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Anyway the first slide shows something that I found really fascinating, it was Lancet 2003 and this gives you an idea of what can be achieved by transplantation. This person is 42 years old, Kelly Perkins was up to what we are used to call Cervino, I don’t know how many of you can do that I certainly can’t. I certainly was not able to do that when I was 42. What is peculiar about this person that she had 7 years before a heart transplantation. So this gives you an idea of what can be achieved.

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The problem however, in transplantation is long-term survival. You know when I tell my friends that we haven’t done so much in the last few years in terms of half life in transplantation, they tell me it is not true now we have much longer survival and they are true in some sense. Instead of 8 years we have now 11 years which is not enough for me. But you may notice that if you don’t have an acute rejection, now you can approach 20 years of half-life which is really something so you have to do as much as you can to avoid acute rejection.

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While you had 60% acute rejection by the use of cyclosporine alone, many other drugs now have abated the percentage of acute rejection down to 10%. We have a lot of new medication. Also we have mycophenolate that has replaced azathioprine.

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Unfortunately, we have shown in several publications that there is no difference between these two drugs in terms of acute rejection and chronic kidney survival but in some sense this is another story just telling you that what is depicted as new not always is really new but we have some challenging compounds and a possibility is that with these compounds we will be able to prolong the survival post-transplant up to 30 maybe 40 years which is what all of us are aiming to.

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The problem however, is that if we will be able to reach 30 years half life, almost 100% of our patients will develop a cancer which is something that we don’t want to have. You see if you have a patient who has diabetes, 70 years old who is looking for a renal transplant 30 years half life after transplant is certainly enough but for babies who have congenital liver disease and are transplanted when they are months old, of course, 30 years is not enough.

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So what we are looking at is immunological tolerance all of us and how to do that.

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The immune system learns about self, it produces cells that would attack every tissue in the body but within the thymus it learns by itself to be harmful, useless or useful. In the thymus you have negative selection that is high affinity interaction between thymic epithelial cells with self-peptide complexes presented in the contest of MHC to T cell receptors and when you have high affinity interaction between these two now you have T cell deletion and those cells die in the thymus due to DNA fragmentation subsequent to enhanced intracellular calcium. Positive selection, however, is for the minority of cells when the interaction with low affinity positively selects T cells for maturation.

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In the thymus in other words, what you see in the medulla is what you become tolerant to.

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The fascinating paper published in Science very recently shows how positive selection of T cells requires their exposure to a repertoire of self-peptides generated by the thymus specific proteasomes. So it became more and more clear why in the thymus you can see self-peptides. Of course, they are presented to T cell receptors by MHC.

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This creates negative selection, those cells which recognise self-peptides die in the thymus and those peptides are generated by proteasomes that are specific for the thymus capable of doing this particular job under regulation of another fascinating system which is Aire. In fact, Aire induces peripheral antigens and permits clonal elimination of tissue-specific T cells. You know, when these particular genes Aire are expressed in the thymus, thymic epithelial cells are capable of presenting a lot of self-peptides to maturing thymocytes. So you have a lot of matching and a few cell, reactive cells going into the periphery but if you have Aire deletion, now the recognition of self-peptide by maturing thymocytes is much less and this creates a mature T cell repertoire reaching the periphery and those are potentially self-reactive cells.

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Many years ago what we did was to present to the thymus self-antigens and at that time we thought that it was important to use tissue-specific antigen and we used glomeruli from incompatible rats transplanted into the thymus before transplantation of the contralateral kidney and this resulted in prolonged survival.

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If you generate in vitro specific rat peptides which correspond to human DQ, or DR antigens and you need at least 8 different peptides, now you can achieve tolerance in rats presenting the appropriate alloantigen peptides in the synthetic form instead of cells. This has been done in collaboration with Mohamed Sayegh in Boston.

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Others have repeated these experiments in monkeys and when they have injected enough cells into the thymus from donor origin, they have used leukocytes instead of glomeruli and when they had allowed enough time to elapse between injection of cells and subsequent transplantation, they achieved a  long-term tolerance which can also be achieved in humans in liver transplantation.

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Those are data from Doctor Starzl in Pittsburgh showing that years post liver transplant you have that number of patients who have been out of immunosuppressive drugs for so many years and they did tolerate the organ without any form of immunosuppression. They were off of immunosuppression because of infection, of cancer or simply because they did not want to take immunosuppression any longer.

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In order to increase this phenomenon that Doctor Starzl called chimerism between donor and recipients some have transplanted donor bone marrow together with kidney to enhance this form of chimerism between donor and recipients and what they found was that, in fact, you have better survival in those who have received the kidney and the bone marrow from the same donor as compared to those who have received kidney alone. But you see what they called tolerance, in fact, is something which you achieve with so many medications which I think is not what we are aiming to exactly.

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So Marina Noris in our lab did this experiment in rats Brown Norway donor, Lewis rat recipient, kidney transplantation, association with peripheral blood cells or bone marrow to see whether indeed you need bone marrow to ameliorate graft acceptance. They found that it is exactly the same, if you do kidney and bone marrow, kidney and peripheral cells in terms of long-term surviving animals. They did 15 days of cyclosporine in order for the donor leukocytes or bone marrow not to be rejected. All the controls with cyclosporine alone reject, so this dose of cyclosporine is not enough to prolong survival.

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However, what Marina showed is that it depends on the number of cells that you are going to inject. If you inject enough cells, now you have prolonged survival in all animals. So having someone who does reject and someone who has prolonged survival allowed us to establish the difference between the two and the difference is that in tolerant animals you have chimerism in the thymus and liver while blood chimerism as suggested by Starzl is irrelevant.

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So, it is not a question that you need the blood cells to tolerate, if anything you have more blood chimerism in rejecting than in tolerizing animals. You really need donor cells in the thymus.

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Thymectomy abrogated the phenomenon and when we did PCR analysis of MHC class II fraction from thymic cell suspensions of tolerant animals we found that in fact, dendritic cells were present in the thymus of tolerant animals.

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You see if you deplete MHC class II cells, actually you abrogate the phenomenon, so the phenomenon is dependent on donor dendritic cells reaching the thymus.

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Now, tolerance versus rejection is the question of cells who are, in fact, activated by transplant antigens and regulatory cells that, in fact, tip the balance towards tolerance versus rejection.

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Regulatory cells are Foxp3 positive. Foxp3 is specifically expressed in naturally arising CD4+ CD25+ regulatory T cells and retroviral transfer of Foxp3 converts naïve T cells toward a regulatory T cell phenotype similar to that of naturally occurring regulatory cells.

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Here is how Foxp3 works upon stimulation of a given cell. Foxp3 acts by occupancy of promoters to suppress the activation of a number of target genes into stimulated cells.

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You have many cells that can be of regulatory type that can be formed in the thymus upon the exposure to appropriate self-antigens but regulatory cells, acquired regulatory cells can also be formed in the periphery upon challenge of naïve T cells with alloantigens in the presence of costimulatory blockade.

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Regulatory T cells in transplant tolerance are probably important in the induction phase and in the maintenance phase of the phenomenon.

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And again, with the same animal transplantation system, a few days of cyclosporine and then blood donor leukocytes to induce tolerance.

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Marina Noris has found that the induction phase of tolerance is mediated by anergic T cells that she has recovered in the thymus and those cells were anergic in that IL-2 partially restored the activity of these cells that were, in fact, unable to respond in vitro to donor antigens.

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She also found that in the early post transplant period you have anergic cells in the graft that are capable possibly of regulating, of generating regulatory cells that do regulate naïve cells and she has documented that regulatory cells resident within the graft promote robust suppression and may sustain long-term graft survival.

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Interestingly, a very recent experiment consistent with this concept is that recruitment of Foxp3 regulatory cells mediating allograft tolerance depends on these particular chemokine receptors. If you have a system in which you tolerise by an anti CD 154 antibody and donor specific transfusion, then you can abrogate the tolerance in animals who lack this particular receptor by gene deletion.

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Marina also found that the maintenance phase of tolerance is associated with Treg in the peripheral lymph nodes. In fact, lymph node cells isolated at 30 or at 60 days post transplant from tolerant rats inhibit the proliferation of naïve T cells to donor alloantigens.

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She found that you can adoptively transfer cells with regulatory activity prolonging kidney survival in an incompatible strain.

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Then new concepts show that those cells can be educated to be tolerising cells even in the periphery.

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In the periphery at the level of the tissue, tubular cells for instance, parenchymal expression of costimulatory blockade molecules, CTLA4Ig or the recently discovered costimulatory signal programmed death-ligand 1 (PD-L1) provides a second level of regulation of immune response locally in the graft. This is really new and fascinating.

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What we are doing today is trying to avoid rejection by modifying the recipient immune system. What we are looking for tomorrow and we are doing these experiments in animals is trying to have the kidney or the liver or the heart or the pancreas become capable of defending itself from rejection. If you infuse therapeutic DNA and you have the organs in your hands to infuse therapeutic DNA, then when cells go there in order to activate a rejection phenomenon they will receive that inhibitory signal.

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We have used this particular fusion protein of the extracellular domain of CTLA4 and Fc domain of IgG. This fusion protein is a competitive inhibitor of CD28 binding to B7-1 or B7-2 and this particular molecule has many fold higher activity for B7-1 and B7-2 than does CD28. You can by gene transfer transfer this particular molecule into the renal allograft.

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And as Doctor Benigni has documented you inhibit in rats acute and more importantly chronic rejection.

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This is associated with suppression of gene expression for TGF-β, IL-10, IFN and IL-2 in graft infiltrating leukocytes and this is also associated with regulatory activity as documented by enhanced Foxp3.

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Another way, absolutely novel, is this tissue costimulatory pathway that can be inhibited rising negative regulatory function. This particular molecule is expressed on endothelial cells and parenchymal cells beside antigen presenting cells. This particular molecule binds to the counter receptor on T cells and inhibits proliferation and cytokine production by antigen specific CD4+ T cells.

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So this is another peripheral way to inhibit tolerance. In fact, if you use hearts that are knockout for this particular molecule, you accelerate graft rejection as compared to wild type.

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How near are we to reaching the point where transplant tolerance will become a clinical reality?

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You can do that as I told you and that has been done in 5 subjects by bone marrow and kidney from one haplotype mismatched living-related donors. It requires a lot of anti-rejection medication in order to have the bone marrow from the donor engrafting the recipient.

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And the risk of infections and aplasia and ultimately death associated with actually available conditioning regimens significantly outweighs the potential benefit of tolerance in solid organ transplantation I believe.

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Doctor Roy Calne has suggested that tolerance-permissive environment can be generated by Campath-1H that is a monoclonal antibody that is directed against CD52 antigen expressed on B and T lymphocytes. This induces complement activation and cell lysis.

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However, for some reasons this fails to induce renal allograft tolerance in humans. Actually you see that even 3 different dosing at different days of Campath-1H is associated with rejection.

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What we have done in humans was to use Campath as induction therapy to minimise maintenance immunosuppression with tolerance-permissive drugs in a steroid free regimen. No steroids, Campath-1H,  just one dose, kidney transplantation and then we have randomised patients to low dose sirolimus and low dose cyclosporine considerably less, 50% than current therapy.

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We found that in sirolimus treated patients you have more regulatory cells and I believe this is one of the first demonstrations that in fact, you have a given regimen that as opposite to cyclosporine regimen can give rise to regulatory cells.

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However, when we look at the decline in GFR in these particular patients with time, we found that those who had more Treg had more decline in GFR. So you generate Tregs but so far it doesn’t translate in better function of your kidney.

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Then you also have the special problem of T and B cell memory cells. T cells from lymphopenic hosts as induced by Campath-1H undergo extensive proliferation. The phenotype and function of the re-emerging T cells is similar to that of memory cells.

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This phenomenon could explain the occurrence of rejection in patients treated with Campath-1H as part of a lymphodepleting strategy in renal transplantation, since memory T cells are resistant to depletion by Campath-1H.

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We had a case of acute rejection, glomerular thrombosis, tubular necrosis, C4d staining and this patient had to be nephrectomised after Campath-1H.

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This is because Campath depletes mature B cells, but no memory B cells and plasma cells.

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Instead in fact, you have an expansion upon challenge with donor antigen of memory clone and plasma cells and this gives rise to antibodies that are responsible for acute rejection.

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When you have more than one challenge to the immune system as with multiple viral challenge now you can have more allo reactive cells that overweigh the threshold of tolerance and now your tolerance regimens are unsuccessful and this is another problem.

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Recently Mo Sayegh and I have been asked by the editor of the Lancet to tell something about what is new in minimisation of immunosuppression. I believe that the main challenge in the development of novel immunosuppressive  protocols in transplantation remains the lack of predictable assays to measure immunosuppression beyond pharmacological monitoring, a process that does not provide an assessment of the status of the immune response.

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We are trying to minimise therapy but really we don’t know what we are doing to our patients today. This was I believe one of the lessons from the recent transplant meeting in Boston.

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The meeting was opened by Hillary Clinton that gave a beautiful lecture on transplantation. I was literally impressed by that. I suspect that when she addresses another audience, she will give a similar beautiful talk and I was really impressed by the competence.

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Maybe our politicians are different I don’t know. We politicians, concluded Hillary Clinton, we absolutely need you, she was addressing transplant physicians. What we also need are partnerships between the government and academic and research institutions but you scientists have to raise your voice and stand up for science, you should participate in the debate personally. I thanks for what are you doing - she told the transplant physicians before leaving - and for everything you did in these 50 years for millions of people.

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Immediately after Alonzo Mourning, this is for Europeans, is a famous basketball player for Miami this is not needed for Americans in the audience, came on stage. He is black, about 7 feet tall, he speaks like an actor who every time when he jokes is waiting for the reaction of the audience. I believe he is really funny. You can listen to him for hours.

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This is him these days.

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This year the Miami won the NBA championship and they won for Alonzo Mourning, especially.

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Alonzo Mourning 3 years ago received a kidney transplant.

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Thank you very much.

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Chairman: Thank you very much Doctor Remuzzi for this highly fascinating lecture. The lecture is open for discussion and while we are waiting for a question, may I ask something? Because you were talking about the possibility experimentally to enter in the marrow dendritic cells of the donor in the thymus and induce tolerance or to induce anergic T cells once again in the thymus. Considering that on the other side of the problem there is the main problem of minimisation of immunosuppression, is it possible to forecast in the human cloning of anergic T cells or cloning of dendritic donor cells into the thymus as a collateral help to pharmacology?

Prof. Remuzzi: Yes, I think so. I think that this is a very nice question. I think that associating cells, either stem cells or dendritic cells or mesenchymal cells and transplantation cells from donor origin is one way of achieving tolerance and we will see whether these two ways together progressive minimisation of therapy and associated cells will be useful. The problem is to find out the appropriate cells that are not rejected, for instance mesenchymal cells, can be as far as I know now the best possible cells. There are a lot of problems to overcome still but this can be the way for the future.

Question: I wonder what is your opinion on the impact of vitamin D status on tolerance and graft survival?

Prof. Remuzzi: There are a lot of studies particularly from Italy by the way. Doctor Adorini is one who is trying to induce tolerance by vitamin D metabolites. They have very interesting experimental data. I don’t think there is any clinical application so far of these concepts but certainly it is one way to go.

Question: As you know, the combined kidney and liver transplantation is more tolerated than kidney alone in the same pathway pattern of donor-recipient relationship. Can you have a comment on this or do you think that this precise apoptosis of the T cells, selective apoptosis of T cells in this case or not?

Prof. Remuzzi: Well, I think nobody knows why the liver allows kidney in some way to be tolerated. More than that this is based on anecdotal experience and feeling more than on strong data. But let’s imagine that it is true which is likely, from my prospective I believe that what may happen is that donor cells leave the organ and colonise the thymus. I believe that dendritic cells of donor antigen have been documented in the thymus of patients having had liver and kidney transplantation and particularly, I believe mesenchymal cells may do this job of leaving the organ and colonising lymph nodes and the thymus. I cannot tell you more than that at the moment but we have just submitted a paper showing that in fact, you can reproduce tolerance in animals by using together with the organ donor mesenchymal cells that are instructed to be tolerogenic in vitro. I believe that this may explain, of course I don’t have a definitive explanation, but this is what I believe may happen.

Question: Excuse me I’m from Iran. We are using now erythropoietin as a stimulation of blood haemoglobin levels and now we do not use blood transfusion for regulating haemoglobin levels but however, in time we lastly used blood transfusion for haemoglobin concentration levels. Do you think that if we use blood transfusion or specifically a donor specific blood transfusion before transplantation as you suggested with your experiment in mice that can help tolerance for our patients who have already received transplant?

Prof. Remuzzi: Yes, you are correct. In the old days before cyclosporine was available people were used to transfusing patients before transplantation. This was associated with a survival of the organ which is comparable to that that we achieve after cyclosporine. The problem is that you create sensitisation in some patients and you may even have worse outcome because of antibodies that are generated by the blood transfusion. The nature of the phenomenon I believe is exactly what Marina has found in rats if she used donor leukocytes, in fact it is capable of protecting those animals from rejection. So the nature of the phenomenon I think is qualitatively similar to what has been observed in the old days by blood transfusion but blood transfusion is not today I believe a way to go because of sensitisation.

Question: I was wondering so you showed that there should be a targeting of cells to the thymus to induce this regulatory network. In your opinion is it regulating a direct or indirect pathway which is going to be most important? Because you can also envision that your injected cells will be represented by local thymic dendritic cells for instance and induce.

Prof. Remuzzi: Yes, we are working on that trying to sort it out. It can be both and it can well be the indirect pathway as well. What I can tell you and what our experiments have shown very clearly is that opposite to what has been suggested in the old days by the Pittsburgh group chimerism in the periphery is absolutely irrelevant to tolerance while the presence of donor cells in the liver and in the thymus are crucial in order to induce tolerance. We are trying to sort out the mechanism and it can well be that it is involved in the indirect pathway is involved because peptide from donor origin can be processed in the thymus and presented throughout indirect pathway. You are right.

Question: I would like to ask the last question to Doctor Remuzzi. Do you think that the level of Treg in the blood is a biomarker for minimisation or for withdrawal of immunosuppressive agents?

Prof. Remuzzi: Well, I think, I don’t know whether there is other data available I don’t think there is as to the best of my knowledge in the literature in humans. You have a lot of experiments in animals showing a relationship between level of Treg cells and organ acceptance. In humans I think we have found a way to dissect the phenomenon and there is a particular therapy, sirolimus without cyclosporine on the cover of just one dose of Campath to create lymphopenia which is capable of allowing the emergence of regulatory cells which is the opposite to cyclosporine but in humans if you see the long-term outcome of the graft, it is much better in those on cyclosporine who don’t have regulatory cells as compared to those who have regulatory cells. Now, the interpretation can be either that regulatory cells don’t have enough to do with acceptance of the transplant. I wouldn’t say that it is irrelevant but it is not so important. The other explanation is that in fact, sirolimus per se is responsible of declining GFR in these particular patients which is also a possibility that I think is difficult to dissect with this kind of experiment and is also difficult to envision other experiments in humans in order to answer that. So, for the moment I think that induction therapy and a very low level of cyclosporine is much better than induction therapy and sirolimus. The negative effect can be by sirolimus itself and the negative effect of sirolimus in the kidney.

Chairman: Thank you very much Doctor Remuzzi.