Tolerance can be infectious - Nature

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Tolerance can be infectious Herman Waldmann Studies focused on the goal of eliciting transplant tolerance led Herman Waldmann to rejuvenate discarded ideas of ‘suppressor T cells’. Such cells are now known to exist.

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s a medical student, I was disappointed to see how inadequate the drugs available were for control of unwanted inflammation. I realized then that a better understanding of the immune system would be needed if we were to find safe and more efficacious drugs, let alone cures. When I began my PhD in 1971 under the guidance of Alan Munro, it seemed that solving how T cells could ‘cooperate’ with other cells might be a good starting point for ‘translational’ opportunities. Moreover, this arena had the potential to identify mechanisms of tolerance that might be exploitable therapeutically. The hypothesis was emerging that the failure of T cells to participate in cooperative events put them at risk of tolerance. That idea derived from diverse sources hoping to explain how the self-tolerance ‘decision’ is made1, why B cells need T cell help2,3 and why linked recognition is needed4,5. The clonal selection theory offered deletion as a neat mechanism to explain both self and acquired immunological tolerance, as extrapolated from the two most popular tolerance models of that period: classical transplantation tolerance in the neonatal mouse6, and low and high zone tolerance to human gamma globulin in the mature adult7. While I was ‘finding my feet’ in immunology, I was very fortunate that Alan arranged for me to meet with Jacques Chiller over a prolonged Mexican lunch in La Jolla. This was a great initiation into thinking about tolerance. One principle became clear to me: a therapeutic agent able to induce tolerance would have to take account of all new T cells that developed long after the drug was cleared from the system. In other words, the drug had to set up a long-term program for tolerance in the immune system. Herman Waldmann is with the Sir William Dunn School of Pathology, Oxford OX1 3RE, UK. e-mail: [email protected]

My increasing convictions as to how the immune system organized itself to maintain self-tolerance was abruptly interrupted in the early 1970s by publications from Wulf Droege8 and Richard Gershon9, who had discovered T cell–mediated ‘suppression’. Gershon used the term ‘infectious tolerance’ to describe the finding that normal lymphocytes are prevented from responding to antigen in the presence of ‘suppressor’ T cells. By using antisera directed against the lymphocytic surface antigens 1 and 2 (Lyt1 and Lyt2), Gershon found that CD8+ T cells were the suppressor cells. Many groups followed with comparable claims in a range of different models. However, within a few years, almost as rapidly as the edifice had grown, so then did it implode. The complex models were not easy to replicate, publications on shed antigen-specific receptors simply ‘dried up’ and I-J, a marker for suppressor cells and their factors, could never be accounted for on a molecular or genetic basis. Suppression became a ‘bad word’. I had become acquainted with many of the authors of those works and could never quite reconcile how such seemingly normal people had become associated with such fragile data. Little did I realize that I would evoke similar reactions in others in due course. The discovery of monoclonal antibodies rejuvenated immunology by providing beautifully precise reagents to ‘dissect’ the complex systems of this field. Not only did they provide tools to track cells with defined functions, but also these same tools seemed to be possible therapeutics. I asked César Milstein if I could spend a sabbatical period in his laboratory to learn how to generate rat monoclonal antibodies to human and mouse white blood cells. Our initial goals were to generate lymphocyte-depleting antibodies, antibodies to all lymphocytes and antibodies to the main subsets of lymphocytes. The hypothesis was that

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The author happily ensconced in his office at the Sir William Dunn School of Pathology, where James Gowans first identified the lymphocyte as the cell responsible for adaptive immunity.

if we could debulk lymphocyte populations to small numbers, then, after exposure to antigen, the chances of interlymphocyte cooperation would be low and so tolerance of the residual cells would be more likely. Any new T cells emigrating from the thymus would consequently lack cooperative partners in the periphery and would as a result be rendered tolerant themselves. Tolerance based on this program would be ‘infectious’ toward new thymic émigrés, because the chance of their finding collaborative partners would be small. CAMPATH-1, an antibody to human lymphocytes (antibody to CD52) now commercially available as alemtuzumab, emerged from that line of thinking10. With a view to using

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e ss ay engraftment of bone marrow stem cells as a path to ‘classical-type’ transplantation tolerance in the adult, we successfully applied this antibody to prevent graft-versus-host disease and marrow rejection in humans. These clinical studies taught that control of strong alloreactivity, albeit in a heavily conditioned recipient, could be achieved by inductive therapy with a monoclonal antibody without the need for maintenance immunosuppression. In parallel preclinical studies, our best lytic combination for ablating mouse T cells proved to be a combination of rat antibodies to CD4 and CD8 (ref. 11). However, we were unable to achieve marrow chimerism (and transplantation tolerance) across complete major histocompatibility complex barriers without some additional host conditioning (with irradiation or drugs)12. Donor-recipient combinations that were less demanding (differing only in multiple minor histocompatibility antigens) proved more conducive to further study13. We could achieve donor chimerism (albeit a very small amount) and transplantation tolerance without any other host conditioning. To check whether T cell ablation was necessary, we tested nonablative subclasses of antibodies to CD4 and CD8. To our surprise, these nonlytic antibodies could achieve the same outcome13,14. As the tolerized CD4+ T cells were available for further analysis, we used the opportunity to determine if antigen-specific T cells had been deleted from the host and were surprised to see that they were still there. These T cells, however, were refractory or anergic to antigen challenge in vitro. We modified our previous conclusions about the mechanism by suggesting that the antibody to CD4 had effectively prevented the T cells from being properly activated, which enabled them to default to tolerance because they had received no help from each other15. The overall cooperation-based hypothesis seemed intact. One experimental finding, however, was not compatible with a simple idea of antigen-specific deletion or anergy through lack of cooperativity. We could not break tolerance by infusing normal lymphocytes. This phenomenon of ‘resistance’ needed an explanation. It was not compatible with a simple deletional or anergy-based model of tolerance. We tried to come up with an idea that incorporated anergic cells as potential regulators able to mediate ‘resistance’. We made an analogy with the civil service16. We reasoned that ineffective ‘anergic’ T cells (civil servants) would accumulate at sites of antigen presentation and there interfere with cooperativity between normal T cells. This could be passive, due to competition for some critical limiting component, or active, due to some ‘subversive’ function of these seemingly inert cells.

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Notably, the proposed mechanism lent itself to ‘infectiousness’, as failure to benefit from cooperation would render new T cells anergic (civil servants) and so on. Our analogy evoked much empathy, particularly from colleagues at the US National Institutes of Health, who could easily relate to it! We later discovered that we could achieve the same sort of tolerance directly to tissue grafts grafted under the ‘umbrella’ of nondepleting coreceptor antibody therapy14. No bone marrow stem cells were needed. That in itself was surprising, as I was hooked on the idea that chimerism was essential to ensure sufficient and long-term T cell deletion or anergy. Yet again, tolerance could not be broken by the infusion of normal lymphocytes. Our suspicions were growing about the involvement of regulatory or suppressor T cells. At that time, we were fortuitously collaborating with a sabbatical visitor to the laboratory. Yi-Chi Kong, an expert in autoimmune thyroiditis, had previously established that thyroiditis-prone mice could be made tolerant to thyroglobulin by injection or infusion of thyroid-stimulating hormone. This tolerance could not be broken by the infusion of normal T cells unless host CD4+ T cells were ablated first. This result suggested that CD4+ T cells were responsible for this form of ‘resistance’17. We wondered how ‘resistance’ could operate lifelong if it were due to regulatory or suppressor T cells. Were those suppressor cells generated at the outset still the ones operating years later? Alternatively, were new cells being recruited or converted to this function, as in our civil service analogy? By making use of tolerized mice whose T cells were marked by expression of human CD52, we found that donor T cells, prevented from rejecting grafts, were themselves rendered tolerant and suppressive. Later studies showed that this ‘infectious’ conversion could continue through many generations of transfer18. We eventually took the plunge and did the classical experiments of adoptive transfer into lymphocyte-depleted hosts. Suppression could be adoptively transferred onto normal T cells. Ablation of CD4+ T cells from the tolerant donor prevented suppression, whereas ablation of CD8+ T cells did not18. From that moment, I knew we were in for trouble with the immunology community still reeling from its earlier skirmishes with suppression. But the show had to go on! This work had been put together for publication and submitted to a high-ranking journal. After many revisions aimed at satisfying skeptical referees, the paper was eventually rejected. We resubmitted it to Science

and received a warmer outcome. When the paper was eventually published in1993, its key findings were well over 3 years old. Our subsequent work on mechanisms provided two key findings. The first was that regulatory T cells induced to one set of antigens can prevent rejection of tissues carrying ‘third-party’ antigens together with the tolerated set19. This linked suppression indicates that regulatory T cells must become focused at sites of antigen presentation and therein halt aggression by T cells with ‘third-party’ specificities. The second was the demonstration that tolerated grafts carry in them functional regulatory T cells20, which indicates that regulatory T cells interact with tissues to endow them with some form of immune privilege. The reaction of immunologists to the paper on ‘infectious tolerance’ was, as we had perhaps expected, mixed. As in all such scenarios, people rarely told us what they really thought, although the body language of some clearly indicated great discomfort. I recall meeting an eminent immunologist (an old friend) at a meeting in Rome some years later. I had heard that she was very much against the idea of suppression, despite the increasing number of publications on the subject. I asked her how she could reconcile her friendly feelings toward me with her public disbelief of suppression. Her answer was brief: “I don’t know!” Perhaps the most extreme and disturbing public statement about suppression came from a Nobel Laureate who published his concern about the resurgence of this subject, as if it had not done enough damage to the field of immunology in the past21! Despite the many skeptics, the field of regulation took on a new life. In parallel with our studies, similar principles were emerging for the control of immunopathology22 and autoimmune disease17,23 by regulatory T cells. Looking back now, we can take comfort that despite the initial adverse reactions, the biology of CD4+ regulatory T cells is now a very hot topic in immunology. Although the models we chose were in the field of transplantation, the principles of infectious tolerance are proving relevant to regulation in autoimmunity, allergy and chronic infectious disease. The ability to ‘tip’ the host’s immune response toward regulation has elicited new waves of research in therapeutics with a whole host of antibodies and drugs showing some utility in this context. The ability to ‘tip’ the immune system away from regulation may spawn new approaches for treating cancer and chronic microbial disease. There have been concerns that lymphocyte ablation would indiscriminately remove regulatory T cells and thus

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e ss ay prove less useful for tolerance. This concern may be unfounded, as the critical issue may be how the immune system reconstitutes. I would argue that physician-controlled reconstitution with a bias toward regulation might become a very feasible procedure in the not-too-distant future. Looking even further to the future, I think that a new field of immunology will develop, that of ‘acquired immune privilege’. The identification of mechanisms by which regulatory T cells interact with tissues to ‘exempt’ those tissues from damage has enormous therapeutic potential. It is now becoming apparent that tissues are not just passive targets, but that in their interaction with regulatory T cells, they can become empowered to resist harmful inflammation. I still recall that lunch with Jacques Chiller—on his part, a generous act to give precious hours to a student he had never met before. Jacques and the many immunologists of that era had to conceptualize ideas on tolerance with very little experimental data at hand; they had no monoclonal antibodies or genetically manipulated mice. The conceptual framework they offered exploited one of the most powerful tools of that era, that of Occam’s razor, a powerful approach that Alan Munro tried to impress on me. I have tried to use that tool to understand tolerance and to think of strategies to exploit it therapeutically. We went into ‘translational mode’ as early as 1980 on the basis of the hypotheses that seemed to make sense. Even though we had to refine those through to ‘civil service’ and active suppression, the principle that for therapeutic purposes the program

needs to be ‘infectious’ has remained the driver. Witnessing the diverse ‘human’ reactions to our unexpected results has made me much more open-minded about assessing other people’s finding on their merits rather than on any religious or dogmatic principle. ACKNOWLEDGMENTS I thank my many collaborators, students and colleagues who have contributed so much to this work, many of whom are mentioned here as authors of key papers. Supported by the Medical Research Council (Programme Grant). COMPETING INTERESTS STATEMENT The author declares competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/natureimmunology/. 1. Bretscher, P. & Cohn, M. A theory of self-nonself discrimination. Science 169, 1042–1049 (1970). 2. Claman, H.N., Chaperon, E.A. & Triplett, R.F. Immunocompetence of transferred thymus-marrow cell combinations. J. Immunol. 97, 828–832 (1966). 3. Mitchell, G.F. & Miller, J.F. Cell to cell interaction in the immune response. II. The source of hemolysinforming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes. J. Exp. Med. 128, 821–837 (1968). 4. Mitchison, N.A. The carrier effect in the secondary response to hapten-protein conjugates. II. Cellular cooperation. Eur. J. Immunol. 1, 18–27 (1971). 5. Rajewsky, K., Schirrmacher, V., Nase, S. & Jerne, N.K. The requirement of more than one antigenic determinant for immunogenicity. J. Exp. Med. 129, 1131–1143 (1969). 6. Billingham, R.E., Brent, L. & Medawar, P.B. Actively acquired tolerance of foreign cells. Nature 172, 603– 606 (1953). 7. Chiller, J.M., Habicht, G.S. & Weigle, W.O. Cellular sites of immunologic unresponsiveness. Proc. Natl. Acad. Sci. USA 65, 551–556 (1970). 8. Droege, W. Amplifying and suppressive effect of thymus cells. Nature 234, 549–551 (1971). 9. Gershon, R.K. & Kondo, K. Infectious immunological tolerance. Immunology 21, 903–914 (1971). 10. Hale, G. et al. Removal of T cells from bone marrow

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for transplantation: a monoclonal antilymphocyte antibody that fixes human complement. Blood 62, 873–882 (1983). 11. Cobbold, S.P., Jayasuriya, A., Nash, A., Prospero, T.D. & Waldmann, H. Therapy with monoclonal antibodies by elimination of T-cell subsets in vivo. Nature 312, 548–551 (1984). 12. Cobbold, S.P., Martin, G., Qin, S. & Waldmann, H. Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance. Nature 323, 164– 166 (1986). 13. Qin, S.X., Cobbold, S., Benjamin, R. & Waldmann, H. Induction of classical transplantation tolerance in the adult. J. Exp. Med. 169, 779–794 (1989). 14. Qin, S.X. et al. Induction of tolerance in peripheral T cells with monoclonal antibodies. Eur. J. Immunol. 20, 2737–2745 (1990). 15. Waldmann, H., Cobbold, S., Benjamin, R. & Qin, S. A theoretical framework for self-tolerance and its relevance to therapy of autoimmune disease. J. Autoimmun. 1, 623–629 (1988). 16. Waldmann, H., Qin, S. & Cobbold, S. Monoclonal antibodies as agents to reinduce tolerance in autoimmunity. J. Autoimmun. 5, 93–102 (1992). 17. Kong, Y.M., Giraldo, A.A., Waldmann, H., Cobbold, S.P. & Fuller, B.E. Resistance to experimental autoimmune thyroiditis: L3T4+ cells as mediators of both thyroglobulin-activated and TSH-induced suppression. Clin. Immunol. Immunopathol. 51, 38–54 (1989). 18. Qin, S. et al. “Infectious” transplantation tolerance. Science 259, 974–977 (1993). 19. Davies, J.D., Leong, L.Y., Mellor, A., Cobbold, S.P. & Waldmann, H. T cell suppression in transplantation tolerance through linked recognition. J. Immunol. 156, 3602–3607 (1996). 20. Graca, L., Cobbold, S.P. & Waldmann, H. Identification of regulatory T cells in tolerated allografts. J. Exp. Med. 195, 1641–1646 (2002). 21. Zinkernagel, R.M. On cross-priming of MHC class I-specific CTL: rule or exception? Eur. J. Immunol. 32, 2385–2392 (2002). 22. Powrie, F. & Mason, D. OX-22high CD4+ T cells induce wasting disease with multiple organ pathology: prevention by the OX-22low subset. J. Exp. Med. 172, 1701–1708 (1990). 23. Sakaguchi, S. & Sakaguchi, N. Thymus and autoimmunity. Transplantation of the thymus from cyclosporin A-treated mice causes organ-specific autoimmune disease in athymic nude mice. J. Exp. Med. 167, 1479–1485 (1988).

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