A human lymphoid cell line with receptors for both sheep red blood cells and complement

CELLITI.AK

IMl\l

UNOLOGY

14, 139-145 (1974)

A Human Lymphoid Cell Line with Receptors for Both Sheep Red Blood Cells and Complement WILLIAM

I$T~~~l

AND RONALD B. HERBERMAN~

Received

Febmary

15,1974

The human lymphoid cell line MOLT 4, from a patient with acute lymphocytic leukemia, was initially considered to be derived from T lymphocytes, on the basis of rosette formation with sheep erythrocytes (E). This cell line has now also been found to form rosettes with sheep erythrocytes sensitized with rabbit antibody and mouse complement (EAC). Evidence is presented that the formation of both E and EAC rosettes is due to two separate receptors on the MOLT cells: (a) E.4C rosettes were formed more rapidly and were more stable than E rosettes ; (b) preincubation of MOLT with an EAC membrane preparation inhibited rosetting with EAC and not with E; (c) MOLT formed rosettes with EAC prepared from trypsinized E, but did not bind to trypsin-treated E alone. The implications of this finding, in regard to the derivation of this cell line, are discussed.

INTRODUCTION Lymphocytes have been divided into two main categories, thymus-derived (T ) cells and bone marrow-derived (B) cells. Evidence has accumulated that human T cells have surface receptors for sheep erythrocytes (E) and form spontaneous E rosettes in vitro (l-5). Human B cells have also been identified by characteristic cell surface markers (1, 6, 7). The B cell receptor for complement component, C3, has been most frequently detected by rosette formation with sheep erythrocytes sensitized with rabbit antibody and mouse complement (EAC). These rosette assays have been used to classify human leukemias and lymphomas, and lymphoid tissue culture cell lines as to their probable derivation. Most of the lymphoid cells from patients with chronic lymphocytic leukemia formed EAC rosettes (7, 8 11 whereas the cells from some but not all patients with acute lymphocytic leukemia (9, 10) and lymphomas (11) formed E rosettes. Most of the human lymphoid cell lines have been thought to be derived from B cells, since they form EAC rosettes (8). However, Minowada it al. (12) described four cell lines, designated as MOI,T 14, derived from a patient with acute lymphocytic leukemia, which formed li rosettes and were therefore presumed to be derived from T cells. In performing studies of the proportions of T and B cells in the peripheral blootl of cancer patients, we attempted to use cell lines, which formed either E or EAC: 1 Present address : Department of Medicine, University of Tennessee, 2 To whom correspondence and reprint requests should be sent. 139 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved

Memphis,

TX.

140

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TABLE

1

VARIATION IN ROSETTE FORMATION WITH MOLT-4 Passage history

CELL LINE

Date of tests

y0 of cells” forming rosettes with E

Obtained

from D. Buell, January

1973

January-May

Obtained

from D. Buell, January

1973

September-December

Obtained

from D. Buell, October

1973

October-December

EAC

1973 $95) 1973 (1~to) 1973 (2f8)

MOLT 4-B December MOLT 4-B, December a Mean

from J. Minowada, 1973 Clone 2 from J. Minowada, 1973

December

1973

December

1973

(22v29) (303-337)

(range).

rosettes, as homogeneous positive controls. It soon became apparent that the MOLT 4 cell line forms both E and EAC rosettes. We thereupon initiated a study on this cell line to determine whether it had two separate membrane receptors. MATERIALS Lymphoid

AND

METHODS

Tissue Cul&.we Cells

The MOLT 4 cell line was obtained from Dr. Donald Buell, National Cancer Institute, and also directly from Dr. Jun Minowada, Roswell Park Memorial Institute (Buffalo, NY) (Table 1). The characteristics of the other cell lines used in this study are given in Table 2. The CCRF-CEM cells were obtained from Dr. Paul Gerber, Bureau of Biologics, Food and Drug Administration. The 70BM-2 cell line was obtained from Dr. George Klein, Karolinska Institutet (Stockholm, Sweeden). All cell lines were maintained in culture at 0.5-2 x 10“ cells/ml in RPM1 1640 medium supplemented with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml of penicillin, and 100 pg/ml of streptomycin. The cultures were fed and split every 2-4 days. Before use in the rosette assays, the cells were washed twice in phosphate-buffered saline, pH 7.4 (PBS), and resuspended in RPM1 1640 at 1 x lo6 cells/ml. E Rosette Assay Sheep erythrocytes were washed twice with PBS and resuspended in RPM1 1640 at 1 x 108 cells/ml, and were then added to an equal volume of tissue culture cells in 13 x loo-mm plastic tubes. The cell mixtures were left undisturbed at room temperature for 2 hr. Then 0.1 ml of 0.2% trypan blue was added and the tubes were gently rocked for 15 set in the cup of a variable-speed vortex mixer (Scientific Products), at its lowest setting, to resuspend the cells. To determine the percent of rosette-forming cells, 200 viable lymphoid cells were counted in the chamber of a hemocytometer. A lymphoid cell had to be surrounded by at least three erythrocytes to be called a rosette.

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TABLE

2

KOSETTE FORMATION WITH OTHER ld~~~~~~~

<‘’ of cells forming .c rosettrs with ---.-~--.-

Characteristics

-.~

CEIL IJWS

E:

l-XC

0

lU0

0

5

70

0

-.~

From patient with Burkitt’s lymphoma; contains EB virus genome From patient with acute lymphocytic leukemia; no EB virus genomc Monkey cell line, transformed by Herpesvirus saimiri

EAC Rosette Assay Washed sheep erythrocytes, suspended in RPM1 1640 at 2 X 108/ml, were incubated for 30 min at 37°C with an equal volume of 19s rabbit anti-E (A, obtained from Cordis Laboratory, Miami, FL and further purified on Sephadex (i-200, used at 1 : 250 dilution in RPM1 1640). The resulting EA was washed twice with PBS and then mixed with an equal volume of fresh BALB/c mouse serum, diluted 1 : 10 in RPM1 1640, and incubated for 30 rnin at 37°C. The EAC complexes lvere washed twice with PBS and then resuspended in RPM1 1640 at 1 X lO*/ml. To form EAC rosettes, 0.1 ml of tissue culture cells and 0.1 ml of EAC were mixed and left undisturbed at room temperature for 2 hr. Cells were then resuspended and counted by the same procedures described above for E rosettes. In tests of peripheral blood lymphocytes of 100 normal individuals, an average of 1170 of lymphocytes formed EAC rosettes. IYthibition Assay z&h Disrupted EAC The EAC suspensions were centrifuged at 2oOg for 5 min and the pellet was suspended in 0.25 ml of Ficoll (9% in water). This resulted in complete lysis of the cells. Then 0.1 ml of Hypaque (34% in water) and 0.25 ml of RPM1 1640 were added. One milliliter of MOLT cells (1 X 106/ml) was centrifuged at 200g for 5 min, and the cell pellet was resuspended in the suspension of disrupted EAC cells and incubated for 2 hr at room temperature. As controls, MOLT cells were incubated with disrupted E or in the Ficoll-Hypaque-RPM1 1640 solution without added EAC. After incubation, cells were washed twice with PBS and then tested in the rosette assays. Preparation

of Tryps-inized E

Three-tenths-milliliter aliquots of E were centrifuged at 2OOg for 5 min, and the pellet was resuspended in 5 ml of a solution containing 0.25% trypsin. After incubation for 4 hr at 37”C, the cells were washed three times with PBS and resuspended in RPMI lG40. These cells were then used to prepare EAC, or tested directly in the E rosette assay.

142

FIG. 1. Kinetics (O---O).

SHORT

of formation

COMMUNICATIONS

of rosettes at 22°C between

MOLT

and EAC

(0-O)

or E

RESULTS Rosette Formation of MOLT

Cells with E and EAC

The MOLT cells were found to form rosettes with both E and EAC. As previously reported by Minowada et al. (la), E rosettes formed at room temperature and at 37°C. Rosette formation with E and EAC was highest at room temperature, and therefore most of the studies were performed at this temperature (approximately 22°C). Figure 1 shows the kinetics of rosette formation. Many EAC rosettes were formed within 15 min and a plateau was reached by 45 min. Rosette formation with E was a slower process, and a plateau was not reached until 90 min. If, instead of the usual gravity sedimentation of the MOLT cell-erythrocytes mixtures, they were centrifuged together as in the routine assays for rosette formation with peripheral blood lymphocytes, no subsequent incubation was needed for maximal rosette formation. VaGation in Rosette Formation with MOLT

Cells

As shown in Fig. 1, the initial studies with the MOLT cells showed a very high proportion of the cells forming both E and EAC rosettes. Later tests, with cells grown under the same conditions, gave considerably lower percentages of rosettes. Therefore, additional flasks of MOLT cells were obtained and tested (Table 1). The four different stocks of cells, even when tested at the same time, gave considerably different results. However, none gave the high percentage of E rosettes which were originally seen. Rosette Formation -z&h Other Lynzphoid Cell Lines As suggested by Mendes et al. (13), some or all of the EAC rosettes obtained with the MOLT cells could have been due to binding to the E receptors. To determine if both types of rosettes would be seen with other lymphoid cell lines, three cell lines were selected for study under the same conditions used for the MOLT cells (Table 2). The 70BM-2 cells gave a high percentage of E rosettes yet formed no rosettes with EAC. Another cell line from a patient with acute lymphocytic leukemia, CCRF-CEM, was studied because it was found to lack detectable EB virus genome (14) and might therefore also be expected to be derived from T cells rather than B cells. This line formed no E rosettes, and 5% of the cells formed

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FACTORS

AFFECTING

143

CO~IMLJNICATIOWS

E AND EAC

ROSETTE

FORMATIOK

WITH

MOLT CELLS “; of cells forminp rosettes with _---. -- --

No mechanical agitation Mechanical agitation of MOLT-erythrocyte MOLT cells preincubated with media control membranes from 0.5 X 10B EAC cells/ml membranes from 1.0 X lOa EAC cells/ml membranes from 1.5 X 108 EAC cells/ml No enzyme treatment of E Trypsin treatment of ED

mixture”

E

E:\C

96 13

98 86

30 27 29 27 86 5

60 52 26 ‘2 97 96

G Five-second agitation in cup of Scientific Products variable-speed vortex turned to highest setting. Values of experimental and control are means of three tests. * Erythrocytes treated with 0.255 crude trypsin for 4 hr at 37°C before preparation of UC or before use in rosette assay.

EAC rosettes. The Raji cell line, previously found to form EAC rosettes (,8), showed 100% rosette formation with EAC and none with E. With several other lymphoid cell lines tested, either E and EAC rosette formation, but not both, were seen. Fa,ctors Affecting

E and EAC

Rosette Fownation

with MOLT

Cells

To obtain further evidence that the formation of both E and EAC rosettes with MOLT cells were independent processes, a series of experiments were performed to look for factors differentially affecting rosette formation (Table 3). The EAC rosettes were found to be considerably more stable. After vigorous agitation. most of the EAC rosettes remained, whereas the percentage of E rosettes dropped to low levels. If binding of EAC was due to a complement receptor on MOLT cells, one would expect to be able to inhibit EAC rosette formation by prior incubation with EAC membrane fragments. The observed results fit this expectation fairly well. Increasing amounts of disrupted EAC resulted in a progressive decrease in EAC rosette formation. Comparable preincubation with disrupted E had no effect on EAC rosette formation (not shown). Erythrocyte rosette formation was not inhibited by prior incubation of MOLT cells with EAC. Bianco et al. (15) recently reported that treatment of E with trypsin interfered with E rosette formation but had no effect on the Forssman antigen sites, and therefore EAC could be prepared from such cells. Rosettes formed with trypsinized EAC therefore would have to be due to attachment to the receptor for complement and not due to interaction with the receptor for E. When this was tested with the MOLT cells, very few rosettes were seen with trypsinized E, but a high proportion of MOLT cells still formed rosettes with trypsinized EAC.

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DISCUSSION The MOLT cell line was the first human lymphoid cell line to be identified as possibly derived from T cells. Since then, Minowada and Moore (16) have found another cell line, 8402, also derived from a patient with acute lymphocytic leukemia, which had similar characteristics. In addition, several monkey lymphoid cell lines derived from Herpesvirus saimiri (HVS) transformed cells have been shown to be form E rosettes, lack surface immunoglobulin, and not form rosettes with EAC (17). This study presented evidence that receptors for EB virus are characteristic of human and monkey B cells, and that receptors for HVS are characteristic for T cells. However, it appears from the present study that human lymphoid cell lines lacking EBV genome can not be presumed to be T cells but may also be “null” cells, lacking both E and EAC receptors. Our findings that the MOLT cell line can form both E and EAC rosettes adds even a more important complication to the categorization of human lymphoid cell lines as derived from B or T cells, on the basis on their surface receptors. While our study was in progress, we learned of similar findings by Jondal and Klein (17). Minowada (18) has also recently confirmed that MOLT forms EAC rosettes. The main issue related to this observation is whether the MOLT cell line actually has two independent receptors, one for E and the other for complement. It was important to rule out the possibility that both E and EAC bond to MOLT via the receptor for E. The evidence for two independent receptors is the following: (a) During the course of these studies, some sublines of MOLT showed decreased ability to form E rosettes, but the percentage of EAC rosettes remained high. In fact, with some tests, the percentage of EAC rosette formation was higher than that of E rosettes. The methods used in this study did not allow us to determine whether an individual cell could form both E and EAC rosettes. However, the tests with a cloned subline of MOLT, which showed approximately equal numbers of E and EAC rosettes, indicated that the results were not due to a mixture of two separate cell populations. (b) Another cell line, 70BM-2, which formed a high percentage of E rosettes, did not form any rosettes with EAC. (c) The binding of EAC to MOLT was shown to be more rapid and to be more resistant to agitation than the binding of E. (d) Preincubation of MOLT cells with disrupted EAC inhibited subsequent rosetting with EAC, but did not interfere with the formation of E rosettes. (e) The MOLT cells formed rosettes with EAC prepared from trypsinized E but did not bind to trypsinized E. (f) The MOLT cells did not form rosettes with EA, prepared as in (8). Jondal and Klein (17) had the same findings. With the above evidence that the MOLT cell line has separate receptors for E and EAC, the derivation of this cell line from T cells is called into question. It is quite possible that the MOLT cell line is derived from a different population of lymphocytes, which have both receptors. Shevach et al. (19) have recently found both receptors on a high percentage of the neoplastic cells of a patient with leukemia and have also found that approximately 2% of normal human lymphocytes have both receptors. Similarly, Bentwich et al. (20) found that 2-40/o of normal lymphocytes formed E rosettes and also had detectable surface immunoglobulin. Although tests for cell surface receptors on normal lymphocytes have been useful for classification, results of such tests on neoplastic lymphoid cells and on lymphoid

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cell lines must be interpreted with caution. These abnormal lymphoid cells may lose normal receptors and appear to be “null” cells (8, 21). Alternatively, these less differentiated lymphoid cells may express receptors not present on the normal cells from which they were derived, similar to the expression of embryonic antigens (22) or production of hormones (23) by tumor cells. Because of these possibilities, it is not possible to determine whether the MOLT cell line is derived from a T cell and is simply expressing an extra receptor, or whether it is derived from a lymphocyte which normally expresses both receptors. REFERENCES 1. Silveira, N. P. A., Mendes, N. F., Tolnai, M. E. A., J. Zmmunol. 108, 1456, 1972. 2. Lay, W. H., Mendes, N. F., Bianco, C., and Nussenzweig, V., Nature (Loxdon) 230. 531, 1972. 3. Brain, I’., Gordon, J., and Willetts, W. A., Cl&t. Exp. Zmmunol. 6, 681, 1970. 4. Wybran, J., Carr, M. C., and Fudenberg, H., J. Clin. Invest. 51, 2537, 1972. 5. Jondal, J., Holm, G., and Wigzell, H., J. Exp. Med. 136, 207, 1972. 6. Bianco, C., Patrick, R., and Nussenzweig, V., J. Exp. Med. 132, 702, 1970. 7. Ross, G. D., Rabellino, E. M., Polley, M. J., and Grey, H. M., J. Clin. Zwvest. 52, 377, 1973. 8. Shevach, E. M., Herberman, R., Frank, M. M., and Green, I., J. C&z. Invest. 51, 1933.

1972. 9. Borella, L., and Sen, L., J. Immunol. 111, 1257, 1973. 10. West, W., and Herberman, R. B., unpublished observations. 11. Smith, J. L., Barker, C. R., Clein, G. P., and Collins, R. D., Lawet 1, 74, 1973. 12. Minowada, J., Ohnuma, T., and Moore, G. E., J. Nat. Cancer Inst. 49, 891, 1972. 13. Mendes, N. F., Tolnai, E. A., Silveira, N. P. i4., Gilbertsen, R. B., and Metzgar, R. S., J. Znznzunol. 111, 860, 1973. 14. Zur Hausen, H., personal communication, 1973. 15. Bianco, C., Weiner, M., and Nussenzweig, V., Fed. Proc. 32, 975, 1973. 16. Minowada, J., and Moore, G. D., In. “Proceedings of the VIth International Symposium on Comparative Leukemia Research” (Y. Ito, Ed.). In press, 1974. 17. Jondal, M., and Klein, G., 1. Exp. Med. 138, 1365, 1973. 18. Minowada, J., personal communication. 19. Shevach, E., Edelson, R., Frank, M., Lutzner, M., and Green, I., Proc. Nat. Acad. Sci.

U.S.A. 71, 863, 1974. 20. Bentwich,

Z., Douglas,

S. D., Siegal,

F. P., and Kunkel,

H. G., Cl&.

Im~mz~nol. Zmmuna-

pathol. 1. 511, 1973. 21. Moore, G. E., and Minowada, J., N. Engl. J. Med. 288, 106, 1973. 22. Ting, C. C., Lavrin, D. H., Shiu, G., and Herberman, R. B., Proc. 69, 1664, 1972 23. Omenn, G. S., Pathobiol. Anwual 3, 177, 1973.

Nat. Acad. Sci. Cr.S..-I.