104

immunologytoday,2¢ovember1980

not over-react to trivial stimuli, b u t would be very far from defenceless. The authors' research is supported by programme and project grants from the Medical Research Council and Cancer Research Campaign. References 1 Pierce, A. E. (1959) Vet. Revs. Annols 5, 17-36 2 Heremans, J. F. (1974) In The Antigens (Sela, M., ed.) Vol. 2, pp. 365-522, Academic Press, New York 3 Tomasi, T. and Bienenstock, J. (1968) Adv. Immunol. 9, 1-96 4 Brandtzaeg, P. and Savilahti, J. (1968) Adv. Exp. Med. Biol. 107, 219-226 5 Brown, W. R. (1978) Gastroenterology75,129-138 6 Porter, P., Noakes, D. E. and Allen, W. D. (1972) Immunology 23, 299-307 7 Nagura, H., Nakane, P. K. and Brown, W. R. (1979) J. lmmunol. 123, 2359-2368 8 Ogra, P. L. and Karzon, D. T. (1969) J. Immunol. 102, 1423-1430 9 Porter, P., Noakes, D. E. and Allen, W. D. (1970) Immunology 18, 909-920

10 Hall, J. G. (1979) BloodCells 5,479-492 11 Orlans, E., Peppard, J., Reynolds, J. and Hall, J. G. (1978)J. Exp, Med. 147, 588-590 12 Lemairre-Coelho, I., Jackson, G. D. F., Vaerman, J-P (1977) Eur. j . Immunol.7, 588-590 13 Jackson, G. D. F., Lemaitre-Coelho, I., Vaerman, J.-P., Bazin, H. and Beckers, A. (1978) Eur. J. Immunot. 8, 123-126 14 Birbeck, M. S. C., Cartwright, P., Hall, J. G., Orlans, E. and Peppard, J. (1979) Immunology37,477-484 15 Mullock, B. M., Hinton, R. M., Dobrota, M., Peppard, J. and Orlans, E. (1979) Biochem. Biophys. Acta 587, 381-391 16 Hall, J., Orlans, E., Reynolds, J., Dean, C., Peppard, J., Gyure, L. and Hobbs, S. (1979) Int. Archs Allerg. Appl. Immunol. 59, 75-84 17 Reynolds, J., Gyure, L., Andrew, E. and Hall, J. G. (1980) Immunology39, 463-467 18 Porter, P., Linggood, M. A., Chidlow, J. (1978) Adv. Exp. Med. Biol. 107, 133-142 19 Hemmings, W. A. (ed.) (1978) Antigen Absorplionby theGut, pp. 1-226, MTP Press, Lancaster 20 Peppard, J., Orlans, E., Payne, A. W. R. and Andrew E. Immunology (in press)

(techniques 1 Assessment of cell-mediated cytotoxicity Benjamin Bonavida and Thomas P. Bradley Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles, California 90024 Several different techniques are used to assess the expression of cellular immunity (reviewed in Ref. l). In this article Benjamin Bonavida and Thomas Bradley discuss ways of measuring one aspect of cellular immunity- the activity of cytotoxic cells on target cells. T h e role of lymphocytes in interactions with target cells which lead to target lysis was not established until the early 1960s. R o s e n a u a n d M o o n 2 were the first to d e m o n s t r a t e the lysis of homologous cells by sensitized lymphocytes in a n in vitro tissue culture system. L y m p h o c y t e s from B A L B / c mice sensitized against L cells from a n allogeneic mouse (C3H), i n d u c e d a striking cytopathic change w h e n coc u l t u r e d with L target-cells. These changes occurred in the absence of a n t i b o d y a n d c o m p l e m e n t . Close contact b e t w e e n the sensitized lymphocytes a n d the target cells was found to be essential for the cytotoxic reaction to occur. I n the t w e n t y years since this demonstration of cell-mediated cytotoxicity (CMC), progress in the direct m e a s u r e m e n t of C M C has been slow, possibly because of the complex n a t u r e of the © Elsevier/North-Holland Biomedical Press 1980

reaction a n d the difficulty in assessing target-cell destruction. In order to make a reasonable assessment of a cytotoxic reaction with a p a r t i c u l a r assay, one should know: (1) the development, differentiation, a n d fate of the cytotoxic cell, (2) the exact n a t u r e of the effector cell involved, (3) whether this effector acts alone or in cooperation with other cells, (4) the biological state of the effector cell before a n d after i n t e r a c t i o n with target-cells, (5) the molecular m e c h a n i s m of the cytotoxic reaction a n d (6) the effect of the target on the cytotoxic celt. F u r t h e r m o r e the assay should provide a m e a s u r e of the absolute frequency of cytotoxic cells present in a mixed population, a m e a s u r e of the affinity a n d avidity of the effector cells to corresponding target-cells a n d a m e a n s to q u a n t i t a t e the cyto-

105

immunology today, November 1980 MONOLAYERS OF

TUMOR

TARGET

CELLS

000

000

IMMUNE LYMPHOCYTES ADDED

NORMAL LYMPHOCYTES ADDED

~:,

Incubate 2 - 3 days Fix and stain Count adherent cells

©©©

DETACHMENT AND LYSIS

0@0

NO DETACHMENT OR LYSIS

Fig. 1 Assessment of cell-mediated cytotoxiclty by enumeration of target cells left after interaction with effector cells. Both the colony inhibition assay (Ref. 3) and microcytotoxic!ty assay (Ref. 4) use this technique.

toxic reaction. To date, none of the assay methods available satisfy all these requirements. With these ideal requirements in mind, we shall briefly discuss the three main categories of methods used to measure CMC. These are: (1) enumeration of residual target-cells after incubation with effector cells, (2) examination of target-cell lysis by radioisotope labelling of target cells and (3) microscopic viewing of single cytotoxic effector cells b o u n d to targets. We will discuss the potential applications of these methods and their limitations and advantages (Table I). E n u m e r a t i o n of r e s i d u a l target cells after i n t e r a c t i o n w i t h effector cells (Fig. 1) Two methods which detect C M C by cell counting are the colony inhibition assay modified by Hellstr6m in 19673 and the microcytotoxicity assay of Tagasuki and Klein 4. In the colony inhibition assay, target cells (1-2 x 103 ) are a d d e d to plastic petri dishes and allowed to grow and become attached to the dish. 12-24 h later, effector cells or control cells are a d d e d to the monolayers, the dishes are further incubated for 3-4 d a y s . a n d the colonies are counted microscopically. If the effector cells have killed or inactivated the target, the number of colonies in the experimental dishes will be less than that in the control plates (Fig. 1). The microcytotoxicity assay of Tagasuki and Klein is based on the ability of effector cells to reduce the n u m b e r of viable target cells left adherent to a plastic surface, after co-culture with effector cells for 48 h or longer. The method is termed 'microcytotoxicity' because it utilizes a small number of cells (less than 500) in a minute volume on a Terasaki microtest culture plate. Briefly, target cells are seeded in the wells the day before the experiment and allowed to form a monolayer. T h e n effector cells are a d d e d to the target cells and incubated at 37°C for 2 days. At the

conclusion of the assay, the wells are washed twice and living cells which remain attached to the floor of the wells are fixed in methanol and stained with Giemsa. The n u m b e r of remaining cells is scored with a bottom-viewing microscope. The percentage reduction in the number of adherent targets is calculated in comparison with control plates incubated without effector cells (Fig. 1). Although these methods offer the advantage of using small numbers of cells, they have several limitations. (1) The target cells used must be able to adhere to the plates. (2) Because the incubation is long, target-cells grow during culturing and cell growth in experimental and control wells may not be the same. Furthermore, because of the length of the assay, it is possible that invitro sensitization of effector cells may take place. The interpretation of results is complex when the populations of effector cells are not pure: several different effector cells m a y be present and various cell-cell interactions might occur in a synergistic and antagonistic manner during the assay. (3) The assay is tedious to establish and to count. However, the microcytotoxicity tests of Tagasuki and Klein can be more easily calculated if counted by computer-assisted image analysisL (4) Since these assays measure detachment of nonviable or inactivated cells from the target monolayer, they may not measure direct target-cell destruction. R a d i o - l a b e l l i n g of target-cells (Fig. 2) A technique for radioisotope labelling of target-cells has several requirements: the isotope should be incorporated into targets in amounts sufficient to label without inducing death; the half-life of a particular isotope should be such that radioactivity can be counted within a convenient time period; the isotope, once released or degraded, should not be re-utilized; PRE-INCUBATION LABEL LABEL TARGET: (5]Cr, 1251UdR)

POST-INCUBATION LABEL UNLABELED TARGET

~A/CUBA TIOA/

LABEL TARGET: (e6Rb, 1251UdR, 14C_LEU 3 H-PRO, 3 H-THYMIDINE) RADIOACTIVITY

I

TARGET

COUNTED

TARGET

I

Fig. 2 Assessment of cell-mediated cytotoxicity by radioisotope - labelled target cells.

106

immunology today, November 1980 Techniques commonlyused to measure CMC.

Technique

Target-cell count Colony inhibition Microcytotoxicity Radiolabelling of targets 51Cr

~2sIUdR 3H-proline 3H-thymidine 86Rb 14C-leucine Single-cell assay

Measurement

Refs

Percent of tumor targets remainingthat form colonies Viabletarget cellsremaining

3

Radioactivityreleased Radioactivityreleased or retained Radioactivityretained Radioactivityretained Radioactivityretained Radioactivityretained Killed target cells bound to a cytotox!ccell

6 8

4

9 10 11 12 15,16 17,7,24

the interaction of the effector cells with the target cells should be unaffected by the radioactive labelling; and the spontaneous release of isotope from the target should be minimal. Several of the current techniques have taken into consideration these prerequisites. In most radioisotope techniques targets are labelled before incubation with effector cells. This method works quite well in systems with high numbers of active cytotoxic cells and with an appropriate target cell. Several problems, however, are encountered in weaker cytotoxic systems. In these cases, labelling targets after incubation has been shown to work more efficiently.

Radio-labelling of target cells prior to use in CIVIC assay (Fig. 2) Assays involving preincubation of labelled targets are either short-term (3 h) or long-term (18-64 h). Short-term assays are simpler: targets are labelled with radioactive chromium (sz Cr) or iodine (12sIUdR) and incubated for 3h (+ lh) with effector cells. One of the first short-term preincubation labelling techniques, adopted by Brunner and his colleagues in 19686, has been the most widely used assay of cyto-

toxicity. Target cells are labelled with sodium 51chromate and at various times after co-culture with the effector lymphocytes the radioactivity released into the medium is counted. With appropriate targets this slCr-release assay measures irreversible damage to the target: release of slCr indicates an advanced state of target-cell deterioration. The method is simple, quick, reproducible, and can be adapted to automation. Its main advantages are the rapid labelling of the targetcells and low spontaneous release of isotope in the short-term. There are also limitations to this technique, however, mainly artifacts and assumptions which are not necessarily valid. For instance: (a) quantitation of the cytotoxic reaction is not always possible; each targetcell may not contain precisely equal amounts of slCr, so that the number of targets killed cannot be calculated from the measured release of 51Cr, (b) cellular factors in the medium may affect the release of slCr from a lysed target, (c) the measured release of isotope does not reflect damage to target-cells which have been inactivated, rather than lysed, by effector cells, and (d) the heterogeneity of the cytotoxic population, in terms of lytic rate and capacity to recycle, makes both qualitative and quantitative analyses quite complex 7. Long-term assays in which targets are labelled before incubation are generally adaptations of the Tagasuki and Klein microcytotoxicity assay described above. I n these assays, target-cells adherent to the bottom of plastic wells are labelled with ~25IUdR8, 3Hproline 9, or 3H-thymidinel0. After incubation with effector cells, the radioactivity remaining in the targetcells which remain attached and viable or the radioactivity released in the supernatant from lysed targets is counted. The advantages of this long-term assay are that: (a) weaker cytotoxic systems mediated by cytotoxic T cells (CTL) or by natural killer (NK) cells can be detected which are undetectable in a short-term assay, (b) a small n u m b e r of targets can be used, as in the microcytotoxicity test, (c) radioactivity can be

TABLE II. Frequency of cytotoxic cells determined by single-celltechniques Cytotoxic cell T lymphocyte

Effector populations

Species

Frequency of cytotoxic cells °70

Refs

Allosensitized peritoneal lymphocytes(in vivo) Allosensitizedspleen cells(in vivo) Allosensitizedspleen cells(in vitro) Lines clonedusing TCGF Nylon-wool-purifiedspleencells Peripheral-bloodlymphocytes Peripheral-bloodlymphocytes Allosensitizedperitoneal lymphocytes(in vivo) Peripheral blood lymphocytes

Mouse

20-50

15,l 6,7,22

Mouse Mouse Mouse Mouse Man Man Mouse

7-13 3-17 10-25 20-30 3 2-3 20-50

7 7 Bonavida, unpublished 25 31 27 24

a

NK cell ADCC~ effectorcell LDCC~ effectorcell

Man

3-5

~raeIey, ----r............

TCGF: T-cell growth factor; ADCC: antibody-dependentcellular cytotoxicity;LDCC: lectin-dependentcellular cytotoxicity(in which lectins (concanavalinA or phytohemagglutinin)induce nonspecificcytolysisof syngeneicor xenogeneictargets by alloimmune lymphocytes (mouse)or normal blood lymphocytes (man))

107

immunology today, November 1980 counted as a measure of cytotoxicity, which is preferable to the tedious counting method of the microcytotoxicity test, and (d) there has been a good correlation between the celt counts done visually and isotope counting at the end of the assay, which suggests that the assay preferentially measures cytotoxic activity rather than inhibition of proliferation. The major drawbacks to this long-term assay are essentially those of the colony inhibition and microcytotoxicity assays mentioned above.

Radio-labelling of target cells after incubation with effector cells Several assays of this type are available. T h e y are based on the fact that only viable targets will take up isotope-labelled essentials like the potassium analog r u b i d i u m (86Rb)ll, amino acids (14C-leucine)12, or nucleosides (3H-thymidine)l°. After the target cells have formed a confluent monolayer in microtiter plates effector lymphocytes are a d d e d to each well and the plates are incubated for 48 h. The plates are washed several times, isotope is a d d e d to each well, the plates are washed again, and detergent is a d d e d to each well. T h e contents are removed and counted in a liquid scintillation spectrometer (for emitters of beta radiation) or in a g a m m a counter. A major advantage of this technique is that spontaneous release is negligible because targets are labelled after incubation. The results correlate well with cell counts of viability and a low n u m b e r of targets is needed. The limitations of this type of assay include the fact that lymphocytes which remain attached to the targets are labelled with isotope, and that viable target cells may detach from the monolayer, falsely increasing the measured extent of cytotoxicity. With all the radiolabelling techniques discussed, one can only compare the lytic activity of one lymphoid celt population with another. It is difficult to discern the frequency of single cytotoxic effectors in a given population. Several factors have h a m p e r e d this direct enumeration of cytotoxic cells. One is the ability of cytotoxic lymphocytes to move and to recycle, each effector cell thus being able to kill multiple target cells during the assay. The effector cell responsible for lysis is therefore hard to detect. However, T h o r n and Henney 13 attempted to demonstrate the frequency of cytotoxic cells in a lymphoid population by an elegant, albeit indirect means. Lymphocytes were allowed to interact with 51Crlabelled targets in the absence of Ca +2 ions; interaction was thus permitted, but not lysis, when Ca +2 was added, cytochalasin A was also added, blocking further lvmohocvte-tar~et interaction~ h !t n !owine~ lymphocytes to 1 v¢ -o~ -t- + t ¢.l . .~.' +°a +I l.l. . . V Wv.i~. tllt I t +v.ev Itl • w e r e .q!rff.qdy 11 ~}/ bound. 11~ this l ! ! O ! ! ! ! l - ! } !.E.L.y L. !.! !.! ~ ~:. . . . .W.d.~ . ! ! [ l ]:[ I-I [ :! ! -L r-2 ":_ - - ~I t , r*~-this technique it was estimated that at the peak of the cytotoxic response, 2.2% of spleen cells were cytotoxic

cells. This frequency, however, is less than that obtained with the single celt technique discussed below (Table II). The problems in interpreting this use of the slCr-release assay were that the enumeration of cytotoxic cells was indirect, multi-target conjugates (a lymphocyte which had bound more than one target) were not accounted for, and all potentially cytotoxic lymphocytes might not have had the opportunity to interact with available target cells. These factors might all have contributed to an underestimation of the frequency of cytotoxic cells in the lymphoic cell population.

Enumeration of single cytotoxic cells b o u n d to targets The isotope release and target adherence assays focus on the target celt and infer the cytotoxic activity of a population of effector cells from target death. An alternative approach is to directly evaluate the effeetor cells, ability first to bind to a particular target cell and then to lyse it. There were originally two methods of viewing effect o t - t a r g e t interactions microscopically. Thus, in studies aimed at delineating the various steps involved in target cell lysis by C T L TM, M a r t z reported the formation of clusters in which one C T L bound to one target. Berke e/al. also reported the formation of stable lymphocyte-target cell conjugates and established a correlation between the n u m b e r of conjugates and the number of killers with the 51Cr-release assay 15. W i t h single-cell manipulation techniques, Zagury et al. sub-

EFFECTOR C E L L S O OO OO O

+ TARGET

CELLS

INCUBATE 5 0 ° C CEN FRIFUGE PELLET

CONJUGATES

RESUSPENDED

FORMED:

/14/CROMAN/PULATION

PLATE ONTO SLIDES IN A G A R O S E

OR

INCUBATE 5 7 ° C STAIN IN TRYPAN BLUE FIX IN FORMALDEHYDE

DISPERSE IN HEMOCYTOMETER

CONOUGATES

VIEWED

MICROSCOPICALLY

@fT ENUMERATE FREQUENCY W! I H L)F AD

/

0[- CO;'4JUG,&TKS TARGETS

Fig. 3 Single cytotoxic effector cell assay,

108

immunology today, November/980

s e q u e n t l y r e p o r t e d that most of the l y m p h o c y t e s in conjugates were killers 16. W e t h e n p r e s e n t e d a p l a q u e t e c h n i q u e for the e n u m e r a t i o n of cytotoxic effector cells 17. In this assay, targets were first fixed on a polyL - l y s i n e - c o a t e d plastic dish, a n d effector cells were t h e n a d d e d to the confluent target-cell monolayer. After incubation, the plates were t r e a t e d with eosin to stain d e a d cells, a n d zones of m o r e t h a n four dead targets ( ' p l a q u e s ' ) were viewed u n d e r the m i c r o s c o p e and e n u m e r a t e d . W h i l e this t e c h n i q u e yielded frequencies of cytotoxic cells w h i c h were m u c h lower t h a n those from assays using effector-target conj u g a t e s ( T a b l e II), it did provide a m e a s u r e m e n t of cytotoxic T l y m p h o c y t e s w i t h p r e s u m a b l y high affinity. T h e p l a q u e t e c h n i q u e ' s l i m i t a t i o n has b e e n its inability to m e a s u r e single target-cell death. It m a y nevertheless be useful in direct e n u m e r a t i o n of killer cells w h e n the target cells are m o r e t h a n 95% viable a n d adherent. T h e p l a q u e t e c h n i q u e is particularly a p p l i c a b l e in studies of a n t i b o d y - d e p e n d e n t cellm e d i a t e d cytotoxicity with red blood cells as targets, in which a zone of lysis is easily discerned 18. W h i l e the t e c h n i q u e of single cell m a n i p u l a t i o n r e p o r t e d by Z a g u r y et al. I6,19 was feasible and reprod u c e d by us 2°, it was tedious a n d difficult to a p p l y in routine e x a m i n a t i o n of killer-cell frequency. Fishelson a n d Berke 21 r e p o r t e d that killer-target conjugates could be viewed in a h e m o c y t o m e t e r a n d used in the detection of C T L but this t e c h n i q u e is limited in its uses and p r o l o n g e d i n c u b a t i o n in the h e m o c y t o m e t e r at 3 7 ° C m a y be s u b o p t i m a l for survival of conjugates. W e have modified the t e c h n i q u e s above by using agarose to i m m o b i l i z e effector-target conjugates formed at 30°C, at which binding, but not lysis, can Occur 23'24. T h e agarose, w h e n gelled, does not h a r m

the cells and prevents effector cells from m o v i n g a w a y from the a t t a c h e d target-cell. T h e viability of the e f f e c t o r - b o u n d target can be assessed microscopically with vital cell dyes after i n c u b a t i o n at 37°C. T h e single-cell assay, in general, involves two steps (Fig. 3). First the effector-target conjugates are formed in small glass culture tubes u n d e r slow centrifugation after a 5 rain i n c u b a t i o n at 3 0 ° C and the pellet is gently resuspended. At this point, conjugates can be m i c r o m a n i p u l a t e d into T e r a s a k i wells I9 or a h e m a c y t o m e t e r 21, or m i x e d w i t h agarose (30°C) and plated on to slides = . After i n c u b a t i o n for various times target-cell d e a t h is seen either with t r y p a n blue uptake 23 or by loss of density w i t h p h a s e - c o n t r a s t m i c r o s c o p y 19. A l t h o u g h the single-cell assays were originally developed to m e a s u r e C T L , they have b e e n a d a p t e d to q u a n t i t a t e N K cells in the m o u s e 25 a n d m a n 26, as well as a n t i b o d y - d e p e n d e n t cytotoxic cells 27 ( T a b l e II). T h e single cell assay offers several a d v a n t a g e s over existing cytotoxic assays : (1) It is simple, cheap, rapid, reproducible, a n d highly sensitive. (2) It provides an e s t i m a t e of the n u m b e r of cytotoxic cells present in the p o p u l a t i o n ( T a b l e II), a n d also the heterogeneity in the cytotoxic potential of the effector cells 7, a l t h o u g h it does u n d e r e s t i m a t e their frequency. It is a s s u m e d that all effectors with cytotoxic potential b i n d to targets but, as S h o r t m a n and Golstein report 28, the m e t h o d of resuspension und o u b t e d l y disrupts some potential conjugates that m i g h t otherwise lead to lysis, so that some cytotoxic effector cells are missed. (3) T h e p h e n o t y p e of the effector cell can be determ i n e d at the single cell level 24.

TABLE III. Comparison among various cytotoxic assays Assay features (Reference)

Cell counting Colony Microcytoinhibition toxicity (3) (4)

Radioisotope labelling ShortLongPlaque term term (6) (8,9,10) (17)

Single effector cell MicromaniLymphocyte-target pulation conjugates (16) (7,21,24)

96 hi b lo lo

3-18 v.hi hi hi

4 1o 1o 1o

3-18 hi/lo hi lo

3-18 hi v.lo lo

+

+

+

+

+ +/

+ -

+

+

+

+ -

Properties

Duration (h) a Reproducible Cost (hi/lo) c Number cells needed

72 hi lo v.lo

96 hi hi lo

Effeetor cell measures

Frequency in population determined Weak cytotoxicity revealed Recycling allowed

.

.

.

.

+ +

+ +

+

+ +/-

+ + +

+ . + -

+/+

Target cell measures

Direct eytotoxicity Cytostasis Adaptable to non-tumor cell Limited to adherent cell

+ .

. ? -

+ . + +/-

.

aDuration of assay includes both the time for effector-target incubation and the time to grow adherent target layers. b High (hi), low (1o), very low (v.lo) and very hi (v.hi) describe the degree of a particular property. c Cost in terms of equipment (radiation counters, micromanipulators etc.) or materials (i.e. radioisotopes).

immunology today, November 7980

(4) T a r g e t cells can be m a r k e d with fluorescent dyes so that they can be easily distinguished from the effector cells 7. T h e assay is thus not limited to targets physiologically different from the effector cell. (5) Besides t u m o r cells in suspension, a d h e r e n t t u m o r lines, blast cells, fibroblasts and n o r m a l splenocytes can all be used as targets. (6) Aspects of the m e c h a n i s m of cytotoxicity can be directly e x a m i n e d . For example, by blocking at various points in the lytic p a t h w a y (pre-target b i n d i n g v. post-target b i n d i n g ) we m i g h t d e t e r m i n e w h a t structures are involved in lysis 29 or m e t a b o l i c requirem e n t s are needed for each step of lysis 3°. F u r t h e r m o r e , the single-celt t e c h n i q u e has e n a b l e d the d e m o n s t r a tion that i n t e r f e r o n - i n d u c e d a u g m e n t a t i o n of N K - c e l l cytotoxicity results from activation of non-cytotoxic p r e - N K cells whose kinetics of lysis is increased. T h e s e findings could not have b e e n revealed by the 51Crrelease assay 26. A n o t h e r a p p l i c a t i o n of the single-cell t e c h n i q u e is the d e m o n s t r a t i o n that a single C T L can kill one type of target specifically a n d a lectin-coated target non-specifically w h e n b o u n d s i m u l t a n e o u s l y to a target of each type 24. Conclusions W e have p r e s e n t e d a general overview of m e t h o d s used to m e a s u r e C M C . W h e n confronted w i t h a p a r t i c u l a r problem, it is often difficult to decide on the m e t h o d of choice. T h i s decision will be b a s e d on the n a t u r e of the cytotoxic reaction, the n a t u r e of the target, and the q u e s t i o n to be resolved. In T a b l e III, we have s u m m a r i z e d several of the p a r a m e t e r s m e a s u r e d and limitations e n c o u n t e r e d w i t h present t e c h n i q u e s in C M C . T h e recently i n t r o d u c e d m e t h o d s for e n u m e r a t i n g the frequency of cytotoxic effector cells by single-cell t e c h n i q u e s have o p e n e d new avenues in the investigation of C M C w h i c h will u n d o u b t e d l y b e a r fruit in the next few years. Work in this laboratory has been supported by grants from the National Cancer Institute, DHEW.

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