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References 1 Ishizaka, K. (1976)Adv. ImmunoL 23, 1-75 2 Levine, B. B. and Vaz, N. M. (1970)Int. Arch. Allergy. Appl. Irnmunol. 39, 156-171 3 Katz, D. H. (1980) Immunology 41, 1-24 4 Jarrett, E. E. E. and Stewart, D. C. (1972) Immuno/ogy23, 749-755 5 Ishizaka, K., Seumura, M., Yodoi, J. and Hirashima, M. (1981) Fed. Proc. Fed. Am. Soc. Eap. BioL 40, 2162-2166 6 Gonzalez~Molina, A. and Spiegel, H. L. (1977)J. Clin. Invest. 59, 616-624 7 Fritsche, R. and Spiegelberg, H. L. (1978)J. Immunol. 121,471-478 8 Yodoi, J. and Ishizaka, K. (1980)J. Immunol. 124, 1322-1329 9 Yodoi, J , Hirashima, M. and Ishizaka, K. (1981)J. Immunol. 126, 877-882 10 Spiegelberg, H. L., O'Connor, R. D., Simon, R. A. and Mathison, D. A. (1979)J. Clin. Invest. 64, 714-720 11 Yodoi, J,, Hirashama, M. and Ishizaka, K. (1981) J. ImmunoL 127, 471-475 12 Hirata, F., Sehiffmann, E., Verkatasurbramanian, K., Solomon, D. and Axelrod, J. (1980)Proc. NatlAcad. Sa'. USA 77, 2533-2536

13 Yodoi, J., Hirashima, M., Hirata, F., DeBlas, A. L. and Ishizaka, K. (1981)J. ImmunoL 127,476-482 14 Hirashima, M., Yodoi, J. and Ishizaka, K. (1981)J. Immunol. 126, 838-842 15 Hirashima, M., Yodoi, J. and Ishizaka, K. (1980)J. lmmunol. 125, 2154-2160 16 Hirashima, M., Yodoi, J., Huff, T. and Ishizaka, K. (1981)J. Immunol. 127, 1810-1827 17 Hirashima, M., Yodoi, J. and Ishizaka, K. (1981)J. Immunol. 127, 1804-1809 18 Yodoi, J., Hirashima, M and Ishizaka, K. (1981)d~ Immunol. 127, 1579-1585 19 Chen, S. S., Bohn, J. W., Liu, F. T. and Katz, D. H. (1981) J. ImmunoL 127, 166-173 20 Kishimoto, T., Hirai, Y., Suemura, M. and Yamamura, Y. (1976)J. Imraunol. 117, 396--404 21 Suemura, M., Kishimoto, T., Hirai, Y. and Yamamura, Y. (1977)J. lmmunoL 119, 149-155 22 Kishimoto, T., Hirai, Y., Suemura, M., Nakanishi, K. and Yamamura, Y. (1978)J. Imnmnol. 121, 2106-2112 23 Hirashima, M., Uede, T,, Huff, T. and Ishizaka, K. (1982)J. Immunol. 128, 1909-1916 24 Ishizaka, K. and Sandberg, K. (1981)J. Immunol. 126, 1692-1696

The single-cell assay in cell-mediated cytotoxicity Benjamin Bonavida, Thomas P. Bradley and Elizabeth A. Grimm Studies on the humoral antibody response weregreatly advanced by the introduction in 1963 of theJerne plaque assay which permitted direct enumeration of individual antibody-producingplasma cel~. Studies of cellular cytotoxicity were limited until recently to the examination of whole populations of oCfectorcells2. Here, Benjamin Bonavida and his colleagues describean assay in agarose which allows the study of single cytotoxiccellf1-5. Cytotoxic T lymphocytes (CTL) must bind to target cells before lysis occurs; cytotoxic cells thus adhere to targetcell monolayersn. Our first attempt to demonstrate killing at the single-cell level took advantage of this observation. When C T L were dispersed on the surface of monolayers and incubated, zones or plaques oflysis in the monolayers were obtained. This method provided only a minimal estimate of the frequency of killers since plaque formation required an effector cell to kill four or more targets in a short-term assay7. Berke et aL 8 and Martz ° demonstrated the binding of effector to target cells in suspension. Lymphocyte-target-cell interactions were studied in liquid medium in a hemocytometer~° or in microwells after micromanipulation", and lysis was determined by the loss of target-cell optical refractiveness. The limitations of these techniques gave rise to the idea of immobilizing the effector-target conjugates which were a prerequisite for target killing, by diluting them in agarose and then incubating them to permit target-cell lysis. After staining, the dead target cells are counted microscopically. Each stained (dead) conjugate target cell indicates that the adjacent lymphocyte was indeed a cytolytic cell. Thus, the frequency of killer cells can be easily estimated t2,'3. The single-cell assay complements previous indirect methods used in measuring cytotoxicity (for review see Benjamin Bonavida and Thomas P. Bradley are in the Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024, USA; Elizabeth A. Grimm is in the Surgery Branch, National Cancer Instittxte, National Institutes of Health, Bethesda, MD 20205, USA.

Ref. 2) by permitting direct examination of the cellular characterisitcs and lytic mechanisms of single effector cells. It was first used to study alloimmune cytotoxic T lymphocytes (CTL) 3 but immediately applied by us and others to other cytotoxic ceils, including natural killer cells (NK) 14'16, antibody-dependent killer cells (K cells in ADCC) 17,1s, and cytotoxic macrophages 19. The assay is applicable in many species such as man, mice, rats, frogs, rabbits and lampreys. It is discussed in detail in references indicated in Table I and applications of the single-cell assay are outlined in Table II.

Enumeration and characterization of cytotoxic cells Frequency of effector cells which bind to targets All single-cell assays rely on the requirement ofeffector cells to interact with and bind to target cells. Centrifugation facilitates binding. The frequencies of effector cells which bind to target cells in several systems are summarized in Table III. In most systems there is some nonspecific binding. Neither its mechanism nor functional relevance are known but in most circumstances binding seems to be related to recognition by the effector cell, and thus a high frequency of specific binding above background level is observed. The recognition event in CTL-mediated lysis is antigen-specific, suggesting that binding is the result of interaction between antigen receptors of T cells and target antigens. Binding in other effectors probably involves different receptors. For instance, mouse C T L recognize antigens primarily encoded in the K and D regions of the M H C but mouse N K cells recognize an entity common to ©1983, ElsevierSciencePublishersB.V.,Amsterdam 0167- 4919/03/$01.00

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TABLE I. Single-cellassays used in binding and cytotoxicity Method Clusters of CTL-bound targets in suspension Conjugates of CTL-bound targets in suspension Cytolytic plaque assay Conjugates in agarose Single target-single effector

Double target-single effector

Single target-multiple effector

System

References

CTL CTL CTL

8,10,11 7

CTL 2-5, 13, 26 LDCC, ODCC 5, 13, 28 NK 13-16, 18, 28 K 13, 16, 17 M~ 19 CTL 5, 13 LDCC, ODCC 5, 13, 28 NK 13, 28, 29 ADCC 13, 29 CTL E. A Grimm eta/., unpubl. obs.

TCGF: T-cell growth factor; ADCC: antibody-dependent cellular cytotoxicity; LDCC: lectin-dependent cellular cytotoxicity [in which lectins (concanavalin A or phytohemagglutinin) induce non-specific cytolysis of syngeneic or xenogeneic targets by alloimmune lymphocytes(mouse) or normal blood lymphocytes(man)I; ODCC: oxidativedependent cytotoxicityin which targets are modified by periodate or neuraminidase and galactose oxidase.

define the effector cell surface phenotype include ferritinlabeled antibodies directed against surface components of choice 25. Electron microscopy has also been used to view u n i q u e m e m b r a n e configurations that might reveal ultrastructured aspects of the lytic reaction 2s.

Preselection of conjugatesfor analysis A n adaptation of the single-cell assay in agarose involves the uses o f a microlocater grid to select a particular conjugate for viewing throughout the various lytic post-binding stage¢. After being immobilized but before incubation, the location of a conjugate on a grid matrix is recorded, so that the conjugate can be relocated in order to view the lytic process. Relocation is also a n advantage when studying effector cells combined with two morphologically similar types of target cell (one being stained with fluorescein dye before incubation) a n d effector cells morphologically indistinguishable from target cells, the latter being fluorescein-dyed. After incubation, the conjugates of interest are relocated a n d the identity of lysed targets assessed by trypan blue dye exclusion. It m a y also be possible to mechanically isolate prelocated killer cells for further study.

Killing of more than one type of target by a single effector cell all N K targets and distinct from the K- a n d D-encoded structures 2°. A D C C requires a n interaction between a receptor o n the effector cell and the Fc region of antibodycoated target cells21. T h e molecular n a t u r e of these various receptors and how they signal the effector cell for lysis is u n k n o w n but b i n d i n g is a requirement for lysis. Since the b i n d i n g frequency can be altered by various factors, the frequency of binders in a cytotoxic system against a particular target m a y have biological and clinical significance. Thus, a correlation m a y be made between the frequency of binders a n d the frequency of killers u n d e r specific conditions.

Frequency of cytolytic effector cells which lyse bound target cells First attempts to determine the frequency of killer cells within a cell population using a 51Cr-release assay and describing lysis in terms of enzyme-snbstrate interactions 22, were inconsistent a n d required m a n y assumptions, m a i n l y that a single effector cell can interact and bind with only one target cell a n d that the rate of lysis by individual ceils is constant23. F r o m the single-cell assay, by direct measurement, we know that effector cells can recycle a n d that ceils are inherently heterogeneous in their lytic efficiency3'4. Target-ceil death is detected either by dye exclusions or by the loss of optical refractiveness '1. These methods corrrespond well to other measures of cell death, such as alteration in cell permeability, detachment from monolayers, failure of division and release of radioactivity2L

SuoCacephenotype of individual ~fector cells The surface phenotype of the effector or target cells in a conjugate can be easily discerned by indirect irnmunofluorescence. In this way mouse cytotoxic T lymphocytes b o u n d simultaneously to both specific a n d non-specific targets were found to be Lyt 2 ÷ (Ref. 5). O t h e r probes to

Is a single effector cell restricted in its specificity to killing only a single type of target cell? This question can be addressed with the single-cell assay. Z a g u r y et aL have shown that multiple targets b o u n d to a single effector cell are killed at different times sequentially2L I n addition, a single C T L kills two simultaneously b o u n d non-identical targets when one target displays the sensitizing antigen a n d the other target is syngeneic to the effector (i.e. not recognized by it) a n d coated with lectin5. This two-target conjugate technique (described above) can also be used to delineate subpopulations within the i m m u n e system. Thus, in m a n , cytotoxic cells able to kill NK-sensitive targets were shown to be distinct from the cells that kill lectin-coated targets ( L D C C ) 2s, while some N K cells could kill antibody-coated, NK-resistant targets 29.

Competition for binding Specificity in cytotoxicity, when assessed by testing the lytic potential of a n effector population against a battery of target cells, does not establish whether the effectors are a mixed or single population. L a n d a z u r i a n d Herberm a n ~°have developed a cold-target cell competition assay using 51Cr release in which unlabeled target cells are added to a constant n u m b e r of labeled target cells and TABLE II. Applications of the single-cellcytotoxicitytechnique 1. Establishment of frequency of cells which bind to target ceils 2. Establishment of frequency of the cytotoxic cells among binders 3. Determination of surface phenotype of the effector or target cells by immunofluorescence 4. Determination of the effector-celland target-cell type specificities using the two-target conjugate assay 5. Investigation of the mechanism of lysis at distinct stages in the lytic process 6. Determination of effector-cellcytotoxic efficiencyand activation 7. Ability ofcytotoxic cells to recycle 8. Application in clinicallyrelated studies

Immunology Today, vol. 4, No. 7, 1983

198 TABLE IIL Frequency of binders and killers Cytotoxic cell T lymphocyte

ADCC a effector LDCC a and ODCC effector

Effector populations Allosensitized peritoneal lymphocytes (in vivo) Allosensitized spleen cells (in vivo) Allosensitized spleen cells (in vitro) Lines cloned using TCGFa Nylon-wool-purified spleen cells Peripheral-blood lymphocytes Large granular lymphocytes Large granular lymphocytes Peripheral-blood lymphocytes Large granular lymphocytes AUosensitized peritoneal lymphocytes (in vivo)

Species Mouse Mouse Mouse Mouse Mouse Man Man Rat Man Man Mouse

% Conjugates b 20-50 15-25 3-10 10-20 20-40 8-15 30-50

% Killersc 20-50 7-13

8-15 30-50 20-50

2-3 20-40

Peripheral-blood lymphocytes

Man

20-35

8-15

3-17

10-25 20-30 3 20-40

20-50

References 3, 5 3 3

3 14, 28, 29 28, 39 40 17, 29 29, 39 5, 13 13, 28

• For abbreviations see Table I. b % Effectors which bind to targets. c % Target-bound effectors which kill attached target. cytotoxicity is measured. If the non-labeled target cells are antigenically related, they compete with the labeled target cells and there is less killing. In an analogous system using single cells, Berke et al. 31 have reported competition at the level of conjugate formation. T h e target cell is labeled

with fluorescein and the inhibiting targets are unlabeled. If there is competition then fluorescent conjugates are fewer. T h e frequency of both antigenically specific and non-specific cells can be examined, as well as the specificity of target antigens.

Cell enrichment C T L can be enriched by first binding them to allogeneic target cells and layering the suspension on a discontinuous fetal calf serum gradient. Conjugated lymphocytes separate from free targets because of the difference in size 12 and the bottom layer contains up to 90% conjugated lymphocytes, an average of 70% of these lysing their attached target. T h e lymphocytes b o u n d to targets can be dissociated and separated from the target cells, thus enabling enrichment ofcytotoxic cells.

Sensitivity of assay In conventional whole-population assays for cytotoxicity a large n u m b e r of cytotoxic cells must be present in the effector population for reasonable chance of detecting cytotoxicity. In the single-cell assay, however, it is theoretically possible to detect a single killer cell from a mixed population of cells, and so a m u c h smaller n u m b e r of cells (< 1 000) is needed 12. Studies o n the m e c h a n i s m of c e l l - m e d i a t e d cytotoxicity

Evaluation o/distinct stages of the lyric process

Fig. 1. Two NK conjugates.

The conjugates (consisting of human peripheral-blood lymphocytes bound to the NK-sensitive targets K562) have been immobilized in agarose, plated onto a glass slide and used in the single-ceUassay. After an incubation for three hours at 37°C, the slide is stained in trypan blue and fixed in formaldehyde. The upper conjugate is an example of a target which has been lysed by the attached lymphocyte (note that the target has been stained blue and has lost refractivity). The lower conjugate consists of a viable target which has not been lysed by the bound lymphocyte (note the high degree of refractivity of the viable target).

Although the m e c h a n i s m underlying cell-mediated cytotoxicity is not known, it is proposed that various stages lead to the cytolytic event. M a r t z has shown that binding, p r o g r a m m i n g for lysis, and a lethal hit stage are distinct ~2. Agents which block binding can be uniquely evaluated with the single-cell assay. W e have investigated the blocking of cytolysis by antibody as a probe to delineate the steps involved in lysis. For example, anti-Lyt 2 antibody and R A T * (rat anti-murine activated T cells) antibody block cell-mediated cytotoxicity in the absence of complement. T h e single-cell assay showed that antiLyt 2 antibodies block effector-target interaction 33 while R A T * blocks cytotoxicity post-binding0~.35. These antisera presumably recognize different molecules on the ef-

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lector cells, each of which m a y be involved in the mechanism o f l y s i ~ . Kinetics of lysis After b i n d i n g to a target, some C T L take longer than others to kill itL The rate of killing by cytotoxic cells is not constant a n d cells at different states of differentiation or m a t u r a t i o n kill at different rates. For example, single-cell techniques have shown that C T L generated in a secondary response are faster killers than their counterparts in the p r i m a r y response3'L The single-cell assay allows examination of agents which enhance cytotoxicity. In the N K system treatment of the effector cells by interferon did not alter the n u m b e r of b i n d i n g cells but raised the frequency of killer cells a n d e n h a n c e d the kinetics of lysis, suggesting that the rate of killing was increased a n d that some cells which b i n d and do not kill (non-lytic, p r e - N K cells) can be triggered by interferon to kill. L ysis of single targets bound by multiple effectors In cultures containing mouse C T L , multiply b o u n d targets can be seen if the effector/target cell ratio is increased b u t the total n u m b e r of b i n d i n g lymphocytes is lower t h a n the n u m b e r observed at the standard 1/ 1 ratio, indicating that b i n d i n g avidity is heterogeneous. W h e n multiply b o u n d targets were incubated in the single-cell assay, and the kinetics of lysis followed, we found that targets b o u n d by one, two or three lymphocytes lysed at rates of 1.3 _ 0.1, 1.9 _ 0.2 a n d 3.3 _ 0.4 per hour, respectively. I n all cases, the lysis followed firstorder e n z y m e kinetics, sugesting that when simultaneously b o u n d to the target, the effector cells operated i n d e p e n d e n d y , with the fastest killer i n d u c i n g lysis. These data suggest a n association between b i n d i n g avidity a n d lytic efficiency (E. A. G r i m m , L. Roos a n d B. Bonavida, unpublished observations). Recycling of effector cells to kill more than a single target The single-cell technique has established that a cell which has killed one target can recycle to kill others. Zagury et al. showed by micromanipulation that single effector cells can kill a n u m b e r of targets in sequence 11, a n d M a r t z 32 earlier detected recycling in a mixed population of effector ceils when targets were added in sequence. The single-cell assay analyses the recycling of individual cells within the mixed effector-cell population. T h e fact that physical m o v e m e n t a n d hence recycling is prevented in the agarose single-cell assay explains why killing in this assay sometimes does not correlate with killing in 51Cr-release assays where recycling occurs. Nature of the lytic lesion and the delivery of the lethal hit Time-lapse cinematography of C T L - t a r g e t cell conjugates d u r i n g the single-cell assay a n d observations of the entire lytic process at the light-microscope level37 showed that the b i n d i n g of effector to target cells was the initial event, b u t continued attachment of the lymphocyte to the target was not necessary for lysis to progress. This initial attachment corresponds directly to the steps termed ' p r o g r a m m i n g for lysis' by Martz ~2. T h e stage of target-

cell death varied in time, from 15 minutes to as long as several hours. D u r i n g death, the target cell undergoes a u n i q u e process of swelling a n d stretching, ('zeosis') which differs in appearance from the killing induced by antibody a n d complement. By electron microscopy a n u m b e r of poorly defined junction-like structures bridging the connection between C T L a n d target cell were observed 26. Such structures might provide a conduit for transfer of lytic molecules or they could be responsible for the very strong b i n d i n g necessary to damage a target cell b y a shearing force. No obvious secretory apparatus was visible at the j u n c t i o n region. Clinical applications T h e i m m u n e system is adversely affected in m a n y diseases and in patients treated with immunosuppressive agents. Conventional cytotoxicity assays cannot indicate the level of defect b u t single-cell assays can determine if the defect is due to: (a) a n absence of cells that can recognize a n d b i n d targets; (b) preservation of b i n d i n g b u t failure oflytic mechanisms; (c) decrease in frequency of killer cells; (d) slowed kinetics of lysis; (e) loss of 'recyclability'. As a n example, N K lytic function in both Chediak-Higashi disease 37 in m a n a n d the beige m u t a n t in mice 3s is deficient, not because b i n d i n g is absent but because the N K cells lack the ability to lyse the b o u n d targets. Changes in immunological status after treatment and studies of ontogeny and phylogeny can also be evaluated with the single-cell assay•

Conclusion T h e single-cell assay is a valuable means of studying different types of cell-mediated cytotoxicity directly a n d allows examination of m a n y aspects of effector function a n d target specificity. In the future, it should be possible to devise computerized i n s t r u m e n t a t i o n able to visualize, record, a n d count b i n d e r a n d killer cells with precision, rapidity and reproducibility.

Acknowledgements Work in this laboratory has been supported by Grants CA12800, CA19753, and CA24314 from the National Cancer Institute, USPHS.

References 1 Jerne, N. K. and Nordin, A. A. (1963) Science, 140, 405-406

2 3 4 5 6 7

Bonavida,B. and Bradley,T. P. (1980)Immunol. Today 5, 104-109 Grimm, E. A. and Bonavida,B. (1979)J. Immunol. 123, 2861-2869 Grimm, E. A. and Bonavida,B. (1979)J. Immunol. 123, 2870-2877 Bradley,T. P. and Bonavida,B. (1981)J. Immunol. 126, 208-213 Berke, G. and Levey, R. (1972)J. Exp. Med. 135,972-984 Bonavida, B., Ikejeri, B. and Kedar, E. (1974) Nature (London) 249, 658-659 8 Be~:ke,G., Gabison, D. and Feldman, M. (1975) Eur. J. Immunol. 5, 813-818 9 Martz, E. (1975)J. Immunol. 115, 261-267 10 Zagury, D., Bernard,J., Thiemess, N., Feldman,M. and Berke, G. (1975) Eur. J. Immunol. 5, 818--822 11 Zagury, D., Bernard,J., Jeannesson, p., Thierness,N. and Duffer,J. (1975) Ann. Immunol. (Paris) 126C, 23 12 Grimm, E. A. and Bonavida,B. (1977)J. lmmunol. 119, 1041-1047 13 Bonavida,B., Bradley, T. P. and Grimm, E. A. Methods Enzymol. (in

press)

14 Silva,A., Bonavida,B. and Targan S. (1980)J. Immunol. 125,479-484 15 Jondal, M. and Merril,J. E. (1981)Eur.J. Immunol. 11,531-535 16 Merril,J. E., Ulberg, M. and Jondal, M. (1981)Eur.J. lmmunol. 11, 535-541

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17 Neville, M., Grimm, E. A. and Bonavida, B. (1980)J. lmmunoL Method. 36, 255-268 18 Targan, S., Grimm, E. A. and Bonavida, B. (1980)J. Clin. Lab. Immunal. 4, 165-168 19 Fischer, D. G., Golightly, M. G. and Koren, H. (1982) in Natural Killer Cells and OtherNatural Effector Cells (Herberman, R. B., ed.), pp. 159-164, Academic Press, New York 20 Roder, J. C., Kiessling, R., Biberfeld, P, and Andersson, B. (1978)J. Immunol. 121, 2509-2517 21 Perlman, P. and Holm, G. (1969) Adv. lmmunoL I1, 117-193 22 Sanderson, C. J. and Taylor, G. A. (1975) Cell Tissue Kiuet. 8, 23-32 23 Thorn, R. M. and Henney, C. S. (1977)J. Immunol. 119, 1973-1977 24 Sullivan, K. A., Berke, G. and Amos, B. (1972) Transplantation 13, 627-628 25 Heitzmann, H. and Richards, F. M. (1974)Proc. NatlAcad. Sci. U.S.A 71, 3537-3541 26 Grimm, E. A., Price, Z. and Bonavida, B. (1979) Celllmmunol. 46, 77-99 27 Zagury, D., Bernard, J., Jeannesson, P., Thiernesse, N. and Cerottini, J.-C. (1979)J. Immunal. 123, 1604-1609 28 Bradley, T. P. and Bonavida, B. (1982) in Natural Killer Cells and Other

The Restless Tide: The Persistent Challenge of the Microbial World by Richard M . Krause, National Foundation for Infectious Diseases, 1981. $ 1 6 . 0 0 (i + 152 pages) Library of Congress No. 82-116089

There is no doubt about the urgent importance of good 'popular science' writing. In a world of half-baked scientific ideas dominated by news values, real science would seem to throw from time to time a flickering light on what m a y in 'this pragmatical pig of a world' be loosely considered truth. I cannot but believe that the ability to distinguish truth from falsehood, which Socrates went on about so, is socially useful, and can be best learnt from the understanding of genuine scientific thought processes. I also believe that it would be desirable for all our masters, from newspaper proprietors and military. dictators to borough council officials and shop stewards, to achieve this ability. Those are the people to w h o m popular science writing should be addressed. Its point is not to demonstrate how clever scientists are, in their remote world of fundamental particles and algebraic formulae and wonder drugs, but to show that there is a continuous thread, and that the imagination is exercised in the same way, from the problems dealt with by Einstein or Crick to those such as how to fell a tree without damaging the rest of the wood, how to locate the source of the 'funny noise' which o n e ' s spouse has complained of in the car engine, or where in the oven to bake a meringue. It is positively bad for non-scientists to be told about science if they do not learn this too. Everyone can think, more or less: science

29 30 31 32 33 34 35 36 37 38 39 40

Natural Effector Cells (Herberrnan, R. B., ed.), pp. 145-152, Academic Press, New York Bradley, T. P. and Bonavida, B.J. ImmunoL (in press) Landazuri, M. O. and Herberman, R. (1972) Nature (London) New Biol. 238, 18-19 Schick, B. and Berke, G. (1979) Transplantation 27, 365-368 Martz, E. (1977) Contemp. Top. lmmunobiol. 7, 245 Fan, J., Ahmed, A. and Bonavida, B. (1980)J. ImmunoL 125, 2444-2453 Hiserodt, J. C. and Bonavida, B. (1981)J. Immunol. 126, 256-262 Effros, R. B., Hiserodt, J. C. and Bonavida, B. (1980)J. l m ~ l . 125, 1879-1884 Wexler, H., Fan. J., Hiserodt, J. C. and Bonavida, B. Cell Immunol. (in press) Klein, M., Roder, J., Haliotis, T., Koreu, S., Jett, J., Herberman, R. B., Katz, P. and Fauci, A. S. (1980)J. Exp. Med. 151, 1049-1058 Roder, J. and Durve, A. (1979) Nature (London) 278, 451--453 Timonen, T., Ortaldo, J. R. and Herbermen, R. B. (1981)J. Exp. Med. 153, 569-582 Reynolds, C. W., Timonen, T. and Herberman, R. B. (1981)J. Immm~l. 127(1), 282-287

is just workaday thinking with a few heuristic rules. Most popular writing about science is purely informative with all the scientific, i.e. critical, thinking simplified out of it; the analytical structure is given but not how it was derived, which is thought to be too 'difficult' for the man-in-thestreet to understand: if you despise your audience you get the audience you deserve. Every schoolteacher knows now that this is bad teaching technique; our schoolchildren are better instructed than the general public. There is also a requirement for a personal voice: as Eric Ashby says 1, the scientific writer should 'convey delight like a kitten playing with a ball of wool ... should tell you a lot about the essayist, his prejudices, his enthusiasms, what he stands f o r . . . ' , qualities which have to be bleached out of technical scientific writing. This ' m a n d a r i n ' style of professional writing is a bad model for the popular science writer, professional though he must be. O n all these criteria I feel that Krause's book does not really hit the jackpot. It is easy reading, it is informa-. tive and urbane, factually sound and convincing. W h y therefore does one have a prevailing sensation of disappointment, of d~3~-vu? Nowhere alas is there an intrusive passion or prejudice, some abrasive opinion to make the reader sit up; anxieties once raised, about overpopulation for example or genetic engineering, are too often soothed by an emollient and woolly optimism - just the subjects upon which public anxieties, however unjustified, need to be taken seriously. T h e r e is also a distressing lack of detail, or detail runs out just where one is getting interested. For example ...'Intensive research is now focused on the m a n n e r in which these genetic factors predispose to the allergic state'. 'Another area of investigation concerns ...' etc. Well, what is that intensive research? It is

not that Krause does not know: apart from being Director of the National Institute of Allergy at Bethesda, he has been personally associated with some of the most elegant research of recent years. It is because the general public must not be exposed to something still speculative and uncertain, poor things. All the best popularizers of science of the m o d e r n period, such as J. B. S. Haldane and Lewis T h o m a s , have not hesitated to deal with the shadowy frontiers of advancing knowledge, and to adduce their own ideas and speculations; Krause is too cautious, or too humble, to present any idea which has not the well-rubbed patina of the market-place. This is a pity, since apart from anything else it is positively dangerous for science to be made to sound too certain and too peaceful rather than a structure of militant hypotheses all of which one expects to discard within ten or twenty years. Otherwise the public will accept (as it does) a pronunciamento by D r This or Professor That, as if it were a truth written with a plume plucked from the wing of the angel Gabriel. Krause, to be fair, does describe a few blind alleys of the past and perplexities as to the future; this is by no means just a flashy journalistic description of scientific breakthroughs. So we have a good description of microbial resistance mechanisms, of the contribution of the magnificent and still underestimated Oswald T. Avery to molecular biology, on heart disease, bilharzia, malaria. This book is strong on information, but not quite strong enough on ideas.

Reference 1 Ashby, E. (1980) Nature (London) 284, 193 P. G. H. GELL P. G. H. Gell is professor in the Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK.

The single-cell assay in cell-mediated cytotoxicity.

Studies on the humoral antibody response were greatly advanced by the introduction in 1963 of the Jerne plaque assay which permitted direct enumeratio...
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