In the more than 60 years since the seminal demonstration of acquired transplantation tolerance in mice by Billingham, Brent, and Medawar, enormous efforts have been dedicated to translating that tantalizing possibility into a clinical reality. Careful and stepwise experiments in rodents and nonhuman primates led to the development of protocols that satisfied the risk-benefit analysis necessary for studies in humans, and although it took longer than everyone hoped and many would have expected, operational tolerance to kidney and liver transplants has now been specifically induced in small numbers of patients. Not surprisingly, much of the early work was performed in human leukocyte antigen (HLA)–matched donor-recipient pairs, but increasing numbers of trials are now enrolling HLA-mismatched transplant recipients as well.
These are tremendous successes of which the field is justifiably proud; however, they are just a start, and if we are to achieve a goal where tolerance is “routine” and not remarkable, more studies are needed. Unfortunately, it has proven challenging to leverage the initial promise of these early results into the next phase, that is, new trials that involve more centers and larger numbers of patients. Recently, an international workshop of approximately 50 physicians and scientists was convened by The Transplantation Society (TTS) in Boston to address the question, “What do we need to have in place to make tolerance induction protocols a ‘standard of care’ for organ transplant recipients over the next decade?” This article summarizes the proceedings of that meeting and outlines consensus findings and recommendations for paths forward.
Overview of Progress to Date and the Problems Faced
Three centers in the United States have enrolled renal transplant patients in tolerance protocols with successful withdrawal from immunosuppression. The Stanford University protocol (1) uses posttransplantation antithymocyte globulin and total lymphoid irradiation. This is followed by the administration of a manipulated donor hematopoietic cell product controlling for the dose of CD34+ and CD3+ cells with weaning of immunosuppression based on the presence of durable mixed chimerism. Overall, 16 of 22 HLA identical subjects have been taken off immunosuppressives. Although the early experience in HLA-mismatched transplantation did not succeed in achieving durable donor chimerism, a new series with increased cell dosing is being used, and persistent chimerism has been achieved. No subjects have lost a graft.
Two trials are being conducted at Northwestern University. A total of 22 patients have been transplanted in a trial of mismatched and unrelated living-donor kidneys using nonmyeloablative conditioning (fludarabine, cyclophosphamide, and low-dose total body irradiation) combined with a manipulated stem-cell product (2). Withdrawal of immunosuppression is conducted during a 1-year period and is based in part on the presence of durable donor chimerism. Of 19 subjects with 1-year follow-up, 12 have achieved durable chimerism and are off immunosuppression. Five subjects did not develop stable engraftment and are on immunosuppression, and two subjects experienced allograft loss. No subjects developed graft-versus-host disease. A separate trial of combined kidney and hematopoeitic stem-cell transplantation in HLA-identical patients uses alemtuzumab-based conditioning and serial infusions of donor hematopoeitic stem cells. Of 20 patients, 10 have been withdrawn from immunosuppression.
A completed Immune Tolerance Network (ITN)–sponsored mixed chimerism trial conducted at Massachusetts General Hospital (MGH) used thymic irradiation combined with antibody-based T-cell depletion (the anti-CD2 mAb MEDI-507), rituximab, and cyclophosphamide, with unmodified donor bone marrow–infused peritransplant (3). Operational tolerance was induced in 7 of 10 subjects (the other 3 lost their grafts), 4 of whom remain off immunosuppression long term at 11.5, 5.2, 5.0, and 4.6 years. Most patients developed “engraftment syndrome,” characterized by severe but transient renal dysfunction in the first week after transplantation. A modified version of the protocol with total body irradiation replacing cyclophosphamide has begun in an effort to prevent engraftment syndrome.
Samsung and Hokkaido Universities (Korea) shared their experience with combined bone marrow and kidney transplantation in five subjects. A modified version of the MGH protocol has been used. Engraftment syndrome has been observed in the initial two patients. Although one of them was off immunosuppression for more than 1 year, the patient recently developed acute rejection. Lastly, the Johns Hopkins group has begun an ITN-sponsored trial in live-donor HLA one haplotype–matched patients with a stem-cell transplantation protocol that uses high-dose posttransplantation cyclophosphamide adapted from their successful experience with sickle-cell disease. An initial subject has been transplanted and has early full donor chimerism.
A panel discussion explored several key points related to tolerance trials. (1) Patient selection: issues explored included exclusion and inclusion criteria, the impact of ethnicity, cognizance and capacity of potential subjects, disease comorbidities, underlying cause of end-stage renal disease, and presensitization. The group all agreed that certain forms of end-stage renal disease with a very high risk of recurrence, such as focal and segmental glomerular sclerosis, and preexisting donor-specific antibody should represent an exclusion to enrollment. (2) Study design: there was a spirited discussion about the need for control groups (possibly with randomization) treated with standard immunosuppressive protocols to address the question of whether trying to achieve tolerance leads to superior patient outcomes over the long term compared with standard of care or drug minimization. Part of this discussion also focused on whether investigators at different institutions would participate in a multicenter trial with a single protocol if the one proposed differed from what they had used previously. (3) Underlying mechanisms responsible for the success or failure in achieving tolerance: most discussants agreed that stable donor chimerism was sufficient to establish donor-specific tolerance. Considerable time was spent debating how transient chimerism might establish a form of regulatory tolerance and how that state might be identified and measured.
Safety Issues in Drug Withdrawal
There was an extensive discussion around the issue of making drug withdrawal as safe as possible. One overarching conclusion of this discussion was the need to be more systematic and standardized in collecting and disseminating clinical and immune monitoring data.
For example, many patients are tolerant already, having been enrolled in a variety of tolerance-induction protocols at different centers. It was agreed that valuable lessons can be learned from these experiences and that wider dissemination and availability of information would help inform future study design.
In keeping with this spirit, a great deal of attention at the meeting was devoted to examining what can already be learned from patients enrolled in the ongoing chimerism clinical trials at MGH, Stanford, Johns Hopkins, and Northwestern (with the first two centers using mixed-chimerism strategies, and the latter two using full-chimerism approaches). Serial monitoring and quantitation of donor chimerism, usually part of a flow cytometry–based immune phenotyping of the peripheral blood, was reported by all four centers and thought to be important, although the loss of chimerism in the peripheral blood did not always predict or lead to graft loss. It was thought that more emphasis on the development of strategies to quantitate the level of chimerism in the graft and bone marrow would be valuable. All centers were performing functional assays, including mixed leukocyte cultures and cell-mediated cytotoxicity, to investigate the evolution of donor-specific unresponsiveness, although there was agreement that these assays were of very limited predictive value. Tracking donor reactive clones through CDR3 sequencing is being investigated by MGH and Northwestern as a strategy for identifying donor reactive T cells, and the preliminary data suggesting that this tool can be applied to transplantation was thought to be extremely promising.
It was noted that each center currently has its own strategy for sample collection and storage. Implementation of standard operating procedure–driven sample collection, preparation, and storage was agreed unanimously (see recommendations later). Similarly, although extensive immune monitoring is being performed by each of the clinical centers, it can be difficult to compare results between centers because of a lack of harmonization of assays and assay conditions. It was agreed that creating a baseline set of standard assays and biomarkers that all centers adhered to would be extremely valuable in enabling cross-trial analyses, a particularly important goal, given the small patient numbers in any individual trial.
Making the Protocols Accessible to Other Patient Groups
All organs are not equal when it comes to tolerance induction. It is well known that some organs are tolerance prone (liver and kidney), whereas others are tolerance resistant (heart and lung). The reasons for these organ-specific differences remain unclear, but organ-specific differences in tolerogenicity need to be taken into account while developing tolerance strategies for extrarenal organs and tissues.
The liver is generally considered the most tolerogenic of transplanted organs, and withdrawal of all immunosuppression may be achieved up to 20% of appropriately selected liver allograft recipients without risk of imminent graft loss. In this workshop, the first successful induction of adult liver allograft tolerance using regulatory T cells was reported by a group in Hokkaido University, Japan. Based on results from nonhuman primate studies (4), regulatory T cells were induced and expanded in vitro by culturing recipient cells (obtained from splenectomy) irradiated donor peripheral blood monocyte cells under costimulatory blockade (using anti-CD80 plus anti-CD86 mAbs). After the recipients were conditioned with cyclophosphamide, the unfractionated cultured cells were infused back into the recipient. It was reported that 7 of 10 recipients were currently off immunosuppression for a duration of 2 to 18 months with stable liver function.
The specific considerations required for tolerance induction of islet allografts were discussed. The need to protect a very limited mass of β cells from humoral responses, alloantibodies, and autoantibodies was shown to be particularly critical for the induction of islet allograft tolerance. In a nonhuman primate system, a predominance of transitional B cells after rituximab and thymoglobulin treatments was associated with long-term islet allograft survival. B-cell tolerance may be critically important in inducing islet allograft tolerance. This may be achieved by using anti-BLyS treatment to control B-cell maturation.
Heart and Lung
The heart and lung are two of the most tolerance-resistant organs. However, studies in swine and nonhuman primate models at MGH revealed that heart allograft tolerance can be induced by cotransplantation of the renal allograft. Preliminary results suggest that tolerance-prone kidney allografts are able to confer unresponsiveness upon tolerance-resistant heart allografts by expanding regulatory T cells or enhancing their function. Understanding how kidney-specific elements amplify regulatory pathways could result in novel strategies to induce heart allograft tolerance in humans. Most recently, successful induction of tolerance was also achieved in major histocompatibility complex–mismatched lung allograft recipients through a mixed-chimerism approach with anti–interleukin-6R mAb. This suggests that inhibition of anti-inflammatory responses may be critical in achieving lung allograft tolerance.
Application of Tolerance Induction for Children
Applying tolerance induction protocols to children was a significant topic of discussion. As is generally recognized, pediatric patients are the population that benefits the most from transplantation in general and tolerance induction in particular. Although a fully functional thymus, present in all children, seems to be especially helpful in promoting tolerance in experimental systems, there are a number of concerns in applying certain components of current tolerance protocols to children. These include the use of cyclophosphamide and radiation, which could affect neurologic development and fertility. However, after weighing the pros and the cons, all participants agreed that it would be ethically proper to apply a tolerance protocol proven to be successful in adults to children at the upper end (≥12 years old) of the pediatric age group.
Funding the Studies
There was unanimous agreement that difficulties in securing funds for clinical trials was a major impediment to progress. The largest share of U.S. federal funding for this area comes from the National Institute of Allergy and Infectious Diseases (NIAID), and Nancy Bridges, Chief of the Transplantation Branch of the Division of Allergy, Immunity and Transplantation NIAID provided an overview of how that agency supported transplant research. Funding includes areas of basic discovery, translational research, and clinical trials (solicited and investigator initiated). Preclinical studies are supported including rodent and nonhuman primate researches. Support for clinical trials includes associated mechanistic studies in adults and children. Dr Bridges pointed out that the ideal concept of tolerance may not be achievable except in a small subset of patients, that there are safety implications in tolerance trials, and that striving for absolute, rather than incremental, goals can sometimes impede progress.
Larry Turka represented the ITN, an international clinical research consortium funded primarily by NIAID. He explained that the ITN had made an internal decision to focus efforts on HLA-mismatched transplants. Areas of interest include kidney, islet, and liver transplantation, with emphasis on pilot clinical trials that are investigator initiated. The ITN has supported the development of immune monitoring assays that can be applied to the clinical trials that are sponsored by the ITN and is opening to partnerships in which they perform mechanistic assays in trials sponsored by other consortia or agencies.
Tim Schroeder, of CTI Clinical Trial and Consulting, addressed issues of infrastructure and cost for tolerance trials, focusing on some of the challenging aspects of these studies, including the limited numbers of patients available, the complexity of protocols, and the need for multidisciplinary approaches and regulatory oversight by institutional review boards, data safety monitoring boards, and the U.S. Food and Drug Administration. Most multicenter kidney transplantation trials require an infrastructure developed by contract research organizations and personnel with regulatory agency experience. The budgets for pivotal trials of drug safety and efficacy are in the range of $50,000 to $100,000 per patient, and costs for tolerance studies may be considerably higher (up to $300,000 per patient) because of the need for close patient monitoring, the use of additional nonstandard procedures and tests, and longer-term follow-up. The marked variation among centers in charges for the same procedures or tests is yet another challenge to overcome when developing trial budgets.
Amy Rosenberg of the U.S. Food and Drug Administration discussed how tolerance induction trials in organ transplantation can apply for expedited review or approval in four areas. These included breakthrough therapies in which there is a substantial improvement over available treatments, accelerated approval in which surrogate markers are used to document substantial improvement, fast track approval in which therapies address an unmet medical need, and priority review in which there is significant improvement in safety or efficacy over available therapies. U.S. Food and Drug Administration approvals would allow medical centers to bill insurers for the therapies. The approvals are usually applied for by commercial entities.
It was clear that the costs of the studies are a barrier to progress and considerable discussion revolved around the conditions that would allow investigators to charge insurance companies and Medicare for the costs of the tolerance induction procedures. There was great enthusiasm to establish a task force to reach out to third-party payers to fund costs of tolerance protocols.
Conclusions and Recommendations
This meeting was conceived to address the issue of how to make tolerance induction protocols a standard of care for appropriate transplant recipients. None of the attendees expected a solution to emerge during the meeting, but rather, all hoped to identify issues and barriers and to begin to craft a path forward, and indeed, by this metric, the workshop was a success. There was a frank and open exchange of ideas on a wide range of topics, and the workshop participants reached five consensus recommendations listed in the following sections. Efforts to implement these are already underway, and the next gathering of this TTS-supported workshop is scheduled for the fall of 2015.
Establish a reporting standards committee to develop the standards by which investigators report the results of tolerance trials. The following metrics might be included:
- Annual self-reported health state
- Strict reporting standard on histologic findings, with external independent review
- Functional organ status over time, for example, estimated creatinine clearance
- Annual days in the hospital and severe adverse events
- Standards for control groups. This is important because drug minimization protocols may have similar outcomes.
- Precise definitions of the cellular and pharmaceutical intervention
Establish standardized protocols for sample collection and storage at agreed time points for all centers undertaking tolerance trials to ensure that comparable samples (in terms of when and how they are obtained, and types of samples collected) from patients enrolled in different protocols are available for future analysis and comparison.
Establish a standard panel of biomarkers and assays agreed upon by the tolerance research community. This will be invaluable for the next phase of clinical investigation to facilitate optimization of current protocols and the design as well as the implementation of multicenter randomized clinical trials.
Children 12 years or older should be eligible for enrollment in tolerance-inducing protocols proven to be safe and successful in adults.
Establish a task force to engage third-party payers in discussions of how to fund costs of tolerance protocols.
The authors thank all of the workshop participants (please see Appendix) for their attendance and participation and especially acknowledge Geneviève Leclerc for outstanding administrative assistance and Francis Delmonico, as the President of TTS, in the formulation of the plan and agenda of the workshop in close collaboration with the Executive Leadership. The authors also thank TTS for organizing the meeting, and Novartis, Astellas, Genentech, Alexion, and Bristol-Myers Squibb for the additional support.
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2. Leventhal J, Abecassis M, Miller J, et al. Chimerism and tolerance
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2012; 4: 124ra128.
3. Kawai T, Cosimi AB, Spitzer TR, et al. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med
2008; 358: 353.
4. Bashuda H, Kimikawa M, Seino K, et al. Renal allograft rejection is prevented by adoptive transfer of anergic T cells in nonhuman primates. J Clin Invest
2005; 115: 1896.
The attendees of the meeting are as follows: M. Abecassis (Northwestern University School of Medicine), S. Alexander (Children’s Hospital at Westmead), J. Allan (Massachusetts General Hospital), S. Bartlett (University of Maryland), G. Benichou (Massachusetts General Hospital), N. Bridges (National Institutes of Health), W. Burlingham (University of Wisconsin), S. Busque (Stanford School of Medicine), A. Chong (University of Chicago), A.B. Cosimi (Massachusetts General Hospital), F. Delmonico (The Transplantation Society), E. Fuchs (The Johns Hopkins University), E. Geissler (University of Regensburg Hospital), G. Hill (Queensland Institute of Medical Research), D. Kaufman (University of Wisconsin), T. Kawai (Massachusetts General Hospital), A. Kirk (Emory University), N. Krieger (Novartis), J. Leventhal (Northwestern University School of Medicine), J. Madsen (Massachusetts General Hospital), J. Markmann (Massachusetts General Hospital), J. Miller (Northwestern University School of Medicine), A. Monaco (Beth Israel Deaconess Medical Center), R. Montgomery (The Johns Hopkins University), A. Naji (University of Pennsylvania), K Newell (Emory University), J.B. Park (Samsung Medical Center), A. Rosenberg (U.S. Food and Drug Administration), G. Ryoichi (Hokkaido University), D. Sachs (Massachusetts General Hospital), B. Sawitzki (Charité Universitätsmedizin Berlin), J. Scandling (Stanford University School of Medicine), T. Schroeder (CTI Clinical Trial and Consulting), J.A. Shizuru (Stanford University School of Medicine), T. Spitzer (Massachusetts General Hospital), S. Strober (Stanford University School of Medicine), M. Suthanthiran (Cornell University), L.J. Swinnen (The Johns Hopkins University), M. Sykes (Columbia University Medical Center), N. Tchao (Immune Tolerance Network), S. Todo (Research Institute of St. Mary’s Hospital), S. Tullius (Brigham and Women’s Hospital), L. Turka (Massachusetts General Hospital), K. Wood (Oxford University), and K. Yamashita (Hokkaido University)