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A modified 51Cr release assay for cytotoxic lymphocytes
Journal oflmmunologicalMethods 6 (1974) 39-51
© North-Holland Publishing Company
A MODIFIED 51Cr RELEASE ASSAY FOR CYTOTOXIC LYMPHOCYTES* Margaret DUNKLEY, R.G. MILLER** and Ken SHORTMAN The Walter & Eliza Hall Institute for Medical Reasearch, Melbourne, Vic. 3050, Australia
Received 26 March 1974, accepted 14 June 1974
An improved 5 ICr release assay for cytotoxic lymphocytes has been developed. The assay system combines the technical advantages of a titration tray method with a modification which enables a more quantitative measure of the cytotoxicity of sensitized cell preparations in a shorter time. A simple mathematical model describing the cytotoxic lymphocyte-target cell interaction has been shown to apply to this system. The advantages and limitations of the assay system are discussed and the recommended procedure is described in detail.
1. Introduction Many attempts at obtaining a quantitative assay for cell-mediated immunity have been based on the s 1Cr release assay of Brunner et al. (1968) which measures cellmediated cytotoxicity against H-2 alloantigens. There have been several attempts to simplify and improve the technical aspects of the procedure, as well as to understand the assay in terms of an exact mathematical model. Thorn et al. (1974) recently developed a modified assay using small titration trays, thus eliminating centrifuging steps and reducing the number of cells required. Miller and Dunkley (1974) using a Brunner type assay have developed a mathematical model for the cytotoxic lymphocyte (CL)-target cell interaction which agrees well with experimental data. The latter study also demonstrated that the assay was limited by the slow rate of 51Cr release from damaged target cells at 37°C. A modification was therefore introduced in which a 1 hr incubation at 45°C gave rapid and complete Sl Cr release from target cells damaged in a prior, short incubation at 37°C. The aim of this paper was to combine the advantages of the titration tray method of Thorn et al. with the modified procedure of Miller and Dunkley, to produce a simple accurate assay for CL.
* This is Publication No. 1998 from the Walter & Eliza Hail Institute. ** Present address: Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada. 39
40
M. Dunkley et al., A modified sl Cr release assay
2. Materials and methods 2.1. Mice
Six to ten week old BALB/c/AnBradley Wehi, and C57BL]6J mice of both sexes were used. 2. 2. Media 2.2.1. Suspension medium
The medium used for preparing cells and for general purposes was a HEPESbuffered balanced salt solution iso-osmotic with mouse serum and containing 10% (v/v) fetal calf serum (Shortman et al., 1972b). 2.2.2. Culture medium
For assay of cytotoxic activity, cells and targets were suspended in a modified, mouse osmolarity, CO2-buffered culture medium prepared as follows: One packet of Minimum Essential Medium powder (Cat. No. F-15 Grand Island Biological Co., Grand Island, New York) was dissolved in 700 ml of distilled water to which was added 25 mg penicillin, 20 mg streptomycin, 0.1 g pyruvate and 127 ml of sodium bicarbonate (2.8% w/v, Commonwealth Serum Laboratories, Melbourne, Australia). The medium was then made up to a total volume of 1050 ml with distilled water to give a solution iso-osmotic with mouse serum and was maintained at pH 7.2 by equilibrium with a 10% CO2 in air-gas mixture. Heat-inactivated fetal calf serum was added (10% v/v) before use. The fetal calf serum was inactivated by incubation at 56°C for 1 hr. 2.3. Cell suspensions
Mice were killed by cervical dislocation, the spleen or thymus removed, chopped into small pieces and pushed gently through a fine stainless steel sieve into cold suspension media. Cell clumps were removed by layering the cell suspension over 1 ml of fetal calf serum and standing for 5 rain. The supernatant fraction (a single cell suspension) was washed once through fetal calf serum and either resuspended in suspension media for injection into mice, or subjected to a damaged cell removal technique (Von Boehmer and Shortman, 1973) before resuspension in culture medium for assay of cytotoxic activity. 2. 4. Production o f sensitized cell populations in vivo
An adoptive transfer to allogeneic hosts was used to generate CL. Approximately 108 C57BL spleen cells or 108 'light density' (density "< 1.077 g/cm 3) thymus cells (Shortman et al., 1972a, b) were injected intravenously into irradiated (700 rads)
M. Dunkley et al., A modified Sl Cr release assay
41
BALB/c mice. After 5 - 6 days the BALB/c mice were killed, the spleens removed and spleen cell suspensions assayed for cytotoxic activity against target cells with the same alloantigenic specificity at the H-2 locus as the BALB/c cells. 2.5. Target cells Unless otherwise stated s ~Cr-labeled P815 mastocytoma cells were used as target cells. The cells were maintained in continuous exponential growth in tissue culture in a modified Dulbecco's medium containing 10% fetal calf serum. Cultures were harvested in the exponential phase of growth and labeled by incubation with sodium sl Cr-chromate (100/ICi per 5 × 10 6 cells) for 20 min at 37°C in 1 ml of the suspension medium. The cells were washed 3 times through 1 ml of heat-inactivated fetal calf serum before resuspension in culture medium for use. Large, dividing B lymphocytes, obtained as a light density fraction from pokeweed mitogen (PWM)-stimulated spleen cells were also tested as possible targets. Spleen cells (60 × 10 6) from BALB/c mice were cultured for 3 - 4 days with 0.4 ml of PWM (Grand Island Biological Co., Grand Island, New York. Batch No. 128248A, reconstituted in 5 ml normal saline and diluted 1:15 with culture medium) in Marbrook flasks (internal chamber diameter 2.1 cm, external volume of medium 50 ml) (Byrd et al., 1973). The ceils were harvested, washed and a density cut carried out using albumin of specific density 1.073 g/cm 3 at mouse osmolarity and pH 5.1 (Shortman et al., 1972b). The cells less dense than 1.073 g/cm 3 were labeled with sodium si Cr-chromate (100 pCi/106 cells) for 4 0 - 5 0 rain at 37°C in 1 ml of suspension medium and washed 3 times through heat-inactivated fetal calf serum before use. 2. 6. Cytotoxicity assay 2. 6.1. 'The standard tray assay' Tile method of Thorn et al. (1974) was used in the initial investigations. The sensitized or normal cell population was suspended in culture medium and diluted by serial 2-fold dilutions in titration trays - (Micro Test II. Tissue culture plate, Falcon plastics, Oxnard, C.A. 93030, U.S.A.) to give 0.1 ml per well. An equal volume of target cells (2.5 X 104 cells) was added to each well to give a final volume of 0.2 ml. The trays were then incubated at 37°C for the required period, placed on ice and 0.1 ml of the supernatant carefully removed from each well with an automatic pipette (Eppendorf, made in Germany). It has been shown that at this stage the cells have settled out completely and removal of the supernatant without cells is easily accomplished (Thorn et al., 1974). 2. 6.2. Final recommended procedure The titration trays were placed on ice. Serial 2-fold dilutions of the sensitized cell population were carried out in quadruplicate using the culture medium, and 2.5
42
M. Dunkley et aL, A modified 51Cr release assay
× 104 s~ Cr-labeled P815 mastocytes (in 0.1 ml) were added to each well (total volume per well = 0.2 ml). The trays were then transferred to a plastic box on ice which was gassed with 10% COz in air, sealed and stood in the cold for 30 min. This procedure allowed the cells to settle out, which is necessary as there is evidence that very few encounters between CL and target cells take place while the cells are in suspension (Miller and Dunkley, 1974). Equilibration of pH also took place at this stage. The trays were placed in water in a CO2 incubator (10% CO2 in air) at 37°C for a short period (usually 1 - 2 hr but this time may be extended for low activity cell preparations). The trays were then transferred to a plastic box containing a layer of water maintained at 45°C by sitting the box in a water bath. The box was gassed (10% CO2 in air), sealed and incubated for 1 hr. This period at 45°C has been shown to be optimal for release of all s l Cr from cells previously damaged at 37°C; whilst no new lytic events between CL and target cells take place (Miller and Dunkley, 1974). The trays were placed on ice and 0.1 ml of the supernatant carefully removed for radioactive counting in a well-type gamma radiation counter. 2. 6. 3. Calculation o f percent specific s ~Cr release
The spontaneous s ~Cr release from the target cells was measured by incubating 2.5 × 104 mastocytes alone in 0.2 ml of culture medium in quadruplicate. The specific s LCr release due to killing by cytotoxic lymphocytes was calculated by subtraction of the mean spontaneous release counts from the mean total counts for each group. This value was expressed as a percentage of the maximum releasable counts minus the spontaneous release. The maximum releasable counts represent the maximum amount of the s~ Cr taken up by the cells which can be released again on cell lysis. This was determined by incubation of 1.25 X l 0 4 mastocytes in 0.05 ml of medium, plus 0.15 ml zaponin (lysing reagent for white blood cell counts, Coulter Electronics Ltd., High St. South, Dunstable, Beds, England; 3 drops in 5 ml of water), for 3 hr at 37°C. The maximum releasable counts were usually 8 0 - 9 0 % of the total counts taken up by the mastocytes.
3. Results 3.1. Spontaneous 5 ~Cr release under various conditions
In initial experiments, we investigated the extent of spontaneous release of 51 Cr by target cells in the titration trays. Using cultured P815 mastocytoma cells, the spontaneous S~Cr release expressed as a percentage of the maximum releasable 51Cr over a period of several months was 23 -+ 6% for 3 - 5 hr at 37°C. This compared well with values obtained in an assay system using small capped tubes, similar to the assay system of Brunner et al. (1968), the value here being 20% or more for 4 hr at 37°C with cultured cells. In vivo passaged cells always gave lower spontaneous release (around 10% under these conditions). When the trays were
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Fig. 1. The effect of non-immune spleen cells on the spontaneous release of s 1Cr from labeled P815 mastocytoma target cells. The spontaneous s 1Cr release is expressed as a percentage of the maximum releasable s l Cr (see Materials and Methods) _+ S.E.M. The assay procedure was the standard titration tray method incubating at 37°C only. Each well contained either 2,5 X l04 labeled P815 mastocytoma cells or 1 X 105 labeled PWM-stimulated BALB/c spleen cells. The lymphocytes added in various concentrations were non-sensitized BALB/c spleen cells. The 'labile mastoeytes' were from a batch of cultured cells giving, for undetermined reasons, a very high background release; this was an exceptional circumstance, the lower curve giving the normal results. The time of incubation was 3 hr for the normal P815 mastocytoma targets and 4 hr for the 'labile' PS15 mastocytes and the PWM-stimulated spleen cell targets.
incubated for 1.5 hr at 37°C followed by 1 hr at 45°C (as in the r e c o m m e n d e d procedure) the spontaneous release was 21 -+- 5%, compared with 15 -+ 4% for 1.5 hr at 37°C, representing a definite but tolerable increase. Very occasionally a batch of cultured P815 mastocytes gave a very high spontaneous release ( u p ' t o 60%, eg. fig. 1); this was assumed to be due to poor culture conditions, since w e have never observed this with mastocytes passaged in D B A / 2 mice. Fig. 1 shows the variation in spontaneous s ~ Cr release in several other situations. Addition o f n o n - i m m u n e cells to labeled P815 m a s t o c y t o m a cells produced no significant effect on the s~ Cr release, even in the exceptional case o f P815 mastocytes with high backgrounds. However, other target cells did not always behave in this way. For example PWM-stimulated spleen cells gave very high spontaneous st Cr release w h e n incubated alone, but addition o f n o n - i m m u n e cells lowered the
44
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Fig. 2, The relationship between specific s J Cr release and the number of sensitized lymphocytes present. The standard tray assay was used, incubating at 37°C only. The values are expressed as percent specific lysis (see Materials and Methods) -+ S.E.M. Sensitized cells were obtained by transfer of C57BL spleen or thymus cells into irradiated BALB/c mice. The target ceils were P815 mastocytoma cells.
spontaneous s i Cr release (fig, 1). Viable and non-viable lymphocytes and red blood cells all had the effect of lowering the spontaneous S~Cr release. Thus for these targets the specific s 1Cr release due to CL can only be investigated if careful paired controls are run using non-immune cell preparations. 3.2. Relationship between sensitized lymphocyte concentration and specific S ~Cr release from P815-mastocytes The relationship between the percentage specific s i Cr release (see Materials and Methods) and the log of the ratio of viable lymphocytes to target cells was determined for the standard tray assay incubating at 37°C only. The results (fig. 2) were similar to those reported by others for ratios up to 20:1, and show the standard near-linear relationship in the middle of the lysis curve. However a striking and unusual result was obtained at higher sensitized lymphocyte concentrations, where, instead of a plateau, an actual drop in the percentage specific lysis was observed. This was unexpected as previous studies had shown that the addition of moderate levels of unsensitized bystander cells had no effect on the percentage lysis (Miller and Dunkley, 1974). This inhibition was obtained whether or not damaged cells were removed from the lymphocyte preparation and thus can be caused by viable
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Fig. 3. The effect of large numbers of non-sensitized cells on the lysis of P815 mastocytoma target cells by sensitized lymphocytes. Two series of dilutions of sensitized C57BL cells were incubated with P815 mastocytoma cells and to one series 5 X 106 normal BALB/c cells were added per well. The values presented are the mean -+S.E.M. cells. The effect also appeared to occur at the same cell concentration and was independent of the incubation time over the 3 - 5 hr period (fig. 2). It was also obtained with activated thymus cells (fig. 2) indicating that it was not caused by activated, antibody-forming B cells blocking the lysis. It is thought that the effect is due predominantly to the limited capacity o f the medium to provide nutrients or oxygen or to cope with waste products or COs production as indicated by a pH change in the most dense (100:1 ratio) cultures. The inhibition may also be due in part to a slowing down o f the CL movement or shielding of target cells due to a physical crowding in the most dense cultures. That this inhibition can be caused by non-sensitized cells regardless o f the number o f sensitized cells present is shown in fig. 3. Here, a large number (5 × 106) of non-activated BALB/c spleen cells were added to each culture. This caused a depression o f lysis over the whole range thus showing that it is only an excess of cell numbers which depresses the lysis. It should be noted that this level of cells did not affect the background release (fig. 1). 3. 3. Application o f the theoretical model to the standard titration tray system A mathematical model describing various phases of the sl Cr release assay, and giving an excellent fit to experimental data in a Brunner-type assay procedure, has been given b y Miller and Dunkley using the following notation:
46 p
M. D u n k l e y et al., A modified sl Cr release assay
= f r a c t i o n a l specific 51Cr release;
N = no. o f sensitized cells; t = t i m e o f i n c u b a t i o n at 37°C; B = a c o n s t a n t c h a r a c t e r i s t i c o f the s 1Cr release process; = a c o n s t a n t = v6; v = f r e q u e n c y o f C L in t h e sensitized p o p u l a t i o n ; 8 = a c o n s t a n t (includes such f a c t o r s as cell velocity a n d size a n d area or v o l u m e o f the c u l t u r e vessel). The general f o r m u l a t i o n leads to t w o p a r t i c u l a r l y i n t e r e s t i n g special cases. A t s h o r t t i m e s a n d small cell n u m b e r s ( N a t , B t ~ 1) (1)
p = ~c~BNt 2
At l o n g t i m e s or in t h e case o f rapid s 1Cr release ( B t >> 1) (2)
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Fig. 4. Testing the theoretical model when varying the concentration of sensitized lymphocytes and incubating at 37°C only for 5 hr. The open circles represent mean values -+ S.E.M. from experimental data. The solid line is the theoretical curve predicted by the equation p = 1 - e - k N . This curve is generated by calculation of k for each p and N value. A mean k is obtained from the region of constant k values, in this case for lymphocyte:target ratios between 12:1 and 0.2:1, and a curve generated by calculating p for each N value using the mean k value.
M. Dunkley et al., A modified Sl C'r release assay
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Fig. 5. Testing the theoretical model when varying the incubation time. The open circles represent the mean -* S.E.M. specific 5 ICr release obtained after incubating 1.5 X 10 s sensitized C57BL spleen cells with 2.5 X 104 P815 mastocytoma cells for various times at 37°C. The solid line is the theoretical curve predicted by p ---et 2. This curve was generated by calculation o f c for each time point and taking a mean where the c values were constant (up to 3 hr). This mean c was used to generate the theoretical curve.
If this model holds for the titration tray assay, it would provide a means of comparing quantitatively the cytotoxicity of different cell preparations and enable comparison between assays done on different days under different conditions. Thus, values of specific SlCr release obtained experimentally when either the number of sensitized lymphocytes or the time of incubation was varied were compared with values predicted by the theoretical model. For long incubation times at 37°C Eq. (2) should be approximately valid. This is demonstrated by the experiment of fig. 4 in which variable numbers of lymphocytes were incubated for 5 hr at 37°C with a fixed number of target cells. The observed lysis had an exponential dependence on the number of sensitized cells present (p --- 1 - e -kN, where: p = specific SlCr release;N = number of lymphocytes, and k is a constant) as predicted. Deviation from this relationship occurred only at high cell numbers (above a lymphocyte:target ratio of 20:1) presumably as crowding effects became important. For short incubation times and small numbers of sensitized cells, Eq. (1) predicts
48
M. Dunkley et al., A modified sl Cr release assay
a quadratic time dependence. This is demonstrated by the experiment of fig, 5 in which the response at a constant low cell number showed that the specific S~Cr release had a dependence on t 2 (p = ct 2, where p = specific s~ Cr release, t = time of incubation at 37°C and c = a constant) up to 3 hr. For longer incubation times the specific lysis appears to have an exponential dependence on t (p = 1 - e - z t , where z a constant, has the value of 0.067 at 3 hr, 0.058 at 4 hr and 0.056 at 5 hr). These observations again agree with the predictions of the theoretical model of Miller and Dunkley. 3. 4. Modification o f the assay to produce c o m p l e t e s ~Cr release f r o m a damaged target cell
Previous studies of Miller and Dunkley (1974) showed that the factor-limiting quantitation o f the assay was the slow s~ Cr release at 37°C from the damaged cells. It was estimated that it took approximately 1 . 5 - 2 hr for all the s ~Cr to leak out o f a damaged cell at 37°C. Thus in the normal 37°C-incubation procedures, at the time of assay targets damaged 2 hr previously would have released all their s ~Cr, whereas targets damaged since that time would have released only portions of their s~ Cr. However it was demonstrated that during an incubation at 45°C there are no further lytic events, but that the remaining releasable s ~Cr contained within target cells damaged during the 37°C-incubation is released within 1 hr. The mathematical model also simplifies under these conditions (see section 3.3.), reducing to p = 1 e - N a t , and thus enabling the direct calculation of a. This is a measure (although not an absolute measure) of the frequency of CL in the sensitized population, The application of the modified procedure (see Materials and Methods for details) is given in fig. 6, where paired trays were compared to estimate the degree of s~ Cr release. The additional 45°C-incubation raised the spontaneous sl Cr release from 19.0 -+ 0,4% to 28,0 +- 0.3% but since variability was low and many replicates of spontaneous release can readily be run, good precision is maintained. The 45°C incubation gave about a 3-fold increase in specific s l Cr release. (The procedure allowed as much release of s~ Cr as about 3 hr at 37°C alone.) The tray, incubated at 37°C only, gave experimental points fitted by p = VzaBNt 2 (for cell numbers up to 2.5 × 10 s ) a s predicted for s m a l l N and short t. The tray incubated for a further hour at 45°C gave experimental points fitted by p = 1 - e - N a t up to a lymphocyte:target cell ratio of 10:1. Beyond this point deviation occurs presumably because of crowding effects mentioned earlier. Thus the 45°C-incubation introduced by Miller and Dunkley (1974) can be used in this system.
M. D u n k l e y et aL, A m o d i f i e d sl Cr release assay
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Fig. 6. The effect of an additional incubation for 1 hr at 45°C on the specific s 1Cr release. ] h e open circles are experimental values (mean -+ S.E.M.) obtained by incubating various n u m b e r s of sensitized spleen cells with 2.5 x 104 P815 m a s t o c y t o m a cells at 37°C for 1.5 hr. The dotted l~ne is t h e fit given by p = Y2 a B N t 2 calculated using the constant ~ value obtained from the experimental points where this value was constant (10:1 and below). The closed circles are experimental points (mean -+S.E.M.) obtained by incubating an identical series for 1.5 hr at 37°C followed b y 1 hr at 45°C: the solid line is the theoretical fit given by p = 1 - e - N a t calculated using the constant ~ value obtained from the experimental points (at the ratio 10:1 and below) where this value was constant.
50
M. D u n k l e y et al., A modified sl Cr release assay
4. Discussion The final modified tray procedure offers a number of advantages for routine assay of CL, especially where large numbers of samples must be processed. Simplicity and speed are inherent in the original tray method of Thorn et al. (1974). All centrifuging steps are avoided and the amount of glass- or plastic-ware required is reduced as the whole procedure, including the serial dilutions of the sensitized cells, takes place in the same wells used for incubation. Large volumes of culture medium are not required and the procedure is economical of target cells and sensitized lymphocytes. The reproducibility of the method is very good despite the small volumes used. The use of the 45°C-incubation after the 37°C-incubation period increases the efficiency of the assay, as every effective CL-target interaction is completely expressed as S~Cr release. This procedure also eliminates variation due to variable release of s ~Cr at 37°C from different batches of target cells. In practical terms this procedure does result in some shortening of the overall incubation time required to give a reasonable 51Cr release. However tile advantage of the 45°C-incubation is primarily in the improved quantitation: the results within a reasonable range are predictable by the mathematical fornmlation and ~xvalues from different assays can be compared directly. One disadvantage of the procedure is the small increase in the spontaneous s i Cr release from the target cells (this could be lowered appreciably by using in vivo transplanted-, rather than cultured-target cells). Since reproducibility was high this did not greatly affect the accuracy of the assay but could be a limiting factor in some experiments. Another limitation of the procedure is the deviation from the theoretical curve at higher cell concentrations. This problem is not unique to the tray assay, although it may be less pronounced in systems using larger volumes of culture medium or larger surface area incubation vessels. In practical terms this means limiting the number of activated cells to approximately 5 × 10 s per well. It should, however, be emphasized that the accuracy and reproducibility of the assay is high, so there is no difficulty working with values of 5 - 3 0 % specific s l Cr release. The recommended approach is therefore to run replicate serial 2 or 3-fold dilutions of the test cell suspensions in the trays, add labeled target cells, incubate 1 - 3 hr at 37°C (the time of incubation depending on the suspected activity of the sensitized cell preparation) then incubate for 1 hr at 45°C. After determining the fractional specific s l Cr release an c~ value is calculated for each dilution using the equation p = 1 - e - N c ~ t (p = fractional specific lysis, N = number of sensitized cells and t = time of incubation at 37°C). In the region where these values are constant a mean c~ value for this sensitized cell preparation is then obtained. This value of c~ is a measure of the relative CL frequency in the sensitized cell population. In common with other methods the absolute number of CL cannot be obtained, but by using this modified procedure a simple linear relationship between ~ and the absolute CL number should pertain.
M. Dunkley et al., A modified 5 1 0 release assay
51
Acknowledgements Drs. R.M. Thorn, J.C. Palmer and L.A. Manson of the Wistar Institute, Philadelphia, U.S.A. are thanked for providing details of their tray method and a copy of the manuscript prior to publication. This work was supported by grants from the Whitehall Foundation, U.S.A., the National Health and Medical Research Council, Canberra, Australia, and the National Cancer Institute of Canada. The research upon which this publication is based was performed pursuant to Contract NO1-CB-23889 with the National Cancer Institute, National Institutes of Health, Department of Health, Education and Welfare, U.S.A.
References Brunner, K.T., J. Mauel, J.-C. Cerottini and B. Chapuis, 1968, Immunology 14,181. Byrd, W.J., H. von Boehmer and B.T. Rouse, 1973, Cell. lmmunol. 6, 12. Miller, R.G. and M.L. Dunkley, 1974, to be published in J. Cell. Immunol. Shortman, K., K.T. Brunner and J.-C. Cerottini, 1972a, J. Exptl. Med. 135, 1375. Shortman, K., N. Williamsand P. Adams, 1972b, J. lmmunol. Methods 1,273. Thorn, R.M., J.C. Palmer and L.A. Manson, 1974, J. Immunol. Methods 4, 301. Von Boehmer, H. and K. Shortman, 1973, J. Immunol. Methods 2,293.
© North-Holland Publishing Company
A MODIFIED 51Cr RELEASE ASSAY FOR CYTOTOXIC LYMPHOCYTES* Margaret DUNKLEY, R.G. MILLER** and Ken SHORTMAN The Walter & Eliza Hall Institute for Medical Reasearch, Melbourne, Vic. 3050, Australia
Received 26 March 1974, accepted 14 June 1974
An improved 5 ICr release assay for cytotoxic lymphocytes has been developed. The assay system combines the technical advantages of a titration tray method with a modification which enables a more quantitative measure of the cytotoxicity of sensitized cell preparations in a shorter time. A simple mathematical model describing the cytotoxic lymphocyte-target cell interaction has been shown to apply to this system. The advantages and limitations of the assay system are discussed and the recommended procedure is described in detail.
1. Introduction Many attempts at obtaining a quantitative assay for cell-mediated immunity have been based on the s 1Cr release assay of Brunner et al. (1968) which measures cellmediated cytotoxicity against H-2 alloantigens. There have been several attempts to simplify and improve the technical aspects of the procedure, as well as to understand the assay in terms of an exact mathematical model. Thorn et al. (1974) recently developed a modified assay using small titration trays, thus eliminating centrifuging steps and reducing the number of cells required. Miller and Dunkley (1974) using a Brunner type assay have developed a mathematical model for the cytotoxic lymphocyte (CL)-target cell interaction which agrees well with experimental data. The latter study also demonstrated that the assay was limited by the slow rate of 51Cr release from damaged target cells at 37°C. A modification was therefore introduced in which a 1 hr incubation at 45°C gave rapid and complete Sl Cr release from target cells damaged in a prior, short incubation at 37°C. The aim of this paper was to combine the advantages of the titration tray method of Thorn et al. with the modified procedure of Miller and Dunkley, to produce a simple accurate assay for CL.
* This is Publication No. 1998 from the Walter & Eliza Hail Institute. ** Present address: Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada. 39
40
M. Dunkley et al., A modified sl Cr release assay
2. Materials and methods 2.1. Mice
Six to ten week old BALB/c/AnBradley Wehi, and C57BL]6J mice of both sexes were used. 2. 2. Media 2.2.1. Suspension medium
The medium used for preparing cells and for general purposes was a HEPESbuffered balanced salt solution iso-osmotic with mouse serum and containing 10% (v/v) fetal calf serum (Shortman et al., 1972b). 2.2.2. Culture medium
For assay of cytotoxic activity, cells and targets were suspended in a modified, mouse osmolarity, CO2-buffered culture medium prepared as follows: One packet of Minimum Essential Medium powder (Cat. No. F-15 Grand Island Biological Co., Grand Island, New York) was dissolved in 700 ml of distilled water to which was added 25 mg penicillin, 20 mg streptomycin, 0.1 g pyruvate and 127 ml of sodium bicarbonate (2.8% w/v, Commonwealth Serum Laboratories, Melbourne, Australia). The medium was then made up to a total volume of 1050 ml with distilled water to give a solution iso-osmotic with mouse serum and was maintained at pH 7.2 by equilibrium with a 10% CO2 in air-gas mixture. Heat-inactivated fetal calf serum was added (10% v/v) before use. The fetal calf serum was inactivated by incubation at 56°C for 1 hr. 2.3. Cell suspensions
Mice were killed by cervical dislocation, the spleen or thymus removed, chopped into small pieces and pushed gently through a fine stainless steel sieve into cold suspension media. Cell clumps were removed by layering the cell suspension over 1 ml of fetal calf serum and standing for 5 rain. The supernatant fraction (a single cell suspension) was washed once through fetal calf serum and either resuspended in suspension media for injection into mice, or subjected to a damaged cell removal technique (Von Boehmer and Shortman, 1973) before resuspension in culture medium for assay of cytotoxic activity. 2. 4. Production o f sensitized cell populations in vivo
An adoptive transfer to allogeneic hosts was used to generate CL. Approximately 108 C57BL spleen cells or 108 'light density' (density "< 1.077 g/cm 3) thymus cells (Shortman et al., 1972a, b) were injected intravenously into irradiated (700 rads)
M. Dunkley et al., A modified Sl Cr release assay
41
BALB/c mice. After 5 - 6 days the BALB/c mice were killed, the spleens removed and spleen cell suspensions assayed for cytotoxic activity against target cells with the same alloantigenic specificity at the H-2 locus as the BALB/c cells. 2.5. Target cells Unless otherwise stated s ~Cr-labeled P815 mastocytoma cells were used as target cells. The cells were maintained in continuous exponential growth in tissue culture in a modified Dulbecco's medium containing 10% fetal calf serum. Cultures were harvested in the exponential phase of growth and labeled by incubation with sodium sl Cr-chromate (100/ICi per 5 × 10 6 cells) for 20 min at 37°C in 1 ml of the suspension medium. The cells were washed 3 times through 1 ml of heat-inactivated fetal calf serum before resuspension in culture medium for use. Large, dividing B lymphocytes, obtained as a light density fraction from pokeweed mitogen (PWM)-stimulated spleen cells were also tested as possible targets. Spleen cells (60 × 10 6) from BALB/c mice were cultured for 3 - 4 days with 0.4 ml of PWM (Grand Island Biological Co., Grand Island, New York. Batch No. 128248A, reconstituted in 5 ml normal saline and diluted 1:15 with culture medium) in Marbrook flasks (internal chamber diameter 2.1 cm, external volume of medium 50 ml) (Byrd et al., 1973). The ceils were harvested, washed and a density cut carried out using albumin of specific density 1.073 g/cm 3 at mouse osmolarity and pH 5.1 (Shortman et al., 1972b). The cells less dense than 1.073 g/cm 3 were labeled with sodium si Cr-chromate (100 pCi/106 cells) for 4 0 - 5 0 rain at 37°C in 1 ml of suspension medium and washed 3 times through heat-inactivated fetal calf serum before use. 2. 6. Cytotoxicity assay 2. 6.1. 'The standard tray assay' Tile method of Thorn et al. (1974) was used in the initial investigations. The sensitized or normal cell population was suspended in culture medium and diluted by serial 2-fold dilutions in titration trays - (Micro Test II. Tissue culture plate, Falcon plastics, Oxnard, C.A. 93030, U.S.A.) to give 0.1 ml per well. An equal volume of target cells (2.5 X 104 cells) was added to each well to give a final volume of 0.2 ml. The trays were then incubated at 37°C for the required period, placed on ice and 0.1 ml of the supernatant carefully removed from each well with an automatic pipette (Eppendorf, made in Germany). It has been shown that at this stage the cells have settled out completely and removal of the supernatant without cells is easily accomplished (Thorn et al., 1974). 2. 6.2. Final recommended procedure The titration trays were placed on ice. Serial 2-fold dilutions of the sensitized cell population were carried out in quadruplicate using the culture medium, and 2.5
42
M. Dunkley et aL, A modified 51Cr release assay
× 104 s~ Cr-labeled P815 mastocytes (in 0.1 ml) were added to each well (total volume per well = 0.2 ml). The trays were then transferred to a plastic box on ice which was gassed with 10% COz in air, sealed and stood in the cold for 30 min. This procedure allowed the cells to settle out, which is necessary as there is evidence that very few encounters between CL and target cells take place while the cells are in suspension (Miller and Dunkley, 1974). Equilibration of pH also took place at this stage. The trays were placed in water in a CO2 incubator (10% CO2 in air) at 37°C for a short period (usually 1 - 2 hr but this time may be extended for low activity cell preparations). The trays were then transferred to a plastic box containing a layer of water maintained at 45°C by sitting the box in a water bath. The box was gassed (10% CO2 in air), sealed and incubated for 1 hr. This period at 45°C has been shown to be optimal for release of all s l Cr from cells previously damaged at 37°C; whilst no new lytic events between CL and target cells take place (Miller and Dunkley, 1974). The trays were placed on ice and 0.1 ml of the supernatant carefully removed for radioactive counting in a well-type gamma radiation counter. 2. 6. 3. Calculation o f percent specific s ~Cr release
The spontaneous s ~Cr release from the target cells was measured by incubating 2.5 × 104 mastocytes alone in 0.2 ml of culture medium in quadruplicate. The specific s LCr release due to killing by cytotoxic lymphocytes was calculated by subtraction of the mean spontaneous release counts from the mean total counts for each group. This value was expressed as a percentage of the maximum releasable counts minus the spontaneous release. The maximum releasable counts represent the maximum amount of the s~ Cr taken up by the cells which can be released again on cell lysis. This was determined by incubation of 1.25 X l 0 4 mastocytes in 0.05 ml of medium, plus 0.15 ml zaponin (lysing reagent for white blood cell counts, Coulter Electronics Ltd., High St. South, Dunstable, Beds, England; 3 drops in 5 ml of water), for 3 hr at 37°C. The maximum releasable counts were usually 8 0 - 9 0 % of the total counts taken up by the mastocytes.
3. Results 3.1. Spontaneous 5 ~Cr release under various conditions
In initial experiments, we investigated the extent of spontaneous release of 51 Cr by target cells in the titration trays. Using cultured P815 mastocytoma cells, the spontaneous S~Cr release expressed as a percentage of the maximum releasable 51Cr over a period of several months was 23 -+ 6% for 3 - 5 hr at 37°C. This compared well with values obtained in an assay system using small capped tubes, similar to the assay system of Brunner et al. (1968), the value here being 20% or more for 4 hr at 37°C with cultured cells. In vivo passaged cells always gave lower spontaneous release (around 10% under these conditions). When the trays were
M. Dunkley et al., A modified s t Cr release assay
43
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Fig. 1. The effect of non-immune spleen cells on the spontaneous release of s 1Cr from labeled P815 mastocytoma target cells. The spontaneous s 1Cr release is expressed as a percentage of the maximum releasable s l Cr (see Materials and Methods) _+ S.E.M. The assay procedure was the standard titration tray method incubating at 37°C only. Each well contained either 2,5 X l04 labeled P815 mastocytoma cells or 1 X 105 labeled PWM-stimulated BALB/c spleen cells. The lymphocytes added in various concentrations were non-sensitized BALB/c spleen cells. The 'labile mastoeytes' were from a batch of cultured cells giving, for undetermined reasons, a very high background release; this was an exceptional circumstance, the lower curve giving the normal results. The time of incubation was 3 hr for the normal P815 mastocytoma targets and 4 hr for the 'labile' PS15 mastocytes and the PWM-stimulated spleen cell targets.
incubated for 1.5 hr at 37°C followed by 1 hr at 45°C (as in the r e c o m m e n d e d procedure) the spontaneous release was 21 -+- 5%, compared with 15 -+ 4% for 1.5 hr at 37°C, representing a definite but tolerable increase. Very occasionally a batch of cultured P815 mastocytes gave a very high spontaneous release ( u p ' t o 60%, eg. fig. 1); this was assumed to be due to poor culture conditions, since w e have never observed this with mastocytes passaged in D B A / 2 mice. Fig. 1 shows the variation in spontaneous s ~ Cr release in several other situations. Addition o f n o n - i m m u n e cells to labeled P815 m a s t o c y t o m a cells produced no significant effect on the s~ Cr release, even in the exceptional case o f P815 mastocytes with high backgrounds. However, other target cells did not always behave in this way. For example PWM-stimulated spleen cells gave very high spontaneous st Cr release w h e n incubated alone, but addition o f n o n - i m m u n e cells lowered the
44
M. Dunkley et al., A modified st Cr release assay I00
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Fig. 2, The relationship between specific s J Cr release and the number of sensitized lymphocytes present. The standard tray assay was used, incubating at 37°C only. The values are expressed as percent specific lysis (see Materials and Methods) -+ S.E.M. Sensitized cells were obtained by transfer of C57BL spleen or thymus cells into irradiated BALB/c mice. The target ceils were P815 mastocytoma cells.
spontaneous s i Cr release (fig, 1). Viable and non-viable lymphocytes and red blood cells all had the effect of lowering the spontaneous S~Cr release. Thus for these targets the specific s 1Cr release due to CL can only be investigated if careful paired controls are run using non-immune cell preparations. 3.2. Relationship between sensitized lymphocyte concentration and specific S ~Cr release from P815-mastocytes The relationship between the percentage specific s i Cr release (see Materials and Methods) and the log of the ratio of viable lymphocytes to target cells was determined for the standard tray assay incubating at 37°C only. The results (fig. 2) were similar to those reported by others for ratios up to 20:1, and show the standard near-linear relationship in the middle of the lysis curve. However a striking and unusual result was obtained at higher sensitized lymphocyte concentrations, where, instead of a plateau, an actual drop in the percentage specific lysis was observed. This was unexpected as previous studies had shown that the addition of moderate levels of unsensitized bystander cells had no effect on the percentage lysis (Miller and Dunkley, 1974). This inhibition was obtained whether or not damaged cells were removed from the lymphocyte preparation and thus can be caused by viable
M. Dunkley et al., A modified 510 release assay
45
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Fig. 3. The effect of large numbers of non-sensitized cells on the lysis of P815 mastocytoma target cells by sensitized lymphocytes. Two series of dilutions of sensitized C57BL cells were incubated with P815 mastocytoma cells and to one series 5 X 106 normal BALB/c cells were added per well. The values presented are the mean -+S.E.M. cells. The effect also appeared to occur at the same cell concentration and was independent of the incubation time over the 3 - 5 hr period (fig. 2). It was also obtained with activated thymus cells (fig. 2) indicating that it was not caused by activated, antibody-forming B cells blocking the lysis. It is thought that the effect is due predominantly to the limited capacity o f the medium to provide nutrients or oxygen or to cope with waste products or COs production as indicated by a pH change in the most dense (100:1 ratio) cultures. The inhibition may also be due in part to a slowing down o f the CL movement or shielding of target cells due to a physical crowding in the most dense cultures. That this inhibition can be caused by non-sensitized cells regardless o f the number o f sensitized cells present is shown in fig. 3. Here, a large number (5 × 106) of non-activated BALB/c spleen cells were added to each culture. This caused a depression o f lysis over the whole range thus showing that it is only an excess of cell numbers which depresses the lysis. It should be noted that this level of cells did not affect the background release (fig. 1). 3. 3. Application o f the theoretical model to the standard titration tray system A mathematical model describing various phases of the sl Cr release assay, and giving an excellent fit to experimental data in a Brunner-type assay procedure, has been given b y Miller and Dunkley using the following notation:
46 p
M. D u n k l e y et al., A modified sl Cr release assay
= f r a c t i o n a l specific 51Cr release;
N = no. o f sensitized cells; t = t i m e o f i n c u b a t i o n at 37°C; B = a c o n s t a n t c h a r a c t e r i s t i c o f the s 1Cr release process; = a c o n s t a n t = v6; v = f r e q u e n c y o f C L in t h e sensitized p o p u l a t i o n ; 8 = a c o n s t a n t (includes such f a c t o r s as cell velocity a n d size a n d area or v o l u m e o f the c u l t u r e vessel). The general f o r m u l a t i o n leads to t w o p a r t i c u l a r l y i n t e r e s t i n g special cases. A t s h o r t t i m e s a n d small cell n u m b e r s ( N a t , B t ~ 1) (1)
p = ~c~BNt 2
At l o n g t i m e s or in t h e case o f rapid s 1Cr release ( B t >> 1) (2)
p = 1 - e -N~t
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Fig. 4. Testing the theoretical model when varying the concentration of sensitized lymphocytes and incubating at 37°C only for 5 hr. The open circles represent mean values -+ S.E.M. from experimental data. The solid line is the theoretical curve predicted by the equation p = 1 - e - k N . This curve is generated by calculation of k for each p and N value. A mean k is obtained from the region of constant k values, in this case for lymphocyte:target ratios between 12:1 and 0.2:1, and a curve generated by calculating p for each N value using the mean k value.
M. Dunkley et al., A modified Sl C'r release assay
47
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Fig. 5. Testing the theoretical model when varying the incubation time. The open circles represent the mean -* S.E.M. specific 5 ICr release obtained after incubating 1.5 X 10 s sensitized C57BL spleen cells with 2.5 X 104 P815 mastocytoma cells for various times at 37°C. The solid line is the theoretical curve predicted by p ---et 2. This curve was generated by calculation o f c for each time point and taking a mean where the c values were constant (up to 3 hr). This mean c was used to generate the theoretical curve.
If this model holds for the titration tray assay, it would provide a means of comparing quantitatively the cytotoxicity of different cell preparations and enable comparison between assays done on different days under different conditions. Thus, values of specific SlCr release obtained experimentally when either the number of sensitized lymphocytes or the time of incubation was varied were compared with values predicted by the theoretical model. For long incubation times at 37°C Eq. (2) should be approximately valid. This is demonstrated by the experiment of fig. 4 in which variable numbers of lymphocytes were incubated for 5 hr at 37°C with a fixed number of target cells. The observed lysis had an exponential dependence on the number of sensitized cells present (p --- 1 - e -kN, where: p = specific SlCr release;N = number of lymphocytes, and k is a constant) as predicted. Deviation from this relationship occurred only at high cell numbers (above a lymphocyte:target ratio of 20:1) presumably as crowding effects became important. For short incubation times and small numbers of sensitized cells, Eq. (1) predicts
48
M. Dunkley et al., A modified sl Cr release assay
a quadratic time dependence. This is demonstrated by the experiment of fig, 5 in which the response at a constant low cell number showed that the specific S~Cr release had a dependence on t 2 (p = ct 2, where p = specific s~ Cr release, t = time of incubation at 37°C and c = a constant) up to 3 hr. For longer incubation times the specific lysis appears to have an exponential dependence on t (p = 1 - e - z t , where z a constant, has the value of 0.067 at 3 hr, 0.058 at 4 hr and 0.056 at 5 hr). These observations again agree with the predictions of the theoretical model of Miller and Dunkley. 3. 4. Modification o f the assay to produce c o m p l e t e s ~Cr release f r o m a damaged target cell
Previous studies of Miller and Dunkley (1974) showed that the factor-limiting quantitation o f the assay was the slow s~ Cr release at 37°C from the damaged cells. It was estimated that it took approximately 1 . 5 - 2 hr for all the s ~Cr to leak out o f a damaged cell at 37°C. Thus in the normal 37°C-incubation procedures, at the time of assay targets damaged 2 hr previously would have released all their s ~Cr, whereas targets damaged since that time would have released only portions of their s~ Cr. However it was demonstrated that during an incubation at 45°C there are no further lytic events, but that the remaining releasable s ~Cr contained within target cells damaged during the 37°C-incubation is released within 1 hr. The mathematical model also simplifies under these conditions (see section 3.3.), reducing to p = 1 e - N a t , and thus enabling the direct calculation of a. This is a measure (although not an absolute measure) of the frequency of CL in the sensitized population, The application of the modified procedure (see Materials and Methods for details) is given in fig. 6, where paired trays were compared to estimate the degree of s~ Cr release. The additional 45°C-incubation raised the spontaneous sl Cr release from 19.0 -+ 0,4% to 28,0 +- 0.3% but since variability was low and many replicates of spontaneous release can readily be run, good precision is maintained. The 45°C incubation gave about a 3-fold increase in specific s l Cr release. (The procedure allowed as much release of s~ Cr as about 3 hr at 37°C alone.) The tray, incubated at 37°C only, gave experimental points fitted by p = VzaBNt 2 (for cell numbers up to 2.5 × 10 s ) a s predicted for s m a l l N and short t. The tray incubated for a further hour at 45°C gave experimental points fitted by p = 1 - e - N a t up to a lymphocyte:target cell ratio of 10:1. Beyond this point deviation occurs presumably because of crowding effects mentioned earlier. Thus the 45°C-incubation introduced by Miller and Dunkley (1974) can be used in this system.
M. D u n k l e y et aL, A m o d i f i e d sl Cr release assay
49
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Fig. 6. The effect of an additional incubation for 1 hr at 45°C on the specific s 1Cr release. ] h e open circles are experimental values (mean -+ S.E.M.) obtained by incubating various n u m b e r s of sensitized spleen cells with 2.5 x 104 P815 m a s t o c y t o m a cells at 37°C for 1.5 hr. The dotted l~ne is t h e fit given by p = Y2 a B N t 2 calculated using the constant ~ value obtained from the experimental points where this value was constant (10:1 and below). The closed circles are experimental points (mean -+S.E.M.) obtained by incubating an identical series for 1.5 hr at 37°C followed b y 1 hr at 45°C: the solid line is the theoretical fit given by p = 1 - e - N a t calculated using the constant ~ value obtained from the experimental points (at the ratio 10:1 and below) where this value was constant.
50
M. D u n k l e y et al., A modified sl Cr release assay
4. Discussion The final modified tray procedure offers a number of advantages for routine assay of CL, especially where large numbers of samples must be processed. Simplicity and speed are inherent in the original tray method of Thorn et al. (1974). All centrifuging steps are avoided and the amount of glass- or plastic-ware required is reduced as the whole procedure, including the serial dilutions of the sensitized cells, takes place in the same wells used for incubation. Large volumes of culture medium are not required and the procedure is economical of target cells and sensitized lymphocytes. The reproducibility of the method is very good despite the small volumes used. The use of the 45°C-incubation after the 37°C-incubation period increases the efficiency of the assay, as every effective CL-target interaction is completely expressed as S~Cr release. This procedure also eliminates variation due to variable release of s ~Cr at 37°C from different batches of target cells. In practical terms this procedure does result in some shortening of the overall incubation time required to give a reasonable 51Cr release. However tile advantage of the 45°C-incubation is primarily in the improved quantitation: the results within a reasonable range are predictable by the mathematical fornmlation and ~xvalues from different assays can be compared directly. One disadvantage of the procedure is the small increase in the spontaneous s i Cr release from the target cells (this could be lowered appreciably by using in vivo transplanted-, rather than cultured-target cells). Since reproducibility was high this did not greatly affect the accuracy of the assay but could be a limiting factor in some experiments. Another limitation of the procedure is the deviation from the theoretical curve at higher cell concentrations. This problem is not unique to the tray assay, although it may be less pronounced in systems using larger volumes of culture medium or larger surface area incubation vessels. In practical terms this means limiting the number of activated cells to approximately 5 × 10 s per well. It should, however, be emphasized that the accuracy and reproducibility of the assay is high, so there is no difficulty working with values of 5 - 3 0 % specific s l Cr release. The recommended approach is therefore to run replicate serial 2 or 3-fold dilutions of the test cell suspensions in the trays, add labeled target cells, incubate 1 - 3 hr at 37°C (the time of incubation depending on the suspected activity of the sensitized cell preparation) then incubate for 1 hr at 45°C. After determining the fractional specific s l Cr release an c~ value is calculated for each dilution using the equation p = 1 - e - N c ~ t (p = fractional specific lysis, N = number of sensitized cells and t = time of incubation at 37°C). In the region where these values are constant a mean c~ value for this sensitized cell preparation is then obtained. This value of c~ is a measure of the relative CL frequency in the sensitized cell population. In common with other methods the absolute number of CL cannot be obtained, but by using this modified procedure a simple linear relationship between ~ and the absolute CL number should pertain.
M. Dunkley et al., A modified 5 1 0 release assay
51
Acknowledgements Drs. R.M. Thorn, J.C. Palmer and L.A. Manson of the Wistar Institute, Philadelphia, U.S.A. are thanked for providing details of their tray method and a copy of the manuscript prior to publication. This work was supported by grants from the Whitehall Foundation, U.S.A., the National Health and Medical Research Council, Canberra, Australia, and the National Cancer Institute of Canada. The research upon which this publication is based was performed pursuant to Contract NO1-CB-23889 with the National Cancer Institute, National Institutes of Health, Department of Health, Education and Welfare, U.S.A.
References Brunner, K.T., J. Mauel, J.-C. Cerottini and B. Chapuis, 1968, Immunology 14,181. Byrd, W.J., H. von Boehmer and B.T. Rouse, 1973, Cell. lmmunol. 6, 12. Miller, R.G. and M.L. Dunkley, 1974, to be published in J. Cell. Immunol. Shortman, K., K.T. Brunner and J.-C. Cerottini, 1972a, J. Exptl. Med. 135, 1375. Shortman, K., N. Williamsand P. Adams, 1972b, J. lmmunol. Methods 1,273. Thorn, R.M., J.C. Palmer and L.A. Manson, 1974, J. Immunol. Methods 4, 301. Von Boehmer, H. and K. Shortman, 1973, J. Immunol. Methods 2,293.