Immunoregulation by leukotrienes and other lipoxygenase metabolites

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302 17 Nepom, J. T., Weiner, H. L., Dichter, M. A., Tardieu, M., Spriggs, D. R., Gramrn, C. F., Powers, M. L., Fields, B. N. and Greene, M. I. (1982)J. Exp. Med. 155, 155-167 18 Islam, M. N., Pepper, B. M., Briones-Urbina, R. and Farid, N. R. (1983) Eur. J. Immunol. 13, 57-63 19 Bona, C. and Moran, T. Ann. Immunol. (Paris) in press 20 Holmberg, D., Forsgreen, S., Forni, L., Ivars, F. and Coutir~o, A. (1984) Proc. Natl Acad. Sci. USA 81, 3175-3179 21 Sire, G-K., Nelson, J. E. and Augustin, A. A. (1984) in Regulationof the Immune System (Sercarz, E., Cantor, H. and Chess, L. eds) Vol. 18, pp. 677-687, Alan R. Liss Publ., New York 22 Schechter, Y., Maron, R., Elias, D. and Cohen, I. R. (1982) Science216, 542-545 23 Wasserman, N. H., Penn, A. S., Freimuth, P. L, Treptow, N., Wentzel, S., Cleveland, W. L. and Erlanger, B. F. (1983) Proc. Natl Acad. SoL USA 79, 4810-4814 24 Augustin, A. A., Sim, G. K. and Bona, C. A. (1983) Surv. ImmunoL Res. 2, 78-87 25 Zanetti, M. and Katz, D. H. in Current Topics in Microbiology and Immunology (Koprowski, H. and Melchers, F. eds) Springer-Verlag in press 26 Brown, C. A., Carey, K. and Colvin, R. B. (1979)J. Immunol. 123 2102-2107 27 Zanetti, M., Marnpaso, F. and Wilson, C. B. (1983).]. ImmunoL 131, 1268-1273 28 Neilson, E. G. and Phillips, M. S. (1982)J. Exp~ Med. 155, 179-189 29 Ben-Nun, A. and Cohen, I. R. (1981)Eur. J. ImmunoL 11,949-952 30 Maron, R., Zerubavel, R., Friedman, A. and Cohen, I. R. (1983) J. ImmunoL 131, 2316-2322

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Zanetti, M. and Bigazzi, P. E. (1981) Eur. J. ImmunoL 11, 187-195 Hahn, B. H. and Ebling, F. M. (1983)J. Clin. Invest. 71, 1728-1736 Germain, R. N. and Benacerraf, B. (1981) Scand. J. Immunol. 13, 1-10 Takei, I., Sumida, T. and Taniguchi, M. (1983)J. Exp. Med. 158, 1912-1923 Sumida, T., Takei, I. and Taniguchi, M. (1984)J. Immunol. 133, 1131-1136 Herzenberg, L. A. (1983) in Progress in Immunology (Yamamura, Y. and Tada, T. eds), Vol. V, pp. 581-589, Academic Press, Japan Inc. Forni, L., Coutinho, A., Kohler, G. andJerne, N. K. (1980) Proc. Natl Acad. Sci. USA 77, 1125-1128 Wilder, M., Franssen, J. D., Collignon, C., Leo, O., Mariame, B., Van de Walle, P., De Groote, D. and Urbain, J. (1980)J. Exp. Med. 150, 184-195 Reth, M., Kelsoe, G. and Rajewsky, K. (1981) Nature (London) 290, 257-259 Trenkner, E. and Riblet, R. (1975)J. Exp. Med. 142, 1121-1132 Bluestone, J. A., Sharrow, S. O., Epstein, S. L., Ozato, K. and Sachs, D. H. (1981) Nature (London) 291, 233-235 Perle, J., Bekkhoucha, F , Desaymard, C., Zaghouani, H. and Stanisldawski, M. (1983).]. Exp. Med. 157, 1573-1593 Zanetti, M., Rogers, J. and Katz, D. H. (1984)J. ImmunoL 133, 240-243 Datta, S. K., Stonar, D. and Schwartz, R. S. (1983) Proc. NatlAcad. Sci. USA 80, 2723-2727 Shenk, R. R., Weissberger, H. Z. and Diclder, H. B. (1984).]'. Immunol. 132, 2709-2714 Rubinstein, L.J., Goldberg, B., Hiernanx, J., Stein, K. E. and Bona, C. A. (1983)J. Exp. Med. 158, 1129-1144

Immunoregulation by leukotrienes and other lipoxygenase metabolites Marek Rola-Pleszczynski! The leukotrienes are a group of biologically active molecules derivedfrom the oxidative metabolism of unsaturated fatty acids via the 5-1ipoxygenase pathway. They are readily synthesized by leukocytes and probably by other cell types following immune or nonimmune stimulation. In addition to their initially described myotropic activities, they very strongly affect several leukocyte functions. Here Marek Rola-Pleszczynski suggests that leukotrienes may play an important role in immunoregulation during inflammatory processes. W k h some analogy to the prostaglandins (PGs), the leukotrienes and other tipoxygenase products possess potent and varied pro-inflammatory activities. They can modulate vascular permeability and attract and activate phagocytes, two hallmarks of an inflammatory reaction. The structure and chemistry of these compounds are summarised in the box on p. 304 and in Fig, 1. Injection of LTB4 into rabbk, rat or human skin causes local erythema and significant, albeit small extravasation of plasma proteins. This, however, is greatly potentiated by coadministration of PGE1, PGE2, PGD 2 or bradykinin. The monohydroxy products 5-, 12- or 15-HETE are 10- to 100-fold less active than LTB43539 12-HETE was the first lipoxygenase product reported to have chemotactic activity for human P M N 4°. The other mono-HETEs and, to a greater extent, the HPETEs are also potent chemotactic and chemokinetic agents for rabbit and human P M N 41. LTB4, however, is 100 times more potent as a chemotactic and chemokinetic agent than any of the HETEs or H P E T E s 42-~4. Both of these activities of LTB4 were stereospecitlc for the cis-trans-trans triene structure 45'46.

Immunology Division, Department of Pediatrics, Universit~ de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada. Q 1985~FJsevierScienoePublishersB.V., Amsterdam 0167- 4919/85/~02.00

When injected intracutaneously in man, intradermally in rabbits or monkeys and intraperitoneally into guinea pigs, LTB4 also causes leukocyte accumulation 36'45'47'48. Increased leukocyte adherence to the vascular endothelium 49, followed by chemokinetic and chemotactic activity appears to be involved. In vitro, LTB4 also causes aggregation of P M N 4z. Some or all of these phenomena may be involved in the observed neutropenia following intravenous injection of LTB4 ~9. At nanomolar concentrations, LTB4 also causes a dosedependent increase in the secretion of lysosomal enzymes 5°'51,while 5-HETE is inactive. This process may be initiated by the ability of LTB~ to affect calcium flux in P M N 52. It is also energy-dependent and LTB4 induces P M N to take up glucose from the medium 53. It is still unclear what role the very early (peak at 30 s) rise in intracellular cyclic A M P (cAMP) levels induced by LTA4 and LTB4 (but not LTC4) 54, or the suggested increase in cyclic G M P (cGMP) concentrations 55induced by hydroperoxy fatty acids, have in these activation processes. As indicated by structure-activity studies 56 and later confirmed by binding experiments 57, human P M N have specific membrane receptors for LTB4. In contrast, other hydroxy fatty acids, such as 5-HETE, may act at least in part through covalent insertion into the membrane 5a.

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TABLE I. Immunoregulatoryeffects of leukotrienes Compound LTB4 LTB4 LTB4 LTB4 LTB4 LTB4 LTB4 LTB4 LTB4

Target cell Human PBML" T4 + lymphocytes Human B cell Human T8 + Human T cells Rat leukocytes(in vivo) Human T cells T8 ÷ lymphocytes Human NK and NCb cells

LTB4 Mouse T cells LTB4 Human T4 + cells LTB4 Human T4 + cells LTB4 Human monocytes LTA4 Human NK and NC cells LTC4,LTD~ Mouse T cells LTD4,LTE~ Mouse T cells LTD4,LTE~ Mouse B cells aPeripheralbloodmononuclearleukocytes. bNaturallycytostaticcells. In spite of technical difficulties in measuring low concentrations of leukotrienes in biological fluids, functionally significant elevations of these mediators have been observed in several disease states characterized by hypersensitivity and inflammation. For instance, in allergic subjects, tear fluid from an eye challenged with the appropriate allergen showed increased levels of LTCJD4/E459. Allergen challenge of lung tissue from asthmatics also elicited the release of these LTs in correlation with bronchial constriction, suggesting that they are probably the most important mediators of smooth muscle contraction in h u m a n allergic asthma 6°. There is also increasing evidence that LTB4 may be an important mediator of leukocyte accumulation in psoriasis 29'61. Similarly, neutrophil accumulation is an important histopathological feature of ulcerative colitis and elevated levels of LTB4 were observed in the mucosa in this disease as well as increased capacity to synthesize both 5-HETE and LTB~ 62. Rheumatoid arthritis and gout are also associated with high numbers of P M N in the synovium, and increased concentrations of LTB4 were reported therein ~8'63. LTB4 concentrations are also increased in sputum or lung fluids in cystic fibrosis 64, in adult respiratory distress syndrome 65 and in neonatal hypoxemia with pulmonary hypertension66. L e u k o t r i e n e regulation of lymphocyte function Since leukotrienes and other lipoxygenase products are readily synthesized by leukocytes in response to extracellular stimuli, it became compelling to assess whether they could be involved in the control of immune reactivity. Furthermore, while corticosteroids inhibit several immune functions, probably in part via their inhibition of phospholipases, most of these effects are not shared by cyclooxygenase inhibitors, suggesting again that the lipoxygenase pathway may play a major role in regulating immune reactions. In 1980, Goodman and Weigle reported that a 15-1ipoxygenase metabolite of arachidonic acid,

Effect Inhibitionof proliferation Inhibitionof proliferation Inhibitionof Ig synthesis Inhibitionof IFN production Inhibitionof LIF production Inhibitionof MLR Enhancementof suppressor cellfunction Proliferationand marker expression Enhancementof cytotoxicityand enhancement of target binding Enhancementof IFN-)"production Enhancementof IFN-),production Enhancementof IL-2 production Enhancementof IL-1 production Enhancementof cytotoxiclty Enhancementof IFN-)"production Inhibitionof proliferation Inhibitionof PFC formation

Ref. 69,71,73 73,74 72 84a 71,83 81 69,71,72,75a 72,74,76 85,86,87 84 84a 84a 78 86 84 68a 68a

tentatively identified as 15-HPETE, inhibited the proliferative response of mouse splenocytes to several mitogens 67. Similarly, 15-HETE and LTD~ 60 and LTE468" were reported to inhibit mitogen-induced [SH] thymidine incorporation into mouse splenocytes. When directly added to h u m a n lymphocyte cultures stimulated with P H A or ConA, we found that LTB, would also cause a modest but variable inhibition of [~H] thymidine uptake, while LTD4 was relatively inactive69. There was no dose-response relationship, however, and in some experiments, LTB4 was actually stimulatory. This may explain the failure to observe a direct effect of LTB4 in some instances 7°. More interestingly, however, LTB4 induced the appearance of suppressor lyrnphocytes during a 3-24 h pre-incubation. These cells could then inhibit the mitogen-induced proliferation of fresh autologous cells in a coculture system 69. LTB4 was active at very low concentrations (peak at 10-12 M) on either unfractionated or nylon wool-enriched T cells which did not need to replicate in order to exert their suppressor effect. Similar findings were later reported by other laboratories71 73. More specifically, the proliferation of the T4 + (helper/inducer) subset of h u m a n T cells was inhibited by LTB~ while the proliferation of the T8 +(suppressor/cytotoxic) subset was enhanced n'74. 15-HPETE exerted a similar effect 73'75. Further analysis of the cellular requirements for LTB4-induced suppressor cell activity revealed that, while only T cells needed to interact with LTB4, the expression of their suppressor activity required the presence of monocytes in the responding cell population TM.Little suppression was seen in the absence of monocytes and this was actually reversed to a helper effect in the presence of the cyclooxygenase inhibitor indomethacin76'77.We postulated that this could be explained by a dual stimulation of monocytes to produce both PGs and interleukin-1 (IL-1) through interaction with LTB4. We observed that monocytes produced 30-100% more IL-1 in the presence of 10-7-10 -s M LTB~ than control monocytes cultured without LTB4 TM. Indomethacin alone induced a similar increment in IL-l-dependent thymocyte proliferation,

Ju,

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Leukotriene structure and chemistry Leukotrienes (and other hydroxyeicosatetraenoic acids, HETEs), are derived from 20-carbon atom fatty acids, of which arachidonic acid is the most common prototype. The metabolism of arachidonic acid first requires its release from membrane phospholipids (Fig. 1). This involves calcium-dependent phospholipase A 2 and C activity 1'2.Leukotrienes are then synthesized within or adjacent to cell membranes via the activity of 5-1ipoxygenase immediately prior to their release. The formation of5-hydroperoxy-6,8,1 1,1 4-eicosatetraenoic acid (5-HPETE) is the initial step in the synthesis of this group of compounds containing a conjugated triene structure 3, from which their name derives. 5-HPETE is then converted to the 5,6-epoxide leukotriene A4(LTA4) , a highly unstable compound which serves as a common intermediate for synthesis of either (5S, 12R)dihydroxy-6,14-cis-8-10-trans-eicosatetraenoic acid (leukotriene B4, LTB44-6) or the thiol ether leukotrienes C4, D4 and E4. Non-enzymatic hydrolysis of LTA4 produces the relatively inactive diastereoisomers 5,6-diHETE and 5,12-diHETE 4-8. The addition of glutathione to LTA4 by glutathione-S-transferase produces LTC4, which can be converted to LTD4 by removal of the terminal glutamine by )'-glutamyl transpeptidase. In turn, LTD4 can be converted to LTE4 by loss of the terminal glycine 9-~4. The long-elusive structure of slow-reacting substance of anaphylaxis appears to be a mixture of LTC4, LTD4 and LTE4. H u m a n neutrophils can rapidly metabolize LTB4 by m-oxidation, leading to the formation of 20-OH-LTB4 and then to 20-COOH-LTB4 I5. Neutrophils and eosinophils can also degrade LTC4, LTD4 and LTE4 by peroxidation and yield the corresponding sulfoxides, sulfones and, to a lesser extent, 6-trans isomers ofLTB415a'~6 Several tissues are known to contain lipoxygenases and to produce hydroxy-eicosatetraenoic acids. They include polymorphonuclear leukocytes (PMN) ~'17, macrophages m9, basophilic leukemia cells 28 and bone-marrow-derived mast cells 21 which produce mainly 5-1ipoxygenase (leukotrienes, 5-HETE, etc.) and 15-1ipoxygenase metabolites, eosinophils which produce mainly 1 5-1ipoxygenase metabolites 22and platelets which produce 12-1ipoxygenase metabolites 23. These or other cell types may be responsible for leukotriene formation in the lung 2., the spleen 25, the kidney 26, the synovium 27 or the epidermis 2a. Human lymphocytes, however, when thoroughly depleted of contaminating neutrophils, monoeytes and platelets, appear to secrete arachidonic acid upon stimulation 29, but no lipoxygenase metabolites 3°. Complex interactions occur between products of the various lipoxygenases. For example, 12-HPETE from platelets enhances the activity of 5-1ipoxygenase of human leukocytes; 12-HETE is inactive ~. In contrast, 15-HPETE and its more stable product 15-HETE are inhibitors of 5-and 12-1ipoxygenases 32. H u m a n leukocytes have also been shown to produce another series of compounds containing a conjugated triene structure via the 15-1ipoxygenase pathway. A 14,15-epoxide intermediate, analogous to LTA4, is formed initially, giving rise to 14,15- and 8,15-diHETEs (also termed 14,1 5- LTB 4 and 8,1 5- LTB4) 33. More recently, a new series of trihydroxytetraenes (5,6,15-triHETE or lipoxin A and 5,14,15triHETE or lipoxin B) has been described, suggesting interaction(s) between the 5- and 15-1ipoxygenase pathways in human leukocytes 34.

but LTB4 and indomethacin were additive in their effect when combined during the culture. These data suggest that LTB4 may modulate monocyte function and interact with lymphocyte activation through induction of a dual, positive and negative signal from monocytes in the form of IL-1 and PGs, respectively. This is further supported by the observations that ibuprofen, a cyclooxygenase inhibitor, inhibits the synthesis of PGE 2 but not IL-1 while the lipoxygenase inhibitors BW755C and eicosatetraynoic acid (ETYA) inhibit IL- 1 production 78a9. While suppressor cell activity resided in the T8 + subset of T cells, it was inducible from both T4 + and T8 ÷ subsets 72'76. We found, however, that T4 + lymphocytes were functionally inducible to become suppressors only when there were monocytes in the respondqr population. T8 + lymphocytes did not require monocytes to become active suppressor cells 76. In-vivo administration of 15-HETE also reduced the

proliferative responses of murine splenotypes to PHA, ConA or allogeneic cells 8°, while a single dose of LTB4 resulted in reduced mixed lymphocyte reactivity in the rat after 72-96 h 8I. The expression of surface markers was also affected by lipoxygenase products, with possible phenotypic correlation with functional effects. For instance, 15-HPETE inhibited rosette formation with sheep erythrocytes 75and decreased the density of Fcy receptors on human T cells and monocytes 82. 15-HETE enhanced the expression of the Lyt-2 ÷ antigen on mouse splenocytes after a 48 h culture 8°. LTB4 also induced the appearance of T8 + cells in a T8-depleted lymphocyte population 72,76. Immunoglobulin (Ig) production in vitro by murine splenocytes was impaired when cells were cultured in the presence of LTD4 or LTE468'68a,while in the human, only LTB4 could induce suppressors of Ig synthesis 72. H u m a n LIF production was also inhibited by LTB~ 71'83.

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Membrane phospholipids j

glucocortiooids

phospholipase~. ......... lipocortin arachidonic acid

8-HETE 9-HETE 11.HETE

COOH

,-U)> t, :

15-1ipoxygenase

/

15-HPETE ......... \

14,15diHETE

8,15diHETE

5,6 or 5,12diHETEs

C

~.~iyclooxygenase~,-'°'~'

N SA IA

PGs TxA 2

5-1ipoxygenase

......... ~./

...."

.."

Lipoxins A,B

t

a t 5-HPETE

reductase,

=bHET E

dehydrase

non enzymatic

1

LTA4

hydrolysis

hydrolase =

LTB4 (5S,12R diHETE)

glutathioneS-transf6rase ,~ -oxidation LTC4 l"~llutamyl l transpeptidase LTD 4 cysteinyl glycinase

[

20-OH-LTB 4

dSRS.A

~ -oxidation

20-COOH LTB4

LTE 4

Fig. 1. The arachidonicacid cascadewith emphasison the lipoxygenase pathways. Broken lines indicate inhibition. Abbreviations: a 5-Hydroperoxy-eicosatetraenoicacid; b 5-Hydroxyeicosatetraenoicacid; c non-steroid anti-inflammatoryagents; dslow-reactingsubstanceof anaphylaxis.

In contrast, LTB4, L T C , and L T D 4 were capable of replacing mouse T H cells or IL-2 in inducing the production of )'-interferon a4. We found that the addition of 10 -1° M LTB 4 to poly I:C-stimulated h u m a n peripheral blood mononuclear leukocytes resulted in significant inhibition of ),-interferon production. Further studies revealed, however, that selective depletion ofT8 + cells with monoelonal antibodies and complement before stimulation with poly I : C and LTB4 resulted in enhanced )'-interferon production while depletion of T4 + cells was associated with suppression of y-interferon production by

LTB484a. We also found that LTB4 induced significant IL-2 production by T4 + cells in t h e presence of indomethacin 84a Natural killer (NK) or natural cytotoxic (NC) cell activity against tumor (K-562, MA- 160) or virus-infected target cells is strikingly affected by 10 8-10-12 M LTB4. W e initially showed that LTB~ and, to a lesser degree LTA4, significantly enhanced both N K and NC cytotoxicity when added to the effector-target coculture aS's6. LTB 4 increased both the binding ofeffectors to target cells and the rate of target cell killing 87but no interferon or IL-2

306

was detectable in the 4 h cytotoxicity assay culture supernatants. On the other hand, when T cells were pulsed for 3 h with LTB4 followed by washing and culture for 24 h, the supernatant also enhanced cytotoxicity of fresh N K or NC cells a7 (probably due to IL-2 production), while the pretreated lymphocytes suppressed the cytotoxic response if cocultured with N K or N C effectors. The generation of murine Tc cells could also be inhibited by administration in vivo of 15-HETE 8°. In a different system, LTB4 augmented neutrophil- a n d eosinophil-mediated, complement-dependent killing of schistosomula aS. Other lipoxygenase products can also affect cytotoxicity. Lipoxin A enhanced N K cell function, although to a lesser degree than LTB 4 (unpublished observations), while 14,15-diHETE inhibited NK function but did not affect antibody-dependent cell-mediated cytotoxicity (ADCC) or Tc cell responses 80. The implication of lipoxygenase products in the cytotoxic process is further suggested by the impairment of cytotoxicity by lipoxygenase inhibitors such as nordihydroguaiaretic acid (NDGA) or BW755C and not by cyclooxygenase linhibitors such as indomethacin 8a'86'9°.In fact, the latter class of inhibitors can 'redirect' the metabolism ofarachidonic acid through the lipoxygenase pathway, giving rise to enhanced LT or HETE production 9' and enhanced cytotoxic activity 85'9~. At least some of the immunomodulatory effect of LTB4 could be ascribed to its action on cyclic nucleotides. While PGs are known to activate adenylate cyclase, giving rise to increased intracellular levels of cAMP and subsequent inhibition of various leukocyte functions, LTs appear to stimulate cGMP 93 or even to functionally replace cGMp84. 9~. It is thus quite evident that lipoxygenase metabolites of arachidonic acid, and especially LTB~, play a major role in regulation of the immune response through numerous and varied effects on several cell types (summarized in Table I). A better understanding of these processes should prove useful for appropriate management of immune derangement. [] References 1 Resch, K. (1976) in Receptors and Recognition, Vol. 1 (Cuatrecasas, P. and Greaves, M. F. eds), pp. 61-117, Chapman & Hall 2 Ben'idge, M. J. (1982) Calcium Cell Funct.3, 1-10 3 Samuelsson, B., Borgeat, P. and Murphy, R. C. (1979) Prostaglandins 17, 785-787 4 Borgeat, P. and Samuelsson, B. (1979) Proc. Natl Acad. ScL USA 76, 2148-2152 5 Borgeat, P. and Samuelsson, B. (1979)J. BioL Chem. 254, 2643-2646 6 Borgeat, P. and Samuelsson, B. (1979)J. Biol. Chem. 254, 7865-7869 7 Radmark, O., Malmsten, C., Samuelsson, B., Clark, D. A., Gots, G., Marfat, A. and Corey, E. J. (1980) Biochem. Biophys. Res. Commun. 92, 954-961 8 Ford-Hutchinson, A. W., Smith, M. J. H. and Bray, M. A. (1981) J. Pharm. Pharmacol. 33, 332 9 Hammarstrom, S. and Samuelsson, B. (1980) FEBSLett. 122, 83-86 10 Murphy, R. C., Hammarstrom, S. and Samuelsson, B, (1979) Proc. Natl Acad. SoL USA 76, 4275-4279 11 Morris, H. R., Taylor, G. W., Piper, P. J., Samhoun, M. N. and Tippins, J. R. (1980) Prostaglandins 19, 185-201 12 Lewis, R. A., Austen, K. F., Drazen, J. M., Clark, D. M., Marfat, A. and Corey, E. J. (1980)Proc. NatlAcad. Sci. USA 7"1, 3710-3714 13 Orning, L., Hammarstrom, S. and Samuelsson, B. (1980) Proc. Natl Acad. Sci. USA 77, 2014-2017 14 Parker, C. W., Falkenheim, S. F. and Humber, M. M. (1980) Prostaglandins 20, 836-886

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T u m - variants: immunogenic variants obtained by mutagen treatment of tumor cells Thierry Boon Mutagen treatment of mouse tumor cells produces at high frequency stable tumor cell variants that are rejected by syngeneic mice. As Thierry Boon discusses here, these 'turn-' variants express new suoCace antigens that can be recognized by cytolytic T lymphocytes. Tum- variants derived from spontaneous mouse tumors for which no immunogenicity had hitherto been demonstrated induce an immune protection against the parental tumor. Finally, he poses some interesting questions for future investigations. Over the past few years, it has become increasingly clear that, by treating mouse tumor cell cultures with mutagenic compounds, it is possible to obtain, at high frequency, tumor cell variants expressing new surface antigens. These variants elicit a rejection response in the syngeneic host 1. They have been named ' t u r n - ' because, unlike the 'turn +' cell from which they are derived, they fail to form progressive tumors. Here we review the main features of the turn- variants. Production of variants with increased immunogenicity W h e n clonal mouse tumor cell lines are exposed in vitro to the potent mutagen N-methyl-N'-nitro-N-nitrosoguanidine ( M N N G ) , the surviving cell population contains variants that are unable to form progressive tumors in normal adult syngeneic animals (Fig. 1). Such turn

Ludwig Institute for Cancer Research, 74 avenue Hippocrate, UCL 74.59, B-1200 Brussels, Belgium and Cellular Genetics Unit, Catholic University of Louvain, Brussels, Belgium.

variants have been obtained from many different mouse tumor cell lines, including teratocarcinoma 2, Lewis lung carcinoma 3, mastocytoma P8154, several spontaneous leukemias 5 and an adenoacanthoma 6. With a dose of M N N G allowing for approximately 0.1% survival of the initial cells, the frequency of turn- variants among the survivors usually ranges from 1 to 20 %. Repeated mutagenic treatments increase the frequency of variants. For instance, the frequency obtained with P815 was equal to 1, 20 and 95% after 1, 3 and 8 exposures to M N N G respectively 7. A large number of variants have been obtained that retain the t u m - phenotype during several months of continuous culture. For most of these variants, no change in morphology or growth rate was observed in vitro, and no gross karyotype alterations were noticed 24. Recently, the proteins of several variants derived from mastocytoma P815 were analysed in two-dimensional gel electrophoresis. From the thousand or more spots that could be clearly distinguished, at most one or two differences were .observed between turn + and turn- cells (K. Willard, ,personal communication). (~ 1985,ElsevierSciencePublishersB.V.,Amsterdam 0167- 4919/85/$02.00