CELLULAR

15, 452-456 (1975)

IAIIII.NOLOGY

SHORT The Effect

COMMUNICATIONS

of Trypsin OMAR

Drl)artment

of

Chrmical

Pathology,

on Cell FAKHRI

Westminster

Surface

Antigens

1

Medical

School,

London,

London,

SWl,

SWl,

England

AND ROBERT

Department

of Dewnatology,

S-H.

Westminster Received

March

TAN

Hospital,

England

20,1974

The effect of trypsin on cell surface antigenic determinants was measured in a mouse plasmacytoma model. While loss of some antigenic determinants could be detected within 1 min, relevant residual antigens remained even after 1 hr. For solid plasmacytoma of such mice, trypsin could be safely used to prepare single-cell suspensions for similar studies.

INTRODUCTION Trypsin solution is widely used to separate cells from tissue culture plates and from living tissues. It is not known exactly how much this treatment damagescell surface antigens (1)) or whether a solid tumor could be safely trypsinised to create a single-cell suspension retaining required antigenicity for studies of tumour immunology. In our experimental model (2) antibodies can be raised in Wistar rats against mouse plasmacytoma MP5563, grown in ascitic form in CzH mice. After these cells (MP 5563) have been exposed to the 7s fraction of the rat antibodies, nonimmune lymphocytes (obtained from Wistar rats) attach to them, in vitro, forming rosettes (Fig. 1). Nonimmune lymphocytes, however, do not attach in the absenceof these specific antibodies which are presumably bound to the surface antigenie sites of MP5563 to which they are directed. The present experiments measure the effects on the surface antigenic sites of treatment with trypsin solutions for variable periods of time. MATERIALS

AND METHODS

Target cells. Plasmacytoma MP5563 was grown in ascitic form in the peritoneal cavity of CJH mice as described previously by Fakhri (3). This provided an easy source of washed single cells in suspension.It was adjusted to contain 2 X lo6 cells/ ml of saline. 1 Present address : Medical Research Centre, University of Baghdad, Baghdad, Iraq. 452 Copyright All rights

@ 1975 by Academic Press, Inc. of reproduction in any form reserved.

SHORT

453

COMMUNICATIONS

FIG. 1. A large target cell coated with rat 7s antibodies surrounded by coopted smaller nonimmune lymphocytes. Prior treatment of the target cell with trypsin can remove binding sites for the rat antibodies and reduce the number of coopted lymphocytes. Magnification, x moo.

Noninwnune lymphocytes. These were obtained from unimmunized male rats 34 months of age by thoracic duct cannulation using the technique of Gowans (4). Harvesting on the second to third days of drainage provided lymphocytes which could be coopted to the target cells (2). The lymphocytes were freshly collected, centrifuged, washed once with normal saline, and then adjusted to contain 20 X lo6 lymphocytes/ml of saline. The rats were immunized by the method previously described Rat 7s antibodies. by Fakhri and Hobbs (5)) and 7s antibodies (free of lytic 19s antibodies) were isolated by recycling through Sephadex G200 and finally adjusted to the original TABLE THE EFFECT

1

OF TKEATMENT OF PLASMACYTOMA 5563 CELLS WITH 170TKYPSIW SOLUTIOK FOR DIFFERENT TIME INTERVALS ON THE COOPTION OF NONIMMUKE LYMPHOCYTES TO THE TUMOUR CELLS Control

Average number of lymphocytes per tumour cell Percentage of normal Percentage of non-rosetteforming cells

3.6 100 2

1 min

2 min

3.5

3.3

97 2

92 4

5 min 3 84 10

10 min 2.6 73 18

1.5 min 2.2 61 24

30 min 1.8 50 38

AT 37°C

60 min 1.0 28 74

454

SIIORTCOMMUNICATIONS TABLE

2

THE EFFECT OF TREATMENT OF PLASMACYTOMA 5563 CELLS WITH 0.1% TKYPSIN SOLUTION .4~ 37°C FOK DIFFERENT TIME INRTERVALS ONTHE COOPTION OF NONIMMUME LYMPHOCYTESTOTHE TUMOUK CELLS Control

1 min

2 min

5 min

10 min

15 min

30 min

60 min

__ Average number of lymphocytes per tumour cell Percentage of normal Percentage of non-rosetteforming cells

3.6

3.6

100 0

3.5

100 2

3.2

97 2

3.1

89 0

86 0

3.0 84 0

2.9 81 8

2.6 73 32

volume of the lymph and kept at -20°C. This fraction was adsorbed with normal C:+H mouse spleen cells, and reacted with MP5563 cells, but not with normal CSH mouse lymphocytes. Trypsin solutions. Trypure, h’ovo, Copenhagen. Experhw&d procedure. One milliliter of 2% trypsin, already at 37”C, was added to 1 ml of tumour cell suspension,also at 37°C (final concentration of 1% trypsin). A timer was started and 0.1-ml sampleswere subsequently taken at different time intervals (1, 2, 4, 10, 15, 30, and 60 min). Each samplewas transferred immediately into a tube containing 1 ml of TC199 with 10% foetal calf serum kept at 4°C to inactivate the trypsin. This was then centrifuged and the supernatant discarded. The cell pellet was suspendedin 0.5 ml of saline and broken into single

1 O:b Trypsin

0 ! 0

FIG. 2. The mean number exposure

of the cells to trypsin

10

20

of lymphocytes solutions.

30

40 Time

attached

50

per

60

turnour

70

cell

80

related

to the duration

of

SHORT

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455

cells by a whirlmixer. To this tumor cell suspension was added 0.1 ml of 7s antibody, 0.1 ml of nonimmune lymphocyte suspension (equivalent to 2 x lo6 lymphocytes), and the final volume was adjusted to 1 ml by the addition of normal saline. The mixture was then left for 2 hr at room temperature. At the end of this period a sample was taken and counted by light microscopy. One hundred tumour cells were examined; the average number of lymphocytes bound per tumour cell was recorded. This was expressed as a percentage of the average number of lymphocytes bound to the control untrypsinized tumour cells set up at the same time. The same experiments were repeated using 0.2% trypsin solution (final concentration of 0.1% trypsin). At each concentration of trypsin, experiments were carried out in triplicate; the average of the three tests was taken. RESULTS Table 1 shows the effect on subsequent rosette formation of a final concentration of 1% trypsin solution and Table 2 that of 0.1% trypsin. The mean number of lymphocytes attached per tumour cell decreased with time, with both concentrations of trypsin (Fig. 2) ; the effect was more marked with the 1% solution. The percentage of non-rosette-forming tumour cells increased with time, and this also was more marked with the higher concentration of trypsin. In neither set of experiments was there complete loss of rosetting, not even after trypsinization for 1 hr. DISCUSSION Cellular and humoral components of the immune reaction are known to cooperate in the immune response (6). Direct evidence of such cooperation on the efferent limb of tumour immunity has been shown by Fakhri and Hobbs (2) who demonstrated rosette formation after incubation of tumour cells MP5563 with nonimmune lymphocytes and 7s antitumour antibodies. The 7s antibodies were effective up to a dilution equivalent to l/100 of the original rat thoracic duct lymph. However, optimum results were obtained using a final dilution equivalent to 1 :lO lymph, to which was added 20 lymphocytes per tumour cell. This type of rosette formation is dependent on the fact that specific 7s antibody becomes attached to the corresponding antigens on the tumour cell surface. In doing so the Fc portion of the antibody is activated. This attracts nonimmune lymphocytes which have surface receptors for activated Fc (2 and 7) as shown by failure of rosette formation when the 7s antibodies were treated with pepsin. In the present experiment, use was made of this model system, the only additional factor being the prior treatment of the cells with trypsin in two different concentrations for varying periods of time at 37°C. 1Ve found that trypsinization of single-cell suspensions of mouse plasmacytoma modifies the ability of these cells to form rosettes when treated with rat specific 7s antibody and nonimmune rat lymphocytes. This is probably due to the damage trypsin produces on cell surface antigens. The damage is slight with 0.1% trypsin, and little further damage occurs when incubation is continued for longer than 30 min (Fig. 2). With the 1% trypsin solution the damage is greater. It is detectable after incubation for only 1 min and, with continued incubation, further damage occurs. However, even after incubation for 60 min, about 25% of the cells are still capable of forming rosettes. although the mean number of lymphocytes attached per tumour cell has decreased to aroutid 25 s of the control values (Fig. 2).

4.56

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The gradual decrease of the number of lymphocytes per tumour cell with the period of trypsinization is probably because surface antigens differ in their ability to withstand trypsinization. This could be the results of either the presence of a multiplicity of antigens on the cell surface, each with a different response to trypsinization, or because the cell surface antigens are situated at different levels on the cell membrane, trypsinization first affecting the superficially placed antigens and subsequently the deeper ones. In this model although some of these antigens are damaged by trypsinization, the immune reaction could still occur against the remaining antigens, and it seems certain that a solid plasmacytoma could be safely trypsinised to produce useful single-cell suspensions. ACKNOWLEDGMENTS One of the authors (0. F.) was supported by the Cancer Research Campaign and the other (R. S-H T.) by the Governor’s Discretionary Fund of the Westminster Group of Hospitals. We also thank Dr. C. Butterworth and Dr. L. Karaca for technical assistance, and Professor J. R. Hobbs and Dr. P. W. M. Copeman for their encouragement.

REFERENCES R. C., Phillips, T. M., Hamilton-Fairley, 1. Lewis, M. G., Ikonopisov, R. L., Nairn, Bodenhams, D. C., and Alexander, P., Brit. Med. J. 3, 547, 1969. 2. Fakhri, O., and Hobbs, J. R., Nature New Biol. 235, 177, 1972. 3. Fakhri, O., Brit. J. Cancer 24, 389, 1970. 4. Gowans, L. J., Brit. J. Exp. Pathol. 38, 67, 1957. 5. Fakhri, O., and Hobbs, J. R., Brit. J. Cancer 24, 853, 1970. 6. Moller, G., Transplant. Proc. 3, 15, 1971. 7. Fakhri, O., McLaughlin, H., and Hobbs, J. R., Eur. J. Cancer 9, 19, 1973.

G..

Short communications. The effect of trypsin on cell surface antigens.

CELLULAR 15, 452-456 (1975) IAIIII.NOLOGY SHORT The Effect COMMUNICATIONS of Trypsin OMAR Drl)artment of Chrmical Pathology, on Cell FAKHRI...
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