297
Immunology Today, vol. 6, No. 10, 1985
in screening for expression of single epitopes recognised by monoclonal antibodies is particularly striking and such a combination of monoclonal antibody and recombinant D N A technologies represents a potent approach to further analysis of mycobacterial antigens. The asd promoter in the plasmid vector ofClark-Curtiss et al. may be essential for enhancement of expression of particular proteins. These authors found no evidence for expression of recombinant genes in the absence of a strong nonmycobacterial promoter but, while the 3~gtl1 vector does contain a/3-galactosidase promoter, preliminary results suggest that expression of some mycobacterial antigens is independent of the//-galactosidase gene (D.B. Young, unpublished). Further study of the antigens coded by the recombinant mycobacterial D N A can proceed by a variety of strategies. In some cases the antigen may he present as a fusion protein linked to fl-galactosidase and purification by immunoaffinity chromatography using anti-fl-galactosidase antibody may be possible. In other cases, where E. coli recognizes mycobacterial transcription or ribosome binding sequences, the antigen may be synthesized as a free protein identical to that found in the native mycobacteria and immunoaffinity chromatography using monoclonal antibodies will be most productive. Alternatively, subcloning of restriction fragments may be used to identify segments of D N A coding for particular epitopes and synthetic peptides based on nucleotide sequences may be produced without recourse to protein purification. With the application of these novel developments in antigen production and purification, detailed investigation of the possible role of individual antigens in stimulation or suppression of cellular immunity can be under-
taken. Isolated proteins or peptides can be tested for their ability to induce T-cell proliferation and delayed-type hypersensitivity reactions, and possible modulation of the course of infection in experimental animals following vaccination with purified components can be assessed. It is hoped that such studies will lead to the development of improved diagnostic reagents for early detection of leprosy and tuberculosis and, in the longer term, to the production of effective anti-mycobacterial vaccines. ~-1 References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
World Health Organization. (1983) Bull. WHO 61, 779-785 Bloom, B. R. and Godal, T. (1983) Rev. Infect. DIS. 5, 765-780 Medical Research Council. (1972) Bull. WHO 46, 371-376 World Health Organization. (1980) WHO TechnicalReport Series, No. 651 Kirchheimer, W. F. and Storrs, E. E. (1971) Int. J. Lepr. 39, 693-702 Fox, W. (1980) in Recent Advances in Respiratory Medicine (D. C. Flenley ed.) p. 183, Churchill-Livingstone, Edinburgh and London Young, D. B. and Buchanan, T. M. (1983) Science 221, 1057-1059 Cho, S. N., Fujiwara, T., Hunter, S. W., Rea, T. H., Gelber, R. H. and Brennan, P. J.. (1984)J. Infect. Dis. 150, 311-322 Ma, Y. and Daniel, T. M. (1983)3. Infect. Dis. 148, 500-509 Coates, A. R. M., Allen, B. W., Hewitt, J., Mitchison, D. A. and Ivanyi, J. (1981) Lancet ii, 167-169 Gillis, T. P. and Buchanan, T. M. (1982) Infect. Immun. 37, 172-178 Ivanyi, J., Sinha, S., Aston, R., Cussel, D., Keen, M, and Sengupta, U. (1983) Clin. Exp. Immunol. 52, 528-536 Young, D. B., Fohn, M . J . , Khanolkar, S. R. and Buchanan, T. M. (1985) Clin. Exp. Immunol. 60, 546-552 Engers, H. D. et al. (1985). Infect. Immun. 48, 603-605 Young, R. A., Bloom, B. R., Grosskinsky, C. M., Ivanyi, J. and Davis, R. W. (1985)Proc. NatlAcad Sci. USA 82, 2583-2587 Young, R. A., Mehra, V., Sweetser, D., Buchanan, T., Clark-Curtiss, J,, Davis, R. W. and Bloom, B. R. (1985) Nature (London) 316, 450-452 Clark-Curtiss, J. E., Jacobs, W. R., Docherty, M. A., Ritchie, L. R. and Curtiss, R. (1985)J. Bacteriol. 161, 1093-1102 DOUGLAS B. YOUNG
M R C Tuberculosis and Related Infections Unit, Hammersmith Hospital, Ducane Road, London W12 OHS, UK.
Immunoregulation of MHC antigen expression It is now apparent that the expression of major histocompatibility complex (MHC) antigens on the cell surface is not constant but a variable phenomenon which may depend on the immune status of the individual. It is often stated that class II M H C antigens can be present constitutively (as on B cells, activated T cells, dendritic cells and macrophages) or they may be induced. In rats, epidermal keratinocytes and intestinal epidermal cells, normally negative for class II antigens, become positive during a graft versus host response 1'2. Similarly, in rats and mice class II antigens are induced on vascular endothelial cells in transplanted hearC and skin ~. Studies in vitro have demonstrated that gamma interferon (IFN-lt) is a potent inducer of class II antigens on human endothelial cells 5 and on a number of human 6 and murine cell lines 7. Supernatant from phytohaemagglutinin (PHA)- or alloantigen-activated T cells causes induction of class II antigens on human umbilical vein 5 presumably by the action of IFN-),, but the possibility that other lymphokines cause M H C induction cannot be excluded. Detailed studies of the distribution of class II antigens in human organs are possible using frozen sections, monoclonal antibodies and immunofluorescent 0 or immunoenzymatic 9'1° techniques. Such studies have
demonstrated considerable variation in antigen expression within organs and between individuals. For example, in a study of 46 normal kidneys, all expressed class II antigen on the endothelium of glomeruli, but in only 60% of the kidneys was tubular epithelium class II positive 9. Similarly, in another investigation 1° vascular endothelium from capillaries was found to be consistently class II positive in all organs (except brain, testis and placenta) but large blood vessels showed variable expression within an organ. The possibility therefore arises that some of this variation in 'normal tissue' is due to immune stimulation of unknown origin and some of it may be caused by the observer, depending on whether the procedures for sampling material is traumatic1°. A recent paper suggests this is the case for expression of class II antigens on canine vascular endothelium 11. The authors treated normal dogs with large doses of cyclosporin A and found that such treatment abrogated the expression of class II antigens on endothelial cells of kidney and skin. The dog resembles humans i n ' normally' expressing class II antigens on the capillary endothelial cells of skin and the glomerular capillary endothelium of the kidney. The exact mode of action of cyclosporin A is unknown, but one © 1985,ElsevierSciencePublishersB.V., Amsterdam 0167 4919/85/$02.00
298
of its effects is to inhibit production of IFN-), by activated T cells ~2. The authors concluded that endothelial expression of class II antigens in species where endothelinm is 'normally' positive is lymphokine dependent. They also suggest that this effect of cyclosporin A on class II expression is one of the explanations for its powerful immunosuppressive properties. The issue of the constitutive nature of endothelial class II antigens is an important one for transplant immunologists. It is known that class II antigens on dendritic cells are powerful stimulators of the anti-allograft response 13, and it has been suggested that class II antigen on endothelial cells may similarly be able to initiate graft rejection 14. If, as the authors suggest, endothelial expression of class II in man is not constitutive, but is lymphokine dependent, it follows that an immunosuppression regimen which includes cyclosporin A should suppress expression of class II on endothelial cells and thus render the graft less immunogenic. Unfortunately, the clinical data do not support this reasoning. M y colleagues and I have results 15 which demonstrate strong expression of class II antigens on the capillary endothelium of the transplanted heart of cardiac transplant patients, at a time when they are receiving high doses of cyclosporin A (18mg/kg) and in the absence of a histologically defined rejection episode. T lymphocytes expressing class II antigens are found in the blood of bone marrow ~6 and cardiac ~7 transplant recipients who are receiving cyclosporin A immun0suppression. It may be that in patients receiving cyclosporin A class II expression is modulated. A comparison between patients receiving cyclosporin A and those on conventional immunosuppression (steroids and azathioprine) might be helpful. It is notable that the dogs in this study were said to have blood levels of cyclosporin A (0.2-0.4 mg/ml) a thousand times in excess of that found in transplant patients (200-400 ng/ml). However, in a personal communication, G. Groenewegen has disclosed that the level quoted in his paper was a printing error and should have read 0.2-0.4 mgL. Thus, dogs and patients absorbed similar amounts of cyclosporin A. It would be helpful to know whether the cyclosporin A effect in dogs is dose dependent. M H C class II expression is notoriously species specific and it is with considerable circumspection that conclusions derived from animal experiments should be applied to humans. Most of the literature on induction of M H C antigens concerns class II antigens, but it is now clear that class I antigens are also inducible in vitro and in vivo. It is often stated that 'all nucleated cells express class I antigens'. Such a statement is based on studies using cell suspension
Immunology Today, vol. 6, No. 10, 1985
as targets and allo- or heteroantisera as cytotoxic reagents. However, immunocytochemical techniques using frozen sections and monoclonal antibodies against non-polymorphic determinants have revealed that in humans a number of tissues, including myocardium and skeletal muscle, show little or no expression of class I ~ antigens rag. ExperimentaP and clinical studies 15 have reporte d induction of class I antigen on the myocardium of the transplanted heart. A recent study has also reported expression of class I antigens on dystrophic human skeletal muscle 2°. It seems likely, therefore, that the question of whether class II antigen expression is constitutive or induced may equally apply to class I antigens. However, as in the case for class II antigens, the expression of class I antigens on various tissues is highly species dependent, and conclusions drawn from experimental studies may not be valid for man. [~ References 1 Lampert, J. A., Suitters, A. J. and Chisholm, P. M. (1981) Nature (London) 293, 149-150 2 Barclay, A. N. and Mason, D. W. (1982)J. Exp. Med. 156, 1665-1676 3 Milton, A. D. and Fabre, J. W. (1985),]. Exp. Med. 161, 98-112 4 De Waal, R. M. W., Bogman, M. J.J., Mass, C. N., Cornelissen, L. M. H., Tax, W . J . M . and Keone, R. A. P. (1983) Nature(London) 303, 426-429 5 Pober, J. S., Gimbrone, M. A., Cotran, K. S., Reiss, L. S., Burakoff, S. J., (1983)J. Exp. Med. 157, 1339-1353 6 Virelizier, J. L., Perez, N., Arenzana-Seisdedos, F. and Devos, R. (1984) Eur. J. Immur~l 14, 106-108 7 Wong, G. H. W., Clark-Lewis, I., Harris, A. W. and Schroeder, J. W. (1984) Eur. J. ImmunoL 14, 52-56 8 Natali, P. G., Martino. C., Quaranta, V., Nicotra, M. R., Frezza, F.,I (1981) Transplantation 31, 75-78 9 Fuggle, S. V., Errasti, P., Daar, S. V., Fabre, J. w., Ting, A. and Morris, P. J. (1983) Transplantation 35, 385-390 10 Daar, A. S., Fuggle, S. V., Fabre, J. W., Ting, A. and Morris, P.J., (1984) Transplantation 38, 293-297 11 Groenewegen, G., Buurman, W. A. and Van der Linden, C.J. (1985)' Nature (London) 316, 361-363 12 Reem, G. H., Cook, L. A. and Vildek, J. (1983) Science221, 63-65 13 Leehler, R. J. and Batchelor, J. R. (1982)J. Exp. Med. 155, 31-41 14 Hirsehberg, H., Bereh, O.J. and Thorsby, E. (1980).]. Exp. Med. 152, 249-255 15 Rose, M. L., Coles, M. I., Griffin, R.J., Pomerance, A. and Yaeous, l M. H. Transplantation in press 16 Forrnan, S.J., Gallagher, M. L, Zaia, J. A., Wright, C. and Blume, K. G. (1984) Transplantation 37, 219-220 17 O'Toole, C. M., Carvalho, G. S., Cranage, M. P. and Large, S. (1984) Clin. Exp. ImmunoL 58, 671-678 18 Daar, A: S., Fuggle, S. V., Fabre, J. W., Ting, A. and Morris, P.J. (1984) Transplantation 38, 287-292 19 Natali, P. G., Bigotti, A., Nicotra, M. R., Viora, M., Manfredi, D. (1984) CancerRes. 44, 4679-4684 20 Appleyard, S. T., Dunn, M.J., Dubowitz, V. and Rose, M. L. (1985) Lancet i 361-363 MARLENE L. ROSE Harefidd Hospital, Harefield, Middlesex UB9 6JH, UK.
Immunology Today, vol. 6, No. 10, 1985
in screening for expression of single epitopes recognised by monoclonal antibodies is particularly striking and such a combination of monoclonal antibody and recombinant D N A technologies represents a potent approach to further analysis of mycobacterial antigens. The asd promoter in the plasmid vector ofClark-Curtiss et al. may be essential for enhancement of expression of particular proteins. These authors found no evidence for expression of recombinant genes in the absence of a strong nonmycobacterial promoter but, while the 3~gtl1 vector does contain a/3-galactosidase promoter, preliminary results suggest that expression of some mycobacterial antigens is independent of the//-galactosidase gene (D.B. Young, unpublished). Further study of the antigens coded by the recombinant mycobacterial D N A can proceed by a variety of strategies. In some cases the antigen may he present as a fusion protein linked to fl-galactosidase and purification by immunoaffinity chromatography using anti-fl-galactosidase antibody may be possible. In other cases, where E. coli recognizes mycobacterial transcription or ribosome binding sequences, the antigen may be synthesized as a free protein identical to that found in the native mycobacteria and immunoaffinity chromatography using monoclonal antibodies will be most productive. Alternatively, subcloning of restriction fragments may be used to identify segments of D N A coding for particular epitopes and synthetic peptides based on nucleotide sequences may be produced without recourse to protein purification. With the application of these novel developments in antigen production and purification, detailed investigation of the possible role of individual antigens in stimulation or suppression of cellular immunity can be under-
taken. Isolated proteins or peptides can be tested for their ability to induce T-cell proliferation and delayed-type hypersensitivity reactions, and possible modulation of the course of infection in experimental animals following vaccination with purified components can be assessed. It is hoped that such studies will lead to the development of improved diagnostic reagents for early detection of leprosy and tuberculosis and, in the longer term, to the production of effective anti-mycobacterial vaccines. ~-1 References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
World Health Organization. (1983) Bull. WHO 61, 779-785 Bloom, B. R. and Godal, T. (1983) Rev. Infect. DIS. 5, 765-780 Medical Research Council. (1972) Bull. WHO 46, 371-376 World Health Organization. (1980) WHO TechnicalReport Series, No. 651 Kirchheimer, W. F. and Storrs, E. E. (1971) Int. J. Lepr. 39, 693-702 Fox, W. (1980) in Recent Advances in Respiratory Medicine (D. C. Flenley ed.) p. 183, Churchill-Livingstone, Edinburgh and London Young, D. B. and Buchanan, T. M. (1983) Science 221, 1057-1059 Cho, S. N., Fujiwara, T., Hunter, S. W., Rea, T. H., Gelber, R. H. and Brennan, P. J.. (1984)J. Infect. Dis. 150, 311-322 Ma, Y. and Daniel, T. M. (1983)3. Infect. Dis. 148, 500-509 Coates, A. R. M., Allen, B. W., Hewitt, J., Mitchison, D. A. and Ivanyi, J. (1981) Lancet ii, 167-169 Gillis, T. P. and Buchanan, T. M. (1982) Infect. Immun. 37, 172-178 Ivanyi, J., Sinha, S., Aston, R., Cussel, D., Keen, M, and Sengupta, U. (1983) Clin. Exp. Immunol. 52, 528-536 Young, D. B., Fohn, M . J . , Khanolkar, S. R. and Buchanan, T. M. (1985) Clin. Exp. Immunol. 60, 546-552 Engers, H. D. et al. (1985). Infect. Immun. 48, 603-605 Young, R. A., Bloom, B. R., Grosskinsky, C. M., Ivanyi, J. and Davis, R. W. (1985)Proc. NatlAcad Sci. USA 82, 2583-2587 Young, R. A., Mehra, V., Sweetser, D., Buchanan, T., Clark-Curtiss, J,, Davis, R. W. and Bloom, B. R. (1985) Nature (London) 316, 450-452 Clark-Curtiss, J. E., Jacobs, W. R., Docherty, M. A., Ritchie, L. R. and Curtiss, R. (1985)J. Bacteriol. 161, 1093-1102 DOUGLAS B. YOUNG
M R C Tuberculosis and Related Infections Unit, Hammersmith Hospital, Ducane Road, London W12 OHS, UK.
Immunoregulation of MHC antigen expression It is now apparent that the expression of major histocompatibility complex (MHC) antigens on the cell surface is not constant but a variable phenomenon which may depend on the immune status of the individual. It is often stated that class II M H C antigens can be present constitutively (as on B cells, activated T cells, dendritic cells and macrophages) or they may be induced. In rats, epidermal keratinocytes and intestinal epidermal cells, normally negative for class II antigens, become positive during a graft versus host response 1'2. Similarly, in rats and mice class II antigens are induced on vascular endothelial cells in transplanted hearC and skin ~. Studies in vitro have demonstrated that gamma interferon (IFN-lt) is a potent inducer of class II antigens on human endothelial cells 5 and on a number of human 6 and murine cell lines 7. Supernatant from phytohaemagglutinin (PHA)- or alloantigen-activated T cells causes induction of class II antigens on human umbilical vein 5 presumably by the action of IFN-),, but the possibility that other lymphokines cause M H C induction cannot be excluded. Detailed studies of the distribution of class II antigens in human organs are possible using frozen sections, monoclonal antibodies and immunofluorescent 0 or immunoenzymatic 9'1° techniques. Such studies have
demonstrated considerable variation in antigen expression within organs and between individuals. For example, in a study of 46 normal kidneys, all expressed class II antigen on the endothelium of glomeruli, but in only 60% of the kidneys was tubular epithelium class II positive 9. Similarly, in another investigation 1° vascular endothelium from capillaries was found to be consistently class II positive in all organs (except brain, testis and placenta) but large blood vessels showed variable expression within an organ. The possibility therefore arises that some of this variation in 'normal tissue' is due to immune stimulation of unknown origin and some of it may be caused by the observer, depending on whether the procedures for sampling material is traumatic1°. A recent paper suggests this is the case for expression of class II antigens on canine vascular endothelium 11. The authors treated normal dogs with large doses of cyclosporin A and found that such treatment abrogated the expression of class II antigens on endothelial cells of kidney and skin. The dog resembles humans i n ' normally' expressing class II antigens on the capillary endothelial cells of skin and the glomerular capillary endothelium of the kidney. The exact mode of action of cyclosporin A is unknown, but one © 1985,ElsevierSciencePublishersB.V., Amsterdam 0167 4919/85/$02.00
298
of its effects is to inhibit production of IFN-), by activated T cells ~2. The authors concluded that endothelial expression of class II antigens in species where endothelinm is 'normally' positive is lymphokine dependent. They also suggest that this effect of cyclosporin A on class II expression is one of the explanations for its powerful immunosuppressive properties. The issue of the constitutive nature of endothelial class II antigens is an important one for transplant immunologists. It is known that class II antigens on dendritic cells are powerful stimulators of the anti-allograft response 13, and it has been suggested that class II antigen on endothelial cells may similarly be able to initiate graft rejection 14. If, as the authors suggest, endothelial expression of class II in man is not constitutive, but is lymphokine dependent, it follows that an immunosuppression regimen which includes cyclosporin A should suppress expression of class II on endothelial cells and thus render the graft less immunogenic. Unfortunately, the clinical data do not support this reasoning. M y colleagues and I have results 15 which demonstrate strong expression of class II antigens on the capillary endothelium of the transplanted heart of cardiac transplant patients, at a time when they are receiving high doses of cyclosporin A (18mg/kg) and in the absence of a histologically defined rejection episode. T lymphocytes expressing class II antigens are found in the blood of bone marrow ~6 and cardiac ~7 transplant recipients who are receiving cyclosporin A immun0suppression. It may be that in patients receiving cyclosporin A class II expression is modulated. A comparison between patients receiving cyclosporin A and those on conventional immunosuppression (steroids and azathioprine) might be helpful. It is notable that the dogs in this study were said to have blood levels of cyclosporin A (0.2-0.4 mg/ml) a thousand times in excess of that found in transplant patients (200-400 ng/ml). However, in a personal communication, G. Groenewegen has disclosed that the level quoted in his paper was a printing error and should have read 0.2-0.4 mgL. Thus, dogs and patients absorbed similar amounts of cyclosporin A. It would be helpful to know whether the cyclosporin A effect in dogs is dose dependent. M H C class II expression is notoriously species specific and it is with considerable circumspection that conclusions derived from animal experiments should be applied to humans. Most of the literature on induction of M H C antigens concerns class II antigens, but it is now clear that class I antigens are also inducible in vitro and in vivo. It is often stated that 'all nucleated cells express class I antigens'. Such a statement is based on studies using cell suspension
Immunology Today, vol. 6, No. 10, 1985
as targets and allo- or heteroantisera as cytotoxic reagents. However, immunocytochemical techniques using frozen sections and monoclonal antibodies against non-polymorphic determinants have revealed that in humans a number of tissues, including myocardium and skeletal muscle, show little or no expression of class I ~ antigens rag. ExperimentaP and clinical studies 15 have reporte d induction of class I antigen on the myocardium of the transplanted heart. A recent study has also reported expression of class I antigens on dystrophic human skeletal muscle 2°. It seems likely, therefore, that the question of whether class II antigen expression is constitutive or induced may equally apply to class I antigens. However, as in the case for class II antigens, the expression of class I antigens on various tissues is highly species dependent, and conclusions drawn from experimental studies may not be valid for man. [~ References 1 Lampert, J. A., Suitters, A. J. and Chisholm, P. M. (1981) Nature (London) 293, 149-150 2 Barclay, A. N. and Mason, D. W. (1982)J. Exp. Med. 156, 1665-1676 3 Milton, A. D. and Fabre, J. W. (1985),]. Exp. Med. 161, 98-112 4 De Waal, R. M. W., Bogman, M. J.J., Mass, C. N., Cornelissen, L. M. H., Tax, W . J . M . and Keone, R. A. P. (1983) Nature(London) 303, 426-429 5 Pober, J. S., Gimbrone, M. A., Cotran, K. S., Reiss, L. S., Burakoff, S. J., (1983)J. Exp. Med. 157, 1339-1353 6 Virelizier, J. L., Perez, N., Arenzana-Seisdedos, F. and Devos, R. (1984) Eur. J. Immur~l 14, 106-108 7 Wong, G. H. W., Clark-Lewis, I., Harris, A. W. and Schroeder, J. W. (1984) Eur. J. ImmunoL 14, 52-56 8 Natali, P. G., Martino. C., Quaranta, V., Nicotra, M. R., Frezza, F.,I (1981) Transplantation 31, 75-78 9 Fuggle, S. V., Errasti, P., Daar, S. V., Fabre, J. w., Ting, A. and Morris, P. J. (1983) Transplantation 35, 385-390 10 Daar, A. S., Fuggle, S. V., Fabre, J. W., Ting, A. and Morris, P.J., (1984) Transplantation 38, 293-297 11 Groenewegen, G., Buurman, W. A. and Van der Linden, C.J. (1985)' Nature (London) 316, 361-363 12 Reem, G. H., Cook, L. A. and Vildek, J. (1983) Science221, 63-65 13 Leehler, R. J. and Batchelor, J. R. (1982)J. Exp. Med. 155, 31-41 14 Hirsehberg, H., Bereh, O.J. and Thorsby, E. (1980).]. Exp. Med. 152, 249-255 15 Rose, M. L., Coles, M. I., Griffin, R.J., Pomerance, A. and Yaeous, l M. H. Transplantation in press 16 Forrnan, S.J., Gallagher, M. L, Zaia, J. A., Wright, C. and Blume, K. G. (1984) Transplantation 37, 219-220 17 O'Toole, C. M., Carvalho, G. S., Cranage, M. P. and Large, S. (1984) Clin. Exp. ImmunoL 58, 671-678 18 Daar, A: S., Fuggle, S. V., Fabre, J. W., Ting, A. and Morris, P.J. (1984) Transplantation 38, 287-292 19 Natali, P. G., Bigotti, A., Nicotra, M. R., Viora, M., Manfredi, D. (1984) CancerRes. 44, 4679-4684 20 Appleyard, S. T., Dunn, M.J., Dubowitz, V. and Rose, M. L. (1985) Lancet i 361-363 MARLENE L. ROSE Harefidd Hospital, Harefield, Middlesex UB9 6JH, UK.
