exhibit fluorochromasia. For our test,the cells were ... - Europe PMC

A FLUOROCHROMATIC TEST FOR IMMUNOCYTOTOXICITY AGAINST TUMOR CELLS AND LEUCOCYTES IN AGAROSE PLATES* BY FRANCO CELADA AND BORIS ROTMANt DEPARTMENT OF TUMOR BIOLOGY, KAROLINSKA INSTITUTET MEDICAL SCHOOL, STOCKHOLM, SWEDEN

Communicated by Albert H. Coons, January 5, 1967

Cytotoxic tests are used extensively in tumor and transplantation immunology because their results can be correlated with the in vivo fate of nucleated cells undergoing immunological aggression. We describe here a new cytotoxic test which can be scaled to analyze many samples at the same time, employs microquantities of serum, and is amenable to quantitative evaluation. The test is based on fluorochromasia, the property of living mammalian cells to accumulate fluorescein intracellularly as a result of the enzymatic hydrolysis of a fluorogenic substrate, thereby becoming brightly fluorescent under blue light.1 This process requires integrity of the cell membrane; cells damaged by antibody and complement action do not exhibit fluorochromasia. For our test, the cells were imbedded in a thin layer of agarose in which there was little overlapping of cells. This procedure was found useful both for optical scanning of fluorochromatic reactions and for keeping the tumor and leucocyte target cells used in our experiments under viable conditions for several days. Materials and Methods.-Cells: The mouse cells employed derived from the YAC lymphoma induced by the Moloney agent in an A/Sn mouse, subsequently converted to ascites form and carried routinely in A/Sn mice. The cells were obtained from tumors which developed 6-9 days after transplantation of 104 tumor cells per mouse.2 The cell suspensions were diluted 4-5 times, spun down, and resuspended to a density of about 2 X 108 cells per ml. All these operations were conducted at 40C. Unless otherwise stated, Eagle's medium with Earle's balanced salt solution3 was used throughout all the procedures. For most experiments, the cell suspension contained less than 10% dead cells estimated by their uptake of trypan blue4 or by their lack of fluorochromasia.1 For the experiments with human leucocytes, fresh blood was defibrinated by shaking for 10 min in an Erlenmeyer flask containing 10-15 glass beads and then mixed with a third of the volume of 3% gelatin in saline. After standing for 30 min at 37°C, the upper layer of the cell suspension was withdrawn. This layer contained roughly equal numbers of leucocytes and erythrocytes. The vast majority (90-95%) of the leucocytes were lymphocytes. After one washing, the cells were resuspended in Eagle's medium and treated exactly as indicated above for the mouse cells. Serology: The following antisera were used: A.CA anti-A/Sn batch XII (a gift from Dr. Erna M6ller), a hyperimmune antiserum obtained by repeated weekly inoculations of normal A tissue into A.CA mice with a titer in hemagglutination of 1:512; an anti-Moloney serum batch 04131 (a gift from Dr. Eva Klein), a serum obtained by immunizing (AxC57BL)F, mice with homogenized YLD-LDA (a tumor induced by the Moloney agent in a C57 leaden mouse and carried routinely in A/Sn X C57 leaden F1 hybrids)-its cytotoxic index was 0.80 at 1:4 dilution; five human antileucocyte typing sera, nos. 22, 102, 463, 528, and 710 (a gift of Dr. J. J. van Rood); human antileucocyte serum Moulene (a gift of Drs. J. Dausset and F. Kourilsky), a cytotoxic serum reacting with 100% of the population. Both fresh and lyophilized guinea pig sera were used as complement for reactions with mouse cells; fresh frozen human and rabbit sera were used with human leucocytes. Chemicals: Agarose (L'Industrie Biologique Frangaise S.A., batch F 3083) was dissolved in distilled water by heating in a boiling water bath. Five-ml aliquots of the solution containing 1.2% agarose were distributed in sterile tubes and stored in the refrigerator. Fluorescein di630

VOL. 57, 1967

'X~:HASo3 ~.:

PATHOLOGY: CELADA AND ROTMAN

631

acetate recrystallized 5 times (Mann Research Lab., Inc.), the fluorogenic substrate, was dissolved in warm acetone at a concentration of 5 mg per ml and kept in the freezer. This solution was diluted 1: 500 in warm (about 350C) phosphate buffered saline' a few minutes prior to its use. Aqueous solutions of the substrate at this concentration tend to flocculate; therefore, fresh solutions were prepared from the stock solution in acetone whenever the interval between their preparation and use was greater than 15 min. Preparation of agarose plates: The 1.2% agarose solution was melted in a boiling water bath and then mixed with an equal volume of Eagle's medium of double concentration which was kept at 42-43oC (the salts in this concentrated Eagle's medium precipitate after prolonged incubation at this temperature). Aliquots of 0.5 ml of the mixture were distributed (with a warm pipette) into sterile tubes previously equilibrated at 42-43OC (the tube with agarose in Eagle's medium can be kept at this temperature for at least 1 hr). A volume ranging from 0.05 to 0.1 ml of cell suspension containing about 2 X 108 cells per ml was added to each agarose tube and the content poured rapidly into a plastic Petri dish (Falcon, 50-mm diameter) which was kept warm by floating it on the water bath at 42-43oC. After a few vigorous, short shakings of the plate to spread the solution evenly, it was removed from the water bath and allowed to cool down to room temperature. During this period of cooling, pieces of filter paper (J. H. Munktells, no. 8) of about 2 X 2 mm containing antisera were deposited on the plates. The pieces of paper were loaded by dipping them in the antiserum and blotting the excess of liquid between two pieces of filter paper. The volume of liquid held by the pieces of paper was estimated by means of a solution of radioactive albumin. The average volume of 10 pieces of paper was 0.25 ,l, with a standard deviation of the mean of 0.03 Al. Figure 1 shows a plate in which pieces of paper of different shapes were used to distinguish between different types and dilutions of antisera. After incubating the plates at 370C under an atmosphere of air-5% C02 for 60 min, 1 ml of complement was added and the plates were returned to the incubator for 30 additional min. Unless otherwise stated, comple-

*

*

FIG. 1.-Agarose plate with filter papers containing antisera. Various shapes of papers were used as a method to code the different sera.


632

PROC. N. A. S.

ment diluted 1:4 was employed. The technique was modified somewhat when human leucocytes were used in order to obtain an optimal antiserum-cell proportion. The density of leucocytes in the agarose was kept between 2 and 4 X 106 cells per ml and the volume plated was increased to 0.7 ml. After complete cooling of the plates (about 20 min), a number of circular holes of 3-mm diameter were punched in the agarose layer using a sharp, stainless steel cylinder (LKB, cat. 6800A) provided with a rubber tubing which served to remove the agarose disk from inside of the cylinder by mouth suction. About 1 ,l of antiserum was placed in each hole and then the plates were incubated at 370C under an atmosphere of air-5% C02 for 30 min. After this incubation, 1 ml of Eagle's medium was added to each plate to rinse the antisera, followed by 1 ml of human or rabbit complement diluted 1:2. Incubation was then resumed for 2 hr. There was no difference in performance between the two sources of complement. The rabbit complement employed was previously absorbed by incubating it at 40C for 20 min with 1 vol of packed human erythrocytes obtained from the sediment in the preparation of the leucocytes used for plating. Fluorochromatic reaction: After the antiserum-complement reaction, the complement was poured off the plates and the agarose layer washed with three successive rinses of 1 ml Eagle's medium each. During this washing, the medium was allowed to remain on the plates for about 5 min between the second and third rinse. This procedure was necessary to eliminate the bulk of the complement since it contains esterases which hydrolyze the fluorogenic substrate. The washing was not so rigorous when complement diluted more than 1:4 was used. After pouring off the last rinse from the plates, 1 ml of a solution containing 10 pug fluorescein diacetate per ml was added to each plate. After 10-15 min incubation at room temperature, the fluorescein diacetate solution was poured off the plates and 1 ml of minimal Eagle's medium containing 10% inactivated (30 min at 560C) calf serum was added. The plates could then be examined under the microscope or alternatively stored in a refrigerator for as long as 20 hr prior to their examination. For experiments involving photographic recording, the phenol red of the Eagle's medium was omitted since it partially absorbs both the blue (exciting) and the green light (fluorescent). A Zeiss microscope provided with a cardioid darkfield condenser was used for the observations. The light of an ordinary 6-v tungsten lamp of the microscope filtered through either a BG-12 or an interference filter transmitting a broad band between 440 and 480 m,4 was used for the excitation. A Wratten 15 was utilized as barrier filter in conjunction with the BG-12 filter. For the interference filter, no barrier filter was necessary. In many instances, the intensity of the fluorochromatic reaction permitted us to examine the plates without either primary or barrier filter. Most of the microscopic observations were conducted in a semidark room in order to increase the sensitivity of the eyes.

Results.-Experiments with mouse lymphoma cells: Preliminary experiments served to compare the fluorochromatic and the trypan blue method using for both a conventional cytotoxic test4 to evaluate the effects caused by specific antiserum and complement. From the data obtained in these experiments, we concluded that fluorochromasia was suitable for cytotoxic tests since it gave results comparable to those obtained with trypan blue (Fig. 2). 1.0

_ 4,

FIG. 2.-Comparison between the fluorochromatic and the trypan blue method. Mouse tumor cells were treated with antiserum and complement in test tubes as in a conventional cyto'@ :2 toxic test.4 After the incubation with complement, part of the cell suspension was used for .2\\scoring the proportion of surviving cells by the U \\ trypan blue technique4 and another portion by L_ fluorochromasia.1 Circles, A.CA anti-A serum; 0.1% squares, normal mouse serum. Black symbols, scoring by fluorochromasia; open symbols, scoring by trypan blue. H

_

i

I

\.1 0.35

.28 .07 .14 Ail undiluted serum

VOL. 57, 1967


633

i(1)

FIG. 3.-Fluorochromatic reaction of mouse tumor cells in agarose plates. (a) Corner of a piece of filter paper containing normal serum and surrounding cells as seen in dark field under white light. (b) The same microscopic field as in (a) observed under blue light and with barrier filter. Only the living cells are fluorescent and can be seen. No dark halo of dead cells is seen near the paper. (c) Corner of filter paper containing antiserum. White light. (d) Same field as in (c), blue light and barrier filter. A dark halo is seen near the paper ( X 80).

Subsequent experimentation with cells imbedded in agarose made use of the fluorochromatic assay because it allowed us to visualize easily the fluorescence of the living cells against a dark background. The presence of dead cells did not affect the observations since they were not visible under the fluorescent microscope. Accordingly, as shown in Figure 3, a positive cytotoxic effect was recognized by an area of dead cells surrounding the paper containing the antiserum. This was seen as a dark halo around the paper contrasting with the background dotted with fluorescent cells. Around the papers with normal serum, no halo of dead cells was observed. (The microscopic picture resembled a dark sky with many bright stars.) Assuming that the extent of the halo of dead cells was due to diffusion of the antiserum in the agarose, we tested the effect of different parameters known to affect diffusion. Accordingly, we found that increasing the concentration of the antiserum or the time of incubation with antiserum increased the width of the halo

(Fig. 4).

6.34 P,3 ATHOLOGY: CELADA AND ROTMAN

.3 0

2000-

2000

1000-

1000

PRoc. N. A. S.

500

5000

250

250-

125

125-

62-

62

s

31 31-

.015

.12 .03 .06 Aill undiluted serum

FIG. 4.-Effect of antiserum concentration and time of incubation with antiserum. Pieces of paper containing 0.25 ,.l of A.CA anti-A/Sn antiserum diluted to different extents were placed on agarose plates containing YAC tumor cells. The plates were incubated for the indicated time and then guinea pig complement was added. From this point on, the procedure was the same for all plates as indicated in the text. The abscissa represents the volume of undiluted antiserum in each paper; the ordinate, the width in microns or the area of dead cells surrounding the paper measured as a dark halo.

1.25

25 5 10 20 Target cells per plate (x106)

FIG. 5.-Effect of concentration of YAC cells in the agarose on the extent of dark halo. Papers containing the same amount of antiserum were tested on plates with the indicated number of cells imbedded in the agarose. The ordinate represents the width of the dark halo around the papers.

The width of the halo was also affected by the temperature of incubation with antiserum. At 40C, about eight hours of incubation were necessary to obtain a halo of 0.8 mm with an antiserum diluted 1:4. At 230C, a comparable halo was obtained after three hours of incubation and, at 370C, it was found after about one hour. Since antiserum must be absorbed by the cells, the effect of different density of cells in the agarose was examined. The width of the halo of dead cells around the positive papers was found to increase with decreasing cell density (Fig. 5). The minimum cell density found to be compatible with the test was about 106 per plate. The effect of dilution of the guinea pig complement as well as different types of complements were investigated. We found that fresh guinea pig complement was effective at dilutions of 1:5 and 1:10 using 30 minutes of incubation. At higher dilution, there were more and more cells remaining alive near the positive papers (Table 1). A longer time of incubation with complement above 30 minutes had no significant effect in the mouse tumor system. The plates for the assay could be stored at 40C for periods up to three days without significant loss of viability. The most important factor was to keep the plates from losing water by evaporation. We stored them in a dessicator under an atmosphere of air-5 per cent CO2 containing water in the bottom. The addition

VOL. 57, 1967


635

TABLE 1 EFFECT OF COMPLEMENT CONCENTRATION ON THE FLUOROCHROMATIC CYTOTOXIC TEST Antiserum

1:5

1:10

A.CA anti-A/Sn 1:4 A.CA anti-A/Sn 1:8 Anti-Moloney 1:1

900 710 300*

780 650 100*

Complement Dilution 1:20

1:40

Control

715* 520* +

715* 520*

0 0 0

0

The figures represent the width in microns of the dark halo of dead cells surrounding papers containing the antiserum. Fresh-frozen guinea pig complement was used. The control was inactivated complement (30 min at 56'C) diluted 1:5. * Some fluorescent cells were scattered throughout the dark halo.

TABLE 2 TITRATION OF Six HUMAN SERA AGAINST THREE LEUCOCYTE SUSPENSIONS IN AGAROSE PLATES Antiserum no.

22 102 463 710 546

Moulene

,-

1:1

500 0 400

B Antiserum Dilution1:2 1:4 1:8 1:16

1:1

F Antiserum Dilution1:2

1:4

1:8

1:16

0 0 0 0 0 0 0 400 300 0 0 0 0 0 0 0 600 500 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1600 1600 1200 600 500 1600 1600 1200 900 800 1600 1600 1000 800 400 1200 800 800 400 400

G Antiserum Dilution 1:1

1:4

0 0 0 0 n.t. 400

0 0 0 0 800 400

The figures represent the width in microns of the halo of dead cells surrounding the hole with antiserum. The leucocytes were obtained from three different persons, Birgitta (B), Franco (F), and Gunilla (G). The antisera had the following specificity: 22 and 463, anti-4a; 102, anti-6a; 710, anti-7c; 528, general, a mixture of anti-4a, 6a, 7b, 8a, and an extra antibody; Moulene, general.

of 5-10 per cent calf serum to the agarose neither interfered with the cytotoxic test nor seemed to improve the viability of the cells under our conditions. Cytotoxicity against human leucocytes: Six human antileucocyte sera, four of them monospecific-typing sera and two reacting with the majority of the population, were tested against leucocytes from several donors. The results obtained with the fluorochromatic method (Table 2) were qualitatively identical with those obtained by the conventional cytotoxic test. The sensitivitiy of the latter, in terms of titer of the antisera, was about half that of our method. In these experiments with human leucocytes, we observed that the monospecific sera, in contrast to the multispecific, did not kill all the cells. 'The few fluorescent survivors scattered throughout the dark area of dead cells did not interfere with the assay. Similar results (Table 1) were observed with the specific mouse reaction in experiments with anti-Moloney serum. This is in agreement with the previous finding that about 10 per cent of nontumoral cells are present in the suspensions of

Moloney tumors.6 We noted a few differences between cytotoxic reactions against mouse lymphoma and those against human leucocytes. First, the latter required higher concentrations (1:2 or 1:4) of either human or rabbit complement coupled with a longer time of complement action (two hours). Second, in order to detect the weak reactions of the human antisera, it was necessary to raise the relative concentration of antiserum in the agarose. This was accomplished by replacing the filter paper containing antiserum by the "hole technique." In addition, this change improved the optical resolution and permitted us to scan the edges of the hole without difficulty for the presence of fluorescent cells. The effects of cell concentration and incubation time in the leucocyte test were essentially the same as for the reaction with mouse tumor cells. The minimum number of leucocytes per plate that we found to be practical for the visual scanning was about 106 per plate.

636


Pxoc. N. A. S.

Discussion.-The results presented above demonstrate that fluorochromasia can be used to detect cell membrane damages caused by the action of specific antiserum and complement. For our test, the peculiar optical properties of the fluorochromatic reaction were combined with the well-established agar methodology yielding a procedure with the following advantageous characteristics: (a) it is a micromethod in which economy of reagent is realized without the need for delicate manipulations; (b) it has higher sensitivity than routine techniques involving about 20 ,Al of antiserum; we obtained unambigous results with 0.25 ,.d of antiserum; (c) it has definite organizational advantages; it is easy to perform and can be extended to test a large number of samples simultaneously; (d) the cells imbedded in agarose could be kept viable for several days before and after performing the test; this property, together with the adaptability to mass testing (20 or more sera can be tested on a single plate), should be of importance for leucocyte-typing since the current methods require that the testing be done during the same day of the bleeding of donors.7 The fluorochromatic test in agarose should also be useful for the assay of transplantation antigens. Small samples obtained during fractionation of an antigenic material could be tested for their capacity to absorb antibodies by placing them on filter papers in contact with a given antiserum. Pieces of paper after electrophoresis could be used directly for the assay. In this antigen test, the disappearance or the reduction of the halo of dead cells would indicate the presence of an antigenically active fraction. Another possibility, using the agarose method, is that the synergistic effect of two different antibodies could be detected in the zone of confluence of their halos. If successful, this might become as useful for cellular antigens as the double diffusion technique is for precipitin reactions. Summary.-A new cytotoxicity assay based on fluorochromasia permits the simultaneous testing of many samples of antiserum and the storage of the test cells over several days. It uses less than 1 pl of serum per test and 1-5 million cells per agarose plate. * This investigation was supported by grants from the Swedish Cancer Society, the Swedish Medical Research Council, the National Cancer Institute (CA-03700), the Damon Runyon Fund (DRY-598), and a U.S. Public Health Service fellowship no. 1-F3-CA-4024-01 from the National Cancer Institute to one of us (B. R.). We are grateful to Professor George Klein for stimulating discussions, to Dr. J. J. van Rood for a generous supply of antileucocyte-testing sera, and to Mrs. Eva Maria Fenyo for performing several conventional cytotoxic tests and for supplying the mouse ascites tumors. t On leave from the Division of Medical Science, Brown University, Providence, Rhode Island. 1 Rotman, B., and B. W. Papermaster, these PROCEEDINGS, 55, 134 (1966). 2 Klein, G., E. Klein, and G. Haughton, J. Natl. Cancer Inst., 36, 607 (1966). 3Eagle, H., Science, 130, 432 (1959). Gorer, P. A., and P. O'Gorman, Transpl. Bull., 3, 142 (1956). Dulbecco, R., and M. Vogt, J. Exptl. Med., 99, 167 (1954). 6 Haughton, G., J. Haot, L. Revesz, and G. Klein, Science, 150, 769 (1965). 7Ceppellini, R., F. Celada, P. L. Mattiuz, and A. Zanalda, Ann. N.Y. Acad. Sci., 120, 335 (1964).