292
Immunology Today, vol. 3, ,No. 11, 1982
(,.-,...
)
Hybridomas produce viruses as well as antibodies In a recent letter to the N e w EnglandJourrzal of Medicine, BartaI et al. 1 expressed concern over the clinical use of mouse monoclonal antibodies because the authors observed by electron microscopy that their hybridomas produced particles characteristic of retroviruses. Many mouse cell lines synthesize retrovirus particles and the plasmacytomas used for hybridoma fusion with spleen cells are no exception. Retroviruses comprise a large group of RNA viruses, isolated from all classes of vertebrate, ranging from bony fish to mammals 2. While many appear harmless, others induce autoimmune and neurological diseases, anaemia, leukaemia, lymphoma and other tumours. Retroviruses can persist in latent form for long periods by an ingenious strategy: upon infection a DNA copy or 'provirus' of the RNA genome is made by the viral enzyme reverse transcriptase and this provirus then becomes inserted into the chromosomal DNA of the host cell. On occasion during vertebrate evolution proviruses have been integrated into germline DNA, with the result that genetic information previously transmitted in an infectious virus becomes a host Mendelian trait 3. The inherited, 'endogenous' viral genomes of most host species are apathogenic but they can be activated to produce infectious viruses again by diverse stimuli, especially in cells of the lymphoid system. Mice have accumulated many copies of Mendelian proviruses and several distinct types of retrovirus may be expressed in hybridoma cells. First, plasmacytomas, especially of B A L B / c mice 4, synthesize
intracysternal A-type particles (Fig. la); these are incomplete, non-infectious particles and their RNA represents the major species of poly-A-tagged RNA in the cytoplasm, more abundant even than immunoglobulin m R N A $. Second, B-type viruses, which cause murine mammary carcinoma have been reported in T-lymphoma lines ~ and might also be expressed in B cells. Third, C-type particles are produced at the cell surface (Fig. lb, c); these are released by some plasmacytomas 7 although in my experience C-type viruses are produced more frequently and abundantly by hybridoma cells. Bartal et al. ~ observed both A-type and C-type particles in hybridomas established by fusing P3U1 plasmacytoma cells with B A L B / c spleen cells. We have observed similar particles in cells derived from fusions of NS-1 and B A L B / c spleen cells. For example, we have examined 12 independently isolated hybridoma clones producing antibodies to vesicular stomatitis virus antigens and 5 hybridomas specific to various human cell surface antigens. The NS-1 parental cells, though full of A-type particles, released no detectable C-type particles, whereas 12 of the 17 hybridomas released large quantities of C-type particles assayed by electron microscopy or by reverse transcriptase activity in the culture medium 8. Activated, endogenous, C-type viruses of mice are frequently infectious and fall into two maior host range groups 2,9, those that infect and replicate in mouse cells (known as ecotropic) and those that replicate only in cells of other species (known as
b
Fig. 1. Electron micrographs of retrovirus particlesseenin thin sections, a. lntracisternal A-type particles in NS-1 cell (x 122,000). b. Budding and mature C-type particles in NS-1 × BALB/c spleen hybridoma (D2) (×122,000). c. C-type particles in P3U1 x BALB/c spleen hybridoma (× 112,000). (a, b kindly provided by D. RobertsonU;c, by C. FeitL) © Elsevier Biomedical Ptess 1982 0 1 6 7 4 919/82/000(} 13(]00/$1.0/)
bnmunology 7))day, vol. ,3, ~¢~. 11, 1982
xenotropic). The xenotropic viruses will grow to yield substantial titres in foreign cells while their serial propagation as infectious agents is severely restricted in the host species in which they are sequestered as Mendelian proviruses. Infectivity studies of our 12 virus-positive hybridomas revealed that 8 produced virus infectious for h u m a n cells (7605L strain of embryonic lung fibroblasts), 2 produced virus infectious for mouse SC-I cells, a n d 2 produced a mixture of ecotropic and xenotropic viruses s. One hybridoma released as m u c h as 10 s-8 xenotropic infectious units per ml culture medium. Xenotropic mouse C-type viruses are not known to be pathogenic, but certain genetic recombinants between ecotropic and xenotropic viruses hasten the development of thymic l y m p h o m a in susceptible mice. Similar recombinant viruses have been reported in plasmacytomas 7. I a m not aware whether any hybridomas have been examined for the production of recombinant viruses. It is not surprising that different hybridomas produce different C-type viruses as each spleen cell fused to the plasmacytoma cell may already have been producing ecotropic, xenotropic or recombinant virus. T h e preponderance of xenotropic virus production in immunologically active cells, however, is consistent with previous studies of virus activation. T r e a t m e n t of B A L B / c spleen cells with B-cell mitogens *°, mixed lymphocyte reactions in vilro u, a n d the induction of graft-versus-host activity in vivo 1~,~2 all activate the production of xenotropic virus. Indeed, the expression of C-type viral envelope antigen gp70 (if not whole virus) on the cell surface, appears to be a regular feature of normal murine lymphoid cell m a t u r a t i o n and Moroni and S c h u m a n n 13 have postulated that retroviraI gp70 plays an essential role in B-lymphocyte differentiation. Bartal el al. ~ raise the question whether the use of mouse monoclonal antibodies in h u m a n s is safe in view of the propensity of hybridomas to produce retroviruses. The clinical use of monoclonal antibodies is, of course, in its infancy and will undoubtedly burgeon as techniques for antigen-targeted imaging and therapy become refined. T h e exploitation of monoclonal antibodies in bone marrow t r a n s p l a n t a t i o n is already becoming practical, e.g. the removal of metastatic tumour cells for autochthonous transplants, and of T cells from allogeneic transplants. Should we not be worried about a possible hazard to health over the production of retroviruses by hybridomas? The easy answer is that no hospital medical ethics committee or national drug administration agency would sanction the clinical use of crude hybridoma fluids. Monoclonal antibodies must be purified and even simple affinity purification procedures should remove or inactivate all rctrovirus particles. However, monitoring whether such procedures were, indeed, efficient could be m u c h more arduous. The infusion of even low titres of virus into patients would clearly be unacceptable.
293 What, then, of the safety of laboratory personnel engaged in the manufacture of monoclonal antibody production through h y b r i d o m a cultivation? The chance of exposure to xenotropic retroviruses is real, though no more so than in the culture of other mouse cells and perhaps less t h a n in xenograft procedures and h u m a n - m o u s e somatic: cell hybridization which I have discussed previously 14. Given good microbiological technique, I think the risks of h u m a n infection, let alone the induction of disease, by murine retroviruses, are very small. Mice and men have lived together for a long time, yet there is no serological evidence that either the h u m a n populace at large or laboratory personnel become infected with murine retroviruses. Cats are also frequently infected with retroviruses infectious for h u m a n cells, a n d they secrete high titres of virus in the saliva 2. Again there is no evidence of natural h u m a n cross-infection. H u m a n plasma contains a complement factor, C l q , which inactivates m a n y species of retrovirus by viral memb r a n e lysis in the absence of antibody ~5 - a n interesting p h e n o m e n o n indicative of the viral gp70 having an immunoglobufin-fike property recognized by C l q but it is not known whether this is protective against natural infection by animal retroviruses. Recently, a h u m a n C-type virus, HTLV, has been isolated from the t u m o u r cells of adult patients with a particular kind of m a t u r e T-cell lymphomaleukaemia 16,~7,~s. H T L V appears to be a natural h u m a n pathogen of world-wide distribution, but with high endemic areas in South-western J a p a n and the Caribbean. H T L V is not related antigenically or genetically to murine a n d other animal retroviruses 2 (it will be interesting to know whether h u m a n C l q binds to H T L V envelope glycoprotein). T h e clinical use of blood donated by asymptomatic H T L V carriers ~9,2° presents a m u c h greater risk to h u m a n health than the administration of purified mouse monoclonal antibodies. R.A. WEISS"
lmtilute of Cancer Research, CT~esler]3early Laboralories, tVulham Road, London SW3 6JB, U.K.
References 1 Bartal, A. H., Feit, C., Erlandson, R. and Hirshaut, Y. (1982) N. EnglJ. Med. 306, 1423 2 Weiss, R,, Teich, N., Varmus, H. and Coffin, J. (1982) R,NA Tumor Virtues, Cold Spring Harbor Laboratory 3 Weiss, R. A. (1982) in Virus Persistence (Malay, B. W. J., Minson, A. C. and Darby, G. K., eds), pp. 267-288, Cambridge University Press 4 Kuff, E. L., Wivel, N. A. and Lueders, K. K. (1968) CancerRe.< 28, 2137-2148 50no, M., Cole, M. D., White, A. T. and Huang, R. C. C. (1980) Cetl2t, 465-473 6 Vaidya, A. B., Long, C. A., Sheffield, J. B., Tamura, A. and Tanaka, H. (1980) Virology 104, 279-293 7 Spriggs, D. R., Diebold, M. A. and Krueger, R. G. (1981) J. ViroL 36, 541-546 8 Weiss, R. A..N. Er~gIJ. Med. (in press) 9 Levy,J. A. (1978) Curr. Top. Microbiol. bnmunol. 79, 109-113 I0 Moroni, C. and Schumann, G. (1975) Nafure (London) 254, 60 61
294
Immunology 7"oday,vol. 3, No. /1, 1982
11 Sherr, C. J., Lieber, M. M. and Todaro, G. J. (1974) Cell 1, 55-58 12 Levy, J. A., Datta, S. K. and Schwartz, R. S. (1977) Clin. Immunol. Immunopathol. 7,262-268 13 Moroni, C. and Schumann, G. (1977) 3&lure (London) 269, 600-601 14 Weiss, R. (1975) Nature (London) 255,445-447 15 Cooper, N. R., Jensen, F. C., Welsh, R. M. and Oldstonc, M. B. A. (1976)J. Exp. Med. 144, 970-984 16 Poiesz, B. J., Ruscetti, F. W., Gazdar, A. F., Bunn, P. A., Minna, J. D. and Gallo, R. C. (1980) Proc. Nail Acad. Sci. U.S.A.
77, 7415-7419 17 Miyoshi, I., Kubonishi, I., Yoshimoto, S., Akagi, T., Ohtsuki, Y., Shiraishi, Y., Nagata, K. and Hinuma, Y. (1981) Nature (London) 294, 770-771 18 Catovsky, D~, Greaves, M. F., Rose, M., Galton, D. A. G., Goolden, A. W. C,., McCluskey, D. R., White, J. M., Lampert, I., Bourikas, G., ireland, R., Bridges, J. M., Blattner, W. A. and Gallo, R. C. (1982) Lancet i, 639-643 19 Miyoshi, 1., Fujishita, M., Taguchi, H., Ohtsuki, Y., Akagi, T., Morimoto, Y. M. and Nagasaki, A. (1982) Lancet i, 683 684 20 Saxinger, W. C. and Gallo, R. C. (1982) Lar~ceIi, 1074
W h e r e do autoantibodies come from? A few years ago, the story of the origin of a u t o i m m u n e thyroiditis seemed to be settledk Roitt and Torrigiani 2 showed that thyroglobulin (Tg) normally circulates in concentrations of 10 to 50 ng ml ~, so maintaining low zone tolerance, i.e. tolerance at the T-cell (but not the B-cell) level. To initiate autoantibody production, it was necessary to replace the deleted T cells; this could be done by giving a polyclonal B-cell activator, or a cross-reactive or altered Tg antigen. Antibodyforming cells accumulated in the thyroid where they caused damage directly or indirectly. But now there is experimental evidence which challenges the view that T-cell deletion is necessarily the basis of self-tolerance to Tg. A recent publication by W e e t m a n et al. 3 describes the distribution of cells engaged in producing antibodies to Tg in rats with a u t o i m m u n e thyroiditis. W e e t m a n el al. induced thyroiditis in the rats in two different ways. Some were injected with rat Tg in Freund's complete adjuvant; these rats developed a transient disease that remitted unless periodic booster injections were given. O t h e r rats were thymectomized (Tx) a n d treated several times with whole-body irradiation (X); these ' T x plus X ' animals developed a sustained severe form of thyroiditis with hypothyroidism. In both forms of the a u t o i m m u n e disease, the major site of autoantibody production was the bone marrow. The majority of ' T x plus X ' animals also h a d anti-Tg plaque-forming cells in the thyroid at the time of examination. The latter finding must be taken with some reservation, however, because timing may be all-important. Antibody-forming cells may be present in the thyroid earlier or later in the course of the disease. Nevertheless, these findings are in stark contrast with the reports of Clinton and Weigle 4, and Boyd a n d Wick 5 which show numerous antibody-forming cells in the thyroids of rabbits with experimentally induced thyroiditis, a n d chickens with spontaneous thyroiditis, respectively. In fact, W e e t m a n el al/' also showed that anti-T c plaque-forming cells could be found in the thyroids of h u m a n s with Hashimoto's thyroiditis. T h e ' T x plus X ' model of a u t o i m m u n e thyroiditis is also particularly instructive in re-evaluating the T-cell deletion concept of self-tolerance to Tg. T h e most logical explanation for the development of autoimmunity in the ' T x plus X ' rats is that thymectomy
and irradiation reduces a population of thymusderived suppressor ceils, and permits Tg-specific helper T ceils to express themselves. Evidence supporting this view was given by A. M. McGregor, one of the W e c t m a n ' s collaborators, at a recent Thyroid Workshop in Edmonton, Alberta, held on 24-25 July 1982. He showed t h a t ' T x plus X ' rats treated with cyclosporin A show a decrease in the helper:suppressor T-cell ratio, a n d do not develop autoimmune thyroiditis. At the same meeting in Edmonton, W. Weigle proffered new findings that point directly to the presence of Tg-reactive T cells in good responder strains of mice. He found that mouse Tg stimulated the lymphnode cells of animals with experimental thyroiditis. These results confirm the earlier publication of Okayasu el al. ('. Furthermore, Weigle showed that the responding cells were Lyt 1+,2-,3 . O f course, the stimulation indices were quite low compared with those obtained with foreign antigens like ovalbumin or heterologous Tg. But we must realize that only two to six of the forty to sixty antigenic determinants on Tg act as autoantigens. Another similar line of evidence has emerged from the studies of K0jima et al. 7 in J a p a n . They found that thyroiditis develops spontaneously in certain strains of mice if the animals are thymectomized on the third day after birth. It seems that neonatal thymectomy preferentially reduces suppressor T cells and permits Tg-specific helper T cells to predominate. It remains to identify and characterize the putative suppressor-cell populations a n d determine whether they are Tg-specific or idiotype-specific. The existence of such populations may well explain why rats given rat Tg in adjuvant recover from thyroiditis spontaneously, as the balance between helper and suppressor function returns to normal. A u t o i m m u n e diseases in h u m a n s often remit spontaneously too. More important, if we can find conditions that favor suppressor-cell proliferation, we will have a new and specific way of treating a u t o i m m u n e disorders. N O E L R. R O S E
D@artment of Immunology and lnjectious Diseases, 7tze Johns Hopkins University Sehool of Hygiene and Public Health, l?,allimore, MD 27205, U.S.A.
Immunology Today, vol. 3, ,No. 11, 1982
(,.-,...
)
Hybridomas produce viruses as well as antibodies In a recent letter to the N e w EnglandJourrzal of Medicine, BartaI et al. 1 expressed concern over the clinical use of mouse monoclonal antibodies because the authors observed by electron microscopy that their hybridomas produced particles characteristic of retroviruses. Many mouse cell lines synthesize retrovirus particles and the plasmacytomas used for hybridoma fusion with spleen cells are no exception. Retroviruses comprise a large group of RNA viruses, isolated from all classes of vertebrate, ranging from bony fish to mammals 2. While many appear harmless, others induce autoimmune and neurological diseases, anaemia, leukaemia, lymphoma and other tumours. Retroviruses can persist in latent form for long periods by an ingenious strategy: upon infection a DNA copy or 'provirus' of the RNA genome is made by the viral enzyme reverse transcriptase and this provirus then becomes inserted into the chromosomal DNA of the host cell. On occasion during vertebrate evolution proviruses have been integrated into germline DNA, with the result that genetic information previously transmitted in an infectious virus becomes a host Mendelian trait 3. The inherited, 'endogenous' viral genomes of most host species are apathogenic but they can be activated to produce infectious viruses again by diverse stimuli, especially in cells of the lymphoid system. Mice have accumulated many copies of Mendelian proviruses and several distinct types of retrovirus may be expressed in hybridoma cells. First, plasmacytomas, especially of B A L B / c mice 4, synthesize
intracysternal A-type particles (Fig. la); these are incomplete, non-infectious particles and their RNA represents the major species of poly-A-tagged RNA in the cytoplasm, more abundant even than immunoglobulin m R N A $. Second, B-type viruses, which cause murine mammary carcinoma have been reported in T-lymphoma lines ~ and might also be expressed in B cells. Third, C-type particles are produced at the cell surface (Fig. lb, c); these are released by some plasmacytomas 7 although in my experience C-type viruses are produced more frequently and abundantly by hybridoma cells. Bartal et al. ~ observed both A-type and C-type particles in hybridomas established by fusing P3U1 plasmacytoma cells with B A L B / c spleen cells. We have observed similar particles in cells derived from fusions of NS-1 and B A L B / c spleen cells. For example, we have examined 12 independently isolated hybridoma clones producing antibodies to vesicular stomatitis virus antigens and 5 hybridomas specific to various human cell surface antigens. The NS-1 parental cells, though full of A-type particles, released no detectable C-type particles, whereas 12 of the 17 hybridomas released large quantities of C-type particles assayed by electron microscopy or by reverse transcriptase activity in the culture medium 8. Activated, endogenous, C-type viruses of mice are frequently infectious and fall into two maior host range groups 2,9, those that infect and replicate in mouse cells (known as ecotropic) and those that replicate only in cells of other species (known as
b
Fig. 1. Electron micrographs of retrovirus particlesseenin thin sections, a. lntracisternal A-type particles in NS-1 cell (x 122,000). b. Budding and mature C-type particles in NS-1 × BALB/c spleen hybridoma (D2) (×122,000). c. C-type particles in P3U1 x BALB/c spleen hybridoma (× 112,000). (a, b kindly provided by D. RobertsonU;c, by C. FeitL) © Elsevier Biomedical Ptess 1982 0 1 6 7 4 919/82/000(} 13(]00/$1.0/)
bnmunology 7))day, vol. ,3, ~¢~. 11, 1982
xenotropic). The xenotropic viruses will grow to yield substantial titres in foreign cells while their serial propagation as infectious agents is severely restricted in the host species in which they are sequestered as Mendelian proviruses. Infectivity studies of our 12 virus-positive hybridomas revealed that 8 produced virus infectious for h u m a n cells (7605L strain of embryonic lung fibroblasts), 2 produced virus infectious for mouse SC-I cells, a n d 2 produced a mixture of ecotropic and xenotropic viruses s. One hybridoma released as m u c h as 10 s-8 xenotropic infectious units per ml culture medium. Xenotropic mouse C-type viruses are not known to be pathogenic, but certain genetic recombinants between ecotropic and xenotropic viruses hasten the development of thymic l y m p h o m a in susceptible mice. Similar recombinant viruses have been reported in plasmacytomas 7. I a m not aware whether any hybridomas have been examined for the production of recombinant viruses. It is not surprising that different hybridomas produce different C-type viruses as each spleen cell fused to the plasmacytoma cell may already have been producing ecotropic, xenotropic or recombinant virus. T h e preponderance of xenotropic virus production in immunologically active cells, however, is consistent with previous studies of virus activation. T r e a t m e n t of B A L B / c spleen cells with B-cell mitogens *°, mixed lymphocyte reactions in vilro u, a n d the induction of graft-versus-host activity in vivo 1~,~2 all activate the production of xenotropic virus. Indeed, the expression of C-type viral envelope antigen gp70 (if not whole virus) on the cell surface, appears to be a regular feature of normal murine lymphoid cell m a t u r a t i o n and Moroni and S c h u m a n n 13 have postulated that retroviraI gp70 plays an essential role in B-lymphocyte differentiation. Bartal el al. ~ raise the question whether the use of mouse monoclonal antibodies in h u m a n s is safe in view of the propensity of hybridomas to produce retroviruses. The clinical use of monoclonal antibodies is, of course, in its infancy and will undoubtedly burgeon as techniques for antigen-targeted imaging and therapy become refined. T h e exploitation of monoclonal antibodies in bone marrow t r a n s p l a n t a t i o n is already becoming practical, e.g. the removal of metastatic tumour cells for autochthonous transplants, and of T cells from allogeneic transplants. Should we not be worried about a possible hazard to health over the production of retroviruses by hybridomas? The easy answer is that no hospital medical ethics committee or national drug administration agency would sanction the clinical use of crude hybridoma fluids. Monoclonal antibodies must be purified and even simple affinity purification procedures should remove or inactivate all rctrovirus particles. However, monitoring whether such procedures were, indeed, efficient could be m u c h more arduous. The infusion of even low titres of virus into patients would clearly be unacceptable.
293 What, then, of the safety of laboratory personnel engaged in the manufacture of monoclonal antibody production through h y b r i d o m a cultivation? The chance of exposure to xenotropic retroviruses is real, though no more so than in the culture of other mouse cells and perhaps less t h a n in xenograft procedures and h u m a n - m o u s e somatic: cell hybridization which I have discussed previously 14. Given good microbiological technique, I think the risks of h u m a n infection, let alone the induction of disease, by murine retroviruses, are very small. Mice and men have lived together for a long time, yet there is no serological evidence that either the h u m a n populace at large or laboratory personnel become infected with murine retroviruses. Cats are also frequently infected with retroviruses infectious for h u m a n cells, a n d they secrete high titres of virus in the saliva 2. Again there is no evidence of natural h u m a n cross-infection. H u m a n plasma contains a complement factor, C l q , which inactivates m a n y species of retrovirus by viral memb r a n e lysis in the absence of antibody ~5 - a n interesting p h e n o m e n o n indicative of the viral gp70 having an immunoglobufin-fike property recognized by C l q but it is not known whether this is protective against natural infection by animal retroviruses. Recently, a h u m a n C-type virus, HTLV, has been isolated from the t u m o u r cells of adult patients with a particular kind of m a t u r e T-cell lymphomaleukaemia 16,~7,~s. H T L V appears to be a natural h u m a n pathogen of world-wide distribution, but with high endemic areas in South-western J a p a n and the Caribbean. H T L V is not related antigenically or genetically to murine a n d other animal retroviruses 2 (it will be interesting to know whether h u m a n C l q binds to H T L V envelope glycoprotein). T h e clinical use of blood donated by asymptomatic H T L V carriers ~9,2° presents a m u c h greater risk to h u m a n health than the administration of purified mouse monoclonal antibodies. R.A. WEISS"
lmtilute of Cancer Research, CT~esler]3early Laboralories, tVulham Road, London SW3 6JB, U.K.
References 1 Bartal, A. H., Feit, C., Erlandson, R. and Hirshaut, Y. (1982) N. EnglJ. Med. 306, 1423 2 Weiss, R,, Teich, N., Varmus, H. and Coffin, J. (1982) R,NA Tumor Virtues, Cold Spring Harbor Laboratory 3 Weiss, R. A. (1982) in Virus Persistence (Malay, B. W. J., Minson, A. C. and Darby, G. K., eds), pp. 267-288, Cambridge University Press 4 Kuff, E. L., Wivel, N. A. and Lueders, K. K. (1968) CancerRe.< 28, 2137-2148 50no, M., Cole, M. D., White, A. T. and Huang, R. C. C. (1980) Cetl2t, 465-473 6 Vaidya, A. B., Long, C. A., Sheffield, J. B., Tamura, A. and Tanaka, H. (1980) Virology 104, 279-293 7 Spriggs, D. R., Diebold, M. A. and Krueger, R. G. (1981) J. ViroL 36, 541-546 8 Weiss, R. A..N. Er~gIJ. Med. (in press) 9 Levy,J. A. (1978) Curr. Top. Microbiol. bnmunol. 79, 109-113 I0 Moroni, C. and Schumann, G. (1975) Nafure (London) 254, 60 61
294
Immunology 7"oday,vol. 3, No. /1, 1982
11 Sherr, C. J., Lieber, M. M. and Todaro, G. J. (1974) Cell 1, 55-58 12 Levy, J. A., Datta, S. K. and Schwartz, R. S. (1977) Clin. Immunol. Immunopathol. 7,262-268 13 Moroni, C. and Schumann, G. (1977) 3&lure (London) 269, 600-601 14 Weiss, R. (1975) Nature (London) 255,445-447 15 Cooper, N. R., Jensen, F. C., Welsh, R. M. and Oldstonc, M. B. A. (1976)J. Exp. Med. 144, 970-984 16 Poiesz, B. J., Ruscetti, F. W., Gazdar, A. F., Bunn, P. A., Minna, J. D. and Gallo, R. C. (1980) Proc. Nail Acad. Sci. U.S.A.
77, 7415-7419 17 Miyoshi, I., Kubonishi, I., Yoshimoto, S., Akagi, T., Ohtsuki, Y., Shiraishi, Y., Nagata, K. and Hinuma, Y. (1981) Nature (London) 294, 770-771 18 Catovsky, D~, Greaves, M. F., Rose, M., Galton, D. A. G., Goolden, A. W. C,., McCluskey, D. R., White, J. M., Lampert, I., Bourikas, G., ireland, R., Bridges, J. M., Blattner, W. A. and Gallo, R. C. (1982) Lancet i, 639-643 19 Miyoshi, 1., Fujishita, M., Taguchi, H., Ohtsuki, Y., Akagi, T., Morimoto, Y. M. and Nagasaki, A. (1982) Lancet i, 683 684 20 Saxinger, W. C. and Gallo, R. C. (1982) Lar~ceIi, 1074
W h e r e do autoantibodies come from? A few years ago, the story of the origin of a u t o i m m u n e thyroiditis seemed to be settledk Roitt and Torrigiani 2 showed that thyroglobulin (Tg) normally circulates in concentrations of 10 to 50 ng ml ~, so maintaining low zone tolerance, i.e. tolerance at the T-cell (but not the B-cell) level. To initiate autoantibody production, it was necessary to replace the deleted T cells; this could be done by giving a polyclonal B-cell activator, or a cross-reactive or altered Tg antigen. Antibodyforming cells accumulated in the thyroid where they caused damage directly or indirectly. But now there is experimental evidence which challenges the view that T-cell deletion is necessarily the basis of self-tolerance to Tg. A recent publication by W e e t m a n et al. 3 describes the distribution of cells engaged in producing antibodies to Tg in rats with a u t o i m m u n e thyroiditis. W e e t m a n el al. induced thyroiditis in the rats in two different ways. Some were injected with rat Tg in Freund's complete adjuvant; these rats developed a transient disease that remitted unless periodic booster injections were given. O t h e r rats were thymectomized (Tx) a n d treated several times with whole-body irradiation (X); these ' T x plus X ' animals developed a sustained severe form of thyroiditis with hypothyroidism. In both forms of the a u t o i m m u n e disease, the major site of autoantibody production was the bone marrow. The majority of ' T x plus X ' animals also h a d anti-Tg plaque-forming cells in the thyroid at the time of examination. The latter finding must be taken with some reservation, however, because timing may be all-important. Antibody-forming cells may be present in the thyroid earlier or later in the course of the disease. Nevertheless, these findings are in stark contrast with the reports of Clinton and Weigle 4, and Boyd a n d Wick 5 which show numerous antibody-forming cells in the thyroids of rabbits with experimentally induced thyroiditis, a n d chickens with spontaneous thyroiditis, respectively. In fact, W e e t m a n el al/' also showed that anti-T c plaque-forming cells could be found in the thyroids of h u m a n s with Hashimoto's thyroiditis. T h e ' T x plus X ' model of a u t o i m m u n e thyroiditis is also particularly instructive in re-evaluating the T-cell deletion concept of self-tolerance to Tg. T h e most logical explanation for the development of autoimmunity in the ' T x plus X ' rats is that thymectomy
and irradiation reduces a population of thymusderived suppressor ceils, and permits Tg-specific helper T ceils to express themselves. Evidence supporting this view was given by A. M. McGregor, one of the W e c t m a n ' s collaborators, at a recent Thyroid Workshop in Edmonton, Alberta, held on 24-25 July 1982. He showed t h a t ' T x plus X ' rats treated with cyclosporin A show a decrease in the helper:suppressor T-cell ratio, a n d do not develop autoimmune thyroiditis. At the same meeting in Edmonton, W. Weigle proffered new findings that point directly to the presence of Tg-reactive T cells in good responder strains of mice. He found that mouse Tg stimulated the lymphnode cells of animals with experimental thyroiditis. These results confirm the earlier publication of Okayasu el al. ('. Furthermore, Weigle showed that the responding cells were Lyt 1+,2-,3 . O f course, the stimulation indices were quite low compared with those obtained with foreign antigens like ovalbumin or heterologous Tg. But we must realize that only two to six of the forty to sixty antigenic determinants on Tg act as autoantigens. Another similar line of evidence has emerged from the studies of K0jima et al. 7 in J a p a n . They found that thyroiditis develops spontaneously in certain strains of mice if the animals are thymectomized on the third day after birth. It seems that neonatal thymectomy preferentially reduces suppressor T cells and permits Tg-specific helper T cells to predominate. It remains to identify and characterize the putative suppressor-cell populations a n d determine whether they are Tg-specific or idiotype-specific. The existence of such populations may well explain why rats given rat Tg in adjuvant recover from thyroiditis spontaneously, as the balance between helper and suppressor function returns to normal. A u t o i m m u n e diseases in h u m a n s often remit spontaneously too. More important, if we can find conditions that favor suppressor-cell proliferation, we will have a new and specific way of treating a u t o i m m u n e disorders. N O E L R. R O S E
D@artment of Immunology and lnjectious Diseases, 7tze Johns Hopkins University Sehool of Hygiene and Public Health, l?,allimore, MD 27205, U.S.A.
