Immunology Today, Vol. 9, No. I, 1988

MHC class I and class II molecules in the early endosomes. Accordingly, in this scheme molecules could be ex- pressed on the cell-surface bound to MHC molecules without obligatory passage through the lysosomes.

Enhancers and enhancer-binding proteins Enhancer sequences are important

in regulation of genes in general, but seem especially important in the im- mune system. S. Tilghman (Prince- ton) used the alpha fetoprotein (AFP) system to investigate the regulation of two closely related proteins, AFP and albumin. Various gene products can regulate enhancer activity, including, in this case, the RAF (regulator of alpha fetoprotein) gene product. RAF is a single mendelian locus, which acts in a dominant, liver-restricted cell- autonomous manner on the expres- sion of AFP. The RAF protein can interact with three AFP enhancers - any two of which are sufficient for the function of the RAF gene on AFP but not albumin expression. No single enhancer is required for AFP expression, so that any two of the three regions function effectively. Since the AFP enhancers do not change ~he ~emporal r~gulation of the albumin promoter, there must be some specific interaction be- tween the AFP enhancers, its promoter and RAF, which is not mimicked for the closely related albumin gene.

An alternative approach was described for the immunoglobulin enhancer. Here the focus was on the proteins which bound to the enhancer sequence directly, rather than on the analysis of trans-acting modifier genes. K. Calame (Univ. California, Los Angeles) showed by deletion experiments five regions (A,B,C,D,E) of sequence with en- hancer function which lie 5' to the transcription start site. All five re- gions were protected from digestion using a chemical nuclease system. Using DNA affinity column tech- niques coupled with gel retardation assays, three of these proteins have been purified. Interestingly, none is lymphoid specific, much less B-cell specific, leaving the octamer-bindlng protein the only tissue-specific en- hancer-binding protein yet known. One of the purified proteins (binding to C) appears to bind to two sites in the enhancer region. Further, none of the enhancer sequences is abso- lutely required, but the sequences

appear to act in an additive way to regulate genes under their control.

Random and site-directed mutagenesis Structure-function experiments

using molecular biology to create structural changes in proteins were the last major theme. Two systems were described, one focusing on im- munoglobulin, the other on MHC class I molecules.

V. Oi (Becton-Dickenson) reported on a series of experiments to test the idea that intersegmental flexibility was solely a property of the Ig hinge region, and that flexibility was the major determinant of the ability of Ig to fix complement. Using a set of chimeric genes, constructed by in- vitro recombination between ~/1 and -y2, he was able to show that flexibil- ity depended on both CH1 and the hinge region and not on the hinge region alone. This was in part due to the site of the disulfide bonds linking heavy and light chains, which differ in -yl and ~/2a. Further, while mol- ecules which possess a complete ,y2a CH2 domain can all fix complement, when ~/1 is substituted within the CH2 region the ability to fix comple- ment is lost. Thus neither of the rules of segmental flexibility that are sup-

posed to govern the ability of Ig to fix complement are as simple as they may have recently appeared.

R. Murray (Univ. North Carolina) described the technique of 'random mutagenesis by oligonucleotides' for the production of large numbers of mutants in a defined segment of any gene. Murray described a library of 2500 point mutants generated in the ~1 domain of the MHC class I gene DP. He sequenced mutants affecting 44 of the 90 amino acids present in the c~1 region of the protein. One of these mutations destroys the ability of the class I molecule to be recog- nized by allogeneic killer cells, but leaves intact its recognition by lym- phocytic choriomeningitis virus- specific DP-restricted killer cells. This mutagenesis technique can be ap- plied to many other situations, where site-directed mutagenesis is impractical due to a need to alter a large number of bases, or where the relevant regions have not yet been identified, and offers great promise for future attempts at genetic man- ipulation.

Jeffrey Frefinger is in the Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27514,"USA.

Sir Peter Medawar 1915-1987 Sir Peter Medawar died on 2 October 1987. He is mourned by an extra- ordinary number and diversity of people; his talents and interests were so broad and included not only science and philosophy but also music and literature. He shared his enthusiasms with family, friends, col- leagues and a more general public who found his writing and broad- casting a source of lucid information and delight.

He was born in 1915 in Brazil; his mother was English, his father a Lebanese businessman. He was edu- cated at Marlborough College and at Oxford, where he met and married his wife Jean, a fellow student. His research career began in Florey's School of Pathology in Oxford, and he was elected a fellow of Magdalen College and appointed a university lecturer in 1938 at the age of 23. He writes of these early years very entertainingly in his autobiography Memoirs of a Thinking Radish pub- lished in 1986. His initiation into ~ 1988. Elsevier Publications. Cambridge 0167 49191881502

from Elizabeth Simpson what was to become his lifelong research interest, transplantation biology, is also vividly described there: it was his response to the compelling need to explore ways of improving the survival of severe burns patients, such as wounded airmen in the war. His work on skin graft rejection in rabbits, and then, under the influence of the American scientist, Ray Owen, in cattle, and also from his observations on skin graft patients, led him to the under- standing that rejection of a graft from a genetically nonidentical donor was due to an immunological response, and not to vague 'physiological incompatibility' as had previously been thought.

He continued his work on skin grafting when he moved to become the Mason Professor of Zoology in Birmingham in 1947, and following his move in 1951 to University Col- lege to become Jodrell Professor of

00

Immunology Today, Vol. 9, No. 1, 1988

Zoology. At this time he was en- gaged with his colleagues, Leslie Brent and Rupert Billingham, on work on induction of tolerance to allografts in neonatal mice, for which he was to be awarded, jointly with Burnet, the Nobel prize for Physiology and Medicine in 1960. The importance of this work lies in the demonstration that the ability to distinguish between 'self' and 'non- self' is an acquired characteristic of the immune system. It was reported in a series of now classic papers in the Proceedings of the Royal Society between 1954-1958. What strikes the reader is not only the elegance and clarity of style but the extraor- dinary capacity to perceive order and meaning in the results. This was part of Peter Medawar's brilliance, the ability to see through the extraneous and superfluousness of any situation to the essential core of an idea - or the lack of it: he was an enemy of cant in any form.

In 1962 he became Director of the National Institute for Medical Re- search (NIMR) at Mill Hill where he continued his work on transplanta- tion tolerance and immunosuppres- sion and where he created a very exciting environment for biomedical research. He had changed the scope of immunology as a result of his work first on skin grafting and then on tolerance. These results and his characteristically clear analyses of them provided a rational approach to clinical transplantation but they also underpinned a new era. Pre- viously, immune responses had essentially been thought of in terms of antibodies; Peter Medawar's in- sistence that 'cell-mediated immun- ity' was qualitatively a different kind of immune response from humoral immunity paved the way for the recognition and exploration of the special and central role of T cells. His own work on the immunosuppres- sive effect of antilymphocyte serum (ALS) in the mid-1960s showed that such a reagent could profoundly modify graft rejection in vivo and set the scene for separating T-cell subsets which were differentially affected by graded doses of ALS. This work foreshadowed the rise of the more discriminating CD4 and CD8 antibodies.

In addition to his scientific re- search and an increasing administra- tive burden, he wrote a number of scholarly books combining his in- terest in science and philosophy; The

Uniqueness of the Individual was published in 1957, The Future of Man in 1960, The Art of the Soluble in 1967 and Induction and Intuition in Scientific Thought in 1969. But to have an idea of the intellectual im- pact of Peter Medawar it is also necessary to know something about the environment he created for sci- entific research. I can speak at first hand about the National Institute for Medical Research which, under his

leadership, had become a Mecca for immunologists by the time I arrived there in the late 1960s. Immuno- logical ideas permeated a number of divisions, not only the Division of Immunology, but also those of Ex-

~ 1 i 1 1 1 ~ 1 l ~ U l l..J i v I vv i . . j .~ / L./I Uv~,-I I ~ l I I I ..-~ I- I ,,y /

Parasitology and Virology. In fact such was the level of excitement and communication that members of all these divisions would ebb and flow into each other's labs, making it impossible to determine from casual inspection who 'belonged' to which division. They talked in their labs, over coffee and lunch in the cafeteria and in the bar after work. Peter Medawar was frequently part of those conversations. He was al- ways generous and supportive of his colleagues. He held back none of his own ideas from them, and delighted in their achievements. With charac- teristic energy he dealt with adminis- trative matters on three days a week, answering letters immediately on reading them, to avoid wasting time in re-reading after laying them on one side. Then, on the other three days (we worked on Saturday morn- ings then), he was in the lab, skin grafting his own experimental mice alongside his own small research team, exchanging ideas as they did so, designing experiments, even

doing his own washing up - he said he thought better when he was doing such tasks. His foresight was of both an intellectual and practical nature. For example, realizing the need for genetically uncontam- inated, disease-free, experimenta! animals, he planned not only a large new SPF breeding unit but also new experimental facilities to match. But in aggregate the cost was not acceptable to the MRC, and although NIMR got its SPF breeding unit the experimental unit to match was not built, despite his advocacy. He did insist, however, in the teeth of some opposition, that when the new cafeteria was built in the late 1960s on that beautiful site at the back of the Ailding, overlooking the green valley and hill opposite, a bar was an essential part of it. He knew that scientists needed a congenial place for a drink, where they could break from lab work at the end of the day for a discussion, before re- turning to the lab to check results.

In the intellectual and physical en- vironment Peter Medawar created at NIMR, a lot of us grew up: very few had been trained in immunology but we absorbed it. as if by osmosis, quickly then entering the fray of discussions with our own ideas and special interests. We had many visi- t o r s - from distinguished elder statesmen of immunology on sab- U O L I L ( : ] I ~ to U l l y l I L r a w ~ I U ~ L U U L L U r d l

fellows, many of whom contrived to stay much longer than their initial fellowships allowed for, plus of course the many transient visitors who came for a few days at a time, giving seminars on their work and joining in the discussions.

It was a shattering blow to the whole community when in 1969 Peter Medawar suffered a severe stroke. During the following 18 months, with the help of his wife, he made an extraordinarily good recov- ery, but one which left him partly paralysed, and in 1971 he resigned the Directorship of NIMR. With characteristic optimism and good humour and with the help of an erstwhile colleague and friend of Mill Hill days, Eugene Lance, then head of the Surgical Sciences Division at the Clinical Research Centre, Sir Peter set up a small research group in transplantation biology at the CRC. He also continued to write books, organize meetings, travel ab;oad, give invited lectures and make broadcasts. This period of his

Immunology Today, Vol. 9, No. I, 1988 , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ , : , ~

life, physically disabled to an extent which might induce mos~ people to give up, was fruitful and rewarding, both to him and to those around him. He retained his charm and wit and enjoyment of life, taking as much pleasure in the successes of his younger colleagues as from the pub- lication of each of his several books written during this time: Advice to a Young Scientist (1979), Pluto's Re- public (1982), The Limits o f Science (1984), Memoirs of a Thinking Rad- ish (1986) and, in collaboration with his wife, The Life Science (1977) and From Aristotle to Zoos (1983). He

Lymphokine-activated killer cell activity

Sir, In their recent review (Immunol. Today, 1987, 8,178-181), R. Herber- man et al. noted that "Some . . . progenitors [of lymphokine- activated killer cells (LAK)]... lack the characteristic granules of LGL [large granular lymphocytes] and are therefore resistant to the lysosomo- tropic agent L-leucine methyl ester". We wish to point out that the effects of L-leucine methyl ester (Leu-OMe) on cytotoxic T lymphocytes (CTL) are far more complex than is implied by this statement.

Amino acid methyl esters and, in particular, Leu-OMe are lysosomo- tropic compounds which were first discovered to be nontoxic inhibitors of lysosomal function in living cells 1.z. However, we observed that Leu-OMe was toxic for blood mono- cytes without apparent toxicity for most blood lymphocytes 3. In the ab- sence of monocytes or granulocytes, Leu-OMe was not toxic to CD16 + LGL over a broad concentration range (1-25 raM) 4 but, like other lysosomotropic agents, reversibly in- hibited the lytic activity of NK cells 4. By contrast, in the presence of fresh- ly isolated monocytes or granulo- cytes, 1 mM Leu-OMe rapidly killed all CD16 + LGL 4. A lysosomal enzyme present in monocytes and granulo- cytes produced the condensation product Leu-Leu-OMe upon incuba- tion with Leu-OMe 5 and Leu-Leu- OMe was directly toxic to CD16 + LGL s,6. Thus, categories such as 'sensitive' or 'resistant' to L-leucine methyl ester are meaningful only if the monocyte or granulocyte con- tent of the incubation mixture is

officially retired from the CRC in 1984 but continued to write and his last book, an autobiography, was published in 1986.

His stature as a creative scientist was recognized from the earliest days and throughout his career: he was made a member of the Royal Society in 1949, received the Royal Medal in 1959, the Copley Medal of the Royal Society in 1969 and in 1987 the Royal Society's Michael Faraday Award. He was knighted in 1965, made a Companion of Honour in !972 and a member of the Order of Merit in 1981.

The legacy that Peter Medawar left consists not only of his own seminal scientific work and ideas, and of his many books embodying science and philosophy, but of the next generation of scientists whom he met, nurtured and influenced. His style was to be optimistic, generous and caring, as well as to achieve. This he has inspired many people to emulate.

Elizabeth Simpson is in the Transplantation Biology Section of the Clinical Research Centre, Watford Road, Harrow HA 1 3UJ, UK.

clearly defined and the cell function being assessed (cell viability or re- versible loss of lysosomal function) is clearly stated.

Concentrations of 50-100 I~M Leu-Leu-OMe but not 1-50 mM Leu- OMe irreversibly removes all natural killer (NK), activated LAK, or acti- vated CTL function from human, rat or mouse lymphoid populations (Refs 4-7, and unpublished). By con- trast, the bulk of B cells and prolif- erative CD4 + T cells are resistant to Leu-Leu-OMe in the 50-250 I~M concentration range at which all NK, LAK and antigen-specific CTL precur- sors are eliminated. After lympho- cyte populations are depleted of all CD16 ÷, NKH1 ÷ LGL and allospecific CTL precursors by treatment with Leu-Leu-OMe, the surviving T ceils are virtually noncytotoxic for tumor targets after culture with interleukin 2 (IL-2) alone, but they can be acti- vated to mediate MHC-unrestricted cytotoxicity following mitogenic stimulation 8. Thus, whether synthe- sized de novo or within the lysosom- al compartment of a myeloid cell exposed to Leu-OMe, Leu-Leu-OMe is useful in the characterization of

lymphocytes with cytolytic function. However, it is essential to distinguish the effects of Leu-OMe and Leu-Leu- OMe on the function and viability of these cells when interpreting the re- sults of experiments performed with these agents.

Dwain L. Thiele Peter E. Lipsky

Department of Internal Medicine, University of Texas Health Science Center, Dallas, TX

75235, USA.

References 1 Goldman, R. and Kaplan, A. (1973) Biochim. Biophys. Acta 318, 205-216 2 Reeves, J.P., Decker, R.S., Crie, J.S. and Wildenthal, K. (1981 ) Proc. NatlAcad. Sci. USA 78, 4426-4429 3 Thiele, D.L., Kurosaka, M. and Lipsky, P.E. (1983) J. Imrnunol. 131,2282-2290 4 Thiele, D.L. and Lipsky, P.E. (1985) J. Immunol. 134, 786-793 5 Thiel~., D.L. and Lipsky, P.E. (1985) Proc. Natl Acad. Sci. USA 82, 2468-2472 6 Thiele, D.L. and Lipsky, P.E. (1986) J./mmunol. 136, 1038-1047 7 Thiele, D.L., Charley, M.R., Calomeni, J.A. and Lipsky, P.E. (1987)J. Imrnunol. 138, 51-57 8 Thiele, D.L. and Lipsky, P.E. (1986) J. Immunol. 137, 1399-1406

Lack of tolerance to a soluble MHC class I antigen

Sir, Recently, D. Gussow and H. Ploegh (Immunol. Today, 1987, 8, 220-222) reviewed the extensive data describ- ing different forms of soluble class I major histocompatibility complex (MHC) antigens. They presented the various mechanisms by which sol- uble class I antigens can be gener- ated, and discussed the lack of evi-

dence to explain the function of the~e molecules. However, they did not address one intriguing aspect of this area of research - the associa- tion of soluble class I antigens with the phenomenon of liver allograft tolerance. In several species, liver allografts are tolerated much better than grafts of skin, heart and kidney 1. In rats, liver allografts are associated with the induction of a state of donor-specific tolerance accompanied by a functional dele-