Journal of Immunological Methods, 17 (1977) 101--116 © Elsevier/North-Holland Biomedical Press

101

A N I M P R O V E D P L A Q U E A S S A Y F O R M O U S E M Y E L O M A (MOPC 3 1 5 ) CELLS FOR USE IN STUDIES OF HUMORAL AND CELL-MEDIATED IMMUNITY

DAVID LEVIN, JIRI JONAK and T.N. HARRIS The Joseph Stokes, Jr. Research Institute of the Children's Hospital of Philadelphia, and the Department of Pediatrics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A. (Received 19 January 1977, accepted 1 April 1977)

Dinitrophenyl-bovine albumin was coupled at room temperature to sheep red blood cells in a procedure which minimized spontaneous lysis and allowed the preparation of large batches and their use for at least 3 weeks. The modified erythrocytes were used as a substrate for detecting local hemolytic plaques in agar by myeloma MOPC 315 cells, which secrete a paraprotein IgA with high affinity for dinitrophenyl ligand. Conditions maximizing the number of plaques formed by a given number of tumor cells were found to include coupling the erythrocytes at 1 mg/ml dinitrophenyl-bovine albumin with a molar ratio of about 50, and incubation with an amino-to-carboxy cross-linking agent, 1-ethyl-3(3 dimethyl aminopropyl) carbodiimide, at 2 mg/ml for 50 min. The method thus developed was employed to measure cellular and antibody-dependent immune reactions against the MOPC 315 cells. The experimental results show comparisons of the plaque technique with other measurements of tumor cell injury. The nature of the assay, which requires only 500 cells per plating, and which tests the synthetic capacity of single cells, suggests its use in experiments which limit the number of target cells, and in immune reactions causing injury, but not necessarily lysis, of the target cells.

INTRODUCTION

T h e ability o f cells secreting h e m o l y t i c a n t i b o d i e s t o p r o d u c e individual p l a q u e s against e r y t h r o c y t e s in gel has f o u n d n u m e r o u s i m p o r t a n t applications. T h e t e c h n i q u e originally e m p l o y e d b y J e r n e a n d N o r d i n ( 1 9 6 3 ) and I n g r a h a m a n d Bussard ( 1 9 6 4 ) m a d e use o f i m m u n e l y m p h o c y t e s secreting a n t i b o d i e s against s h e e p red b l o o d cell ( S R B C ) antigens. It was t h e n e x t e n d e d to l y m p h o c y t e s s t i m u l a t e d b y o t h e r antigens p r o d u c i n g p l a q u e s against S R B C c o u p l e d t o t h e s a m e antigens (Moller, 1 9 6 5 ; M e r c h a n t et al., 1 9 6 6 ; Walsh et al., 1 9 6 7 ; G o l u b et al., 1 9 6 8 ) . T h e availability o f p l a s m a c y t o m a s s e c r e t i n g a n t i b o d y - l i k e m o l e c u l e s ( P o t t e r a n d L e o n , 1 9 6 8 ) w i t h s t r o n g affinity t o various h a p t e n s e x t e n d e d t h e t e c h n i q u e t o include t u m o r cells w h i c h c o u l d be p l a q u e - f o r m i n g cells ( P F C ) against 8 R B C c o u p l e d t o t h e a p p r o p r i ate h a p t e n s ( Y a m a d a et al., 1 9 7 0 ; G h a n t a et al., 1 9 7 2 ) . A l t h o u g h t h e use o f

102 the plaque technique to measure immune reactions with t u m o r cells as targets had the potential of a powerful analytic tool, this assay has not been routinely utilized because of the difficulties encountered with coupling red" blood cells to the appropriate haptens in a reproducible manner, and in a way which would preserve the coupled cells from spontaneous lysis. In this paper it will be shown that with m y e l o m a MOPC 315 cells, which secrete IgA-like molecules with a strong affinity to DNP (Eisen et al., 1968), it is possible to carry out routine plaque assays with stable DNP-coupled SRBC. The method will be applied to measurements of cellular and humoral immune reactions and compared to results obtained by other technics. MATERIALS AND METHODS MOPC 315 tumor line

MOPC 315 cells were maintained for 3 years by serial subcutaneous passages of the t u m o r in BALB/c mice (Flow Laboratories, Dublin, VA). For experimental purposes, single cell suspensions from ascites growth of the t u m o r were used. For this purpose 10--15 million viable cells from minced solid t u m o r were injected into mice which had received 0.5 ml of Pristane (2 ,6 ,10 ,14-tetramethyl pentadecane, Aldrich Chemical Co., Milwaukee, WI) 3--4 weeks prior to transfer to accelerate t u m o r growth (Potter et al., 1972; Potter and Walters, 1973). The ascites cells were used on day 6--9 after transfer. They were purified by banding on a Ficoll--Hypaque layer (Boyum, 1968), washed and maintained in RPMI 1640 supplemented with 8% heatinactivated, IgG-free fetal calf serum (FCS). DNP conjugated bovine albumin

DNP conjugation was carried out by a modified procedure of Eisen (1964). Bovine albumin powder, fraction V from bovine plasma (Armour Co., Phoenix, AZ), was dissolved, extensively dialysed against distilled water, filtered and kept at 4°C as a stock solution of 25% w/v. The reaction mixture was prepared by dissolving 70 mM 2,4 dinitrobenzene-sulfonic acid or its sodium salt in distilled water with the pH adjusted to 11. The solution was made 2% in bovine plasma albumin (BPA), and 100 mM K2CO3 added as solid. After readjusting the pH to 10.8, the reaction was allowed to proceed at 37°C. It was stopped b y extensive dialysis against distilled water in the cold. The solution was concentrated by air evaporation, filtered and stored a t - - 2 0 ° C . Protein concentration was determined by the Lowry m e t h o d { 1 9 5 1 ) w i t h freshly dissolved BPA as a standard, and DNP concentration measured by O. D. at 360 nm, taking the extinction coefficient E360 1M 17,530. Reaction time of 8--16 h resulted in 45--55 residues of DNP bemg conjugated per albumin molecule (assumed molecular weight of 69,000 daltons). =

103

Coupling o f SRBC to DNP BPA All manipulations were carried out at room temperature. SRBC in Alsever's solution at approximately 20% packed volume, were washed 4 times with isotonic PBS {2.32 g Na2HPO4, 0.49 g KH2PO4, 16 g NaC1 in 1000 ml H20), resuspended in 0.8 volume (v) mixed with 0.2 v of 0.03% v/v glutaraldehyde in PBS and rotated for 15 min in a tilted rotator. Then 1 v of 0.15 M phosphate buffer pH 7.2 was slowly admixed, and the SRBC were centrifuged and resuspended in the phosphate buffer at 0.6 v. DPN-BPA at 5 mg/ ml in 0.2 v buffer was added, and after rotating for 10--15 min was followed by 0.2 v of ECDI (1-ethyl-3(3-dimethyl-amino propyl) carbodiimide, Sigma, St. Louis, MO) at 10 mg/ml and additional rotation for 50 min. The coupled SRBC were washed once with phosphate buffer and resuspended stepwise in Alsever's solution containing 0.4% w/v BPA, in which the cells were washed twice and stored in the cold at 20% packed volume. The entire process resulted in very little hemolysis. The coupled cells were usable for at least 3 weeks, provided that transfers from one buffer to another, involving changes in pH or reductions in BPA concentration, were carried out gradually. Before use, the coupled SRBC were washed once in Alsever's containing 0.4% BPA, followed by a wash in RPMI buffered with 10 mM HEPES (N-2hydroxethylpiperazine-N'-2-ethanesulfonic acid, Sigma), also containing 0.4% BPA, and finally resuspended at 12% packed volume in HEPES-buffered RPMI with 4% FCS.

Antisera Ascites fluid from MOPC 315 t u m o r growth in BALB/c mice was collected, and the IgA was purified by the m e t h o d of Goetzl and Metzger (1970) w i t h o u t prior reduction and alkylation. One mg of purified 315 protein was injected per rabbit in complete Freund adjuvant, followed by 3 injections of the same w i t h o u t adjuvant, at 3-week intervals. Anti-IgA serum was collected 3 weeks after the last injection. Secondary CBA anti-BALB/c ascitic fluid globulin was obtained by immunization with BALB/c spleen cells and use of a peritoneal irritant to produce ascites, as described (Harris and Harris, 1975). Several similar preparations were pooled and used as a standard to m o n i t o r sensitivity of MOPC 315 cells to anti-BALB/c alloantibody. Xenogeneic, allogeneic and syngeneic anti MOPC 315 sera were prepared as follows: rabbits were given 2 injections of 30 X 106 MOPC 315 cells at a 3week interval and bled 15--18 days after the second injection. The serum was collected and absorbed with BALB/c spleen cells to a cytotoxic titer of less than 23. To obtain allogeneic anti-MOPC 315 t u m o r antibody, C3H mice were injected with the t u m o r cells and with the peritoneal irritant, and tapped for ascites fluid between 8 and 20 days. The anti-H-2 d antibodies were absorbed with BALB/c spleen cells and the globulin fraction prepared

104 by a m m o n i u m sulfate precipitation. Syngeneic anti-tumor ascitic fluid was obtained from B A L B / c mice primed with MOPC 315 cells which had been inactivated with 0.02% v/v glutaraldehyde (Frost and Sanderson, 1975) for 10 min at 10 million/ml cells in PBS. The mice were re-injected subcutaneously 3 weeks later with l 0 s live t u m o r cells and given the peritoneal irritant. Ascitic fluid was collected at various intervals thereafter (9--20 days).

Cell-mediated immune reactions BALB/c mice bearing MOPC 315 solid t u m o r for 14--18 days were sacrificed, their spleens teased, washed and resuspended in cold RPMI with 10% FCS. The cells were added in 0.1 ml volumes to 60,000 MOPC 315 cells in Linbro plates (Linbro Chem. Co. New Haven, CT) making a final volume of 0.25 ml and an effector to target cell ratio of 100 : 1. After incubation for 18--20 h at 37°C in 5% CO2, the contents of the wells were removed into tubes, washed 3 times and resuspended in 0.5 ml medium. A sample was withdrawn from each tube for analysis of plaque forming cells (PFC), and the remaining cells were incubated for 3 h to allow for de novo IgA synthesis.

Antibody mediated immune reactions To determine titers of antisera by plaque reduction, short term assays were usually conducted. Samples of 0.1 ml of the suspension of MOPC 315 cells in RPMI with 8% FCS were added to tubes containing 0.1 ml of twofold serum dilutions, and incubated for 30 min at 37 ° in 8% CO2. The cells were washed twice and resuspended in medium containing final dilutions of 1 : 30 or 1 : 70, respectively, of guinea pig (GP) or rabbit serum as sources of c o m p l e m e n t (C'). Following an additional incubation for 30 min, the cells were washed once, and a sample containing 500 cells from the initial culture was removed from each tube for estimation of PFC.

[3H] thymidine incorporation and cytotoxicity For long term assays, 50,000 MOPC cells in volumes of 50 X were added to wells of a Linbro plate containing equal volumes of antiserum dilutions. After 30 min at 37 ° C, 22 X of GPC' and RC' were added to give final dilutions of 1 : 60 and 1 : 90, respectively. In some cases 0.2 pCi of [3H]thymidine (TdR) in 0.1 ml was added 4 h after C', and the incubation was continued for a total of 18 h. The well contents were transferred into tubes, and samples of washed cells were removed for the plaque test. The remainder was precipitated, washed with TCA and resuspended in a mixture of 2 parts LSC Scintillent (Yorktown, NY), 1 part Triton X-100 (Rohm and Haas, Philadelphia, PA), and enough water to clear the samples. Parallel samples receiving

105 22 ~ of 0.5% Trypan blue in PBS and 50 mM-EDTA were mixed and scored for viability by d y e exclusion. Serum titers (50% endpoints) were determined by the interpolated fractional dilution which gave 50% in plaque reduction, [aH]TdR incorporation or viable cell count, compared to control samples containing no antibody. Titer values were expressed as the exponents of base 2.

Radioirnmunoassay for IgA The method e m p l o y e d was modified from that of Klinman and Taylor (1969). Briefly, samples of medium from MOPC 315 cultures were mixed in tubes with 45 pg of e-DNP-lysine b o u n d to acetyl cellulose (Miles Yeda, Israel) and incubated in 0.3 ml volumes at room temperature for 30 min with occasional shaking. The immunoadsorbent was washed twice with PBS containing 1% v/v FCS, and the purified rabbit anti-IgA, labeled with ~2sI (McConahey and Dixon, 1966), was added to the tubes and allowed to bind to the adsorbed IgA for 30 min. After 3 washings in the cold, the b o u n d L:sI was counted in a Packard gamma counter. The amount of L:sI b o u n d was linear for the range'of 1 to 15 ng of IgA.

Plaque assay To 500 MOPC 315 cells in 0.6 ml medium were added 0.2 ml of 12% DNP-coupled SRBC and 0.7 ml of 1.4% agarose (L'Industrie Biologique Francaise) in Earle's HEPES (EH) kept at 46°C. After mixing with a warm pipette, 1 ml was delivered onto a 60 × 15 mm plate (Falcon Plastics, Oxnard, Ca) containing a layer of 2 ml solid agar in EH. The fresh agar layer was overlaid with 1.5 ml RPMI and incubated for 3 h at 37°C in 8% CO2. The medium was replaced with 2 ml EH containing 1 : 1000 dilution of rabbit anti-IgA serum and incubated for 30 min at 37°C, followed by overnight incubation in the cold. The liquid layer of the plates, equilibrated to room temperature, was further replaced with 1.5 ml EH containing 1 : 20 dilution of GPC' that had been absorbed with SRBC. Following incubation for 1.5-2 h at 37°C. the plaques were counted by projection on a graded paper. T h e 2 dilution in the plating and t h e a r e a of the plate read was such that of the 500 cells initially used, the plaques produced by an estimated 280 cells were actually counted. RESULTS

Coupling of DNP-BPA to SRBC Early experiments were done to modify the coupling procedure by ECDI introduced b y Johnson et al. (1966) with the following objectives: minimal lysis of the SRBC during the coupling reaction and prolonged storage; mini-

106 mal amounts of ECDI and DNP-BPA for the o p t i m u m concentration of SRBC; and preparation of large batches of coupled cells for use as a standard reagent in several repeated experiments. The first objective, to minimize spontaneous hemolysis, was fulfilled by mild fixation of the SRBC with glutaraldehyde prior to coupling, a procedure suggested by Suzuki et al. (1974). The coupling reaction was done at room temperature. This allowed the use of the ECDI at much lower concentrations than the 200 mg/ml used for o p t i m u m coupling in the cold (Yamada and Yamada 1969). At 8 mg/ml of ECDI, and 1 mg/ml DNP-BPA, there was considerable spontaneous lysis, and considerable cross linking on the remaining intact cells, as judged by their slow lysis in distilled water. Higher concentrations of DNP-BPA prevented such hemolysis, b u t it also led to turbid supernates following centrifugation of the SRBC, indicating that ECDI was extensively cross-linking the BPA in solution. ECDI was therefore used in a range below 8 mg/ml. Fig. 1 shows that ECDI at 2--4 mg/ml approached the o p t i m u m concentration for subsequent plaque production. This range of concentration did not cause spontaneous lysis during the coupling. Fig. 1 also shows a slight advantage of using the DNP-BPA at 2 mg/ml in comparison with 1 mg/ml. With the ECDI at 2 mg/ml, the effects of varying the concentration of DNP-BSA in the range below 1 mg/ml were examined. The results, shown in fig. 2, indicated that for optimal plaque formation the DNP-BSA should not be used below 1 mg/ml. The figure also shows that pre-fixation with glutaraldehyde at 0.01% v/v caused a decrease in the subsequent number of plaques. However, this treatment preserved the cells for three weeks without considerable hemolysis, whereas coupled SRBC which had not been fixed with glutaraldehyde began to show lysis after 2 days of storage in the cold. In later preparations the concentration of glutaraldehyde was lowered to 0.006% v/v, to compromise between the opposing effects. Fig. 2 also indicates the significance of using highly substituted DNP-BPA. This is reemphasized in fig. 3, where it can be seen that residues in the range of 16--30 per albumin molecule b o u n d to SRBC were insufficient for promoting hemolytic plaques b y MOPC 315 cells. The efficiency of plaque formation increased with increasing number of residues, until it leveled off at about 50 residues of DNP per albumin molecule. The reaction time necessary for obtaining o p t i m u m coupling of DNP-BPA to SRBC was examined at concentrations of 2 mg/ml and 1 mg/ml of the ECDI and BPA. The results of such an experiment are seen in fig. 4, which shows the number of detectable plaques as a function of reaction time with ECDI. There were also qualitative changes in the appearance of the plaques which were helpful in choosing the conditions for o p t i m u m coupling. In the reaction carried out at 1 mg/ml of ECDI and of DNP-BPA, the appearance of the plaques changed with increasing time of coupling, from very faint to faint. Increasing the concentration of ECDI to 2 mg/ml gave faint plaques at 5 and 10 min reaction time, and starting at 20 min, the plaques were large

107

140 4 m g / m l ECDI 120

2 ma/ml ECDI

I/ml ECDI

I00

U LI. {1. 8 O rO U n 0 6O

40

aO

6

o15

i DNP47BPA(mg/rnl)

Fig. 1. Effects on plaque production of the concentrations of DNP47BPA and ECDI in the coupling reaction. S R B C at 20% packed volume fixed in 0.01% v/v glutaraIdehyde, centrifuged and resuspended in DNP47BPA. Tubes placed in rotator for 10 rain. Then ECDI was added and rotation continued for 50 rain, The concentrations indicated are final.

and clear. When the DNP-BPA was also increased to 2 mg/ml, fairly clear plaques were obtained after 5 min of reaction, and these were clearer after an additional 5 min of coupling. Starting at about 20 min reaction time the plaques became sharper and smaller. This was presumably due to excessive coupling, leading to sharp localization of the limited IgA produced b y the MOPC 315 cells. This interpretation was supported by another observation, that the plaque size increased as the concentration of SRBC on the plates was lowered. For the concentrations chosen, 2 mg/ml ECDI and 1 mg/ml DNP-BPA, the plaques were rather faint after coupling for 5 and 10 min, and from then on they appeared large and clear. Finally, after adopting standard conditions o f 2 mg/ml ECDI, 1 mg/ml DNP-BPA and 50 min reaction time, the coupling was performed simultaneously on six different shipments of SRBC obtained in the course of six weeks. All samples gave a reproducible

108

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DNP-BPA (mg/ml) Fig. 2. Sensitivity o f m o d i f i e d S R B C t o local h e m o l y s i s in agar as a f u n c t i o n o f glutarald e h y d e p r e f i x a t i o n a n d o f t h e c o n c e n t r a t i o n o f DNP-BPA in t h e c o u p l i n g r e a c t i o n . T h e closed a n d o p e n triangles are for DNP47BPA w i t h o u t a n d w i t h 0.01% v/v g l u t a r a l d e h y d e p r e f i x a t i o n , respectively. T h e closed a n d o p e n circles are for DNP54.3BPA w i t h o u t a n d w i t h g l u t a r a l d e h y d e p r e f i x a t i o n . E C D I c o n c e n t r a t i o n was 2 m g / m l a n d t h e r e a c t i o n cont i n u e d for 50 rain. i

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DNP/BPA m010r roti0 Fig. 3. S u s c e p t i b i l i t y o f S R B C t o local h e m o l y s i s in agar as a f u n c t i o n o f D N P / B P A m o l a r r a t i o in t h e p r o t e i n c o u p l e d t o t h e cells. S R B C p r e f i x e d w i t h 0 . 0 0 6 % v/v g l u t a r a l d e h y d e a n d t r e a t e d w i t h 1 m g / m l DNP-BPA, f o l l o w e d b y 50 m i n i n c u b a t i o n at r o o m t e m p e r a t u r e w i t h 2 m g / m l ECDI.

109 260

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Fig. 4. Sensitivity o f SRBC to local hemolysis as a function of time allowed for the coupling reaction. SRBC prefixed with 0.006% v/v glutaraldehyde and coupled to

DNPs4.3BPA under the following conditions: (~ 1 rng/ml DNP-BPA, 1 rng/ml ECDI; (~) 1 mg/ml DNP-BPA, 2 mg/ml ECDI; (~) 2 mg/ml DNP-BPA, 1 mg/ml ECDI; (~) 2 mg/ml DNP-BPA, 2 mg/ml ECDI. result in the quantity of plaques, although one sample differed in the quality of the plaque appearance. To determine the stability of RBC coupled by this method, coupled RBC which had been prepared 1, 12 and 24 days before a test were plated in triplicate with the same suspension of MOPC 315 cells. The number of plaques obtained were 176 -+ 4.5, 156 + 7 and 157.3 -+ 5.4, respectively. Therefore, a preparation of coupled cells could be used for 3 weeks w i t h o u t appreciable loss in the number of plaques.

Inhibition of plaques by DNP The various coupled SRBC preparations from which the data in fig. 3 were obtained were also used in a plaque test in which e-DNP-lysine was present in the plates during the period of IgA synthesis b y MOPC 315 cells and during the reaction with rabbit anti-IgA. The number of plaques was reduced to

110

50% of co n tr o l level by 5 × 10 -5 M/1 e-DNP-l:ysine. The result was independent o f the DNP-BPA molar ratios. However, when the hapten DNP was i n t r o d u c e d in the form of DNP-BPA, at the same ratios as the coupled SRBC in the test plates, the 50% reduction levels decreased from 40 t o 0.8 × 10 -7 M/1 DNP as the molar ratio of DNP to BPA decreased from 65.7 to 32.8. The reason for such a pattern of plaque inhibition is n o t clear, and it is presently u n d er study. However, it has been observed that multivalent DNP hapten on an albumin molecule binds IgA more strongly than m onoval ent e-DNPlysine. This was shown in the following experiment: various concentrations of e-DNP-lysine were mixed with 45 pg of cellulose, coupled t o the same hapten at a ratio of 6 ng e-DNP-lysine per pg of b r o m a c e t y l cellulose. Then 10 ng o f purified 315 IgA was added, followed by 12SI-labeled anti-IgA, and t h e samples were processed as described under Methods. In the same experiment, e-DNP-lysine was replaced by DNP-BPA with 54 DNP/albumin molecule. The r ed u ct i on of counts to 50% level of samples w i t h o u t DNP was obtained with 8.5 × 1 0 -4 M/1 e-DNP-lysine and 1.1 × 10 -6 M/1 DNP-BPA. This indicates th a t IgA binds to DNPs4BPA about 800 times more strongly than to e-DNP-lysine. It was t her e f or e not surprising t o find in preliminary experiments th at MOPC 460 cells, which secrete IgA with ~ the affinity of IgA f r o m 315 (Jaffe et al., 1969), also f or m ed plaques with DNP-BPA coupled 8RBC. Cellular a n t i - t u m o r reaction

F r o m BALB/c mice bearing progressive MOPC 315 solid tumors, spleen cells were obtained to test for reaction against the t u m o r cells in vitro, as described u n d er Methods. It was found, as shown in table 1, that washed spleen cells from such animals in an overnight mixed culture with MOPC 315 cells considerably reduced the n u m b e r of PFC com pared t o such cultures with spleen cells from n o n - t u m o r bearing mice, or from the same n u m b e r of MOPC 315 cells incubated alone. The plaques were quite similar in appearance in the three different cases. These results, which are based on e n u m e r a t i o n of individual cells producing de novo IgA of sufficient a m o u n t t o cause a h e m o l y t i c plaque, were c o mp ar ed with direct measurements by radioimmunoassay of de novo IgA released into culture. Table 1 shows t ha t the results of the t w o measurements were in close agreement. In experiments 2, 4 and 5 there was also a p r o n o u n c e d reduction of PFC in cultures with normal spleen cells as compared to cultures with MOPC 315 alone. This was also reflected in the radioimmunoassay. H u m o r a l i m m u n e reactions

The usefulness o f the plaque assay t o measure relative concentrations of anti MOPC 315 a n t i b o d y in sera was explored. It was observed t hat normal

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4

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115±

179±

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998

682

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780

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10614 ± 1212

IgA ( c p m )

PFC

PFC

IgA ( c p m )

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I m m u n e spleen cells

1

Exp. #

70

58

56

40

48

PFC

63

54

54

40

53

IgA

% Reduction

7 3 1 5 ± 25

153-+

180+- 1 8

232-+ 15

233 +- 1 8

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13362-+

188

395

486

526

1 2 2 8 4 -+ 1 1 0 0

IgA ( c p m )

MOPC 3 1 5 cells a l o n e

Cell m e d i a t e d r e d u c t i o n of p l a q u e s a n d o f s e c r e t i o n o f igA. P e r c e n t r e d u c t i o n was c a l c u l a t e d as 1 - - ( P F C or IgA ( I m m u n e s p l e e n c e l l s ) ) / ( P F C or IgA ( n o r m a l s p l e e n cells)) x 100. T h e n u m e r i c a l values are p r e s e n t e d t o g e t h e r w i t h t h e s t a n d a r d deviations. All d a t a obtained from between 3 and 6 experiments.

TABLE 1

112

sera interfered with plaque development. Simple tests indicated that this was mainly due to competition between normal immunoglobulin and IgA on the DNP-coupled SRBC for the binding of the rabbit anti IgA which was added to facilitate the hemolytic reaction by the IgA (Yamada et al., 1970). Also, some sera were tested by radial immunodiffusion against DNP-BPA and were found to contain significant amounts of anti-DNP antibodies which could be

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128

512

Fig. 5. Plaque suppressing titer (50% endpoints) of anti H-2 d globulin in relation to concentration of MOPC 315 target cells. MOPC 315 cells incubated for 30 rain with CBA anti-BALB/e globulin, washed twice and incubated for an additional 30 rain in 1 : 30 GPC or 1 : 75 RC. After additional 30 mm of incubation the cells were washed once and diluted appropriately for the plaque test. Two sources of GPC t were used. The open and t

t

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.

.

.

.

closed d a t a p o i n t s r e p r e s e n t e x p e r i m e n t a l results o b t a i n e d u n d e r similar c o n d i t i o n s o n c o n s e c u t i v e days. T h e line b e t w e e n t h e s e p o i n t s r e p r e s e n t s t h e m e a n s of t h e t w o experiments.

113

adsorbed with DNP-Sepharose. This non-specific interference with plaque development was removed b y simply washing the serum-treated MOPC 315 cells prior to plating. Since only 500 cells were to be plated, for convenient enumeration of plaques, the question arose whether this requirement for a low number of cells could be utilized to increase the sensitivity of detecting antibody in sera. Unfortunately, MOPC 315 cells when cultured alone at less than 4000 cells/ml were very susceptible to injury on incubation at 37°C in liquid medium, the control suspensions showing marked reduction in plaques produced per hundred cells. Using higher numbers of cells, and CBA antiBALB/c ascitic globulin as the effector antibody, it was found, as shown in fig. 5, that a decrease in cells by a factor of 50 increased the sensitivity of the test b y about a factor of 5, or slightly more than t w o titer units. Using different batches of GPC' or RC' changed the base-line titer, but not the relative sensitivity with respect to change in cell number. The same anti-BALB/c globulin was used on 250,000 MOPC 315 cells/ml, which made it feasible to compare the titers obtained by plaque reduction, by [3H]TdR incorporation, and by direct determination of cytotoxicity by Trypan blue exclusion. Table 2 shows that there was no detectable difference between the measurements. Preliminary experiments with the plaque m e t h o d were carried out with xenogeneic, allogeneic and syngeneic anti-tumor antibodies. Table 3 com-

TABLE 2 Titer of CBA anti-BALB]c globulin (50% endpoint, log2) vs MOPC 315 target cells by different methods. MOPC 315 cells were incubated in Linbro plates in serial 2-fold dilutions of antibody at 50,000 cells in 0.2 ml. Tests for Trypan blue exclusion and [aH]TdR incorporation were carried out on parallel cultures. Small samples for the plaque test were removed from either set of cultures. The short and long term incubations were initially carried out together, in plastic tubes with double the amounts. One half of each sample was then transferred into Linbro wells for additional incubation and the remainder analyzed immediately.

Exp. #

1 2 3 3 4 4 5 5

Period of Incubation with antibody and C t

(75 (75 (75 (18 (75 (18 (75 (18

min) min) rain) hrs) rain) hrs) rain) hrs)

Trypan blue exclusion

Reduction in plaque formation

Reduction in [ 3H ]TdR incorporation

GPC r

RC'

GPC'

RC'

GPC'

RC'

9.1 9.4 9.5 9.2 9.3 8.8 8.6 8.9

14.2 14.0 13.7 13.0 14.0 13.9 13.9 14.0

9.2 9.0 8.8 ---9.0 9.8

14.3 13.5 --13.9 -14.1 14.0

---9.3 -8.5 -8.6

---13.4 -13.5 -13.6

114 TABLE 3 Comparison between titers (log2) of different antibody preparations. MOPC 315 cells, taken on the 7th or 8th day of ascitic growth were incubated at 1000 cells/0.2 ml for 30 min at 37°C, with serial dilutions of antibody. The cells were washed, resuspended in 0.2 ml of 1/30 GPC', incubated as before, washed and plated with DNP-BSA coupled SRBC as described under Methods. Exp. #

1

2 3 4

Anti-H-2d

Anti tumor

CBA anti-BALB/c

Syngeneic

Allogeneic

Xenogeneic

13.6 13.9 13.3 13.5

6.3 7.4 6.0 6

7.7 7.4 7.6 7.4

9.0 8.8 8.7 8.5

pares the titers of these preparations with the titer of CBA anti-BALB/c ascitic globulin used as a standard. While the non-syngeneic preparations gave reproducible titers with different batches of ascites-grown MOPC 315 cells, the syngeneic anti-tumor gave variable results. F u r t h e r m o r e , while the others gave sharp reductions in PFC within 2--3 two-fold dilutions, the reduction of PFC with the syngeneic anti-tumor ascitic fluid was very gradual as a function of dilution. DISCUSSION The Jerne plaque technique (Jerne and Nordin, 1963) was e x t e n d e d for the use o f MOPC 315 cells in a plaque assay. The procedure for coupling DNP-conjugated BPA t o SRBC (Johnson et al., 1966) was modified to allow preparation o f large batches with economical use of reagents and good stability o f the coupled cells. This procedure may be com pared t o t h a t of Weyer and Bussard (1974), where SRBC were directly coupled to trinitrophenol. Although the present m e t h o d involves indirect coupling and the added step of preparing DNP-BPA, it has the advantage of prolonged usefulness o f the cells, c o m p a r e d t o 5--7 days r e p o r t e d by Weyer and Bussard. In addition, the immunological properties of DNP are b e t t e r d o c u m e n t e d , and some relevant theoretical problems can be handled with the present system. One such problem deals with t he relationship between the reciprocal of a n t i b o d y affinity constant and the c o n c e n t r a t i o n of hapten required to inhibit 50% o f plaques p r o d u c e d against red blood cells coupled to that hapten. In a s tudy of plaque f o r m a t i o n by MOPC 315 cells and the inhibition by e-DNP-lysine, Yamada et al. (1970), showed t hat the concent rat i on (Is0) o f the m o n o v a l e n t ligand necessary t o inhibit 50% of the plaques was 3.5--5 times larger than the reciprocal of the affinity constant ( l / K ) of 315 IgA towards this hapten. Similarly, Miller and Segre (1972) obtained a value

115

for Is0 5--7.5 times 1/K for these t u m o r cells. These results agree with the theoretical predictions of Jerne et al. (1974) that Is0 = 2 ( l / K ) is a good estimate under certain conditions. The present data, which were based on optimizing the efficiency of plaque formation, showed that Is0 may be as high as 50--75 times 1/K. According to the theory, the above equation will overestimate the value of 1/K if a very high a t t a c h m e n t rate of antibody molecules to red cells is combined with a very low detachment rate. In such cases it was shown that the radius of the plaque size will remain fairly constant and will decrease by a factor of 2 as the number of antibody molecules on the red cells is decreased by 3--5 powers of 10. This would keep the number of detectable plaques constant independent of a wide range of antibody synthesis. It is therefore possible that our experimental conditions of coupling DNP to SRBC, and the prolonged exposure to the developing serum following the synthesis of IgA by MOPC 315 cells, optimized the efficiency of plaque formation, as was intended. By changing these conditions and obtaining data on the mean concentration of hapten per red cell, rate of synthetic activity of the t u m o r cells, and other pertinent parameters, it should be possible to test the t h e o r y experimentally with t u m o r cells producing an IgA of homogeneous affinity. In the plaquing procedure described under Methods, MOPC 315 cells were allowed to synthesize for 3 h at 37°C. Anti-IgA was then added and the plates were incubated for 30 min at 37°C, and overnight in the cold. Under these conditions, the number of plaques following the addition of complem e n t was constant within a range of dilution of anti-IgA serum between 1 : 50 and 1 : 20,000. At 1 : 40,000 dilution, the plaques became diffuse and decreased in number. Incubation of the plates with anti IgA for 2 h at 37°C required the use of the serum diluted at most 1 : 400, to obtain recognizable plaques. For economical use of the serum, overnight incubation with the anti-IgA at 1 : 1000 was adopted for use. When shorter periods were allowed for IgA synthesis, and the C' was added together with the anti-IgA to the plates, the effectiveness of anti-IgA became limited to a narrow range of dilutions, being cytotoxic at the low range of dilutions and insufficient at the higher range. This may explain the strong functional dependence of PFC on anti-IgA concentration observed by Weyer and Bussard (1974). It was important to overcome such a dependence to allow plating of antibody treated cells, since the slightest carryover of antibody reacting with anti-IgA into plates would have changed the effective concentration of the anti-IgA, and would have led to non-specific reduction in PFC. A number of applications of the plaque technique have been presented to demonstrate the reliability of the method, as compared to other assays, and its potential usefulness in the analysis of cellular and humoral immune reactions. Since in these studies we are mostly interested in the growth inhibition of the target cells, the plaque assay, which reveals damage on the cellular level, is a more general indicator of injury than measurements dependent on

116 target-cell lysis, since the l a t t e r m a y r e p r e s e n t o n l y a partial e f f e c t in s o m e i m m u n e r e a c t i o n s (Cleveland et al., 1 9 7 4 ; G r e e n b e r g et al., 1 9 7 5 ) . It is still left t o d e m o n s t r a t e t h a t t h e r e d u c t i o n o f M O P C 3 1 5 P F C b y i m m u n e spleen cells, p r e s e n t e d earlier, is e n t i r e l y or p a r t l y d u e t o target-cell d e s t r u c t i o n . In t h e e x p e r i m e n t s with a n t i b o d y it was s h o w n t h a t l o w e r i n g t h e n u m b e r o f t a r g e t cells did n o t increase c o n s i d e r a b l y the 50% e n d p o i n t o f t h e s e r u m . H o w e v e r , it was o b s e r v e d (Harris et al., 1 9 6 7 ) t h a t l y m p h - n o d e cells in the range o f 1 . 2 5 - - 4 0 million p e r ml, w h e n t r e a t e d w i t h a l l o a n t i b o d i e s c a u s e d increased titers o f a p p r o x i m a t e l y 1 p o w e r o f 2 f o r each t w o - f o l d d i l u t i o n o f cells. It is possible t h a t t h e curves o f fig. 5, p r e s e n t i n g t h e r e l a t i o n s h i p b e t w e e n globulin t i t e r a n d cell n u m b e r , will s t a r t t o d r o p in t h e range o f such high c o n c e n t r a t i o n s o f cells. This will h a v e t o be d e t e r m i n e d e x p e r i m e n t a l l y . REFERENCES Boyum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97) 77. Cleveland, P.H., C.F. McKhann, K. Johnson and S. Nelson, 1974, Int. J. Cancer 14,417. Eisen, H.N., E.S. Simms and M. Potter, 1968, Biochemistry 7, 4126. Eisen, H.N., 1964, Methods in Medical Research Vol. 10 (Year Book Medical Publishers, Chicago) pp. 94--102. Frost, P. and C.J. Sanderson, 1975, Cancer'Res. 35, 2646. Ghanta, V.K., N.M. Hamlin, T.G. Pretflow and R.N. Hiramoto, 1972, J. Immunol. 109, 810. Goetzl, H.J. and H. Metzger, 1970, Biochemistry 9, 1267. Golub, E.S., R.I. Mishell, W.O. Weigle and R.W. Dutton, 1968, J. Immunol. 100, 133. Greenberg, A.H., L. Shen and G. Medley, 1975, Immunology 29, 719. Harris, S., C.A. Ogburn and T.N. Harris, 1967, J. Immunol. 99, 721. Harris, T.N. and S. Harris, 1975, Transplantation 19, 318. Ingraham, J.S. and A. Bussard, 1964, J. Exp. Med. 119,667. Jaffe, B.M., H.N. Eisen, E.S. Simms and M. Potter, 1969, J. Immunol. 103,872. Jerne, N.K. and A.A. Nordin, 1963, Science 140, 405. Jerne, N.K., C. Henry, A.A. Nordin, H. Fuji, A.M.C. Koros and I. Lefkovits, 1974, Transplant. Rev. 18,130. Johnson, H.M., K. Brenner and H.E. Hall, 1966, J. Immunol. 97,791. Klinman, N.R. and R.B. Taylor, 1969, Clin. Exp. Immunol. 4, 473. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randal, 1951, J. Biol. Chem. 193, 265. McConahey, P.J. and F.J. Dixon, 1966, Int. Arch. Allergy 29, 185. Merchant, B. and T. Hraba, 1966, Science 152, 1378. Miller, G.W. and D. Segre, 1972, J. Immunol. 109, 74. Moller, G., 1965, Nature 207, 1166. Potter, M., M.A. Leon, 1968, Science 162,369. Potter, M., J.G. Pumphrey and J.L. Walters, 1972, J. Nat. Cancer Inst. 49, 305. Potter, M. and J.L. Waiters, 1973, J. Nat. Cancer Inst. 51,875. Suzuki, T., S. Tanaka and Y. Kawanishi, 1974, Immunochemistry 11,391. Walsh, P., P. Maurer and M. Egan, 1967, J. Immunol. 98,344. Weyer, J. and A.E. Bussard, 1974, Ann. Immunol. Inst. Pasteur 125C, 947. Yamada, H. and A. Yamada, 1969, J. Immunol. 103, 357. Yamada, H., A. Yamada and V.P. Hollander, 1970, J. Immunol. 104,251. This s t u d y was s u p p o r t e d b y U.S. Public H e a l t h Service G r a n t s CA 1 4 4 8 7 , C A 1 7 1 8 1 a n d AI 1 1 4 6 6 , a n d b y g r a n t IM-3B o f t h e A m e r i c a n C a n c e r S o c i e t y .