Immunology Today

January 1982

Interferon

From clones to clinic J o h n Morse r rep,rl.s ~?ore arl interTmliona/ cong~e.ss ,m interferort, held in San Francisco, Orloher 21-2&

Progress towards an understanding of pected is still there'. The unforseen the molecular biology of interferon has has emerged in the effects of inter- been spectacular but the pace of ferons (IFNs) on virus growth and on clinical research has been slower. I. lymphocyte circulation in ~,iro, their Gresser (Villeneuf) opened the con- role in inducing disease, and their gress with the Kurt Paucker memorial ability to return the transformed phe- lecture on the theme that ' the unex- notype to normal. The congress was to

Induced ceils

18S mRNA known to code for IFN- 7

Reverse transcdptase; DNA polymerase I I I I I I I Double-stranded DNA

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O Plasmids with DNA insert

Plasmids inserted into E. coil

nitrocellulose discs

Induced Non induced Colonies lysed. DNA sticks to surface

(~" <%?["{~"~ DNA hybridized with radioactive DNA probe k ;~ j from induced . . . . . . . induced cells. Autoradiography

I?ndUN2dc2PeCg flc r/FINn ~/id e n t i fie d" ~ DNAe i$$~d I / ~ N_n ~ production assayed

Cloning of 3,-interferon

Pregnancy

Protection and prevention A m a m m a l m a k e s a n i m m u n e response to he r a l loant igenic fetus, bu t does not reject it. Are there lessons in this for the control of graft re ject ion? A n d can i m m u n e responses be d i rec ted to the control of fe r t i l i ty , by l i m i t i n g g a m e t e p roduc t ion or the surv iva l of ear ly embryos? Ques t ions like this are at the hear t of the cu r r en t surge of interest in the i m m u n o l o g y of re- p roduc t ion . A conference on the subject was he ld in Banff, C a n a d a , in M a y . B h a g i r a t h S i n g h a n d Phi l l ip Gambe l repor t here on some of the highl ights . R. Billingham (Dallas) opened the meeting with a review of recent find- ings in transplantation immunology, emphasizing the role of the endo- thelium and passenger lymphocytes in graft rejection, and ~)arious attempts to avoid rq}ection by modifying them. However, the fetal allograft is sited at the placenta , and the p lacenta ' s immunological privilege seems to be

c cmld. on p.3

hear of addi t ions to this list of unexpected and paradoxical .phe- nomena.

The power of the new molecular techniques was vividly illustrated by D. Goeddel (Genentech, San Francisco) in his account of the cloning and expression of IFNs. Yields of 6 x 10 '~ IFN-131 and IFN-% in bacteria and 3 x 10 ¢' molecules/cell in yeast (8% of protein) were reported. Goeddel also described in some detail ( s u m m a r i z e d in ' t he figure) the strategy used by the Genentech team to clone for the first time the DNA sequence coding for IFN-y, the IFN species produced by lymphoid cells after st imulation by antigens or mito- gens. m R N A was extracted from lymphocytes treated with the IFN

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Immunology To@ vol..3, No. 1 1982

From clones to clinic cun[d.

inducers staphylococcal enterotoxin B and thymosin at, purified and used to construct a library of complementary cDNA. Clones were identified which hybridized only to the probe derived from induced Iymphoeytes. All of these clones had the same restriction map but varied in length. The longest was sequenced and shown to encode a polypeptide 166 amino acids long, of which 20 amino acids could he a signal sequence. The sequence also contained a long 3' untranslated region and two glycosylation sites. When the fragment was expressed it was shown to code for IFN-y. The fragment has been used as a probe to investigate the gene structure, and preliminary evidence suggests that there may he only one IFN-y gene, which contains an intron.

These results implied that IFN-y exists as a single species but those who had tried to purify IFN-y agreed that their preparations showed hetero- geneity. Yip (New York) had found two forms with moh wt of 20,000 and 25,000 and a small amount with tool. wt 45,000 and suggested that IFN-y was derived from a multigene family. W. Stewart (Tampa) found the same molecular heterogeneity and sugges- ted there were IFN types additional to those known at present.

The availability of cloned human IFN sequences has allowed investiga- tions of the expression of IFN genes in eukaryotic cells - both cloned genes transferred into cells and the endoge- nous IFN genes already there. P. Pitha (Baltimore) and J. Collins (Braun- schweig) have both made a series of mouse LTK cells cotransformed with TK + and human IFN-[3 gene sequences. Both found that the genes were inducible with virus but their results on the control of IFN-[3 expres- sion dill?red. Pitha has Mso studied the control of IFN expression by endogenous genes in human cells and observed that the decay of IFN-[3 syn- thesis after induction requires con- tinuing protein synthesis. She has some evidence that the size of the IFN- [3 mRNA was shortened by 100 nucleotides from 1100 to 1000 bases

during the course of induction. P. Sehgal (New York) discussed the mul- tiple IFN-[3 mRNA species detectable in human fibroblasts by separation on denaturing gels and microinjection into Xenopus oocytes. Only one of these (13,) hybridizes with the cloned IFN-13 gene. Use of mouse/human hybrids has allowed the assignment of the novel [3s to chromosome 2 or 5 while [3, is on chromosome 9, thus resolving the argument about chromosome alloca- tion.

Two new types of study on the IFN receptor were reported. D. Slate has made monoclonal antibodies to the human IFN c~ and 13 receptor. Mice were inoculated with mouse/human hybrids that contain only human chromosome 21 (which codes for the IFN receptor). The monoclonals block the action of IFN-~ and IFN-[3 but not IFN-y, suggesting that the IFN-a and IFN-[3 share a receptor which is different from the receptor for IFN-y.

Two groups (Estell (San Francisco) and Zoon (Bethesda)) have used dif- tErent protocols to study the binding of labelled IFN-c~ to cells and so calculate the number of receptors on a cell. Their results were very similar: 300-1100 IFN receptors/cell. Human IFN-[3 inhibited the binding of IFN-c~, but neither 2¢:lerminal nor c-terminal fragments of IFN-c~ competed with the binding of intact IFn-a.

The use of mouse models to study the mode of action of IFN was the sul~ject of several papers. O. Haller (Zurich) described work on the Mx locus, which specifically affects the effectiveness of IFN towards ortho- myxo viruses in mice, but not other viruses. The effects of the My locus were observed both in vivo and in vitro, suggesting that the antiviral state is composed of distinct functions, one of which is modified or regulated by the Mx gene.

The defence of mice against Herpes simplex virus (HSV) was discussed by H. Kirschner (Heidelberg). He has shown a correlalion between natural killer (NK) cell activity and HSV resistance in different strains of mice. He believes that NK activation is a secondary effect of IFN and not the p r imary difference between the

strains, and thus may be less relevant for antiviral defense than is early production of IFN. N. Stebbing (San Francisco) has shown that the hybrid human IFN Le A/D (%/a~) is active in mice. It protects against L1210 leukemia, but appears not to act directly on L1210 cells. Nor were these cells a target for NK lysis. He suggested that indirect effects, possibly antibody-dependent cell- mediated lysis, were important in the antitumor effect.

IFN produced by bacteria is now being used in clinical trials. J. Gutter- man (Houston) and ttorning (Stan- ford) reported on their experience with IFN-% (Le-A). Its side effects were similar to those of partially purified IFN prepared from leuko- eytes, but since more was available much larger doses could be admin- istered. The maximum serum level increased with increasing doses, while the half life remained the same. Lymphocyte numbers decreased 24 h after treatment with IFN while cell- surface 132-microglobulin rose at 24 h and returned to normal 72 h after treatment. In this phase I trial of 16 patients seven demonstrated objective evidence of tumor regression during the study.

L. Levine (Waltham) described the successful use of IFN against acute viral infections such as herpes sim- plex, fulminant hepatitis and ence- phalitis, and D. Tyrrell (Harrow) its application against rhinovirus 9. But the most unexpected clinical results were described in a paper by a group from Roswell Park Memorial Institute who have been conducting a trial on IFN-13 against multiple sclerosis. The patients had been randomized before entry to the trial and treated with IFN-[32 intrathecally. The treated group showed an improvement in the number of exacerbations (p 0.01) and in clinical symptoms (p 0.036), com- pared with the control group. The authors expressed cautious optimism for this use of IFN. (see Science 1981, 214, 1026)

Jr~h~7 Mor.~er ia irt lhe Departme~Tt ~/ Bzo/ogical Scleme~, Univer~@, ~/ Warwick, (,'~Jverdry C'V4 7AL, U.K.

Immunology Today vol. 3, No. 1 1982 3

Protection and prevention - contd.

the fetus's chief protection fi'om the maternal immune system. The mother makes both humoral and cellular immune responses to the paternally derived fetal M H C antigens - re- actions that, as T. Gill (Pittsburgh) pointed out, may be important to the success of a viviparous pregnancy. How this happens, however, is not yet clear. There is evidence that allo- antigens are expressed on mouse eggs a n d ear ly e m b r y o s (S. Heyne r , PhiladeLphia). M H C antigens first appear on the inner cell mass which forms the embryo proper, and later are found on the trophoblast. A few minor h is tocompat ib i l i ty an t igens have been studied, and as a rule they tend to be expressed at all t imes in embryonic development except at the two-cell stage. Heyner suggested that these antigens may provide signals for diiterentiation. S. WachteI (New York) suggested that H-Y antigen might have a similar role. This anti- gen seems to be directly involved in primary sex differentiation and is re- markably well conserved dur ing evolution, always in association with the heterogametic sex, be it male or female.

M. Edidin (Baltimore) described the signals controlling differentiation of cell types within the mouse terato- carcinoma cell line Ter C. This cell line expresses certain ant igens - 'oncofetal an t igens ' - which are present only on testicular cells and sperm and are completely absent from the adult female. Oncofetal antigens are also found in the early embryo but disappear a few days after implan- tation. Kemler (Paris) discussed the results of his studies with the terato- carcinoma cell line which carries the F9 antigen. Antibodies to the F9 anti- gen prevent compaction, which occurs at a specific stage of early embryonic development. It should be noted that Kemler 's monoclonal antibodies crossreacted with adult and embry- onic t i ssues . R e g a r d l e s s of the function of the oncofetal antigens, it is possible that antibodies to these anti- gens might inhibit pregnancy in mice.

The placenta Placental tissue may contain clues

to the survival of the fetal allograft. Within it fetally derived trophoblastic tissue is in direct contact with the maternal blood circulation. T. Weg-

m a n n (Edmonton), reviewing tropho- blastic tissue's unique immunological status, mentioned the recent studies of J. Rossant and colleagues with recip- rocal embryo transplants of Mus caroli a n d Mus rnusculus. (Cellular and Molecular Aspects of Implantation, S. R. Glasser and D. Bullock, eds. Plenum, in press.) They have shown that a xenogeneic inner cell mass can gen- erate an adult mouse, but only if pro- tected by a trophoblast of the same species as the mother. This obser- vation has been exploited to produce interspecific chimeras.

Where in the placenta are pater- nally derived M H C antigens ex- pressed? D. Billington (Bristol, U . K ) has demonstrated their presence in vitro on dissociated cells from spon- giotrophoblast in the murine pla- centa. These cells are susceptible to killing in vitro by cytotoxic cells but not in vivo, even when the pregnant mouse is primed against fetal antigens. He also found strain differences in the h u m o r a l i m m u n e r e s p o n s e of pregnant mice to the fetal allograft. B. Singh (Edmonton) , us ing mono- clonal antibodies, has detected class I M H C antigens (H-2K and H-2D anti- gens) on the mouse placenta but not class 1I antigens (/-region-coded anti- gens). The placenta can thus ap- parently absorb out antifetal anti- bodies from the maternal blood as it bathes the placenta, thereby preventing these potentially detri- mental antibodies from entering the fetal circulation. The Fc portion of the anti-paternal H-2 antibody was not required for this binding. Radioauto- graphic studies have revealed paternal M H C antigens on the lateral aspects of the placenta, where the yolk sac inserts, and also on the spongio- trophoblast layer which is in direct contact with maternal circulation. P. Faulk (East Grinstead, U.K.) defined two new trophoblastic antigens, TA-1 and TA-2, in the human placenta, which are potent inhibitors of the mixed lymphocyte reaction. Although he could not locate M H C antigens on the trophoblast tissue between the maternal and fetal circulations in h u m a n placenta, he did identify a series of new antigens, the T L X series, which exist on both trophoblast and leucocytes. He provided interesting evidence that M H C disparity in- fluences fetal survival, i.e., women who have chronic abortions have a greater sharing of HLA antigens with their husbands than would be pre-

dicted by chance alone. Faulk has been able to bring three pregnancies successfully to term by immunizing these women with cells compatible for blood group antigens but incom- patible for HLA antigens.

The placenta has also been seen as providing a means for setting the maternal immune response into a balance between rejection and 'facili- tation', which, normally, allows the fetus to survive to term. G. Voisin (Paris) holds this view and presented data supporting the action of blocking factors and suppressor cells in main- taining this balance.

However, it must be remembered, as J. Enders (Davis, Calitornia) em- phasized, that information about the structure of the placenta during im- plantat!on and its subsequent matur- ation is available from a number of different species Stress on observa- tions made in mouse and man misses a greater part of the spectrum of pla- cental diversity.

Immunoactive cells and substances in pregnancy

There is a growing list of preg- nancy-associated substances which suppress or inhibit immune respon- ses. 17[3-estradiol can do both, in physiological concent ra t ions (W. Stimson, Glasgow). Stimson has iso- lated two other pregnancy-associated substances, a prostaglandin synthe- tase inhibitor which could account for the d iminished it~flammatory re- sponse associated with pregnancy, and a 131-microglobulin which has an additional potential in monitoring ovarian cancers. One of the most ex- tensively studied and controversial p r e g n a n c y - r e l a t e d i m m u n o r e g u - latory substances is alpha-fetopro- tein. Discussing its properties, T. Tomasi (Albuquerque) explained that technical factors in its preparation, such as the presence or absence of copper, may account for the variable results in its immunosuppres s ive propert!es reported in the literature. He also reported that murine am- niotic fluid inhibits antigen-presenting ceils.

Natural killer cells are absent in neonates - a significant observation because they appear to have a pre- ference for 'embryonic-type' antigens, H. Wigzall (Uppsala) postulated that either a lpha-fe toprote in or a de- ficiency of macrophages mediating interferon production could be re- sponsible for this. Killing by T cells

directed to tetal antigens may be regu- lated locally, at the maternal-fetal interface. Clark (Hamilton, Canada) presented evidence for non-specific suppressive activity among cells from lymph ~aodes draining the pregnant uterus and for a soluble suppressor activity in the draining lymph nodes in allogeneic pregnancies. But W. Jones (Bedford Park, Australia) and P. Matangkasombut (Bangkok, Thai- land) have found little evidence for generalized non-specific suppression during human pregnancy which could affect the survival of the fetus. The complex mechanisms involved in the success of pregnancy are still largely obscure and effects seen at the 'whole- animal' level must be distinguished from the pe rhaps more crucial mechanisms active at local level which allow fetal survival.

Regulation of ferti l i ty The natural consequences of both

natural and induced immune re- sponses against spermatozoa, ova and proteins associated with pregnancy are abortion and autoimmune dis- orders of the reproductive system. Induced immunity is intended to regulate fertility without inducing autoimmunity. K. Tung (Albuquer- que) discussed various models of auto- immunity involving the testis and con-

cluded that multiple pathogenic mechanisms are operating in tes- ticular autoimmunity in infertile black mink, A-line beagles and W18 back- cross mice. The production of anti- sperm antibodies after vasectomy is genetically controlled. He suggested that vaccination against pregnancy in a population might increase the re- productive capacity of those indi- viduals who are low responders to the vaccine. The consequences of auto- immuni ty after vasectomy were discussed in greater detail by N. Alexander (Beaverton, Oregon). She described the presence of antisperm autoantibodies in vasectomised mon- keys and arterial damage presumably due to immune complex formation and deposition on vascular tissues.

Human chorionic gonadotropin (hCG), a protein hormone necessary for the maintenance of pregnancy in its earliest stage, has been a can- didate pregnancy vaccine for some years. G. Talwar (New Delhi, India) has used the beta chain of hCG as a vaccine in animals and in women and he reviewed some of the difficulties associated with its poor immuno- genicity in certain individuals. He also described other potential approaches for fertility regulation, including the use of luteinising hormone-releasing hormone and the use of intrates-

Immunology Today vol. 3, .No. 1 1982

ticular iI!jections of BCG vaccine. V. Stevens (Columbus, Ohio) presented a detailed immunochemicaI study of the C terminal peptide of the beta chain of hCG and his efforts to make it immunogen ic enough to inhibi t fertility. E. Goldberg (Evanston, Illinois) presented data on the struc- ture and immunochemistry of a sperm-specific antigen, LDH-X, a variant of the enzyme lactate dehydro- genase. Immunization of baboons against this sperm-specific isozyme reduces their fertility. Antibodies to a synthetic peptide fragment of LDH-X can bind to the native enzyme on sper- matozoa, and this might be a usefial way to block fertilization. Antigens on the zona pellucida can also be used to immunize against fertility (N. Dun- bar, New York) and recent methods for the preparation of individual zonal glycoproteins should refine this ap- proach in the near future.

The abstracts of papers presented at this meeting have been published (o7. Repro& [ramun. 1981 (suppl.) S1-$46). The com- plete proceedings will appear in Reprodue- tzve Immunology Eds. T. G. Wegmann and T. J. Gill), Oxford University Press, New York 1982.

Bhagirath Singh and Phillip I. Gambel are in the Deparlment of Immunology and MRC Group on hnmunoregulation, Universily of Alberta, Ed- monton, Alberta, Canada T6G 2117

X] ) The state of transfer factor

Myer R. Salaman

Department of Immunology, St. Mary's Hospital Medical School, London W2 1PG, U . K

The condi t ion of t ransfer factor (TF) has been giving cause for concern for some years now. Here is an immunologica l phenomenon , first repor ted in 1954, which seems s t ra ight forward enough and has received a great deal of at tent ion. Yet in 1982 we are still arguing about it at a most basic level. Happi ly , a recent con t r ibu t ion fi'om Borkowsky and Lawrence l offers a way forward fi'om this impasse.

Skin react ions of the delayed type (peaking at 24-48 h) are dependen t on ci rculat ing T lymphocytes of the appropr i a t e sub-set that are sensitive to the test ant igen and the capaci ty to r e spond can thus be t rans- ferred in a sui table an imal sys tem by injection of

© Elsevi~i giomedicM Press 1982 0167-4919/82/0000-0000/$275

viable blood leucocytes. T h e chal lenge of T F has been the claim that , in man, t ransfer of delayed hyper- sensitivity from sensitive donor to negative recipient can be b rought about by a dialysable extract of these cells. But is sensitivity really t ransfer red specifically by a factor in the extract? An al ternat ive view is that in the recipient the extract may amplify a pre-exis t ing low-level sensitivity in a m a n n e r unre la ted to the sensitivities of the donor. Some would concede nei ther of these a t t r ibutes !

This curious state of affairs is not ha rd to explain. Because no bt-vivo animal model or irt-~ilro sys tem yields the striking results r epor ted in man, it has been