assessment of cell-mediated cytotoxicity

104

not over-react to trivial stimuli, bu 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.

R e f e r e n c e s 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) Gastroenterology 75,129-138 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

immunology today, 2¢ovember 1980

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) Immunology 37,477-484 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) Immunology 39, 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 Absorplion by 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.

The role of lymphocytes in in teract ions with target cells which lead to target lysis was not established unt i l the early 1960s. Rosenau an d M o o n 2 were the first to demons t ra te the lysis of homologous cells by sensitized lymphocytes in an in vitro tissue cul ture system. Lymphocytes from B A L B / c mice sensitized against L cells from an allogeneic mouse (C3H), induced a str iking cytopathic change when co- cul tured with L target-cells. These changes occurred in the absence of an t ibody and complement . Close contact be tween the sensitized lymphocytes an d the target cells was found to be essential for the cytotoxic react ion to occur. In the twenty years since this demonstration of cell-mediated cytotoxicity (CMC), progress in the direct measu remen t of C M C has been slow, possibly because of the complex na tu re of the © Elsevier/North-Holland Biomedical Press 1980

react ion and the difficulty in assessing target-cell destruction.

In order to make a reasonable assessment of a cytotoxic react ion with a par t icu la r assay, one should know: (1) the development, differentiation, and fate of the cytotoxic cell, (2) the exact na tu re of the effector cell involved, (3) whether this effector acts alone or in cooperat ion with other cells, (4) the biological state of the effector cell before and after in te rac t ion with target-cells, (5) the molecular m e c h a n i s m of the cytotoxic react ion and (6) the effect of the target on the cytotoxic celt. Fur the rmore the assay should provide a measure of the absolute f requency of cytotoxic cells present in a mixed popula t ion , a measure of the affinity and avidity of the effector cells to correspond- ing target-cells and a means to quan t i t a t e the cyto-

immunology today, November 1980

MONOLAYERS OF TUMOR TARGET CELLS

0 0 0 0 0 0 IMMUNE LYMPHOCYTES

ADDED

~:, Incubate 2 -3 days Fix and stain

Count adherent cells

© © © 0 @ 0 DETACHMENT AND LYSIS NO DETACHMENT OR LYSIS

NORMAL LYMPHOCYTES ADDED

Fig. 1 Assessment of cell-mediated cytotoxiclty by enumera- tion 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) enumerat ion of residual target-cells after incubation with effector cells, (2) examinat ion of target-cell lysis by radioisotope labelling of target cells and (3) microscopic viewing of single cytotoxic effector cells bound to targets. We will discuss the potential applicat ions of these methods and their l imitations 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 ce l l s after i n t e r a c t i o n w i t h ef fector ce l l s (Fig. 1)

Two methods which detect C M C by cell counting are the colony inhibit ion assay modified by Hells tr6m in 19673 and the microcytotoxicity assay of Tagasuki and Klein 4. In the colony inhibit ion assay, target cells (1-2 x 103 ) are added to plastic petri dishes and allowed to grow and become at tached to the dish. 12-24 h later, effector cells or control cells are added to the monolayers, the dishes are further incubated for 3-4 days . and 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 abil i ty of effector cells to reduce the number 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 'microcytotoxici ty ' 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. Then effector cells are added to the target cells and incubated at 37°C for 2 days. At the

105

conclusion of the assay, the wells are washed twice and living cells which remain at tached to the floor of the wells are fixed in methanol and stained with Giemsa. The number of remaining cells is scored with a bottom-viewing microscope. The percentage reduc- tion 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 incubat ion 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 in- vitro sensitization of effector cells may take place. The interpretat ion of results is complex when the populations of effector cells are not pure: several different effector cells may be present and various cell-cell interactions might occur in a synergistic and antag- onistic 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 non- viable 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 -ce l l s (Fig. 2) A technique for radioisotope labelling of target-cells

has several requirements: the isotope should be incor- porated into targets in amounts sufficient to label without inducing death; the half-life of a part icular isotope should be such that radioactivity can be counted within a convenient t ime period; the isotope, once released or degraded, should not be re-utilized;

PRE-INCUBATION LABEL LABEL TARGET:

(5]Cr, 1251UdR)

~A/CUBA TIOA/

POST-INCUBATION LABEL UNLABELED TARGET

LABEL TARGET: (e6Rb, 1251UdR, 14C_LE U

3 H-PRO, 3 H-THYMIDINE)

RADIOACTIVITY COUNTED

I TARGET TARGET I

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

106

Techniques commonly used to measure CMC.

Technique Measurement Refs

Target-cell count Colony inhibition

Microcytotoxicity Radiolabelling of targets

51Cr ~2sIUdR

3H-proline 3H-thymidine 86Rb 14C-leucine

Single-cell assay

Percent of tumor targets 3 remaining that form colonies

Viable target cells remaining 4

Radioactivity released 6 Radioactivity released 8

or retained Radioactivity retained 9 Radioactivity retained 10 Radioactivity retained 11 Radioactivity retained 12 Killed target cells bound to 15,16

a cytotox!c cell 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 target- cells 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) quanti tat ion of the cytotoxic reaction is not always possible; each target- cell 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. In these assays, target-cells adherent to the bottom of plastic wells are labelled with ~25IUdR8, 3H- proline 9, or 3H-thymidinel0. After incubation with effector cells, the radioactivity remaining in the target- cells 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 number of targets can be used, as in the microcytotoxicity test, (c) radioact ivi ty can be

TABLE II. Frequency of cytotoxic cells determined by single-cell techniques

Cytotoxic Frequency of cell Effector populations Species cytotoxic cells °70 Refs

T lymphocyte Allosensitized peritoneal Mouse 20-50 15, l 6,7,22 lymphocytes (in vivo)

Allosensitized spleen cells (in vivo) Mouse 7-13 7 Allosensitized spleen cells (in vitro) Mouse 3-17 7 Lines cloned using TCGF a Mouse 10-25 Bonavida, unpublished Nylon-wool-purified spleen cells Mouse 20-30 25 Peripheral-blood lymphocytes Man 3 31 Peripheral-blood lymphocytes Man 2-3 27 Allosensitized peritoneal Mouse 20-50 24

lymphocytes (in vivo) Peripheral blood lymphocytes Man 3-5

NK cell

ADCC ~ effector cell LDCC ~ effector cell

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

TCGF: T-cell growth factor; ADCC: antibody-dependent cellular cytotoxicity; LDCC: lectin-dependent cellular cytotoxicity (in which lectins (concanavalin A or phytohemagglutinin) induce nonspecific cytolysis of syngeneic or xenogeneic targets by alloimmune lymphocytes (mouse) or normal blood lymphocytes (man))


counted as a measure of cytotoxicity, which is prefer- able to the tedious counting method of the microcytotoxicity test, and (d) there has been a good correlat ion 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. They are based on the fact that only viable targets will take up isotope-labelled essentials like the potassium analog rubid ium (86Rb)ll, amino acids (14C-leucine)12, or nucleosides (3H-thymidine)l°. After the target cells have formed a confluent monolayer in microti ter plates effector lymphocytes are added to each well and the plates are incubated for 48 h. The plates are washed several times, isotope is added to each well, the plates are washed again, and detergent is added to each well. The contents are removed and counted in a liquid scintillation spectrometer (for emitters of beta radiation) or in a gamma 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 number of targets is needed. The l imitations of this type of assay include the fact that lymphocytes which remain at tached 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 radiolabell ing techniques discussed, one can only compare the lytic activity of one lymphoid celt populat ion with another. It is difficult to discern the frequency of single cytotoxic effectors in a given population. Several factors have hampered this direct enumerat ion of cytotoxic cells. One is the abil i ty 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, Thorn and Henney 13 a t tempted to dem- onstrate the frequency of cytotoxic cells in a lymphoid populat ion by an elegant, albeit indirect means. Lymphocytes were allowed to interact with 51Cr- labelled 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 v o~ + . . . . +° + . . . . v.i~. +v.ev w e r e .q!rff.qdy 1¢ - - t - t ¢ l ~ ' a I l l V W t l l t I t I t l •

bound. 1~ this . . . . . . ~: . . . . . . . . : - : - - : - - ~ *~-- 1 1 1 l ! ! O ! ! ! ! l - ! } ! E L y L ! ! ! ! ~ W d ~ ! ! [ l ] [ I I [ ! ! L r 2 " _ I t , r ~ } /

this technique it was est imated that at the peak of the cytotoxic response, 2.2% of spleen cells were cytotoxic

107

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 enumera- tion of cytotoxic cells was indirect, multi- target conjugates (a lymphocyte which had bound more than one target) were not accounted for, and all potential ly cytotoxic lymphocytes might not have had the opportunity to interact with available target cells. These factors might all have contr ibuted to an underest imation of the frequency of cytotoxic cells in the lymphoic cell population.

Enumerat ion 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 populat ion of effector cells from target death. An alternative approach is to directly evaluate the effeetor cells, abil i ty first to bind to a par t icular target cell and then to lyse it.

There were originally two methods of viewing effec- to t - ta rge t interactions microscopically. Thus, in studies aimed at delineating the various steps involved in target cell lysis by C T L TM, Mar tz reported the formation of clusters in which one CTL bound to one target. Berke e/al. also reported the formation of stable lymphocyte-target cell conjugates and established a correlation between the number of conjugates and the number of killers with the 51Cr-release assay 15. With single-cell manipula t ion techniques, Zagury et al. sub-

EFFECTOR O O O O O O

CONJUGATES

/14/CR OMAN/P U- LATION

OR DISPERSE IN

HEMOCYTOMETER

CELLS + TARGET CELLS

INCUBATE 50°C

C EN FRIFUGE

PELLET RESUSPENDED

FORMED:

PLATE ONTO SLIDES IN AGAROSE

INCUBATE 57°C

STAIN IN TRYPAN BLUE

FIX IN FORMALDEHYDE

CONOUGATES VIEWED MICROSCOPICALLY

@fT / ENUMERATE FREQUENCY 0[- CO;'4JUG,&TKS

W! I H L)F AD TARGETS

Fig. 3 Single cytotoxic effector cell assay,

108

sequent ly repor ted that most of the lymphocytes in conjugates were killers 16. We then presented a p laque t echn ique for the enumera t ion of cytotoxic effector cells 17. In this assay, targets were first fixed on a poly- L- lys ine-coa ted plastic dish, and effector cells were then added to the confluent target-cel l monolayer . After incubat ion, the plates were t rea ted with eosin to stain dead cells, and zones of more than four dead targets ( 'p laques ' ) were viewed under the microscope and enumera ted . Whi le this t echn ique yie lded fre- quencies of cytotoxic cells which were m u c h lower than those from assays using ef fec tor- target con- juga tes (Tab le II), it did provide a m e a s u r e m e n t of cytotoxic T lymphocytes wi th p re sumab ly high affinity. T h e p laque technique ' s l imi ta t ion has been its inabi l i ty to measure single target-cel l death. It m a y nevertheless be useful in direct enumera t i on of killer cells when the target cells are more than 95% viable and adherent . T h e p laque t echn ique is par t icular ly appl icable in studies of an t ibody-dependen t cell- med ia t ed cytotoxici ty wi th red blood cells as targets, in which a zone of lysis is easily d iscerned 18.

Whi le the t echn ique of single cell man ipu la t ion repor ted by Zagury et al. I6,19 was feasible and repro- duced by us 2°, it was tedious and difficult to apply in rout ine examina t i on of killer-cell f requency. Fishelson and Berke 21 repor ted that ki l ler- target conjugates could be viewed in a hemocy tome te r and used in the detect ion of C T L but this t echn ique is l imited in its uses and p ro longed incuba t ion in the h e m o c y t o m e t e r at 37°C m a y be subopt imal for survival of conjugates.

We have modif ied the techniques above by using agarose to immobi l ize ef fec tor- target conjugates formed at 30°C, at which binding, but not lysis, can Occur 23'24. T h e agarose, when gelled, does not h a r m

TABLE III. Comparison among various cytotoxic assays

immunology today, November/980

the cells and prevents effector cells f rom moving away from the a t t ached target-cell . T h e viabil i ty of the e f fec to r -bound target can be assessed microscopica l ly wi th vital cell dyes after incuba t ion at 37°C.

T h e single-cell assay, in general , involves two steps (Fig. 3). First the ef fec tor - ta rge t conjugates are formed in small glass cul ture tubes under slow centri- fugat ion after a 5 rain incuba t ion at 30°C and the pellet is gent ly resuspended. At this point , conjugates can be m i c r o m a n i p u l a t e d into Terasak i wells I9 or a hemacy tome te r 21, or mixed wi th agarose (30°C) and pla ted on to slides = . After incuba t ion for various t imes target-cel l dea th is seen e i ther wi th t rypan blue uptake 23 or by loss of dens i ty wi th phase-cont ras t microscopy 19.

Al though the single-cell assays were or iginal ly developed to measure C T L , they have been adap ted to quan t i t a te N K cells in the mouse 25 and m a n 26, as well as an t ibody-dependen t cytotoxic cells 27 (Table II). T h e single cell assay offers several advantages over existing cytotoxic assays :

(1) It is simple, cheap, rapid, reproducible , and highly sensitive.

(2) It provides an es t imate of the n u m b e r of cytotoxic cells present in the popu la t ion (Table II), and also the heterogenei ty in the cytotoxic potent ia l of the effector cells 7, a l though it does unde res t ima te their frequency. It is a ssumed that all effectors with cytotoxic potent ia l b ind to targets but, as S h o r t m a n and Gols te in report 28, the m e t h o d of resuspension un- doubtedly disrupts some potent ia l conjugates that might otherwise lead to lysis, so that some cytotoxic effector cells are missed.

(3) The pheno type of the effector cell can be determined at the single cell level 24.

Cell counting Radioisotope labelling Single effector cell Assay features Colony Microcyto- Short- Long- Plaque Micromani- Lymphocyte-target

inhibition toxicity term term pulation conjugates (Reference) (3) (4) (6) (8,9,10) (17) (16) (7,21,24)

Properties Duration (h) a 96 72 3-18 96 4 3-18 3-18 Reproducible hi b hi v.hi hi 1o hi/lo hi Cost (hi/lo) c lo lo hi hi 1o hi v.lo Number cells needed lo v.lo hi lo 1o lo 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 + / - + - - + / - -

a Duration 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 109

(4) Ta rge t cells can be marked with f luorescent dyes so that they can be easily dis t inguished f rom the effector cells 7. T h e assay is thus not l imi ted to targets physiological ly different f rom the effector cell.

(5) Besides t umor cells in suspension, adheren t t umor lines, blast cells, f ibroblasts and no rma l splen- ocytes can all be used as targets.

(6) Aspects of the m e c h a n i s m of cytotoxici ty can be direct ly examined. For example , by blocking at various points in the lytic pa thway (pre- target b ind ing v. post - target b inding) we might de te rmine what s t ructures are involved in lysis 29 or metabol ic requirements are needed for each step of lysis 3°. Fur the rmore , the single-celt t echn ique has enabled the demons t ra - t ion that in ter feron- induced augmen ta t i on of NK-ce l l cytotoxici ty results f rom act ivat ion of non-cytotoxic p r e - N K cells whose kinetics of lysis is increased. These findings could not have been revealed by the 51Cr- release assay 26. Ano the r appl ica t ion of the single-cell t echn ique is the demons t r a t ion that a single C T L can kill one type of target specifically and a lec t in-coated target non-specif ical ly when bound s imul taneous ly to a target of each type 24.

C o n c l u s i o n s We have presen ted a general overview of me thods

used to measure C M C . W h e n confronted wi th a par t icu la r problem, it is often difficult to decide on the m e t h o d of choice. Th is decis ion will be based on the na ture of the cytotoxic reaction, the na tu re of the target , and the ques t ion to be resolved. In T a b l e III, we have summar i zed several of the pa ramete r s measu red and l imitat ions encounte red wi th present t echniques in C M C . T h e recent ly in t roduced methods for e n u m e r a t i n g the f requency of cytotoxic effector cells by single-cell t echniques have opened new avenues in the invest igat ion of C M C which will undoub ted ly bear fruit in the next few years.

Work in this laboratory has been supported by grants from the National Cancer Institute, DHEW.

References 1 Bloom, B. R. andJ. R. David (ed) (1976), In Vitro Methods in

Cell Mediated and Tumor Immunity, Academic Press 2 Rosenau, W. and H. D. Moon (1961) JNCI 27,471-477 3 Hellstrom, J. (1967) Int. J. Cancer2, 65-68 4 Takasugi, M. and E. Klein (1970) Transplantation 9, 219-

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5 Takasugi, M., M. R. Mickey and P. Terasaki (1973) Natl. Cancer Inst. Monograph 37, 77-84

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8 Cohen, A. M., J. F. Burdick and A. S. Ketchaum (1971) J. Immunol. 107, 895-898

9 Bean, M. A., H. Rees, G. Rosen and H. F. Oettgen (1973) Natl. Cancer Inst. Monograph 37, 41-48

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13 Thorn, R. M. and C. S. Henney (1976) Nature (London) 262, 75-77

14 Martz, E. (i975)J. hnmunol. 115, 261-267 15 Berke, G., D. Gabeson and M. Feldman (1975) Europ. J.

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2861-2869 24 Bradley, T. P. and B. Bonavida (1981)). Irnmunol. 127 (in

press)

25 Roder, J. C., R. Kiessling, P. Biberfield and B. Anderson (1978) J. lmmunol. 121, 2509-2516

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27 Neville, M., E. A. Grimm and B. Bonavida (1980)J. lmmunol. Methods (in press)

28 Shortman, K. and P. Golstein (1979) J. lmmunol. 123, 833- 839

29 Fan, J., A. Ahmed and B. Bonavida (1980)J. lmmunol 126, (in Press)

30 Martz, E. (1977) Contemp. Top. Immunol. Vol. 7 (Stutman, O., ed.) Plenum Press, N.Y. 301-361

31 Targan, S., E. A. Grimm and B. Bonavida (1980) Clinical and Laboratory Immunol. (in press)

assessment of cell-mediated cytotoxicity

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