tremls

process is integrin activation, and the role of cytokines such as the chemokine family. Thus the simple expression of an adhesion mol- ecule is only one of the factors in determining the specificity of leuko- cytes for a tissue. Indeed the search for agents that block adhesion events might now be paralleled by an effort to modify the action of particular chemokines or other types of cytokines. Concepts of cell adhesion apply equally well to many systems, such as leukocyte interactions, development and can- cer metastasis. It is gratifying that immunology is at the forefront in establishing these new concepts.

Charles Mackay and Beat Imbof are at the Basel btstitute for Immunology, Grenzacberstrasse 487, CH-4005 Basel, Switzerland.

References 1 Lawrence, M.B. and Springer, T.A. ( 1991 ) Cell 65, 859-873 2 yon Andrian, U.H., Chambers, J.D., McEvoy, L.M. et al. (1991) Proc. Natl Acad. Sci. USA 88, 7538-7542 3 Mackay, C.R., Kimpton, W.G., Brandon, M.R. etal. (1988)J. Exp. Med. 167, 1755-1765 4 Streeter, P.R., Berg, E.L., Rouse, B.T. et al. (1988) Nature 331, 41--46 5 Lasky, L.A., Singer, M.S., Dowbenko, D. et al. (1992) Cell 69, 927-938 6 Berg, E.L., Robinson, M.K., Warnock, R.A. etal. (1991)J. Cell. Biol. 114, 343-349 7 Dustin, M.L. and Springer, T.A. (1989) Nature 341,619--624 8 Yednock, T.A., Cannon, C., Fritz, L.C. et al. (1992) Nature 356, 63--66 9 Giinthert, U., Hofmann, M., Rudy, W. etal. (1991) Cell 65, 13-24

10 Sy, M.S., Guo, Y.J. and Stamenkovic, I. ( 1991 ) J. Exp. Med. 174, 859-866 11 Birch, M., Mitchell, S. and Hart, I.R. (1991) Cancer Res. 51, 6660-6667 12 Plantefaber, L.C. and Hynes, R.O. (1989) Cell 56, 281-290 13 Seftor, R.E., Seftor, E.A., Gehlsen, K.R. et al. (1992) Proc. Natl Acad. Sci. USA 89, 1557-1561 14 Chan, B.M., Matsuura, N., Takada, Y. et al. (1991) Science 251, 1600-1602 15 Miyake, K., Medina, K.L., Hayashi, S. et al. (1990) J. Exp. Med. 171,477-488 16 Miyake, K., Weissman, I.L., Greenberger, J.S. et al. (1991)]. Exp. Med. 173, 599-607 17 Butcher, E.C. (1991) Cell 67, 1033-1036 18 Shimizu, Y., Newman, W., Tanaka, Y. (1992) lmmunol. Today 13, 106-112

British Society for Immunology- epitope mapping workshop

Frank C. Hay, Meinir G. Jones, Andy Soltys and Angela Horsfall

Improvements in technology have taken the subject of epitope map- ping away from the realms of the skilled peptide chemist and simpli- fied the procedures so much that even immunologists can now re- liably synthesize hundreds of pep- tides in a few days. Ronald Frank (Braunschweig) has developed a very user friendly system based on ce!lu!ose chromatography paper as the support for solid-phase peptide synthesis. A sheet of paper about .t.. size ,,¢ ~ microtitre nlate is used to produce 96 'Spots' of peptides attached to two 13 alanine residues. An especially helpful feature is the incorporation of bromophenol blue indicator dye into the coupling reaction. As coupling proceeds the dye changes colour, providing on- line monitoring of the efficiency of the reaction. At most, an hour is required to couple, wash and deblock an amino acid allowing

A recent workshop * discussed the improvements in technology [or methods in epitope mapping. Here, Frank Hay and colleagues

report on the developments in this area.

hexamer peptides to be easily pro- duced in a single day, and with tuck the key epitope is revealed by enzyme linked immunosorbent assay (EL!SA) the next day. Using appropriate coupling reactions, the spots can be punched out and the peptides eluted for T-cell assays, with about 50 nM peptide per ShOt.

Much of the work presented used the PEPSCAN pin technique introduced by Geysen et al. A par- ticular problem has been the high backgrounds seen with human sera. Trebbick et al. 1 reasoned that, for a particular epitope, the reaction

*This workshop was held at the British Society for Immunology neeting held in London 25-29th November, 1992.

1993, Elsevier Science Publishers Ltd, IlK. 0167-5699193/$06.00

mixture in a well with a peptide- pin would be a combination of a small amount of specific antibody binding with high affinity, and a large amount of non-specific anti- body binding with low affinity. In whole serum the non-specific anti- body so overloads the system that virtually every peptide pin becomes loaded with similar amounts of antibody. If the pin is washed and eluted with acid a mixture is obtained which is enriched for spe- cific antibody. On reapplying this mixture to a cleaned pin the laws of mass action predict that the high affinity antibody will bind almost as well as before but, as the non- specific immunoglobulin is now at a much lower concentration than in whole serum, it only binds in low amounts. Davis and colleagues (Cambridge) showed that this pro- cedure works well if an amplifi- cation system (AMPAK) is used to

Immunology Today 102 Vol. 14 No. 3 1993

trends

reveal the specific antibody, giving hope to those of us trying to dis- cover epitopes for human anti- bodies. It may be prudent to run a conjugate-only assay on the pins to check that they have been efficiently stripped of bound immunoglobulin before reincubating with eluted anti- bodies (fresh sonication buffer helps).

D. Whitehouse and colleao, ues (London), using PEPSCAN, found an interesting neighbourhood effect that can complicate mapping. HVLPF constituted a minimal epi- tope in horse alcohol dehydrogen- ase, in which Pro and Phe were essential, intriguingly Glu, within or flanking the epitope, reduced its reactivity.

PEPSCAN may be used to probe idiotopes as well as epitopes and N. Staines (London) reported that a major idiotope in the 16/6 family is VATISG situated in the FR2/CDR2 region. Intriguingly this is homolo- gous to sequences in bacterial and mammalian heat shock proteins.

Linear peptides are ideal for probing epitopes for T cells, and the availability of simply prepared peptides from pins er spots is enabling the fine definition of epi- topes with relative ease. Most work has been with T-cell lines or clones but increasingly the repertoire expressed by peripheral blood T cells is being examined. Traditional limiting dilution analysis accurately estimates the precursor frequency of responding cells but its use to screen numerous peptides is

restricted by the volumes of blood needed to assay at multiple cell concentrations for each peptide. Chiron Mimotopes have intro- duced a protocol based on the Poisson distribution that takes maxi- mum advantage of all the negative results that occur within a peptide screening assay. Discussion of this protocol produced the main area of controversy at the workshop with A. Vyakarnum (London), question- ing the validity of the algorithm in establishing single hit kinetics. However a stout defence was mounted by D.J.M. Lewis (London), who presented data obtained from a study of human T cell responses after oral immunization with a cholera toxin B subunit. Using 10 pools of overlapping 12-mer pin- derived peptides and five matched 30-mer conventional peptides, ex- cellent correlation was seen for the immunodominant regions ident- ified. M.G. Jones (London) simi- larly found the system effective for mouse T cells and identified GEPGIAGFKGEQ as an important epitope in collagen-induced arthritis.

Do the same epitopes induce T-cell proliferation and cytokine release? A. Vyakarnum suggested that there might be subtle differ- ences and that cytokine release might be possible in the absence of significant proliferation. H.M. Danieis (London) also had results that suggested that the cytokine pattern may be different with vari- ation in the epitope.

How degenerate is T-cell recog- nition of peptides? D.P. Harris (London) reported on a cross- reactive T-cell response from mice immunized with M. tuberculosis or M. leprae peptides. Surprisingly the core residues involved in the cross- reactivity were remarkably dissimi- lar, sharing only one asparagine residue in common. Using replace- ment analysis with PEPSCAN, the response was abolished by substi- tutin~ the asparagipe with alanine, suggesting that the mechanism responsible for crossreaction is de- pendent on a single amino acid. Per- haps there is still a lot to be learnt about T-cell recognition of antigen.

Overall the success with which workshop partic,pants were now synthesizing peprides and finding epitopes brings close the day when workers will be able routinely to synthesize precise solid-phase epi- topes for use in irnmunoassays with the same ease with which they now coat ELISA plates with antigens.

Frank C. Hay, Memir G. Jones and Andy Soltys are at the Division of Immunology, Department of Cellular and Molecular Sciences, St George's Hospital Medical School, London, UK SW17 ORE; Angela Horsfall is at the Kennedy Institute of Rheumatology, London~ UK W6 7D W.

Reference 1 Trebbick, E. et al. (1992)J. lmmunoL Meth. 139, 155-166

Immunology Today's sister journal, Parasitology Today, is producing a special issue in May 1993 entit 'ed "The Management o f Drug Resistance in Parasitic Infecitior~s". The reviews which m a y be of interest to ~mmunologists include:

*Funding of malaria drug research - B. Schuster

°Management of malarial disease in aJreas of drug resistance - A. Schapira

*Mechanisms of drug resistance in Leishmania - M. Ouellette

*Drug resistance in African trypanosomes - C. Bacchi

°Drug resistance in schistosomes - D. Cioli

This issue will cover a range of organisms in a variety of hosts, and will be a source of up-to-the-minute information on drug resistance in humans, animals and vectors of parasitic infections.

Immunology Today 1 0 3 Vol. 14 No. 3 1993