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Comparison of multiple assays for detecting human antibodies directed against surface antigens on normal and malignant human tissue culture cells
Journal of Immunological Methods, 17 (1977) 225--239 © Elsevier/North-Holland Biomedical Press
225
COMPARISON OF MULTIPLE ASSAYS FOR DETECTING HUMAN A N T I B O D I E S D I R E C T E D A G A I N S T S U R F A C E A N T I G E N S ON NORMAL AND MALIGNANT HUMAN TISSUE CULTURE CELLS
STEVEN A. ROSENBERG, SUSAN SCHWARZ, HEDI ANDING, CORNELIA HYATT and G. MELVILLE WILLIAMS Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 and Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.
(Received 13 January 1977, accepted 27 April 1977)
Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cell lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual end-point cytolysis assay and the IChromium release assay were equally sensitive in measuring complement mediated antibody cytotoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal ceils from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody.
INTRODUCTION A variety o f m e t h o d s exist for m e a s u r i n g a n t i b o d y reactivity against cell surface antigens on i n t a c t h u m a n cells and have been used to search for t u m o r specific reactivity in patients with sarcomas ( M o r t o n and Malmgren, 1 9 6 8 ; Eilber and M o r t o n , 1 9 7 0 ; W o o d and M o r t o n , 1 9 7 0 ; Priori et al., 1 9 7 1 ; B l o o m , 1 9 7 2 ; M o o r e et al., 1 9 7 3 ; Mukherji and Hirshaut, 1 9 7 3 ; Byers et al., 1975). Prior to initiating studies on the serologic reactivity o f h u m a n s to a u t o l o g o u s s a r c o m a s in tissue culture we have investigated the sensitivity o f a variety o f assays suitable for d e t e c t i n g a n t i b o d y directed against surface antigens on several tissue culture cell lines. In o r d e r to c o m p a r e assays in a s t a n d a r d fashion, a single h u m a n serum c o n t a i n i n g extensive reactivity against h i s t o c o m p a t i b i l i t y antigens was tested against t w o m a l i g n a n t and t w o n o r m a l h u m a n tissue culture lines originating f r o m t w o patients. T h r e e c y t o t o x i c i t y assays and a h e m a d s o r p t i o n assay have been used to titrate this allogeneic serum against the five tissue culture lines.
226 METHODS Tissue culture
Two of the tissue culture lines used in these experiments, JA-Sk, JA-F, were initiated in our laboratory from a 15-year old patient with osteogenic sarcoma. JA-Sk was initiated from the normal skin of the a m p u t a t e d lower extremity. The skin was taken f r om a site far from the t u m o r and was carefully dissected free from subcutaneous fat. JA-F was initiated from a solid piece of osteogenic sarcoma obtained from the primary t um or. The t u m o r was carefully dissected free of normal connective tissue and a small piece of the tissue to be p u t in culture was sent to pathology to confirm the presence of relatively 'pure' tumor. The tissues were extensively washed in sterile Hank's Balanced Salt Solution (HBSS) and then minced with a scissors into 1--2 m m 3 fragments. These pieces were again rinsed with HBSS and exposed for about 15 to 30 sec to a c o n c e n t r a t e d antibiotic solution containing 5000 pg/ml of kanamycin {Flow Laboratories, Rockville, Md.). The pieces were then suspended in Eagle's Minimal Essential Medium (E-MEM) supplemented with 20% fetal calf serum (Flow Laboratories, Rockville, Md.) with 50 gg/ml of b o t h kanamycin and chlortetracycline and 10 pg/ml of fungizone (referred to hereafter as complete medium). A b o u t 20 pieces of tissue were suspended in 5--10 ml of c o mp lete med ium and were allowed to settle to the b o t t o m of plastic tissue culture flasks (no. 3042, Falcon Plastics, Oxnard, Calif.). The volume of complete medium added was sufficient to partially cover but n o t float the pieces f r o m the plastic surface. The tissue culture flasks with caps loosely applied were incubated in a moist incubator at 37°C in an atmosphere of 5% CO2 in air. After 7 days or sooner if the pH of the m e di um dr opped below 7.1 as indicated by the phenol red dye in the medium, the medium was aspirated taking care n o t to dislodge the pieces attached to the plastic surface. An equivalent a m o u n t of fresh med iu m was then added. Cells generally became c o n f l u e n t with bot h skin and t u m o r cells within 3--5 weeks of initiation of the culture. In the experiments described in this paper the skin culture designated JA-Sk, a fibroblastic cell line, was used in the first 8 tissue culture transplant generations. Tissue cultures from t um or , JA-F, were used in the first 11 tissue culture transplant generations. Chromosomal k a r y o t y p e studies on JA-Sk revealed a normal chr om os om al c o m p l e m e n t of 46 chromosomes. JA-F was aneuploid and though the modal c h r o m o s o m e n u m b e r was 46, approximately 50% of all metaphase plates examined had an abnormal c h r o m o s o m e n u m b e r compatible with a malignant cell line. A - 8 9 4 5 and A L O - 9 5
Tissue culture lines A-8945 and ALO-95 were initiated from osteogenic sarcoma and normal skin respectively, obtained from a 10-year old black
227 female. Both lines were fibroblastic in appearance. These cultures were kindly supplied to us by the Cell Culture L a b o r a t o r y , University of California, Naval Biomedical Research L a b o r a t o r y , Oakland, California under a contract to the Special Virus Cancer Program o f the National Cancer Institute. The osteogenic sarcoma and normal skin cultures were sent to us in the 6th and 8th tissue culture passage generation respectively and were used by us between this generation and the 18th tissue culture transplant generation. Chromosomal k a r y o t y p e analysis on A-8945 osteogenic sarcoma line revealed m a n y abnormal metaphase plates including pulverized and minute chromosomes as well as significant endo-reduplication compatible with a malignant cell line. The normal skin line ALO-95 had a normal chrom osom al k a r y o t y p e .
Subculture o f monolayer cultures The four tissue culture lines described above grow as monolayers adherent t o the surface of plastic tissue culture flasks. T he adherent m o n o l a y e r cultures were washed with 10 ml o f HBSS prior to adding 10 ml of 0.25% Trypsin in Dulbecco's Balanced Salt Solution, containing 0.0005 molar versene w i t h o u t calcium or magnesium. These cultures were incubated at 37°C with i n t e r m i t t e n t mixing for approximately 10 rain or until the m o n o l a y e r lifted fr o m the flask surface. The cell suspension was then poured into a centrifuge tube, complete medium added, and centrifuged for 7 min at 4°C at 260 g. The pellet was resuspended in 20--30 ml o f complete medium and the cells from a single m o n o l a y e r flask were generally divided into 2 or 3 flasks depending u p o n the gross characteristics of the cell line. Cells generally adhered to the plastic surface within a few hours after trypsinization and began growth by 24 h later. All tissue culture media were changed 2 times per week.
Allogeneic human serum A highly reactive allogeneic antiserum, obtained from a patient with Waldenstrom's macroglobulinemia who had received multiple blood transfusions, was kindly supplied to us by Dr. Dean Mann, NCI. Serum from this patient was c y t o t o x i c to l y m p h o c y t e s obtained from > 9 9 % of the population when tested by standard histocompatibility antigen typing techniques. This serum was dialyzed at 4°C against distilled water to precipitate macroglobulins and the residual serum was stored in aliquots at --20°C. This allogenic serum, called Io, was used in serial dilutions in the assays to be described. Normal serum used as controls in m a ny of the assays were obtained from normal blood donors who had never received transfusions or undergone surgery. Blood was allowed to clot at r o o m t e m p e r a t u r e for 20 min and serum collected following centrifugation at 1500 g for 10 min.
228
Complement Rabbit serum obtained by cardiac punc ture of New Zealand White rabbits was used as c o m p l e m e n t in all experiments. Blood was allowed to clot at r o o m t e m p e r a t u r e for 10 min and at 4°C. Sera from multiple rabbits were screened to obtain a large pool of sera with high c o m p l e m e n t titer that was non-toxic at a 1 : 4 dilution to hum an cells.
Assays for antibody activity S~Chromium release cytotoxicity test The 5~Chromium release c y t o t o x i c i t y test was perform ed in microtiter plates (no. 3040, Falcon Plastics, Oxnard, Calif.) by techniques similar to those previously described (Rosenberg and Schwarz, 1974). Single cell suspensions of each of the tissue culture target lines were prepared by gentle trypsinization as described above. Previous experiments d e m o n s t r a t e d that trypsinization did n o t affect the ability of antibodies to cause cell lysis in this assay and cells harvested mechanically using a rubber policeman worked equally well. A p p r o x i m a t e l y 2 × 106 target cells were suspended in 200 #c of SlChromium (Amersharn/Searle, Arlington Hgts., Ill.) in 0.3 ml of medium consisting of RPMI 1640 plus 10% heat inactivated fetal calf serum. Following incubation for 45 min at 37°C the cells were washed twice with 15 ml m e d iu m by centrifugation at 500 g for 10 min and resuspended. Following the second wash, cells were suspended in 1 ml of medium and the viability and cell n u mb e r evaluated by counting in 0.2% t r y p a n blue. Cells were diluted to 10 s live cells per ml. 20 X of serum of appropriate dilution was added to the wells of these f i a t - bot t om microtest plates and the plates were v o r t e x e d vigorously to distribute the fluid equally along the surface of the well. Using a Hamilton syringe with a repeating dispenser unit (Hamilton C o m p a n y , Reno, Nevada) 10 X of S~Chromium labelled cells containing 10 "~ live cells were added to each well. Cells and serum were incubated for 1 h at 37°C. Following this incubation 200 ~ of complete medium was added to all wells and the plates were centrifuged at 5 0 0 g for 5 min. Supernatant m ed iu m was flicked from the plate by sharp inversion of the wrist and this wash was repeated two further times. These washes were found essential to eliminate a n t i c o m p l e m e n t a r y factors present in some sera used at low dilutions. Following the third wash, 10 ~ of a rabbit c o m p l e m e n t were added to each well with a Hamilton syringe. These plates were then gently vortexed and incubated for 45 min at 37°C. Following this incubation 200 ~ of complete med ium was added to all wells and the plates were centrifuged at 800 g for 10 min. 100 ~ was removed from each well and transferred to a test tu b e for gamma counting. 10 X of ~lChromium labelled cells in 200 ~ of m ed iu m were frozen and thawed in a dry-ice acetone mixture four times in a test tube. These test tubes were centrifuged at 800 g for 10 min and 100 ~. collected to determine the m a x i m u m releasable chrom i um present in these
229 cells. The p er cen t lysis was calculated using the following formula: c pm test sample -- cpm c o m p l e m e n t control X100 % Lysis = cpm freeze-thaw -- cpm c o m p l e m e n t control Using this assay the mean coefficient o f variation of the triplicate measurements o f cpm S~Chromium release of 100 random measurements from 10 consecutive experiments was 6.07 i 0.43% (SEM).
Visual endpoin t rnierocy to toxicity test A m i c r o c y t o t o x i c i t y test similar to that described by Wood and Morton (1970) was employed. Following trypsinization and washing of the target cells, cells were suspended in 10 ml of complete medium and the viability and cell n u m b e r evaluated by counting in 0.2% t rypan blue. Cells at the appropriate dilution were then plated into ultramicrotiter plates (no. 3034, Falcon Plastics, Oxnard, Calif.). Uniform distribution of cells within the 60 wells of the plate was achieved by flooding the entire plate with the cell suspension. By gently tipping the plate and aspirating all excess medium, a p p r o x i m a t e l y 15--20 ~ was left remaining in each well. These plates were then incubated overnight at 37°C in a moist atmosphere of 5% CO2 to allow the cells to adhere tightly to the b o t t o m of the wells. For the 4 cell lines used in these experiments from 1--5 × 104 cells per ml were plated in each well in order to achieve a ppr oxi m a t e l y 100 cells in each well by the following day. After overnight incubation, the plates were gently flooded with HBSS which was removed by pouring of f excess fluid and then flicking the plate. Following two such washes, 10 ~ of the appropriate serum dilution was added to each well with an O x f o r d pipette. Plates were incubated at 37°C for 2 h. Serum was then flicked from the plates and the plates washed twice with HBSS. Following the second wash, 10 ~ of rabbit c o m p l e m e n t was added to each well with an O x f o r d pipette. After an additional one h o u r incubation at 37°C, 8 ml o f complete medium was added and the plates were allowed to incubate overnight. After ap p r o x i m at el y 18 h, medium was poured from the plates and the plates were flicked. Ten ml of HBSS were used to wash the plates three m ore times prior to flooding with a fixative solution (Perfix, Applied Biosciences, Fairfield, N.J.) for 15 min. The plates were then washed with running tap water and stained with 1 mg percent crystal violet for 10 min. Excess stain was washed away with warm water and the plate allowed to dry. The n u m b e r of residual cells left in each well were c o u n t e d under a microscope at 100 × magnification. In all experiments each serum dilution was checked in sextuplicate on each plate. All plates also contained 6 wells each containing only med iu m and containing m e di um plus com pl em ent .
230 The percent c y t o t o x i c i t y was calculated as follows: % cytotoxicity .
cells in c o m p l e m e n t control well -- cells in test well . . . ×100. cells in c o m p l e m e n t control well
Using this assay, the mean coefficient of variation of 100 sets of cell counts selected r andom l y from 10 experiments was 15 ± 0.5 (SEM).
Trypan blue cytotoxicity test C y t o t o x i c i t y as measured by t r ypan blue dye exclusion was p e r f o r m e d by techniques modified from those previously described by Sachs et al. (1971). Following trypsinization of the target cell as described above, cells were evaluated for n um ber and viability by counting in 0.2% t r y p a n blue. Cells were generally greater than 95% viable and were resuspended to a final concentration o f 5 × 105 cells per ml. 25 X of complete medium was added to the wells of a r o u n d - b o t t o m microtiter plate (no. 1-221-24-1, Cooke Laborat o r y Products, Alexandria, Virginia). 25 X of the appropriate serum dilution was added to appropriate wells and 25 X of target cells containing 1.25 × 104 live cells were added to each well. The plates were mixed on a vortex and incubated for 15 min at 37°C. Following this incubation 150 X of complete m ed iu m was added and the plates were spun at 200 g for 5 min. The plate was 'flicked' to remove supernatant and 25 X of rabbit c o m p l e m e n t was added. The plates were again mixed and incubated for 30 min at 37°C. Following this incubation the plates were centrifuged at 800 g for 10 min and the supernatant removed from the wells. 5 X of a 0.2% solution of t r y p a n blue was added to each well and the cells were then resuspended by vigorous up and down pipetting. Cells were then placed on a glass slide and the number of cells b o th excluding and staining with t rypan blue was evaluated. The p er cen t c y t o t o x i c i t y was evaluated as the percentage of the cells that stained with tr y p an blue or exhibited cell lysis as d e m o n s t r a t e d by 'ballooning' of the cell membrane. In all tests, cells were incubated with media alone and with c o m p l e m e n t alone as controls.
Mixed hemadsorption assay The h e m a d s o r p t i o n assay was p e r f o r m e d by techniques modified by Williams et al. (1976) from the m e t h o d s described by Fagraeus et al. (1961), Milgrom et al. (1965), and by Tachibana et al. (1970). Indicator cells were prepared from fresh h u m a n Rh-D positive blood. The red blood cells were washed three times in 0.01 M phosphate buffered saline containing 0.85% NaC1 (PBS) and a 4% suspension was prepared. 20 ml of this red blood cell suspension plus 50 ~ of a highly reactive anti-D positive antiserum (Ripley; kindly provided by Dr. Marion Walker) were mixed and incubated for 30 min. Following 3 washes in PBS these IgG coated red blood cells (Ripley
231 cells) were stored at 4°C to be used within 7 days. Ripley cells were coated with anti-IgG by adding an equal volume of 1 : 10 dilution of rabbit antih u m a n immunoglobulin (Cappel Labs., Inc., Downington, Pa.) to a 2% suspension. Following a 30-min incubation at 37°C the cells were washed three times in complete medium and diluted to form a 0.2% volume suspension. To p er f o r m the hem a ds or pt i on assay tissue culture target cells were trypsinized and a p p r o x i m a t e l y 2 X 104 cells in 0.2 ml were added to each well of a U - b o t t o m microtiter tray (no. 1-221-24-1, Cooke L a b o r a t o r y Products, Alexandria, Virginia). These plates were incubated for 24--48 h until a confluent m o n o l a y e r of adherent cells was obtained. At this time, the supern atan t medium was gently aspirated from the target cells and the cells washed one time with RPMI-1640 w i t h o u t serum. 0.2 ml of the a n t i b o d y at appropriate dilutions in complete medium containing 50 mM Hepes buffer was added to each well. Following incubation for 1 h at 37°C the cells were washed three times with RPMI-1640 and 0.2 ml of indicator e r y t h r o c y t e s prepared as described above were added to the wells and incubated for 30 min at 37°C. The plates were sealed using pre-cut mylar sealing tape (no. 1-220-30-1, Cooke L a b o r a t o r y Products, Alexandria, Virginia) and inverted 3--5 times by a gentle rocking motion. The n u m b e r of red blood cells a dh er en t to the m o n o l a y e r was then evaluated under a microscope using 50 X magnification. Confluent m onol a ye r s of attached red cells were read as 4+ and one or two red cells attached to occasional cells on the plate were read as 1+. A negative reaction was one in which no red cells were adherent to the target cell monolayers. The he m adsorpt i on titer was read as the highest dilution at which a 2+ reading was obtained. In general, the grades of reactivity declined with increasing dilution such that a ten-fold dilution separated 3--4+ activity f r om 1+ activity. Improved quantitation was possible by radiolabeling the indicator red blood cells with 5~Chromium. In this modification, 200 mc of SIChromium were added to the m i xt ur e of 2% Ripley cells and the 1 : 10 antiglobulin dilution, t h e r e b y producing radiolabeled antiglobulin-coated red blood cells. Following visual reading, the tray was washed two times with PBS and 0.2 ml o f distilled water was added to each well. Ten minutes later, when hemolysis was complete 0.1 ml samples were removed and assayed for radioactivity as described in the S~Chromium release assay. Radioactivity as an index o f indicator cell adherence was pl ot t ed against serum dilution and the titer was determined as the serum c onc e nt rat i on resulting in 50% o f the maximal radioactivity bound to the cultures. RESULTS
Optimization of assays In developing these assays for use with hum an serum and hum an target cells, differing times of a n t i b o d y and c o m p l e m e n t incubations were investi-
232
gated. An example of these optimization experiments using the visual endp oin t m i c r o c y t o t o x i c i t y assay is shown in figs. 1 and 2. On separate microtiter plates, cells were exposed to a 1 : 40 dilution of Io antibody for 60 or 120 min and then exposed to c o m p l e m e n t for either 30, 60, or 120 min. Lysis due to c o m p l e m e n t alone (compared to media) or to serum plus complement (compared to c o m p l e m e n t alone) was calculated separately for each of these six plates (fig. 1). Lysis due to c o m p l e m e n t alone was less than 15% in all plates. At all three times of c o m p l e m e n t exposure, greater lysis was obtained with a 120-rain ant i body incubation than with 60 min of antibody incubation. Time of c o m p l e m e n t exposure between 30 and 120 min appeared n o t to be a critical factor. Following incubation with c o m p l e m e n t , plates were incubated for varying times in medium prior to washing and counting of residual cells (fig. 2). The time of p o s t - c o m p l e m e n t incubation did n o t appear critical. Comparison of cell counting on unstained preparations using phase contrast microscopy as described by Wood and M or t on (1970) gave identical results to those obtained when cells were c o u n t e d after fixation and staining with crystal violet (table 1). As a result of these experiments, we selected a 120-min antibody incubation, 60-rain c o m p l e m e n t incubation, and 16 h (overnight) of post-complem e n t incubation for routine per f or m ance of this assay. All plates were fixed and stained prior to evaluation. Similar optimization experiments led to the selection of the parameters
120
60' Ab Incubation -:~- 120' Ab incubation
11o ~r
100
I
90 F
~
70 r
P Z
60 p
5
so r
a_
40 t
~_~,_
. . . . .
~'~ .[
Serum Complement
L'
Complcnlent Aone
1
3o F
0
0.5
1.0
15
20
25
TIME OF COMPLEMENT iNCUBATION {Hours)
Fig. 1. T h e e f f e c t o f t h e t i m e o f a n t i b o d y i n c u b a t i o n a n d c o m p l e m e n t i n c u b a t i o n o n t h e lysis o f o s t e o s a r c o m a tissue c u l t u r e cells. In all p l a t e s Io a n t i b o d y was u t i l i z e d at a 1 : 40 dilution.
233 100
f j..~.4t
so u~
6o
}z Lu (.3
5o
>,
or-
Serum + Complement
4o 30 Complement Alone
20 ~ lO
I 2
4
6
8
1
1
16
18
20
;2 2i4 26 28
TIME OF POST INCUBATION (Hours)
Fig. 2. The effect of the time of incubation, following a 2-h incubation with complement, on the lysis of osteogenic sarcoma cells by Io serum.
listed in Methods for the 5~Chromium release microcytotoxicity assay. Two points are of special interest with regard to the SlChromium release assay. ; These four tissue culture target cell lines incorporated sufficient S~Chromium to allow the use of 103 target cells in these assays. This provided a small increase in sensitivity compared to the use of 104 target cells (necessary when fresh lymphocyte target cells are used). Secondly, no difference in spontaneous 51Chromium release or sensitivity was obtained when cells were harvested from monolayer cultures mechanically using a rubber policeman or when they were harvested using dilute trypsin solutions as described in Methods above. A variety of factors were investigated to optimize the hemadsorption assay. The persistence of a large number of adherent cells throughout the assay was essential. Experiments were carried out using various media with and without Hepes buffer and fetal calf serum. Only 1 of 9 cell lines toler-
TABLE 1 Effect of staining on cell counts in microcytotoxicity plates No. of cells plated/well
1000 500 250 125 63
No. of cells counted (mean _+ S.E.) Before staining
After staining
120 +__7 72__+2 45 __4-4 13 __4"2 6__+1
117 -+. 4 72__+3 40 + 4 12 __+3 6__+1
234 ated exposure to high dilutions of inactivated hum an serum in the absence of fetal calf serum, and complete medium containing 20% fetal calf serum was used as the medium for carrying out antibody dilutions. In addition, the presence of at least 5% fetal calf serum at the step of indicator-target cell adherence was m a n d a t o r y . In the absence of serum, the sensitivity generally declined which was mainly due to m o n o l a y e r d e t a c h m e n t (fig. 3). Further, in the absence of fetal calf serum, the indicator e r y t h r o c y t e s occasionally adhered non-specifically to the plastic surface of the wells. Experiments designed to evaluate the role of Hepes buffer showed that, while it clearly stabilized the pH during the making of serial dilutions in media, there was no i m p o r t a n t effect on the sensitivity or the reproducibility of the assay. Parameters used in the t r y p a n blue assay were the same as those determined by Sachs et al. (1971).
Comparison of assays Example experiments with the visual e n d p o i n t m i c r o c y t o t o x i c i t y and SlChromium release assays are shown in tables 2 and 3. Comparison of the f o u r assays on the four different hum an tissue culture lines using a single allogeneic serum is presented in fig. 4 and table 4. Of note: 1. SlChromium release and m i c r o c y t o t o x i c i t y assays p e r f o r m e d as described in Methods were appr oxi m at el y equally sensitive and far more sensitive than the described assay using t r y p a n blue uptake as the c y t o t o x i c end-
slCr LABELLED INDICATOR CELL HEMADSORBTION ASSAY WITH AND WITHOUT CALF SERUM 2,0001
114)* T -.------~(4) 9
5 o~/oFetal Calf Serum
1,500
1
~
T
I~ \l~(2)
---
No Fetal Calf Serum
/ ~\
~,111 \ (o)
2,000
(o)
8,000
32,000
SERUM DILUTION -1 Fig. 3. The effect of fetal calf serum on the m e a s u r e m e n t of m i x e d cell h e m a d s o r p t i o n using Io antiserum and tissue cultured t u m o r cells. In this e x p e r i m e n t chromium-labelled red cells were utilized to quantitate the degree of red b l o o d cell adsorption.
235 TABLE 2 M i c r o c y t o t o x i c i t y assay using Io s e r u m o n n o r m a l a n d m a l i g n a n t h u m a n cell lines f r o m t h e same p a t i e n t . A-8945 (osteosarcoma)
ALO-95 (normal} No. cells * Medium alone Complement alone
1 3 8 . 0 _+ 9.5 132.4 ± 14.2
Io 1 : 6 4 0 1 : 1280 1:2560 1 : 5120 1 : 10240
0 6 . 5 ± 1.0 38.3±11.7 83.5 ± 17.7 121.2 ± 13.6
% Lysis * ---
No. cells 115.5 ± 9.9 104.2 ± 10.4
100 9 5 . 1 ± 0.7 7 1 . 1 ± 8.4 36.9 ± 13.4 8.5 ± 10.2
0 0 1 2 . 2 ± 3.1 58.5 ± 14.4 117.0 _+ 18.5
% Lysis --100 100 8 8 . 3 ± 2.9 43.8 ± 13.8 - - 1 2 . 3 ± 17.7
* M e a n ± 1 s t a n d a r d error.
point. The assays appeared equally reproducible a n d f o r all a s s a y s t h e i n d i vidual titers were within 1 tube dilution of the average titer presented in table 2. 2. The hemadsorption assay revealed antibody binding to target cells at approximately ten-fold higher dilutions than that seen using any of the three cytotoxicity assays. 3. B o t h t h e s k i n a n d o s t e o s a r c o m a cells from patient JA were far less r e a c t i v e i n all a s s a y s t e s t e d t h a n w e r e s k i n a n d o s t e o s a r c o m a lines from a different patient (ALO-95 and A-8945).
TABLE 3 C h r o m i u m - 5 1 release assay using Io s e r u m o n t w o h u m a n cell lines.
M e d i u m alone
ALO-95 (normal)
A-8945 (osteosarcoma)
cpm *
cpm
% lysis *
% lysis
339 ± 51
--
415
--
Complement alone Freeze-thaw controls
383 ± 9 2601 ± 90
---
389 3099
---
Io 1 1 I 1 1 1 1 1
1595 _+ 51 1 4 9 6 _+ 58 1 4 8 8 + 54 1421 _+ 34 1046_+ 10 587 ± 13 372 _+ 30 3 7 6 _+ 17
: : : : : : : :
160 320 640 1280 2560 5120 10240 20480
* M e a n ± 1 s t a n d a r d error.
54.7 ± 4.0 50.2 _+ 4.6 50.0 _+ 4.3 46.8 _+ 2.7 28.9_+0.8 9.2 _+ 1.0 --0.5 ± 2.4 --0.3 ± 1.3
1664 _+ 35 1684 _+ 74 1653 _+ 93 1422 ± 31 1078_+ 5 634 ± 48 4 2 9 _+ 37 4 3 0 _+ 33
47.1 _+ 1.3 47.8 + 4.8 46.6 + 6.0 38.1 ± 2.0 25.4_+0.3 9.0 _+ 3.0 1.5 _+ 2.4 1.5 ± 2.1
236 Chromium-51 Release Assay 100 •
100 r
I c, JA-Sk
c2"-"c~<~,
• JA-F
\
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:i
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F
ALO 95
60 i
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Microcytotoxicity Assay 100 ~ .....
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L ............. Trypan Blue Assay
\ 80~
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20 1
512 2048 8192 256 1024 4096
'\'\ 8
32 16
128 64
512 2048 8192 256 1 0 2 4 4096 16384
SERUM DILUTION-'
Fig. 4. A c o m p a r i s o n o f t h e s ] C h r o m i u m r e l e a s e assay ( u p p e r ) , t h e m i c r o c y t o t o x i c i t y assay ( m i d d l e ) a n d t h e t r y p a n b l u e assay ( l o w e r ) u t i l i z i n g Io a n t i s e r u m a n d n o r m a l a n d m a l i g n a n t cell lines f r o m t w o p a t i e n t s .
4. 100% lysis w a s n o t a c h i e v e d w i t h any cell line w h e n using t h e 5'Chrom i u m release assay (fig. 3); that is, even t h e m o s t c o n c e n t r a t e d Io a n t i s e r u m did n o t release as m u c h S1Chromium f r o m t h e cell as w a s released by freezing
237 TABLE 4 Comparison of assays on human tissue culture cells. Human cell line
Microcytotoxicity *
s 1Cr release *
TB *
Hemadsorption **
JA-Sk JA-F ALO-95 A-8945
320 320 5120 5120
320 320 2560 2560
64 64 256 256
8100 (2) 8100 (1) ---
(5) *** (7) (3) (2)
(2) (2) (2) (2)
(2) (2) (2) (2)
* (50% lytic dilution) -1 . ** Highest serum dilution with positive hemadsorption) -1 . *** Number in parentheses is number of determinations in separate experiments.
a n d t h a w i n g t h e cells. In eleven e x p e r i m e n t s on t h e f o u r cell lines, m a x i m a l S ' C h r o m i u m release ranged f r o m 35 to 79% o f t h e f r e e z e - t h a w c o n t r o l s ( m e a n = 57%). F r e e z i n g and t h a w i n g released a p p r o x i m a t e l y 80% o f t h e t o t a l c h r o m i u m in the cells. T h e m a x i m a l a m o u n t o f c h r o m i u m released by t h e cells varied f r o m d a y to d a y a n d c o u l d n o t be c o r r e l a t e d w i t h the age o f t h e S ' C h r o m i u m or b y a n y r e c o g n i z a b l e c u l t u r e c o n d i t i o n s such as the degree of c o n f l u e n c y o f the cell m o n o l a y e r w h e n h a r v e s t e d , t i m e f r o m last feeding, t i m e f r o m last t r y p s i n i z a t i o n or o t h e r factors. 5. Five n o r m a l h u m a n sera t e s t e d o n these cell lines using the h e m a d s o r p t i o n assay gave titers of < 1 : 10 in all f o u r assays. 66% o f 155 n o r m a l h u m a n sera tested using the visual e n d p o i n t m i c r o c y t o t o x i c i t y assay s h o w e d sign i f i c a n t lysis against the h u m a n skin lines w h e n t e s t e d u n d i l u t e d b u t were n o t lytic at d i l u t i o n s g r e a t e r t h a n I : 8. DISCUSSION A v a r i e t y o f assays have b e e n used to d e t e c t a n t i b o d y r e a c t i v i t y to surface antigens on n o r m a l and m a l i g n a n t h u m a n cells g r o w n in tissue culture. In this p a p e r we have a t t e m p t e d to c o m p a r e t h e sensitivity o f t h r e e d i f f e r e n t m i c r o c y t o t o x i c i t y assays and a h e m a d s o r p t i o n assay to m e a s u r e t h e reactivity o f a single a l l o a n t i s e r u m against f o u r s e p a r a t e h u m a n tissue c u l t u r e cell lines derived f r o m t w o p a t i e n t s . In c o m p a r i n g c y t o t o x i c i t y assays based on visual c o u n t i n g o f cells with t h o s e using S ' C h r o m i u m release, similar a n t i b o d y titers were d e t e c t e d . A l t h o u g h less t e d i o u s , the S ' C h r o m i u m release assay e x h i b i t e d c o n s i d e r a b l e d a y to d a y v a r i a t i o n in the m a x i m a l a m o u n t o f S ' C h r o m i u m released b y saturating a n t i b o d y c o n c e n t r a t i o n s . T h e releasable S l C h r o m i u m varied f r o m 35 to 79% o f the f r e e z e - t h a w c o n t r o l s in d i f f e r e n t e x p e r i m e n t s . We w e r e u n a b l e to s t a n d a r d i z e the m a x i m a l releasable c h r o m i u m in these assays d e s p i t e m a n i p u l a t i o n o f a v a r i e t y o f variables such as age o f c h r o m i u m , age o f tissue c u l t u r e cells, etc. This f e a t u r e r e p r e s e n t e d a significant d i s a d v a n t a g e o f this assay and a p p e a r e d u n i q u e to the use of tissue culture t a r g e t cells since we
238 did n o t see these variations when using fresh l y m p h o c y t e target cells. This variation in maximal releasable chromium led to some confusion in determining antibody titers based on the antibody concentration necessary to lyse 50% of the cells. In table 4 we have used the m a x i m u m lysis in each individual S'Chromium experiment and have taken as the serum titer, the dilution necessary to achieve one-half of that a m o u n t of lysis. The ambiguities involved in these manipulations have led us to favor the visual endpoint assay for routine work. 100% lysis was routinely seen when using high antibody concentrations in the visual endpoint microcytotoxicity assay. The visual endpoint and S'Chromium release assays gave very similar titers using the Io serum on all four h u m a n cell lines (table 4 and fig. 4). Trypan blue as a measure of c y t o t o x i c i t y was significantly less sensitive than either of the two former methods. It should be kept in mind, when comparing assay sensitivities, that the visual endpoint technique utilized 100 target cells as compared to 1000 and 12,500 cells for the chromium release and trypan blue assays respectively. Practical considerations prevented the use of fewer cells in the latter two assays. The hemadsorption technique provided significantly greater sensitivity than any of the cytotoxicity assays for measuring the reactivity of Io-serum with all two cell lines obtained from patient JA (table 4). Exact quantitation with this assay is difficult because of the objective nature of visually estimating red blood cell binding to the tissue culture cells. We have not attempted to increase the objectivity of this assay by using S'Chromium labelled red blood cells as described by Thompson et al. (1973) and by Sundqvist and Fagraeus (1972). Some of the difference in titers that we have seen may be due to the increased efficiency of IgM antibodies in cytotoxicity assays as opposed to the design of our hemadsorption assay to detect IgG antibodies only. The predominance of IgG antibodies in the Io serum may account, in part for the greater sensitivity when using the hemadsorption assay. It should be emphasized that each of the assays described here reflects different aspects of the cell-antibody interaction and that the use of multiple assays provides more information than the use of any single assay alone. It is of interest that both normal and malignant cell lines from the same individual showed different degrees of reactivity compared to similar normal and malignant lines from a different individual. Thus, the normal and malignant cell lines from patient JA were far less sensitive to lysis than the normal and malignant cell lines ALO-95 and A-8945 from the same patient (table 4 and fig. 3). At Io serum concentrations of 1/1,000 in the visual endpoint m i c r o c y t o t o x i c i t y assay no lysis of JA-Sk or JA-F was seen, whereas, 100% lysis of ALO-95 and A-8945 was evident. Since Io serum detected alloantigens it is possible that the JA lines expressed greater amounts of these antigens than did the ALO-95 and A-8945 cell lines, though the broad reactivity of the Io serum would seem to make this unlikely. Also possible, is that the ALO-95 and A-8945 lines are more 'lysable' for other reasons, such as increased complement binding, decreased cell adhesivity to plastic, etc. It is
239 clear, h o w e v e r , t h a t t h e a p p a r e n t t u m o r specificity seen b y a c o m p a r i s o n o f a t u m o r cell line f r o m o n e p a t i e n t w i t h a n o r m a l cell line f r o m a n o t h e r individual m a y be d u e to f a c t o r s o t h e r t h a n t h e p r e s e n c e o f t u m o r specific a n t i b o d i e s . Intrinsic d i f f e r e n c e s in t h e ' r e a c t i v i t y ' o f cells in a n y given assay, m u s t b e c o n s i d e r e d in in vitro studies of h u m a n t u m o r i m m u n e responses. REFERENCES Bloom, E.T., 1972, Cancer Res. 32,960. Byers, V.S., Levin, A.S., Johnston, J.O. and Hackett, A.J., 1975, Cancer Res. 35, 2520. Eilber, F.R. and Morton, D.L., 1970, J. Nat. Cancer Inst. 44,651. Fagraeus, A. and Espmark, J.A., 1961, Nature 190,370. Klein, E., 1970, Transplantation 9,219. Moore, M., Witherow, P.J., Price, C.H.G. and Clough, S.A., 1973, Int. J. Cancer 12,428. Morton, D.M. and Malmgren, R.A., 1968, Science 162, 1279. Mukherji, B. and Hirshaut, Y., 1973, Science 181,440. Priori, E.S., Wilbur, J.R. and Dmochowski, L., 1971. J. Nat. Cancer Inst. 46, 1299. Rosenberg, S.A. and Schwarz, S., 1974, J. Nat. Cancer Inst. 52, 1151. Sachs, D.H., Winn, H.J. and Russell, P.S., 1971, J. Immunol. 107,481. Sundqvist, K.G. and Fagraeus, A., 1972, Immunology 22,371. Tachibana, T., Worst, P. and Klein, E., 1970, Immunology 19,809. Thomson, D.M.P., Steele, K. and Alexander, P., 1973, Brit. J. Cancer 27, 28. Williams, G.M., 1976, in preparation. Wood, W.C. and Morton, D.L., 1970, Science 170, 1318.
225
COMPARISON OF MULTIPLE ASSAYS FOR DETECTING HUMAN A N T I B O D I E S D I R E C T E D A G A I N S T S U R F A C E A N T I G E N S ON NORMAL AND MALIGNANT HUMAN TISSUE CULTURE CELLS
STEVEN A. ROSENBERG, SUSAN SCHWARZ, HEDI ANDING, CORNELIA HYATT and G. MELVILLE WILLIAMS Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 and Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.
(Received 13 January 1977, accepted 27 April 1977)
Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cell lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual end-point cytolysis assay and the IChromium release assay were equally sensitive in measuring complement mediated antibody cytotoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal ceils from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody.
INTRODUCTION A variety o f m e t h o d s exist for m e a s u r i n g a n t i b o d y reactivity against cell surface antigens on i n t a c t h u m a n cells and have been used to search for t u m o r specific reactivity in patients with sarcomas ( M o r t o n and Malmgren, 1 9 6 8 ; Eilber and M o r t o n , 1 9 7 0 ; W o o d and M o r t o n , 1 9 7 0 ; Priori et al., 1 9 7 1 ; B l o o m , 1 9 7 2 ; M o o r e et al., 1 9 7 3 ; Mukherji and Hirshaut, 1 9 7 3 ; Byers et al., 1975). Prior to initiating studies on the serologic reactivity o f h u m a n s to a u t o l o g o u s s a r c o m a s in tissue culture we have investigated the sensitivity o f a variety o f assays suitable for d e t e c t i n g a n t i b o d y directed against surface antigens on several tissue culture cell lines. In o r d e r to c o m p a r e assays in a s t a n d a r d fashion, a single h u m a n serum c o n t a i n i n g extensive reactivity against h i s t o c o m p a t i b i l i t y antigens was tested against t w o m a l i g n a n t and t w o n o r m a l h u m a n tissue culture lines originating f r o m t w o patients. T h r e e c y t o t o x i c i t y assays and a h e m a d s o r p t i o n assay have been used to titrate this allogeneic serum against the five tissue culture lines.
226 METHODS Tissue culture
Two of the tissue culture lines used in these experiments, JA-Sk, JA-F, were initiated in our laboratory from a 15-year old patient with osteogenic sarcoma. JA-Sk was initiated from the normal skin of the a m p u t a t e d lower extremity. The skin was taken f r om a site far from the t u m o r and was carefully dissected free from subcutaneous fat. JA-F was initiated from a solid piece of osteogenic sarcoma obtained from the primary t um or. The t u m o r was carefully dissected free of normal connective tissue and a small piece of the tissue to be p u t in culture was sent to pathology to confirm the presence of relatively 'pure' tumor. The tissues were extensively washed in sterile Hank's Balanced Salt Solution (HBSS) and then minced with a scissors into 1--2 m m 3 fragments. These pieces were again rinsed with HBSS and exposed for about 15 to 30 sec to a c o n c e n t r a t e d antibiotic solution containing 5000 pg/ml of kanamycin {Flow Laboratories, Rockville, Md.). The pieces were then suspended in Eagle's Minimal Essential Medium (E-MEM) supplemented with 20% fetal calf serum (Flow Laboratories, Rockville, Md.) with 50 gg/ml of b o t h kanamycin and chlortetracycline and 10 pg/ml of fungizone (referred to hereafter as complete medium). A b o u t 20 pieces of tissue were suspended in 5--10 ml of c o mp lete med ium and were allowed to settle to the b o t t o m of plastic tissue culture flasks (no. 3042, Falcon Plastics, Oxnard, Calif.). The volume of complete medium added was sufficient to partially cover but n o t float the pieces f r o m the plastic surface. The tissue culture flasks with caps loosely applied were incubated in a moist incubator at 37°C in an atmosphere of 5% CO2 in air. After 7 days or sooner if the pH of the m e di um dr opped below 7.1 as indicated by the phenol red dye in the medium, the medium was aspirated taking care n o t to dislodge the pieces attached to the plastic surface. An equivalent a m o u n t of fresh med iu m was then added. Cells generally became c o n f l u e n t with bot h skin and t u m o r cells within 3--5 weeks of initiation of the culture. In the experiments described in this paper the skin culture designated JA-Sk, a fibroblastic cell line, was used in the first 8 tissue culture transplant generations. Tissue cultures from t um or , JA-F, were used in the first 11 tissue culture transplant generations. Chromosomal k a r y o t y p e studies on JA-Sk revealed a normal chr om os om al c o m p l e m e n t of 46 chromosomes. JA-F was aneuploid and though the modal c h r o m o s o m e n u m b e r was 46, approximately 50% of all metaphase plates examined had an abnormal c h r o m o s o m e n u m b e r compatible with a malignant cell line. A - 8 9 4 5 and A L O - 9 5
Tissue culture lines A-8945 and ALO-95 were initiated from osteogenic sarcoma and normal skin respectively, obtained from a 10-year old black
227 female. Both lines were fibroblastic in appearance. These cultures were kindly supplied to us by the Cell Culture L a b o r a t o r y , University of California, Naval Biomedical Research L a b o r a t o r y , Oakland, California under a contract to the Special Virus Cancer Program o f the National Cancer Institute. The osteogenic sarcoma and normal skin cultures were sent to us in the 6th and 8th tissue culture passage generation respectively and were used by us between this generation and the 18th tissue culture transplant generation. Chromosomal k a r y o t y p e analysis on A-8945 osteogenic sarcoma line revealed m a n y abnormal metaphase plates including pulverized and minute chromosomes as well as significant endo-reduplication compatible with a malignant cell line. The normal skin line ALO-95 had a normal chrom osom al k a r y o t y p e .
Subculture o f monolayer cultures The four tissue culture lines described above grow as monolayers adherent t o the surface of plastic tissue culture flasks. T he adherent m o n o l a y e r cultures were washed with 10 ml o f HBSS prior to adding 10 ml of 0.25% Trypsin in Dulbecco's Balanced Salt Solution, containing 0.0005 molar versene w i t h o u t calcium or magnesium. These cultures were incubated at 37°C with i n t e r m i t t e n t mixing for approximately 10 rain or until the m o n o l a y e r lifted fr o m the flask surface. The cell suspension was then poured into a centrifuge tube, complete medium added, and centrifuged for 7 min at 4°C at 260 g. The pellet was resuspended in 20--30 ml o f complete medium and the cells from a single m o n o l a y e r flask were generally divided into 2 or 3 flasks depending u p o n the gross characteristics of the cell line. Cells generally adhered to the plastic surface within a few hours after trypsinization and began growth by 24 h later. All tissue culture media were changed 2 times per week.
Allogeneic human serum A highly reactive allogeneic antiserum, obtained from a patient with Waldenstrom's macroglobulinemia who had received multiple blood transfusions, was kindly supplied to us by Dr. Dean Mann, NCI. Serum from this patient was c y t o t o x i c to l y m p h o c y t e s obtained from > 9 9 % of the population when tested by standard histocompatibility antigen typing techniques. This serum was dialyzed at 4°C against distilled water to precipitate macroglobulins and the residual serum was stored in aliquots at --20°C. This allogenic serum, called Io, was used in serial dilutions in the assays to be described. Normal serum used as controls in m a ny of the assays were obtained from normal blood donors who had never received transfusions or undergone surgery. Blood was allowed to clot at r o o m t e m p e r a t u r e for 20 min and serum collected following centrifugation at 1500 g for 10 min.
228
Complement Rabbit serum obtained by cardiac punc ture of New Zealand White rabbits was used as c o m p l e m e n t in all experiments. Blood was allowed to clot at r o o m t e m p e r a t u r e for 10 min and at 4°C. Sera from multiple rabbits were screened to obtain a large pool of sera with high c o m p l e m e n t titer that was non-toxic at a 1 : 4 dilution to hum an cells.
Assays for antibody activity S~Chromium release cytotoxicity test The 5~Chromium release c y t o t o x i c i t y test was perform ed in microtiter plates (no. 3040, Falcon Plastics, Oxnard, Calif.) by techniques similar to those previously described (Rosenberg and Schwarz, 1974). Single cell suspensions of each of the tissue culture target lines were prepared by gentle trypsinization as described above. Previous experiments d e m o n s t r a t e d that trypsinization did n o t affect the ability of antibodies to cause cell lysis in this assay and cells harvested mechanically using a rubber policeman worked equally well. A p p r o x i m a t e l y 2 × 106 target cells were suspended in 200 #c of SlChromium (Amersharn/Searle, Arlington Hgts., Ill.) in 0.3 ml of medium consisting of RPMI 1640 plus 10% heat inactivated fetal calf serum. Following incubation for 45 min at 37°C the cells were washed twice with 15 ml m e d iu m by centrifugation at 500 g for 10 min and resuspended. Following the second wash, cells were suspended in 1 ml of medium and the viability and cell n u mb e r evaluated by counting in 0.2% t r y p a n blue. Cells were diluted to 10 s live cells per ml. 20 X of serum of appropriate dilution was added to the wells of these f i a t - bot t om microtest plates and the plates were v o r t e x e d vigorously to distribute the fluid equally along the surface of the well. Using a Hamilton syringe with a repeating dispenser unit (Hamilton C o m p a n y , Reno, Nevada) 10 X of S~Chromium labelled cells containing 10 "~ live cells were added to each well. Cells and serum were incubated for 1 h at 37°C. Following this incubation 200 ~ of complete medium was added to all wells and the plates were centrifuged at 5 0 0 g for 5 min. Supernatant m ed iu m was flicked from the plate by sharp inversion of the wrist and this wash was repeated two further times. These washes were found essential to eliminate a n t i c o m p l e m e n t a r y factors present in some sera used at low dilutions. Following the third wash, 10 ~ of a rabbit c o m p l e m e n t were added to each well with a Hamilton syringe. These plates were then gently vortexed and incubated for 45 min at 37°C. Following this incubation 200 ~ of complete med ium was added to all wells and the plates were centrifuged at 800 g for 10 min. 100 ~ was removed from each well and transferred to a test tu b e for gamma counting. 10 X of ~lChromium labelled cells in 200 ~ of m ed iu m were frozen and thawed in a dry-ice acetone mixture four times in a test tube. These test tubes were centrifuged at 800 g for 10 min and 100 ~. collected to determine the m a x i m u m releasable chrom i um present in these
229 cells. The p er cen t lysis was calculated using the following formula: c pm test sample -- cpm c o m p l e m e n t control X100 % Lysis = cpm freeze-thaw -- cpm c o m p l e m e n t control Using this assay the mean coefficient o f variation of the triplicate measurements o f cpm S~Chromium release of 100 random measurements from 10 consecutive experiments was 6.07 i 0.43% (SEM).
Visual endpoin t rnierocy to toxicity test A m i c r o c y t o t o x i c i t y test similar to that described by Wood and Morton (1970) was employed. Following trypsinization and washing of the target cells, cells were suspended in 10 ml of complete medium and the viability and cell n u m b e r evaluated by counting in 0.2% t rypan blue. Cells at the appropriate dilution were then plated into ultramicrotiter plates (no. 3034, Falcon Plastics, Oxnard, Calif.). Uniform distribution of cells within the 60 wells of the plate was achieved by flooding the entire plate with the cell suspension. By gently tipping the plate and aspirating all excess medium, a p p r o x i m a t e l y 15--20 ~ was left remaining in each well. These plates were then incubated overnight at 37°C in a moist atmosphere of 5% CO2 to allow the cells to adhere tightly to the b o t t o m of the wells. For the 4 cell lines used in these experiments from 1--5 × 104 cells per ml were plated in each well in order to achieve a ppr oxi m a t e l y 100 cells in each well by the following day. After overnight incubation, the plates were gently flooded with HBSS which was removed by pouring of f excess fluid and then flicking the plate. Following two such washes, 10 ~ of the appropriate serum dilution was added to each well with an O x f o r d pipette. Plates were incubated at 37°C for 2 h. Serum was then flicked from the plates and the plates washed twice with HBSS. Following the second wash, 10 ~ of rabbit c o m p l e m e n t was added to each well with an O x f o r d pipette. After an additional one h o u r incubation at 37°C, 8 ml o f complete medium was added and the plates were allowed to incubate overnight. After ap p r o x i m at el y 18 h, medium was poured from the plates and the plates were flicked. Ten ml of HBSS were used to wash the plates three m ore times prior to flooding with a fixative solution (Perfix, Applied Biosciences, Fairfield, N.J.) for 15 min. The plates were then washed with running tap water and stained with 1 mg percent crystal violet for 10 min. Excess stain was washed away with warm water and the plate allowed to dry. The n u m b e r of residual cells left in each well were c o u n t e d under a microscope at 100 × magnification. In all experiments each serum dilution was checked in sextuplicate on each plate. All plates also contained 6 wells each containing only med iu m and containing m e di um plus com pl em ent .
230 The percent c y t o t o x i c i t y was calculated as follows: % cytotoxicity .
cells in c o m p l e m e n t control well -- cells in test well . . . ×100. cells in c o m p l e m e n t control well
Using this assay, the mean coefficient of variation of 100 sets of cell counts selected r andom l y from 10 experiments was 15 ± 0.5 (SEM).
Trypan blue cytotoxicity test C y t o t o x i c i t y as measured by t r ypan blue dye exclusion was p e r f o r m e d by techniques modified from those previously described by Sachs et al. (1971). Following trypsinization of the target cell as described above, cells were evaluated for n um ber and viability by counting in 0.2% t r y p a n blue. Cells were generally greater than 95% viable and were resuspended to a final concentration o f 5 × 105 cells per ml. 25 X of complete medium was added to the wells of a r o u n d - b o t t o m microtiter plate (no. 1-221-24-1, Cooke Laborat o r y Products, Alexandria, Virginia). 25 X of the appropriate serum dilution was added to appropriate wells and 25 X of target cells containing 1.25 × 104 live cells were added to each well. The plates were mixed on a vortex and incubated for 15 min at 37°C. Following this incubation 150 X of complete m ed iu m was added and the plates were spun at 200 g for 5 min. The plate was 'flicked' to remove supernatant and 25 X of rabbit c o m p l e m e n t was added. The plates were again mixed and incubated for 30 min at 37°C. Following this incubation the plates were centrifuged at 800 g for 10 min and the supernatant removed from the wells. 5 X of a 0.2% solution of t r y p a n blue was added to each well and the cells were then resuspended by vigorous up and down pipetting. Cells were then placed on a glass slide and the number of cells b o th excluding and staining with t rypan blue was evaluated. The p er cen t c y t o t o x i c i t y was evaluated as the percentage of the cells that stained with tr y p an blue or exhibited cell lysis as d e m o n s t r a t e d by 'ballooning' of the cell membrane. In all tests, cells were incubated with media alone and with c o m p l e m e n t alone as controls.
Mixed hemadsorption assay The h e m a d s o r p t i o n assay was p e r f o r m e d by techniques modified by Williams et al. (1976) from the m e t h o d s described by Fagraeus et al. (1961), Milgrom et al. (1965), and by Tachibana et al. (1970). Indicator cells were prepared from fresh h u m a n Rh-D positive blood. The red blood cells were washed three times in 0.01 M phosphate buffered saline containing 0.85% NaC1 (PBS) and a 4% suspension was prepared. 20 ml of this red blood cell suspension plus 50 ~ of a highly reactive anti-D positive antiserum (Ripley; kindly provided by Dr. Marion Walker) were mixed and incubated for 30 min. Following 3 washes in PBS these IgG coated red blood cells (Ripley
231 cells) were stored at 4°C to be used within 7 days. Ripley cells were coated with anti-IgG by adding an equal volume of 1 : 10 dilution of rabbit antih u m a n immunoglobulin (Cappel Labs., Inc., Downington, Pa.) to a 2% suspension. Following a 30-min incubation at 37°C the cells were washed three times in complete medium and diluted to form a 0.2% volume suspension. To p er f o r m the hem a ds or pt i on assay tissue culture target cells were trypsinized and a p p r o x i m a t e l y 2 X 104 cells in 0.2 ml were added to each well of a U - b o t t o m microtiter tray (no. 1-221-24-1, Cooke L a b o r a t o r y Products, Alexandria, Virginia). These plates were incubated for 24--48 h until a confluent m o n o l a y e r of adherent cells was obtained. At this time, the supern atan t medium was gently aspirated from the target cells and the cells washed one time with RPMI-1640 w i t h o u t serum. 0.2 ml of the a n t i b o d y at appropriate dilutions in complete medium containing 50 mM Hepes buffer was added to each well. Following incubation for 1 h at 37°C the cells were washed three times with RPMI-1640 and 0.2 ml of indicator e r y t h r o c y t e s prepared as described above were added to the wells and incubated for 30 min at 37°C. The plates were sealed using pre-cut mylar sealing tape (no. 1-220-30-1, Cooke L a b o r a t o r y Products, Alexandria, Virginia) and inverted 3--5 times by a gentle rocking motion. The n u m b e r of red blood cells a dh er en t to the m o n o l a y e r was then evaluated under a microscope using 50 X magnification. Confluent m onol a ye r s of attached red cells were read as 4+ and one or two red cells attached to occasional cells on the plate were read as 1+. A negative reaction was one in which no red cells were adherent to the target cell monolayers. The he m adsorpt i on titer was read as the highest dilution at which a 2+ reading was obtained. In general, the grades of reactivity declined with increasing dilution such that a ten-fold dilution separated 3--4+ activity f r om 1+ activity. Improved quantitation was possible by radiolabeling the indicator red blood cells with 5~Chromium. In this modification, 200 mc of SIChromium were added to the m i xt ur e of 2% Ripley cells and the 1 : 10 antiglobulin dilution, t h e r e b y producing radiolabeled antiglobulin-coated red blood cells. Following visual reading, the tray was washed two times with PBS and 0.2 ml o f distilled water was added to each well. Ten minutes later, when hemolysis was complete 0.1 ml samples were removed and assayed for radioactivity as described in the S~Chromium release assay. Radioactivity as an index o f indicator cell adherence was pl ot t ed against serum dilution and the titer was determined as the serum c onc e nt rat i on resulting in 50% o f the maximal radioactivity bound to the cultures. RESULTS
Optimization of assays In developing these assays for use with hum an serum and hum an target cells, differing times of a n t i b o d y and c o m p l e m e n t incubations were investi-
232
gated. An example of these optimization experiments using the visual endp oin t m i c r o c y t o t o x i c i t y assay is shown in figs. 1 and 2. On separate microtiter plates, cells were exposed to a 1 : 40 dilution of Io antibody for 60 or 120 min and then exposed to c o m p l e m e n t for either 30, 60, or 120 min. Lysis due to c o m p l e m e n t alone (compared to media) or to serum plus complement (compared to c o m p l e m e n t alone) was calculated separately for each of these six plates (fig. 1). Lysis due to c o m p l e m e n t alone was less than 15% in all plates. At all three times of c o m p l e m e n t exposure, greater lysis was obtained with a 120-rain ant i body incubation than with 60 min of antibody incubation. Time of c o m p l e m e n t exposure between 30 and 120 min appeared n o t to be a critical factor. Following incubation with c o m p l e m e n t , plates were incubated for varying times in medium prior to washing and counting of residual cells (fig. 2). The time of p o s t - c o m p l e m e n t incubation did n o t appear critical. Comparison of cell counting on unstained preparations using phase contrast microscopy as described by Wood and M or t on (1970) gave identical results to those obtained when cells were c o u n t e d after fixation and staining with crystal violet (table 1). As a result of these experiments, we selected a 120-min antibody incubation, 60-rain c o m p l e m e n t incubation, and 16 h (overnight) of post-complem e n t incubation for routine per f or m ance of this assay. All plates were fixed and stained prior to evaluation. Similar optimization experiments led to the selection of the parameters
120
60' Ab Incubation -:~- 120' Ab incubation
11o ~r
100
I
90 F
~
70 r
P Z
60 p
5
so r
a_
40 t
~_~,_
. . . . .
~'~ .[
Serum Complement
L'
Complcnlent Aone
1
3o F
0
0.5
1.0
15
20
25
TIME OF COMPLEMENT iNCUBATION {Hours)
Fig. 1. T h e e f f e c t o f t h e t i m e o f a n t i b o d y i n c u b a t i o n a n d c o m p l e m e n t i n c u b a t i o n o n t h e lysis o f o s t e o s a r c o m a tissue c u l t u r e cells. In all p l a t e s Io a n t i b o d y was u t i l i z e d at a 1 : 40 dilution.
233 100
f j..~.4t
so u~
6o
}z Lu (.3
5o
>,
or-
Serum + Complement
4o 30 Complement Alone
20 ~ lO
I 2
4
6
8
1
1
16
18
20
;2 2i4 26 28
TIME OF POST INCUBATION (Hours)
Fig. 2. The effect of the time of incubation, following a 2-h incubation with complement, on the lysis of osteogenic sarcoma cells by Io serum.
listed in Methods for the 5~Chromium release microcytotoxicity assay. Two points are of special interest with regard to the SlChromium release assay. ; These four tissue culture target cell lines incorporated sufficient S~Chromium to allow the use of 103 target cells in these assays. This provided a small increase in sensitivity compared to the use of 104 target cells (necessary when fresh lymphocyte target cells are used). Secondly, no difference in spontaneous 51Chromium release or sensitivity was obtained when cells were harvested from monolayer cultures mechanically using a rubber policeman or when they were harvested using dilute trypsin solutions as described in Methods above. A variety of factors were investigated to optimize the hemadsorption assay. The persistence of a large number of adherent cells throughout the assay was essential. Experiments were carried out using various media with and without Hepes buffer and fetal calf serum. Only 1 of 9 cell lines toler-
TABLE 1 Effect of staining on cell counts in microcytotoxicity plates No. of cells plated/well
1000 500 250 125 63
No. of cells counted (mean _+ S.E.) Before staining
After staining
120 +__7 72__+2 45 __4-4 13 __4"2 6__+1
117 -+. 4 72__+3 40 + 4 12 __+3 6__+1
234 ated exposure to high dilutions of inactivated hum an serum in the absence of fetal calf serum, and complete medium containing 20% fetal calf serum was used as the medium for carrying out antibody dilutions. In addition, the presence of at least 5% fetal calf serum at the step of indicator-target cell adherence was m a n d a t o r y . In the absence of serum, the sensitivity generally declined which was mainly due to m o n o l a y e r d e t a c h m e n t (fig. 3). Further, in the absence of fetal calf serum, the indicator e r y t h r o c y t e s occasionally adhered non-specifically to the plastic surface of the wells. Experiments designed to evaluate the role of Hepes buffer showed that, while it clearly stabilized the pH during the making of serial dilutions in media, there was no i m p o r t a n t effect on the sensitivity or the reproducibility of the assay. Parameters used in the t r y p a n blue assay were the same as those determined by Sachs et al. (1971).
Comparison of assays Example experiments with the visual e n d p o i n t m i c r o c y t o t o x i c i t y and SlChromium release assays are shown in tables 2 and 3. Comparison of the f o u r assays on the four different hum an tissue culture lines using a single allogeneic serum is presented in fig. 4 and table 4. Of note: 1. SlChromium release and m i c r o c y t o t o x i c i t y assays p e r f o r m e d as described in Methods were appr oxi m at el y equally sensitive and far more sensitive than the described assay using t r y p a n blue uptake as the c y t o t o x i c end-
slCr LABELLED INDICATOR CELL HEMADSORBTION ASSAY WITH AND WITHOUT CALF SERUM 2,0001
114)* T -.------~(4) 9
5 o~/oFetal Calf Serum
1,500
1
~
T
I~ \l~(2)
---
No Fetal Calf Serum
/ ~\
~,111 \ (o)
2,000
(o)
8,000
32,000
SERUM DILUTION -1 Fig. 3. The effect of fetal calf serum on the m e a s u r e m e n t of m i x e d cell h e m a d s o r p t i o n using Io antiserum and tissue cultured t u m o r cells. In this e x p e r i m e n t chromium-labelled red cells were utilized to quantitate the degree of red b l o o d cell adsorption.
235 TABLE 2 M i c r o c y t o t o x i c i t y assay using Io s e r u m o n n o r m a l a n d m a l i g n a n t h u m a n cell lines f r o m t h e same p a t i e n t . A-8945 (osteosarcoma)
ALO-95 (normal} No. cells * Medium alone Complement alone
1 3 8 . 0 _+ 9.5 132.4 ± 14.2
Io 1 : 6 4 0 1 : 1280 1:2560 1 : 5120 1 : 10240
0 6 . 5 ± 1.0 38.3±11.7 83.5 ± 17.7 121.2 ± 13.6
% Lysis * ---
No. cells 115.5 ± 9.9 104.2 ± 10.4
100 9 5 . 1 ± 0.7 7 1 . 1 ± 8.4 36.9 ± 13.4 8.5 ± 10.2
0 0 1 2 . 2 ± 3.1 58.5 ± 14.4 117.0 _+ 18.5
% Lysis --100 100 8 8 . 3 ± 2.9 43.8 ± 13.8 - - 1 2 . 3 ± 17.7
* M e a n ± 1 s t a n d a r d error.
point. The assays appeared equally reproducible a n d f o r all a s s a y s t h e i n d i vidual titers were within 1 tube dilution of the average titer presented in table 2. 2. The hemadsorption assay revealed antibody binding to target cells at approximately ten-fold higher dilutions than that seen using any of the three cytotoxicity assays. 3. B o t h t h e s k i n a n d o s t e o s a r c o m a cells from patient JA were far less r e a c t i v e i n all a s s a y s t e s t e d t h a n w e r e s k i n a n d o s t e o s a r c o m a lines from a different patient (ALO-95 and A-8945).
TABLE 3 C h r o m i u m - 5 1 release assay using Io s e r u m o n t w o h u m a n cell lines.
M e d i u m alone
ALO-95 (normal)
A-8945 (osteosarcoma)
cpm *
cpm
% lysis *
% lysis
339 ± 51
--
415
--
Complement alone Freeze-thaw controls
383 ± 9 2601 ± 90
---
389 3099
---
Io 1 1 I 1 1 1 1 1
1595 _+ 51 1 4 9 6 _+ 58 1 4 8 8 + 54 1421 _+ 34 1046_+ 10 587 ± 13 372 _+ 30 3 7 6 _+ 17
: : : : : : : :
160 320 640 1280 2560 5120 10240 20480
* M e a n ± 1 s t a n d a r d error.
54.7 ± 4.0 50.2 _+ 4.6 50.0 _+ 4.3 46.8 _+ 2.7 28.9_+0.8 9.2 _+ 1.0 --0.5 ± 2.4 --0.3 ± 1.3
1664 _+ 35 1684 _+ 74 1653 _+ 93 1422 ± 31 1078_+ 5 634 ± 48 4 2 9 _+ 37 4 3 0 _+ 33
47.1 _+ 1.3 47.8 + 4.8 46.6 + 6.0 38.1 ± 2.0 25.4_+0.3 9.0 _+ 3.0 1.5 _+ 2.4 1.5 ± 2.1
236 Chromium-51 Release Assay 100 •
100 r
I c, JA-Sk
c2"-"c~<~,
• JA-F
\
"Y~,
:i
80
• A8945
F
ALO 95
60 i
"-~\ \ \ ~
2oi
40
2o!
\ , k& \,~
Microcytotoxicity Assay 100 ~ .....
_
--
l
"~
80 ~
6O
U~
40I L)
'\
20
\
[
\
\
'L' 1
o!
k
20 F
X'\T
L ............. Trypan Blue Assay
\ 80~
lo0 i
\ \
6oi
\ \
,
r
\'~
6o
°[
\
40
\'\, ,\
~\
20
8
32 16
128 64
20 1
512 2048 8192 256 1024 4096
'\'\ 8
32 16
128 64
512 2048 8192 256 1 0 2 4 4096 16384
SERUM DILUTION-'
Fig. 4. A c o m p a r i s o n o f t h e s ] C h r o m i u m r e l e a s e assay ( u p p e r ) , t h e m i c r o c y t o t o x i c i t y assay ( m i d d l e ) a n d t h e t r y p a n b l u e assay ( l o w e r ) u t i l i z i n g Io a n t i s e r u m a n d n o r m a l a n d m a l i g n a n t cell lines f r o m t w o p a t i e n t s .
4. 100% lysis w a s n o t a c h i e v e d w i t h any cell line w h e n using t h e 5'Chrom i u m release assay (fig. 3); that is, even t h e m o s t c o n c e n t r a t e d Io a n t i s e r u m did n o t release as m u c h S1Chromium f r o m t h e cell as w a s released by freezing
237 TABLE 4 Comparison of assays on human tissue culture cells. Human cell line
Microcytotoxicity *
s 1Cr release *
TB *
Hemadsorption **
JA-Sk JA-F ALO-95 A-8945
320 320 5120 5120
320 320 2560 2560
64 64 256 256
8100 (2) 8100 (1) ---
(5) *** (7) (3) (2)
(2) (2) (2) (2)
(2) (2) (2) (2)
* (50% lytic dilution) -1 . ** Highest serum dilution with positive hemadsorption) -1 . *** Number in parentheses is number of determinations in separate experiments.
a n d t h a w i n g t h e cells. In eleven e x p e r i m e n t s on t h e f o u r cell lines, m a x i m a l S ' C h r o m i u m release ranged f r o m 35 to 79% o f t h e f r e e z e - t h a w c o n t r o l s ( m e a n = 57%). F r e e z i n g and t h a w i n g released a p p r o x i m a t e l y 80% o f t h e t o t a l c h r o m i u m in the cells. T h e m a x i m a l a m o u n t o f c h r o m i u m released by t h e cells varied f r o m d a y to d a y a n d c o u l d n o t be c o r r e l a t e d w i t h the age o f t h e S ' C h r o m i u m or b y a n y r e c o g n i z a b l e c u l t u r e c o n d i t i o n s such as the degree of c o n f l u e n c y o f the cell m o n o l a y e r w h e n h a r v e s t e d , t i m e f r o m last feeding, t i m e f r o m last t r y p s i n i z a t i o n or o t h e r factors. 5. Five n o r m a l h u m a n sera t e s t e d o n these cell lines using the h e m a d s o r p t i o n assay gave titers of < 1 : 10 in all f o u r assays. 66% o f 155 n o r m a l h u m a n sera tested using the visual e n d p o i n t m i c r o c y t o t o x i c i t y assay s h o w e d sign i f i c a n t lysis against the h u m a n skin lines w h e n t e s t e d u n d i l u t e d b u t were n o t lytic at d i l u t i o n s g r e a t e r t h a n I : 8. DISCUSSION A v a r i e t y o f assays have b e e n used to d e t e c t a n t i b o d y r e a c t i v i t y to surface antigens on n o r m a l and m a l i g n a n t h u m a n cells g r o w n in tissue culture. In this p a p e r we have a t t e m p t e d to c o m p a r e t h e sensitivity o f t h r e e d i f f e r e n t m i c r o c y t o t o x i c i t y assays and a h e m a d s o r p t i o n assay to m e a s u r e t h e reactivity o f a single a l l o a n t i s e r u m against f o u r s e p a r a t e h u m a n tissue c u l t u r e cell lines derived f r o m t w o p a t i e n t s . In c o m p a r i n g c y t o t o x i c i t y assays based on visual c o u n t i n g o f cells with t h o s e using S ' C h r o m i u m release, similar a n t i b o d y titers were d e t e c t e d . A l t h o u g h less t e d i o u s , the S ' C h r o m i u m release assay e x h i b i t e d c o n s i d e r a b l e d a y to d a y v a r i a t i o n in the m a x i m a l a m o u n t o f S ' C h r o m i u m released b y saturating a n t i b o d y c o n c e n t r a t i o n s . T h e releasable S l C h r o m i u m varied f r o m 35 to 79% o f the f r e e z e - t h a w c o n t r o l s in d i f f e r e n t e x p e r i m e n t s . We w e r e u n a b l e to s t a n d a r d i z e the m a x i m a l releasable c h r o m i u m in these assays d e s p i t e m a n i p u l a t i o n o f a v a r i e t y o f variables such as age o f c h r o m i u m , age o f tissue c u l t u r e cells, etc. This f e a t u r e r e p r e s e n t e d a significant d i s a d v a n t a g e o f this assay and a p p e a r e d u n i q u e to the use of tissue culture t a r g e t cells since we
238 did n o t see these variations when using fresh l y m p h o c y t e target cells. This variation in maximal releasable chromium led to some confusion in determining antibody titers based on the antibody concentration necessary to lyse 50% of the cells. In table 4 we have used the m a x i m u m lysis in each individual S'Chromium experiment and have taken as the serum titer, the dilution necessary to achieve one-half of that a m o u n t of lysis. The ambiguities involved in these manipulations have led us to favor the visual endpoint assay for routine work. 100% lysis was routinely seen when using high antibody concentrations in the visual endpoint microcytotoxicity assay. The visual endpoint and S'Chromium release assays gave very similar titers using the Io serum on all four h u m a n cell lines (table 4 and fig. 4). Trypan blue as a measure of c y t o t o x i c i t y was significantly less sensitive than either of the two former methods. It should be kept in mind, when comparing assay sensitivities, that the visual endpoint technique utilized 100 target cells as compared to 1000 and 12,500 cells for the chromium release and trypan blue assays respectively. Practical considerations prevented the use of fewer cells in the latter two assays. The hemadsorption technique provided significantly greater sensitivity than any of the cytotoxicity assays for measuring the reactivity of Io-serum with all two cell lines obtained from patient JA (table 4). Exact quantitation with this assay is difficult because of the objective nature of visually estimating red blood cell binding to the tissue culture cells. We have not attempted to increase the objectivity of this assay by using S'Chromium labelled red blood cells as described by Thompson et al. (1973) and by Sundqvist and Fagraeus (1972). Some of the difference in titers that we have seen may be due to the increased efficiency of IgM antibodies in cytotoxicity assays as opposed to the design of our hemadsorption assay to detect IgG antibodies only. The predominance of IgG antibodies in the Io serum may account, in part for the greater sensitivity when using the hemadsorption assay. It should be emphasized that each of the assays described here reflects different aspects of the cell-antibody interaction and that the use of multiple assays provides more information than the use of any single assay alone. It is of interest that both normal and malignant cell lines from the same individual showed different degrees of reactivity compared to similar normal and malignant lines from a different individual. Thus, the normal and malignant cell lines from patient JA were far less sensitive to lysis than the normal and malignant cell lines ALO-95 and A-8945 from the same patient (table 4 and fig. 3). At Io serum concentrations of 1/1,000 in the visual endpoint m i c r o c y t o t o x i c i t y assay no lysis of JA-Sk or JA-F was seen, whereas, 100% lysis of ALO-95 and A-8945 was evident. Since Io serum detected alloantigens it is possible that the JA lines expressed greater amounts of these antigens than did the ALO-95 and A-8945 cell lines, though the broad reactivity of the Io serum would seem to make this unlikely. Also possible, is that the ALO-95 and A-8945 lines are more 'lysable' for other reasons, such as increased complement binding, decreased cell adhesivity to plastic, etc. It is
239 clear, h o w e v e r , t h a t t h e a p p a r e n t t u m o r specificity seen b y a c o m p a r i s o n o f a t u m o r cell line f r o m o n e p a t i e n t w i t h a n o r m a l cell line f r o m a n o t h e r individual m a y be d u e to f a c t o r s o t h e r t h a n t h e p r e s e n c e o f t u m o r specific a n t i b o d i e s . Intrinsic d i f f e r e n c e s in t h e ' r e a c t i v i t y ' o f cells in a n y given assay, m u s t b e c o n s i d e r e d in in vitro studies of h u m a n t u m o r i m m u n e responses. REFERENCES Bloom, E.T., 1972, Cancer Res. 32,960. Byers, V.S., Levin, A.S., Johnston, J.O. and Hackett, A.J., 1975, Cancer Res. 35, 2520. Eilber, F.R. and Morton, D.L., 1970, J. Nat. Cancer Inst. 44,651. Fagraeus, A. and Espmark, J.A., 1961, Nature 190,370. Klein, E., 1970, Transplantation 9,219. Moore, M., Witherow, P.J., Price, C.H.G. and Clough, S.A., 1973, Int. J. Cancer 12,428. Morton, D.M. and Malmgren, R.A., 1968, Science 162, 1279. Mukherji, B. and Hirshaut, Y., 1973, Science 181,440. Priori, E.S., Wilbur, J.R. and Dmochowski, L., 1971. J. Nat. Cancer Inst. 46, 1299. Rosenberg, S.A. and Schwarz, S., 1974, J. Nat. Cancer Inst. 52, 1151. Sachs, D.H., Winn, H.J. and Russell, P.S., 1971, J. Immunol. 107,481. Sundqvist, K.G. and Fagraeus, A., 1972, Immunology 22,371. Tachibana, T., Worst, P. and Klein, E., 1970, Immunology 19,809. Thomson, D.M.P., Steele, K. and Alexander, P., 1973, Brit. J. Cancer 27, 28. Williams, G.M., 1976, in preparation. Wood, W.C. and Morton, D.L., 1970, Science 170, 1318.