228
immunology today, December 1981
The expression of Ir genes
Macrophage or T cell the question
that is
Zoltan A. Nagy and Jan Klein Max-Planck-Institut f/.ir Biologie, Abteilung Immungenetik, Corrensstrasse 42, 7400 Tiibingen, F.R.G. At the fourth International Congress of Immunology held in Paris in 1980, Rosenthal was able to state without a single dissenting voice from the audience that 'the immune-response genes function at the level of the macrophage.' The long debate about the site of Ir-gene expression thus appeared to have been closed: animals are non-responders because 'of an intrinsic defect in Ir-gene product function in the nonresponder m a c r o p h a g e ' and ' m a c r o p h a g e s and lymphocytes must share genetic identity at some portion of the major histocompatibility complex (MHC) for successful detection of the antigenic signal borne by the macrophage '1. A survey of current literature confirms the impression that these three notions put forward by Rosenthal and his colleagues Ir-gene function at the level of the macrophage, macrophage defect in non-responder animals, and requirement for M H C compatibility in macrophage T-cell interactions - are generally accepted. Despite the danger of becoming lonely wolves howling in the desert, we insist here that all three notions are incorrect. We suggest that the notions are based on wrong interpretations of experimental data; we propose that Ir genes are not expressed at the level of the macrophage but at the level of T cells, that there is nothing wrong with the macrophages in nonresponder animals, and that macrophages can interact with MHC-incompatible T lymphocytes. The data most frequently cited in favor of Ir-gene expression in macrophages stem from the notorious F~ experiment first done by Shevach and Rosenthal 2. The essence of the experiment is this. T cells from an F 1 hybrid produced between a responder and a nonresponder strain respond to an antigen presented to them by the responder macrophages but do not respond to the same antigen presented by nonresponder macrophages. Since the T cells, so the argument goes, are always the same, whereas the macrophages vary, it must be the latter that are responsible for the differences in response. But this interpretation is wrong in principle. Consider an alternative Elsevier/North-Holland Biomedical Press 1981 0167 4919/81/0000 0(}00/$2.75
interpretation, namely that the low responder strain is a low responder because it lacks T cells capable of recognizing the antigen in the context of the low responder M H C molecules. If so, then the same defect must also occur in the F~ hybrids and one cannot therefore expect the hybrid to respond to the antigen presented in the context of low responder M H C molecules. So the F~ hybrid experiment can also be interpreted as evidence for a T-cell defect, but by proposing such an interpretation one would make the same error as the proponents of the macrophage defect have made. The truth is that the F~ experiment does not sway the argument one way or the other. The correct way of testing the macrophage defect would have been by asking high responder T cells to co-operate with non-responder macrophages but until now such an experiment was not thought to be feasible since non-responder macrophages differ from high responder T cells in their M H C and, according to the second notion, allogeneic T cells and macrophages cannot interact. But is this notion correct? There is an impressive array of publications purportedly providing evidence for the failure of T cells to collaborate with allogeneic macrophages; without exception the evidence is again based on erroneous interpretation of the data. As an example we shall describe one of the first experiments in this series. Rosenthal and Shevach (1973) immunized guinea pigs of strains 2 and 13 with an antigen, obtained T ceils from these animals, co-cultured them with macrophages pulsed with the same antigen, and measured the antigen-induced T-cell proliferation as reflected in the incorporation of 3H-thymidine. They observed that strain 2's T cells could be significantly stimulated by strain 2 but not by strain 13 macrophages and vice versa, and they therefore concluded that, to collaborate, T cells and macrophages must be histocompatible. However, since 1974, T cells have been known to recognize the antigen and the M H C molecule of the antigen-presenting cell and once stimulated by a particular combination of antigen and
immunologytoday, December197,I M H C they can be restimulated only with this p a r t i c u l a r c o m b i n a t i o n 3. I n the e x p e r i m e n t , Rosenthal and Shevach stimulated T cells with an antigen in the context of one set of M H C molecules and then asked the T cells to respond to the same antigen but in the context of another set of molecules. The fact that the T cells did not respond merely confirms that the concept of the so-called M H C restriction is correct; it has no bearing on the question of whether T cells can recognize an antigen in the context of an allogeneic M H C molecule if they are primed by the antigen in the context of this molecule. Virtually all the subsequent experiments have had the same design in principle: the T cells were primed by the antigen in the context of one M H C molecule and were restimulated with macrophages carrying another M H C molecule4-9: the T cells, of course, invariably failed to respond to this restimulation. In the few cases where the experiments were done in the right way ~°,11 the T cells appeared to collaborate with the allogeneic macrophages. In our laboratory we have recently concluded a large series of experiments in which we exposed T cells to allogeneic macrophages (in the absence of antigen), after three days removed alloreactive cells with BUdR ph~s light treatment, added antigen to the culture for 7 days and after a three-day restimulation with the same antigen on fresh macrophages measured the proliferation of the remaining cells. In some 110 experiments testing some 40 different allogeneic combinations, macrophages were never seen not to present antigen to M H C - d i s p a r a t e T cells ~2. Clearly, when one does the experiment the right way the answer is u n a m b i g u o u s : umprimed T cells can be primed by antigen on allogeneic macropkages. In terms of specificity, the allogeneic priming is no different from the priming by antigens on syngeneic macrophages: primed T cells are then capable of recognizing the antigen in the context of the allogeneic M H C molecules expressed by the macrophages and not in the context of M H C carried by the T-cell donor, nor in the context of any other allogeneic macrophages. The response is also antigen-specific. This system then allows us to answer the question of whether low responder macrophages are defective in presenting the particular antigen. The answer is a very clear no. In all the conceivable high r e s p o n d e r - n o n responder combinations, macrophages can always present antigen to allogeneic T cells 13. There can thus
229 be nothing wrong with the non-responder macrophages: they process the antigen as well as the high responder macrophages and a combination of nonresponder M H C and antigen can be recognized by the right T cells. In the non-responder individuals, T cells capable of recognizing the non-responder M H C in combination with a particular antigen are apparently either absent or prevented from being activated by the antigen. Although our data do not prove the T-cell defect in non-responder strains unambiguously, they do rule out the macrophage defect and with it bury all explanations of Ir-gene control of a n t i g e n - M H C interactions in macrophages. We are aware that we are swimming against the tide, but we do so because we are convinced that the waters are moving the wrong way: if one wants to know the reason for M H C - c o n t r o l l e d non-responsiveness one should forget about macrophages and go back to the T cells.
Acknowledgement We thank Ms. Rosemary Franklin for her help in preparing this manuscript
References 1 Rosenthal, A. S., Thomas, J. W., Schroer, J. and Blake,J. T. (1980). In M. Fougereau and J. Dausset (eds.)Immunology80, p. 458, Academic Press, New York 2 Shevach, E. M. and Rosenthal, A. S. (1973),7. Exp. Med. 138, 1213 3 Zinkernagel, R. M. and Doherty, P. C. (1975)J. Exp. Med. 141, 1427 4 Rosenthal, A. S. and Shevach, E. M. (1973) J. Exp. Med. 138, 1194 5 Erb, P. and Feldmann, M. (1975)J. Exp. Med. 142, 460 6 Kappler, J. W. and Marrack, P. C. (1976) NaO~re(London) 262, 797 7 McDougal,J. S. and Cort, S. P. (1978)J. lmmunol. 120, 445 8 Singer, A., Hathcoek, K. S. and Hodes, R.J. (i979)`7. Exp. Med. 149, I208 9 Yano, A., Schwartz, R. H. and Paul, W. E. (1977)J. Exp. Med. 146, 828 10 Kapp, J. A., Pierce, C. W. and Benacerraf, B. (1973) `7. F.xp. Med. 138, 1121 11 Cosenza, H. and Leserman, L. D. (1972),7. ImmunoI. 108,418 12 Ishii, N., Baxevanis, C., Nagy, Z. A. and Klein, J. (1981) .7. Exp. Med. 154, 978 13 lshii, N., Baxevanis,C., Nagy, Z. A. and Kleirl,J. (1981) (submitted for publication)
immunology today, December 1981
The expression of Ir genes
Macrophage or T cell the question
that is
Zoltan A. Nagy and Jan Klein Max-Planck-Institut f/.ir Biologie, Abteilung Immungenetik, Corrensstrasse 42, 7400 Tiibingen, F.R.G. At the fourth International Congress of Immunology held in Paris in 1980, Rosenthal was able to state without a single dissenting voice from the audience that 'the immune-response genes function at the level of the macrophage.' The long debate about the site of Ir-gene expression thus appeared to have been closed: animals are non-responders because 'of an intrinsic defect in Ir-gene product function in the nonresponder m a c r o p h a g e ' and ' m a c r o p h a g e s and lymphocytes must share genetic identity at some portion of the major histocompatibility complex (MHC) for successful detection of the antigenic signal borne by the macrophage '1. A survey of current literature confirms the impression that these three notions put forward by Rosenthal and his colleagues Ir-gene function at the level of the macrophage, macrophage defect in non-responder animals, and requirement for M H C compatibility in macrophage T-cell interactions - are generally accepted. Despite the danger of becoming lonely wolves howling in the desert, we insist here that all three notions are incorrect. We suggest that the notions are based on wrong interpretations of experimental data; we propose that Ir genes are not expressed at the level of the macrophage but at the level of T cells, that there is nothing wrong with the macrophages in nonresponder animals, and that macrophages can interact with MHC-incompatible T lymphocytes. The data most frequently cited in favor of Ir-gene expression in macrophages stem from the notorious F~ experiment first done by Shevach and Rosenthal 2. The essence of the experiment is this. T cells from an F 1 hybrid produced between a responder and a nonresponder strain respond to an antigen presented to them by the responder macrophages but do not respond to the same antigen presented by nonresponder macrophages. Since the T cells, so the argument goes, are always the same, whereas the macrophages vary, it must be the latter that are responsible for the differences in response. But this interpretation is wrong in principle. Consider an alternative Elsevier/North-Holland Biomedical Press 1981 0167 4919/81/0000 0(}00/$2.75
interpretation, namely that the low responder strain is a low responder because it lacks T cells capable of recognizing the antigen in the context of the low responder M H C molecules. If so, then the same defect must also occur in the F~ hybrids and one cannot therefore expect the hybrid to respond to the antigen presented in the context of low responder M H C molecules. So the F~ hybrid experiment can also be interpreted as evidence for a T-cell defect, but by proposing such an interpretation one would make the same error as the proponents of the macrophage defect have made. The truth is that the F~ experiment does not sway the argument one way or the other. The correct way of testing the macrophage defect would have been by asking high responder T cells to co-operate with non-responder macrophages but until now such an experiment was not thought to be feasible since non-responder macrophages differ from high responder T cells in their M H C and, according to the second notion, allogeneic T cells and macrophages cannot interact. But is this notion correct? There is an impressive array of publications purportedly providing evidence for the failure of T cells to collaborate with allogeneic macrophages; without exception the evidence is again based on erroneous interpretation of the data. As an example we shall describe one of the first experiments in this series. Rosenthal and Shevach (1973) immunized guinea pigs of strains 2 and 13 with an antigen, obtained T ceils from these animals, co-cultured them with macrophages pulsed with the same antigen, and measured the antigen-induced T-cell proliferation as reflected in the incorporation of 3H-thymidine. They observed that strain 2's T cells could be significantly stimulated by strain 2 but not by strain 13 macrophages and vice versa, and they therefore concluded that, to collaborate, T cells and macrophages must be histocompatible. However, since 1974, T cells have been known to recognize the antigen and the M H C molecule of the antigen-presenting cell and once stimulated by a particular combination of antigen and
immunologytoday, December197,I M H C they can be restimulated only with this p a r t i c u l a r c o m b i n a t i o n 3. I n the e x p e r i m e n t , Rosenthal and Shevach stimulated T cells with an antigen in the context of one set of M H C molecules and then asked the T cells to respond to the same antigen but in the context of another set of molecules. The fact that the T cells did not respond merely confirms that the concept of the so-called M H C restriction is correct; it has no bearing on the question of whether T cells can recognize an antigen in the context of an allogeneic M H C molecule if they are primed by the antigen in the context of this molecule. Virtually all the subsequent experiments have had the same design in principle: the T cells were primed by the antigen in the context of one M H C molecule and were restimulated with macrophages carrying another M H C molecule4-9: the T cells, of course, invariably failed to respond to this restimulation. In the few cases where the experiments were done in the right way ~°,11 the T cells appeared to collaborate with the allogeneic macrophages. In our laboratory we have recently concluded a large series of experiments in which we exposed T cells to allogeneic macrophages (in the absence of antigen), after three days removed alloreactive cells with BUdR ph~s light treatment, added antigen to the culture for 7 days and after a three-day restimulation with the same antigen on fresh macrophages measured the proliferation of the remaining cells. In some 110 experiments testing some 40 different allogeneic combinations, macrophages were never seen not to present antigen to M H C - d i s p a r a t e T cells ~2. Clearly, when one does the experiment the right way the answer is u n a m b i g u o u s : umprimed T cells can be primed by antigen on allogeneic macropkages. In terms of specificity, the allogeneic priming is no different from the priming by antigens on syngeneic macrophages: primed T cells are then capable of recognizing the antigen in the context of the allogeneic M H C molecules expressed by the macrophages and not in the context of M H C carried by the T-cell donor, nor in the context of any other allogeneic macrophages. The response is also antigen-specific. This system then allows us to answer the question of whether low responder macrophages are defective in presenting the particular antigen. The answer is a very clear no. In all the conceivable high r e s p o n d e r - n o n responder combinations, macrophages can always present antigen to allogeneic T cells 13. There can thus
229 be nothing wrong with the non-responder macrophages: they process the antigen as well as the high responder macrophages and a combination of nonresponder M H C and antigen can be recognized by the right T cells. In the non-responder individuals, T cells capable of recognizing the non-responder M H C in combination with a particular antigen are apparently either absent or prevented from being activated by the antigen. Although our data do not prove the T-cell defect in non-responder strains unambiguously, they do rule out the macrophage defect and with it bury all explanations of Ir-gene control of a n t i g e n - M H C interactions in macrophages. We are aware that we are swimming against the tide, but we do so because we are convinced that the waters are moving the wrong way: if one wants to know the reason for M H C - c o n t r o l l e d non-responsiveness one should forget about macrophages and go back to the T cells.
Acknowledgement We thank Ms. Rosemary Franklin for her help in preparing this manuscript
References 1 Rosenthal, A. S., Thomas, J. W., Schroer, J. and Blake,J. T. (1980). In M. Fougereau and J. Dausset (eds.)Immunology80, p. 458, Academic Press, New York 2 Shevach, E. M. and Rosenthal, A. S. (1973),7. Exp. Med. 138, 1213 3 Zinkernagel, R. M. and Doherty, P. C. (1975)J. Exp. Med. 141, 1427 4 Rosenthal, A. S. and Shevach, E. M. (1973) J. Exp. Med. 138, 1194 5 Erb, P. and Feldmann, M. (1975)J. Exp. Med. 142, 460 6 Kappler, J. W. and Marrack, P. C. (1976) NaO~re(London) 262, 797 7 McDougal,J. S. and Cort, S. P. (1978)J. lmmunol. 120, 445 8 Singer, A., Hathcoek, K. S. and Hodes, R.J. (i979)`7. Exp. Med. 149, I208 9 Yano, A., Schwartz, R. H. and Paul, W. E. (1977)J. Exp. Med. 146, 828 10 Kapp, J. A., Pierce, C. W. and Benacerraf, B. (1973) `7. F.xp. Med. 138, 1121 11 Cosenza, H. and Leserman, L. D. (1972),7. ImmunoI. 108,418 12 Ishii, N., Baxevanis, C., Nagy, Z. A. and Klein, J. (1981) .7. Exp. Med. 154, 978 13 lshii, N., Baxevanis,C., Nagy, Z. A. and Kleirl,J. (1981) (submitted for publication)
