anil lieavy chains could be demonstrated in the others. These findings are not in complete agreement with results from MRBC rosette formation tests in normal peripheral blood lymphcKytcs. Either of two hypotheses could explain these discrepancies: (1) non-lgM-bearing but MRBC rosette-forming lymphocytes may exist at so low concentration in peripheral blood that they are untletectable; (2) an alternative and more attractive hypothesis is that iti vitro culture conditions may modify the cell membrane so
158
J. Bertoglio, B. Laidjim & J. F. Dore
that MRBC 'receptors' are expressed. .Another finding is that, although only normal peripheral blood lymphocytes and cells from chronic lymphatic leukaemia may form MRBC rosettes, whilst cells from acute lymphoblastic leukaemia may not. no correlation arose between MRBC rosctting capacity and the normal or Icukaemic origin of the lines. The latter hypothesis would also account for this finding. En/yme treatment has been extensively used lo understand the mechanism of various types of rosettes. Surprisingly, MRBC binding to LCL cells appeared almost unaflected by trypsin treaimeni of cells even though the concentration ti>cd has been reported to completely inhibit other types of rosettes. It is noteworthy, although it may not be related at alt to this finding, that the la antigen molecule on B lymphocytes has also been described as rather resistant to trypsin treatment [1]. It was not possible to use higher concentrations of trypsin because of poor viability obtained and clustering iif cells induced by DNA rcsiiUio.. In contrast, neuraminidase treulnicni of LCL cells gave interesting results: neuraminidase has been reported to enable nearly all normal B lymphocytes to form MRBC rosettes [10]. and these findings are partly confirmed, in that cells from six B-cell lines that did not form spontaneous rosettes were induced to form MRBC rosettes, after pretreatment with neuraminidase. However, four tnore B lines were not atTected by eitlier of the two neuraminidase concentrations used. Potter & Moore [23] have rai-sed the possibility that in peripheral blood, immunoglobulin-positive MRBC-negative cells may represent non-B cells carrying adsorbed immunogiobulins. Such an explanation can be ruled out for in vitro cultured cell lines, and our experiments suggest that true B lymphocytes that do not form MRBC rosettes may exist. On the basis of this marker, three groups couki be identified among B lymphoid lines; since such lines do not represent resting B-cells but cells at an arrested state of differentiation it is questioned whether these groups, and especially groups I and II. which do notfitwell with groups determined on the basis of other membrane markers, may represent very close and so far unrecognized steps in the dilferentiation process of B-cells. Cell cycle dependency of most of the membrane markers of LCL have been widely
investigated. Such a phenomenon could account for the finding that in none of the cultures studied could tbe percentage of cells spontaneously forming rosettes be shown to exceed 50"/,,. In one line the percenlage of MRBC rosette-forming ceils has been shown to vary during growth of unsynchroni/ed cultures, decreasing just after subculture and increasing until saturation density has been reached in tbe culture. Since cells are known to gather in GI phase when reaching saturation density, these results would suggest some GI dependency of the expression of this marker. The relative decrease of the percentage of roselte-lorming cells after 96 h of culture may have reflected starvation effects. Lack of sufficient media nutrients and 'crowding' could have been affecting macromolecular synthesis and conM:t|uently MRBC rosetting capacity. In another tine such variations could not be demonstrated, so it could be hypothesized that the cell cycle dependency of MRBC rosetting capacity may itself depend on the cell cycle characteristics of each line. Further experiments, using synchronized cultures and complete analysis of the cell cycle characteristics of each line, are now needed.
ACKNOWLEDGMENTS The authors thank Mrs Christiane Pinatel for her skilful technical assistance. This work was partially supported by a grant from f)clcgation G6neralc a la Recherche Scientifique et Technique (75.7.1369).
REFERENCES 1 Anderson, J.K- & M c t / ^ r . R.S. Dctcciiun and purliiil characterization of humun T lymphocyte mcnihriinc untigcii.s with ie rosetle tesis: elfeci of enxymulic modification of sheep erylhrocytes (E) and the specificity of neuraminidase-lreaicd F. Cell. Immunol. 24, 97. 9 Gupta. S.. Good. R.A. & Sicecl. F.P. Rosette formation with mouse erylhrtKyies. 111. Studies in patients with primary immunodeficiency and lymphoproliferalive disorders. Clin. i.\p. Immunol. 26. 204. 1976. 10 Gupta. S.. Good. H.A. & Siegla. F.P. Rosetle formation with mouse erythrocyies. II. A marker for human B and non-T 1> mpht>cytes. Clin. exp. Immunol. 25. 319. 1976. 11 Gupta. S. & Grieco. M.H. Roseitc formation with mouse eryihrocyies: probahle marker for human B lymphocytes. Int. Arch. Allergy iippl. Immunol. 49. 734. 1975, 12 Gupta, S.. Pahawa. R.. O Rcilly. R.. Gootl. R.A. & Siegal. F.P. Ontogeny of the lymphtx-yte subpopulations in human lelal liver. Prtic. nat. Acad. Sei. (USA). 73. 919. 1976. 13 Hotiig, R.. Bitting. R.. Matsu>uma. M. & Terasaki. P.I. B lymphocyte alloantigen specificities present on cultured lymphoblasioid cell line^ J. Immunol. 117. l.^tJO. 1976. 14 Killandcr. D.. Klein. F. & Levin. A. Expression of memhraiie-hound IgM ami HL-A iintigcns on lymphohlastoid tells m dilltreru stages of the toll cycle. Europ. J. hnmunol. 4, 327. 1974. 15 Klein. E.. Ben-Bassat. H.. Beumann. H.. Ralph, P.. Zeuthen. J.. Polliack. A. & Vanky. F. Properties of the K562 cell line, derived from u patient with chronic mycloid leukemia. Int. J. Cancer, 18. 421. 1976. 16 Klein. G . Lindahl. T.. Jondal, M.. Liebold. W.. Mcnezes. J.. Nilsson. K. & Sundstrbm. C. Continuous lymphoid cell lines with characteristics of Bcetts (hone marrow-derived), lacking the EpsteinBarr virtis Rcnome and derived from ihree human
17
18
19
20
21
22
23
24
25 26
27
159
lymphomas. Proc. nai. Acad. Sci. (VSA), 71, 3283. 1974. Krakowka. S,. Olscn. R. & Cockerell. G. The elTcct of cell synchronization upon the detection of T and B lymphoid cell receptors on two contitiuous lymphoid cell lines. In Vitro. 13. 119. 1977. Kwork. S.Y. & Litwin. S.D, Immunoglobulin expression of cells from human lymphoblastoid lines. Cell. Immunol. 25. 256. 1976. Lozzio. C B.. Lo?7io. B.B.. Yang. W.K., lehiki. A.T. & Bamherger. E.G. Absence of thymusderived lymphocyte markers in myelogenous leukaemia (Ph, ' } cell line K562. Cancer Res. 36, 4657. 1976 Minowada. }. Markers of human leukaemia lymphoma cell lines reflects haematopoietic cell difIcreiiliation. p. 137 in Serrou, B. & Ronenl'eUI. C. (cds.) Human Lymphocyte Differentiation: irs Applivaiion to Cancer. North-Holland. Amsterdam, 1978. Minowadu. J.. Ognuma. T. & Moore. nius derived lymphocyies. J. nai. Cancer hist. 49. 89t. 1972. Pellegrino. M..A.. Ferrone. S. & Theiililopoulo-i. A.N. Rosetle formation of human lymphoid cells with monkey red blood cells. J. hnmunol. 115. 1065. 1975. Potler. M.R. & Moore. M. Chnrtictcn/iilion und separation of human lymphocytes forming mouse red cell roseltes. J. immumtl. Methods. 19. 125. 1978. Rosetifeld.C.Goutner, A.. Venuat. A.M.. C'hoquet. C Pico. J.L.. Dore. J.F.. l.iaheiif. A.. I>urandy. A.. Desgranges, C. & de The. G. An elTective human Icukaemic cell line: Reh. Europ. J. Cancer, 13, 377. 1977. Stathopoutos. G. A Da%ics. A.J.S Human lymphocytes and mouse red cells, l.umit. 1, 1078. 1976 Staihopoulos. (i. & Elliott. l-.V. Formation of mouse or sheep red-blood-celt rosettes hy lymphocytes from normal und Icukaemic individuals. Lancet. I. MO. 1974. Young. W.J.. Utsingcr.P.D.. Hutt, L.M.. Buchanan, P.D.. Korn. J.H., Fuller. C.R., l.ogue. M. & Pagano. }.$. Subpopulation.s of human lymphoblastoid cell lines; correlation with the e\pression of surface receptors and content of Epstein-Barr virus genome. Scand. J. hnmtmol. 5, 795. 1976.
Received 5 February 1979 Received in revised form 25 April 1979
Mouse Erythrocyte Rosette Formation by Human Lymphoid Cell Lines J. BERTOGLIO, B. LALDJIM & J. F. Laboraioirc d'fmmunologie et de Canccrologie Exp^rimentale, INSERM FRA 24. Centre Lton B^rard. Lyon, France
Bertoglio. J.. Laldjim, B. A Dori. J.F. Mouse Frythrocyle Rosette Formalion by Human Lymphoid Cell Lines. .Stand J. tmmiinfl. 10. !5.l 159. 1^79. Lymphoid cell lines have been studied with respect to mouse erythrocyte rosctic formation, a marker for B cells. Celts from six B-cclt lines out of fifteen tested were able lo form mouse er>throc>te rosettes. When this capacity was studied after neuraminidase pretreatment of the cells, il defined three groups of B tymphoid cell line* according to their reactivity. However, these groups could not he correlated toany of the other H-cell markers, and especially mouse erythrocyte rosette formalion appeared unrelated to ihe class of the immitnoglobulin present on the cell surface. Non B-cell lines were never found to form such rosettes. The capacity of B-cell tines to form mouse erythrocyte roscUes was further demonstrated to be relatively insensitive to trypsin treatment, and results are presented suggesting some degree of cell cycle dependency of the expression ol' this murlter. J. Berioglio. Lahorotoire J'tmmunotogle ft df Candroh^ie Expfrimentate, INSERM FRA 24, Centre Leon B^rard. 2S rue iMcnnev. Mi7i Lvon Cfdvx 2. France.
It has been recently demonstrated that rosette formation wi(h mouse erythrocytes is a marker for hutiian B-type lymphocytes [II. 25]. Previous reports from this laboratory [4] and from other investigators [10] would suggest that only a subpopulation of normal B lymphocytes possesses this membrane property. Furthermore, among B-type malignant haemopathies, only cells from typical chronic lymphatic leukaemia have been reported to form mouse erythrocyte rosettes [9. 26]. and this property has been considered useful in making a dilferential diagnosis between chronic lymphatic leukaemia and lymphoma [3. 5]. As an approach to further define subpopulations of normal B cells, we have investigated tiiouse erythrocytes (MRBO rosette formation by human lymphoid cell lines (LCD. The vast majority of long-term cell lines derived from peripheral blood of cither normal donors or patients with various lymphoproHferative disorders share characteristics with B lymphocytes [13. Ift.24]. They usually e.\hibit one or more normal B-cell markers, which have already 0300-9475/79/0800-0153 S02,00
been used in attempts to characterise homogeneous populations of human cell lines [27]; these litics are assumed to represent the in vitro counterpart of the normal B-cell subsets they originated from, or various steps in the differentiation process [20]. The results presented below indicate that some human lymphoid B-celt lines tnay form MRBC rosettes and that this property may help in subciassifying them in three groups. Furihermore. it appeared that MRBC rosette formation is not associated with membrane expression of any given class of imtnunoglobulin and tnay depend on the cell cycle phases. MATERIAL AND METHODS Lymphoid tell lines Eighteen lymphoid cell lines established cither from normal donors or from paiients with various malignant haemopathies have been used iTable I). The wdlcharactcri/ed Molt-4. Raji, P3J and Dnudi lines were obtained from G. de Th6 at the I ARC. Lyon: the K562-4 line, initially established hy B. Lo7.7io. und the RI'MI 74S1. MIO and 8.122 hncs were obtained by
r 1979 Blackwell Scientific Publications
153
154
J. Hertoiilio, B. Laidjim A J. F. Dore
courlcsv ol" N. Carpentier, Geneve; ihe LHN-1.^. Pon. Del and Reh-6 lines were obtained rVom ICIG in Villcjuif; and Cot-3, Boe and PTL line.s were established in this laboratory. C20I and C2O2 were established Ironi normal cord blood lymphocyies by lipstein-Barr virus^(lW5 strain) infeciion ;ind were u gifl from C. Desgranges (lARC. Lyon), and ihc KM.3 line was obtained Ironi U. Schneider (hrlangcn). Suspension cultures ol" ihese cell lines were propagated in cotnpiele culture medium consisting of RPMI IA40 wiih 10-15",. hcai-inactivmed Tetal calf serum. 50|xgslreptomycin/inl. and KKt units penicillin ml. Membrune marker determination was routinely performed 11 h after ihe last suhi.ijlttjre. For MRBC rosette formation studies according to the cell growth, cells were seeded al 10" cells ml after having been maintained for 24 h at siituration density. 4 • II)" cells were then harvested every 24 h and processed tor roseite formalion. Cell viability was assessed by trypan blue exclusion. Enzymatic treatment of cells Seuraminiduse treatment. 4 • 10" washed cells were treated for 30 min al .17 C with either 2.5 or 5 units'K>^ cells of Vihrio cholera neuraminidase (Bcrhingwerke). The cells were then washed thrice in Hanks' balanced salt solution (HBSS) and processed for roscilc formation. Trypsin treatment of cells. ATlcr ihree washes in serum-free medium, 20 •: 10" cultured cells were incubaled for 5-10 min at 37 C with O.OI", lo O.T^ Irypsin solution (Gibco), then washed thrice in cold complete medium, adjusted to 4 ' 10" cells/ml. and processed for rosette formation. Membrane tnarkers Detection of C3 receptor by EAC rosette formation. EAC complex formation was performed by incubating a0.5''o sheep erythrocyte (SRHC) suspension for 30 min at 37 C in an equal volume of a subiigglutinaling dilution of rabbit anti-shccp erythrocyte IgM puritied by chroma to graphy on a Sephadex G-200 column. After three wiisbcs. sheep erylhrocyle-IgM complexes were adjusted to 5 • 10" ml and further incubated tbr 30 min at 37 C with fresh C57Bt6 scrum diluted 1:5 in barbital fVeronal*) bulTer (0.005 M. pH 7..1). After three more wa-shes. EAC complexes were adjusted to KP/ml. stored at 4 C. and used within a week. EAC rosette formalion was performed bj incubating 10" cells in 50 \i\ of HBSS with 50 (il of t A C complex suspension for .10 min at M C. After proper dilution, the percentage of rosette-forming cells was counted in an hiicmocyiomcier. Each lest was done in triplicate atui controls were performed using unsensili^^cd SRBC and SKBC" IgM complexes. ImmtinofluoreMituf detection of surface immiinogli'htdins. 10" cells were pelleted in 3 ml plastic test lubes and resuspended in 50 jil of a proper dilution of tiuorescein isothiocyanate (KITC)-lnbelled globuhn fraction from monospeciflc goat antisera to heavy or light chain (Meloy Laboratories Inc.). After 30 min incubation on ice. three washes were performed in a large excess of cold HBSS. The cells were suspended in biilVercd gl>ccrol, layered on microscope slides, and the percentage iif positive cells counted by using a Leitz onhoplan microscope.
Formation of mouse frythrofvte rti.\pttes. The technique originally described by Stathopoutos |26I for demonstration of mouse crythrocyie rosetlcs was jpplicd. Briefly, to 25O|j.l of a cell suspension (4 •• lO'/ml in HBSSi 50 [xl of MRBC-ahsorbed tetal calf serum and 250 i.tt of a !"„ suspension in HHSS of freshly drawn C57B16 mouse erylhrocytes were added. The tni.ntiire was incubated for 10 min al 37"C. centrifugcd 5 min at 400 g. and further incubated for I h at 4 C Each test was done in triplicate and the percentage of cells forming rosettes with mouse crythrocytes was then counted in a haemocytomctcr after gentle resuspension. Any cell bearing three or more erylhrocytes bound to its membrane was recorded as M R BC-rosette positive.
RESULTS \fou.\i' erythrocyte rosette formation by human lympliohla.stoid cell titles Results of ihe mouse eryilirocyic rosette formation test, together with those of the standard tests for B-cell markers from eighteen LCL are presetited in Table 1. It lirst appeared that both the Molt-4 line, which is thought to be of T-cell origin [2. 21], and the K562 line of myeloid origin, which laeks any of the T or B usual markers 115. 19]. did not form rosettes with mouse erythrocytes. Nine other cell lines, whose B nature cottid be assessed with regard to other markers, did not form MRBC rosettes. Only cells frotn six out of fifteen B-cell lines under study appeared lo form a significant percentage of rosettes with MRBC. In none of these lines could more than 50% cells be demonstrated to form roseltes with the technique used. It is noteworthy that not only cells from lines exhibiting membrane ii-ehain showed this property, so thai among these six mouse erythrocyte roselting lines all types of" heavy and light chains were represented. For each considered line, the percetitage of rosetting cells was relatively reproducible provided the cells were harvested at a constant time after they have been subcultured. Furthermore MRBC rosette-forming capacity appeared to have good stability in long-term culture, since two batches of the normal-derived LHN-t.l line, frozen at a 3 year interval, formed the same percentage of MRBC roseltes when thawed on the same day and further cultured for 2 weeks before being tested.
Mouse Erythrocyte Rosettes on Lymphoid Lines
155
TABLK I. MRBC rosctie formation by LCL
Cell line
Origin*
LHN-13 C-201 C-202 KM-3 REH PTL MOl.T-4 RI»Mi-MIO R I'M 1-8.122 COT-3 BOE K 562-4 RPMI-7481 DEL PON P?J DALDI RAJI
PBL CBL CBL c-ALL c-ALL c-ALL T-ALl. AML AML AML AMML CML CML LS LS BL BL BL
r-AC-RKCJ M-RFC§
Membrune imniunoglobulint Y
a
0 0 0 0
80
0
0
0
0
0
0
0
(1
22 17 0 0 60 0 20 0 0 0 0 30 0 0 0 100
0 0
1)
I)
hO
0
0
(1
60 0 0 7 30 0 0
0
0
11
0
0 0 0 0 0
K
>.
0 — 8 0 0 0 0
0 0 0 0 0 — 0 0 0
70 — 0 0 0 80 0 80 0 0 50 0 30 -_ 70 0 100
0
0
0 — — 0 0
60 0 0
0
80 0 0 0 0 — 0 0 0 5
38 61 49 0 0 57 65 85 90
9 0 0 0 0 16 0 0 22
68
43
80 0 37 0 45 0 27 75
I« 0 0 0 0 0 0 50
• T or t - A L L acute lymphoblastic leukaemia wiih T or non-T. non-U menibnine phcnoiypc; CML=chronic myelogenous leukaemia; BL - Burkitl's lymphomii: LS lymphosarcoma^ A M L acute myeloblastic leukaemia: A M M L acute myelomonocytic leukaemia; CBL-EBV-transformed cord blood lymphocytes; PBL peripheral blood lymphocylcs from a normal donor. t Percentage of cells reactive wiih monospecific antisera. J Percentage of cells forming rosettes with FAC complexes. § Percentage of cells forming rosettes with mouse erytbrocytcs.
Effect of enzymatic treatment of lymphoid cell lines on rosette formation To attain further insight Into the nature of MRBC binding of LCL. we invesligated the effects of trypsin and neuraminidase treatment of
the cells
an
MRBC
rosette
formation.
Trypsin treatment of three MRBC rosetting lines showed that this membrane property is quite resistant to such an enzyme treattnent (Table II). None of the enzyme concentrations
used (0.01-0.1°-;;) was able to destroy the capacity v>\' Raji cells to form MKBC rosettes. Furthermore, the trypsin concentrations needed to completely inhibit MRBC rosette formation by Col-3 and PTL litie were much more important than those usually ellicicnt in oiher types of T- or B-cell markers; Peliegrino ci al. [22], for instance, have reported that rosette formation with monkey erythrocytes could be inhibited by 0.01";, trypsin concentration, and we have found that this concentration was also
TABLE (I. The effect of trypsin treatment of lymphoid cell lines on the percenta^ of rosette-forming
Trypsin concentration ("„ of stock solution) Cell line RAJI COT-3 PTL
0
0.01
55 54
40 33
17
17
Mean of two experiments.
0.02
0.05
0.1
44 20 —
40 4
42 0 —
8
156
J. Berfoglio, B. Laidjim A J. F. Dore
sufficienl to abolish EAC rosette formation by the LCL under study (dala not sliovvn). Almost all types of rosettes that have been described between human lytnphocytes and erythrocytes from various animals have been reported to be modified by neuraminidase treatment of either erythrocytes or lymphocytes [8]. Lytiiphocyte pretreatment wiih neuraminidase has been described to increase dramatically the percentage of MRBC rosetteforming lymphocytes In peripheral blood [10, 23J. In this study neuraminidase treatment of cells from cultured cell lities generated various types of results aecording to the capacity of the untreated cells from the lines to spontaneously form MRBC rosettes. Three groups of B-LCL could be identified by this procedure (Table 111). Group 1 is composed of LCLs whose cells formed spontaneous MRBC rosettes. In this group neuraminidase treatment had no significant elfect or slightly increased the percentage of rosetting cells, Group If is composed of LCLs whose cells formed MRBC rosettes only when pretreated with neuraminidase. and
group III contains the fines whose ceffs could not be demonstrated to form such rosettes even after neuraminidase treatment. No correlation seemed to appear between Ifiese three groups atid the expression of other B-cell markers. f--vidence that neuraminidase treatment did not induce rosette formation tfirough nonspecific interactions could be drawn frotn the observations that neitfier the T-type Moft-4 nor the non-lymphoid K562 could be indueed to form MRBC rosettes; furthermore, we verified that this treatment did not enable B-celi lines to form rosettes with sheep erythrocytes. Effect of cell growth on MRliC rosette fortnation hy LCL The expression of most of tlie membrane properties of euliured cells has been reported to depetid on the cell cycle [7, 14. 17. 18]. This phenomenon could account for ihe rather broad variations in the ability to bind MRBC of cells from some of the lines under study, when repeatedly tested at difl"erent times. As
Ml. The effect of neuraminidase treatment of lymphoid cell lines on MRBC rosette formation
Units of 111
Cell line Group 1 COT-3 RAJI PTL BOF
RPM 1-8322 1-HN-I.T Group II P3-J DAUDI REH
C-201 C-202 DEL Group III RPMI-7481 RPMI-641()
2.5
43 SO 16 16 22 9
56 4S 29 18 52 17
51 47 35
I9.S 39.3 28 12 27 8
11 38 33 32 29 13
1 0 0 0
1 0 1 0
0
0 0
0 t)
0 0 0 0 0 0
PON
0
KM-3
0
Non-B LCL K 562-4 MOlT-4
ISC 10" cells
0
0 0
0
5
34 56 32
Mottse Erythrocyte Rosettes on Lymplwid Lines
157
-40
-30
-20
-40
-30
-20
24 TIME AFTER
48 72 SUBCULTURE
96
FIG. I. Relationship heiween mouse cryihrocytc (MRBCt rosettes (•) nnti growth ol' cells in culture (0). (.A) Cells from PTL line; (U) cells from Col-3 line.
iin approach to study such variaiions of the expression of MRBC roseltc formation, we studied the percentage of rosette-forming cells according to the culture conditions for two ditTercnt LCL. In six ditTcreni experiments using Ihe PTL line, the percentage of rosetteforming cells appeared to significantly decrease within 24 li after Ihe culture had Ix'en split from saturation densily down to 10" cells.nil. As the cells progressively multiplied, ihc percentage of roscite-form ing cells increased and eventually reached its starting value (Fig. lA). In conirast. no variation could be demonstrated under the same conditions using Col-3 tine (Fig. lBj.
DISCUSSION Several reports have now established thai MRBC rosette formation is characteristic of a subpopulation of normal B-cells [4, II, 25]. and it first did not appear unlikely that MRBC rosetting lymphocytes were those that exhibited membrane immunoglobulin |i-chain [10]. The study of patients with imtiiunodeliciencies has conftrnied these results, and the study of both inali^nanl haemopathic!: and fetal liuman liver
has suggested that MRBC rosette formation may occur at some given stage of maturation [6. 12]. In the present study we investigated the formation of MRBC rosettes by hutnan lymphoid cell lines. The results from both the T-typc Moli-4 line and from the non-lympholdderivod K562 line are in agreement wiih ihe B nature of MRBC rosetting capacity. However, even though all of the MRBC rosette-forming lines are B-derived, not all the B-derived lines may form rosettes with MRBC. Furthermore, live non-rosetting lines have been shown to possess membrane ^-chains, and among the MRBC rosette-form ing lines, two had no delectable or very few membrane immunoglobulins. whilst all types of light anil lieavy chains could be demonstrated in the others. These findings are not in complete agreement with results from MRBC rosette formation tests in normal peripheral blood lymphcKytcs. Either of two hypotheses could explain these discrepancies: (1) non-lgM-bearing but MRBC rosette-forming lymphocytes may exist at so low concentration in peripheral blood that they are untletectable; (2) an alternative and more attractive hypothesis is that iti vitro culture conditions may modify the cell membrane so
158
J. Bertoglio, B. Laidjim & J. F. Dore
that MRBC 'receptors' are expressed. .Another finding is that, although only normal peripheral blood lymphocytes and cells from chronic lymphatic leukaemia may form MRBC rosettes, whilst cells from acute lymphoblastic leukaemia may not. no correlation arose between MRBC rosctting capacity and the normal or Icukaemic origin of the lines. The latter hypothesis would also account for this finding. En/yme treatment has been extensively used lo understand the mechanism of various types of rosettes. Surprisingly, MRBC binding to LCL cells appeared almost unaflected by trypsin treaimeni of cells even though the concentration ti>cd has been reported to completely inhibit other types of rosettes. It is noteworthy, although it may not be related at alt to this finding, that the la antigen molecule on B lymphocytes has also been described as rather resistant to trypsin treatment [1]. It was not possible to use higher concentrations of trypsin because of poor viability obtained and clustering iif cells induced by DNA rcsiiUio.. In contrast, neuraminidase treulnicni of LCL cells gave interesting results: neuraminidase has been reported to enable nearly all normal B lymphocytes to form MRBC rosettes [10]. and these findings are partly confirmed, in that cells from six B-cell lines that did not form spontaneous rosettes were induced to form MRBC rosettes, after pretreatment with neuraminidase. However, four tnore B lines were not atTected by eitlier of the two neuraminidase concentrations used. Potter & Moore [23] have rai-sed the possibility that in peripheral blood, immunoglobulin-positive MRBC-negative cells may represent non-B cells carrying adsorbed immunogiobulins. Such an explanation can be ruled out for in vitro cultured cell lines, and our experiments suggest that true B lymphocytes that do not form MRBC rosettes may exist. On the basis of this marker, three groups couki be identified among B lymphoid lines; since such lines do not represent resting B-cells but cells at an arrested state of differentiation it is questioned whether these groups, and especially groups I and II. which do notfitwell with groups determined on the basis of other membrane markers, may represent very close and so far unrecognized steps in the dilferentiation process of B-cells. Cell cycle dependency of most of the membrane markers of LCL have been widely
investigated. Such a phenomenon could account for the finding that in none of the cultures studied could tbe percentage of cells spontaneously forming rosettes be shown to exceed 50"/,,. In one line the percenlage of MRBC rosette-forming ceils has been shown to vary during growth of unsynchroni/ed cultures, decreasing just after subculture and increasing until saturation density has been reached in tbe culture. Since cells are known to gather in GI phase when reaching saturation density, these results would suggest some GI dependency of the expression of this marker. The relative decrease of the percentage of roselte-lorming cells after 96 h of culture may have reflected starvation effects. Lack of sufficient media nutrients and 'crowding' could have been affecting macromolecular synthesis and conM:t|uently MRBC rosetting capacity. In another tine such variations could not be demonstrated, so it could be hypothesized that the cell cycle dependency of MRBC rosetting capacity may itself depend on the cell cycle characteristics of each line. Further experiments, using synchronized cultures and complete analysis of the cell cycle characteristics of each line, are now needed.
ACKNOWLEDGMENTS The authors thank Mrs Christiane Pinatel for her skilful technical assistance. This work was partially supported by a grant from f)clcgation G6neralc a la Recherche Scientifique et Technique (75.7.1369).
REFERENCES 1 Anderson, J.K- & M c t / ^ r . R.S. Dctcciiun and purliiil characterization of humun T lymphocyte mcnihriinc untigcii.s with ie rosetle tesis: elfeci of enxymulic modification of sheep erylhrocytes (E) and the specificity of neuraminidase-lreaicd F. Cell. Immunol. 24, 97. 9 Gupta. S.. Good. R.A. & Sicecl. F.P. Rosette formation with mouse erylhrtKyies. 111. Studies in patients with primary immunodeficiency and lymphoproliferalive disorders. Clin. i.\p. Immunol. 26. 204. 1976. 10 Gupta. S.. Good. H.A. & Siegla. F.P. Rosetle formation with mouse erythrocyies. II. A marker for human B and non-T 1> mpht>cytes. Clin. exp. Immunol. 25. 319. 1976. 11 Gupta. S. & Grieco. M.H. Roseitc formation with mouse eryihrocyies: probahle marker for human B lymphocytes. Int. Arch. Allergy iippl. Immunol. 49. 734. 1975, 12 Gupta, S.. Pahawa. R.. O Rcilly. R.. Gootl. R.A. & Siegal. F.P. Ontogeny of the lymphtx-yte subpopulations in human lelal liver. Prtic. nat. Acad. Sei. (USA). 73. 919. 1976. 13 Hotiig, R.. Bitting. R.. Matsu>uma. M. & Terasaki. P.I. B lymphocyte alloantigen specificities present on cultured lymphoblasioid cell line^ J. Immunol. 117. l.^tJO. 1976. 14 Killandcr. D.. Klein. F. & Levin. A. Expression of memhraiie-hound IgM ami HL-A iintigcns on lymphohlastoid tells m dilltreru stages of the toll cycle. Europ. J. hnmunol. 4, 327. 1974. 15 Klein. E.. Ben-Bassat. H.. Beumann. H.. Ralph, P.. Zeuthen. J.. Polliack. A. & Vanky. F. Properties of the K562 cell line, derived from u patient with chronic mycloid leukemia. Int. J. Cancer, 18. 421. 1976. 16 Klein. G . Lindahl. T.. Jondal, M.. Liebold. W.. Mcnezes. J.. Nilsson. K. & Sundstrbm. C. Continuous lymphoid cell lines with characteristics of Bcetts (hone marrow-derived), lacking the EpsteinBarr virtis Rcnome and derived from ihree human
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lymphomas. Proc. nai. Acad. Sci. (VSA), 71, 3283. 1974. Krakowka. S,. Olscn. R. & Cockerell. G. The elTcct of cell synchronization upon the detection of T and B lymphoid cell receptors on two contitiuous lymphoid cell lines. In Vitro. 13. 119. 1977. Kwork. S.Y. & Litwin. S.D, Immunoglobulin expression of cells from human lymphoblastoid lines. Cell. Immunol. 25. 256. 1976. Lozzio. C B.. Lo?7io. B.B.. Yang. W.K., lehiki. A.T. & Bamherger. E.G. Absence of thymusderived lymphocyte markers in myelogenous leukaemia (Ph, ' } cell line K562. Cancer Res. 36, 4657. 1976 Minowada. }. Markers of human leukaemia lymphoma cell lines reflects haematopoietic cell difIcreiiliation. p. 137 in Serrou, B. & Ronenl'eUI. C. (cds.) Human Lymphocyte Differentiation: irs Applivaiion to Cancer. North-Holland. Amsterdam, 1978. Minowadu. J.. Ognuma. T. & Moore. nius derived lymphocyies. J. nai. Cancer hist. 49. 89t. 1972. Pellegrino. M..A.. Ferrone. S. & Theiililopoulo-i. A.N. Rosetle formation of human lymphoid cells with monkey red blood cells. J. hnmunol. 115. 1065. 1975. Potler. M.R. & Moore. M. Chnrtictcn/iilion und separation of human lymphocytes forming mouse red cell roseltes. J. immumtl. Methods. 19. 125. 1978. Rosetifeld.C.Goutner, A.. Venuat. A.M.. C'hoquet. C Pico. J.L.. Dore. J.F.. l.iaheiif. A.. I>urandy. A.. Desgranges, C. & de The. G. An elTective human Icukaemic cell line: Reh. Europ. J. Cancer, 13, 377. 1977. Stathopoutos. G. A Da%ics. A.J.S Human lymphocytes and mouse red cells, l.umit. 1, 1078. 1976 Staihopoulos. (i. & Elliott. l-.V. Formation of mouse or sheep red-blood-celt rosettes hy lymphocytes from normal und Icukaemic individuals. Lancet. I. MO. 1974. Young. W.J.. Utsingcr.P.D.. Hutt, L.M.. Buchanan, P.D.. Korn. J.H., Fuller. C.R., l.ogue. M. & Pagano. }.$. Subpopulation.s of human lymphoblastoid cell lines; correlation with the e\pression of surface receptors and content of Epstein-Barr virus genome. Scand. J. hnmtmol. 5, 795. 1976.
Received 5 February 1979 Received in revised form 25 April 1979
