Murine models of transplantation tolerance through mixed chimerism: advances and roadblocks

Abstract

Organ transplantation is the treatment of choice for patients with end‐stage organ failure, but chronic immunosuppression is taking its toll in terms of morbidity and poor efficacy in preventing late graft loss. Therefore, a drug‐free state would be desirable where the recipient permanently accepts a donor organ while remaining otherwise fully immunologically competent. Mouse studies unveiled mixed chimerism as an effective approach to induce such donor‐specific tolerance deliberately and laid the foundation for a series of clinical pilot trials. Nevertheless, its widespread clinical implementation is currently prevented by cytotoxic conditioning and limited efficacy. Therefore, the use of mouse studies remains an indispensable tool for the development of novel concepts with potential for translation and for the delineation of underlying tolerance mechanisms. Recent innovations developed in mice include the use of pro‐apoptotic drugs or regulatory T cell (T_reg) transfer for promoting bone marrow engraftment in the absence of myelosuppression and new insight gained in the role of innate immunity and the interplay between deletion and regulation in maintaining tolerance in chimeras. Here, we review these and other recent advances in murine studies inducing transplantation tolerance through mixed chimerism and discuss both the advances and roadblocks of this approach.

OTHER ARTICLES PUBLISHED IN THIS REVIEW SERIES

Immune tolerance in transplantation. Clinical and Experimental Immunology 2017, 189: 133–4.

Transplantation tolerance: the big picture. Where do we stand, where should we go? Clinical and Experimental Immunology 2017, 189: 135–7.

Operational tolerance in kidney transplantation and associated biomarkers. Clinical and Experimental Immunology 2017, 189: 138–57.

Immune monitoring as prerequisite for transplantation tolerance trials. Clinical and Experimental Immunology 2017, 189: 158–70.

Transplantation tolerance: don't forget about the B cells. Clinical and Experimental Immunology 2017, 189: 171–80.

Chimerism‐based tolerance in organ transplantation: preclinical and clinical studies. Clinical and Experimental Immunology 2017, 189: 190–6.

Regulatory T cells: tolerance induction in solid organ transplantation. Clinical and Experimental Immunology 2017, 189: 197–210.

Still in search of the Holy Grail

Transplantation is still the most effective treatment option for patients with end‐stage organ failure 1, but exposes the recipients to a life‐long dependency on immunosuppressive drugs. The necessity of permanent medication puts transplant recipients at risk of a wide range of possible complications 2. Immunosuppression increases the susceptibility to infections and cancers, while under‐immunosuppression may provoke episodes of rejection. In a substantial fraction of transplant recipients late graft loss cannot be prevented by current means, which further increases the demand for donor organs, which are a scarce and limited resource 3. Therefore, transplant immunologists still crave for a state where the recipient immune system accepts donor antigens specifically and permanently while remaining otherwise fully immunologically competent. Numerous strategies have been elaborated in rodent models to achieve this desired state, from which mixed chimerism (i.e. co‐existence of donor and recipient haematopoietic stem cells after a donor haematopoietic stem cell transplantation) emerged as a promising approach 4.

Rodent models have served historically as a useful tool to investigate the basic principles of allorecognition and graft rejection. The availability of defined mouse strains enabled the identification of major histocompatibility complex (MHC) and its role for transplant rejection 5. The landmark experiments by Peter Medawar in 1953 finally paved the way for mouse models as the most favourable instrument in search for the ‘Holy Grail’ of tolerance induction. Medawar could show that tolerance towards alloantigens can be induced deliberately by inoculating prenatal mice with a mixture of allogeneic cells 6. Those recipients accepted donor skin for an extended period of time while promptly rejecting skin from third parties. This finding constitutes a milestone in the history of transplantation tolerance, despite being clinically inapplicable. The first step towards practical feasibility came in 1955, when Joan M. Main and Richmond I. Prehn achieved donor‐specific tolerance by transplanting allogeneic bone marrow successfully into lethally irradiated mice 7. Initial euphoria was soon dampened by the finding that, depending on the strain combinations, most of the mice developed lethal graft‐versus‐host disease (GVHD) 8. Removing donor T cells from the bone marrow inoculum could avert GVHD, although at the expense of reduced engraftment rates 9. On the contrary, supplementing the graft with donor T cells promoted bone marrow engraftment by counteracting host immunity 10. Alternatively, high doses of fractionated total lymphoid irradiation were able to induce chimerism without any clinical evidence of GVHD 11, especially when combined with T cell‐depleting agents 12. Nevertheless, several chimeras still succumbed to radiation‐independent toxicities. Full chimeras also featured specific defects in controlling viral infections 13 ascribed to the discrepancy between positive selection of donor T cells by host thymic epithelial cells and antigen presentation by peripheral donor antigen‐presenting cells (APCs) 14. To overcome this obstacle, Ildstad and Sachs inoculated a mixture of T cell‐depleted host and donor bone marrow into lethally irradiated mice. Mixed lymphohaematopoietic chimerism developed in all lineages without signs of GVHD and skin allografts were specifically accepted long‐term 15. This observation established mixed chimerism as potent approach for the induction of transplantation tolerance.

Targeting T cells to induce mixed chimerism

Because irradiation entails severe side effects, strategies were sought to minimize the required dose for bone marrow engraftment. To achieve this goal, it was necessary to control more specifically the mechanisms responsible for allogeneic bone marrow rejection. After T cells were identified as the major contributors of MHC mismatched bone marrow rejection 16, efforts were made to target them with a higher precision. Elimination of host T cells with depleting antibodies allowed reducing the dose of total body irradiation (TBI) to 6 Gy 17. As T cell‐depleting antibodies primarily target peripheral T cells, local irradiation of the thymus was employed to enable a further reduction of the necessary TBI to a non‐myeloablative dose of 3 Gy 18. Increasing the number of bone marrow cells (‘megadose’ bone marrow transplantation) could eliminate the need for TBI, but not the need for thymic irradiation 19, 20. However, profound T cell depletion is also risky, as it renders the recipient vulnerable to severe infections until T cell repopulation is sufficient again after several weeks. This is a matter of concern, as most patients undergoing transplantation are of adult age and therefore exhibit a reduced thymic function leading to prolonged periods of immunoincompetence 21. Consequently, T cell‐specific antibodies were modified to prevent their activation without depleting them. Blocking the co‐receptors CD4 and CD8 was sufficient to induce chimerism and skin graft tolerance over minor antigen barriers, but failed to do so in the setting of MHC disparities 22. The use of co‐receptor blocking antibodies has recently regained importance for the treatment of autoimmune disorders. Inducing mixed chimerism through concomitant blockade of CD3 and CD8 has been used recently to reverse autoimmunity in non‐obese diabetic (NOD) mice 23. However, the emergence of co‐stimulation blockers (targeting the CD28 and CD40 pathways) brought significant progress, as they provided an effective strategy to induce mixed chimerism without the need for T cell depletion in recipients conditioned with 3Gy TBI 24, and even obviated the need for any irradiation when high enough marrow doses were administered 25, 26.

Co‐stimulation blockade and then…?

Co‐stimulation blockade per se is not sufficient to allow engraftment of conventional (i.e. clinically realistic) donor bone marrow doses in the absence of irradiation. As tolerance‐inducing protocols should ideally be non‐cytotoxic for the sake of patient safety, further strategies are needed to control co‐stimulation blockade‐resistant mechanisms of rejection. The mechanistic target of rapamycin (mTOR) inhibitor was effective in allowing a further reduction in the required TBI dose when given together with co‐stimulation blockade 27. Intriguingly, calcineurin inhibitors, in contrast, impaired tolerance induction under these circumstances by blocking peripheral deletion of alloreactive T cells 27. Interrupting inducible T cell co‐stimulator (ICOS) and CD134 (OX40) signalling has also been shown to synergize with CD28 and CD40L blockade in preventing T cell activation and prolonging allograft survival 28, 29. In line with this, engraftment of ICOS^–/– bone marrow was significantly higher compared to wild‐type grafts (WT) 30, while the effect of OX40L blockade was only marginal 31, 32. Combining CD40L blockade with the pro‐apoptotic small molecule ABT‐737, a Bcl‐2 inhibitor, induced stable mixed chimerism and long‐term allograft survival without the need for further cytoreductive conditioning. Under these circumstances calcineurin inhibitors were required to prevent the resistance of activated T cells to ABT‐737 by reducing the expression of the anti‐apoptotic factor Bcl‐2A1 33, 34. Several Bcl‐2 family inhibitors, including the ABT‐737 derivative navitoclax, are under clinical development as anti‐cancer drugs and could be valuable components of chimerism‐based tolerance regimens.

Cell therapies coming of age

Cell therapies have come into focus in recent years, as they provide several appealing advantages over small‐molecule or biological pharmaceuticals 35. Regulatory T cells (T_regs) eminently aroused the interest of the whole transplant field due to their critical role in maintaining self‐tolerance. Despite significant progress in this field, there is still an ongoing debate about the preferable origin of the cells (donor versus recipient) and the optimal preparation (isolation, activation, expansion) of the product 36, 37. While it remains unclear whether T_reg transfer by itself will be sufficient to induce transplant tolerance, it has emerged as an exceptionally potent strategy to promote bone marrow engraftment. Transfer of polyclonal, in‐vitro‐activated T_regs from the recipient led to engraftment of conventional doses of allogeneic bone marrow without the need for any cytoreductive conditioning when given together with a short course of co‐stimulation blockade [α‐CD40L + cytotoxic T lymphocyte antigen 4‐immunoglobulin (CTLA4‐Ig)] and rapamycin. Durable tolerance towards fully mismatched skin and heart allografts was achieved in more than 90% of the recipients 38, 39. Notably, recipient T_regs were superior to donor or third‐party T_regs in this model 40. An adapted form of this protocol has been translated successfully to non‐human primates 41. Recently, retinoic‐acid induced alloantigen‐specific T_regs from the recipient were reported to induce chimerism and tolerance in a non‐myeloablative bone marrow transplantation (BMT) model using CTLA4‐Ig and rapamycin. Considerable levels of chimerism were achieved which gradually declined over time, and long‐term tolerance was obtained in approximately 35% of the recipients 42.

Regulatory cell therapies are being tested currently in a considerable number of clinical trials, but their application as a clinical routine is still elusive. Therefore, it would be desirable to replace cell therapies by pharmaceutical agents that expand T_regs selectively in vivo or mimic their effector function. Interleukin (IL)‐2 complexes increased T_reg numbers efficiently in vivo 43, 44, but were not able to replace T_reg therapy in a co‐stimulation blockade‐based BMT model. Rather, IL‐2 complexes enhanced bone marrow rejection by stimulating CD8 and natural killer (NK) cells expressing the low‐affinity IL‐2 receptor 44. In another study, a non‐lytic IL‐2 fusion protein (Fc silenced) promoted allogeneic bone marrow engraftment under CD40L blockade and rapamycin. This effect was even more pronounced when combined with a transforming growth factor (TGF)‐β fusion protein 45. The discrepancy between both administration forms of IL‐2 may be related to the distinct binding behaviour to the IL‐2 receptor as well as to differences in the Fc portion 46, 47. Other pharmaceuticals have been proposed to expand T_regs in vivo in order to ameliorate GVHD 48, 49, but have not been tested so far in the effort to induce mixed chimerism.

Space requirement for stem cells

The development of irradiation‐free protocols has raised the question of whether distinct niches need to be emptied actively in the recipient for stem cells contained in conventional doses of donor bone marrow to engraft 50. Several models achieving sustained levels of mixed chimerism without creating ‘space’ for donor bone marrow argue against the requirement for emptying niches 33, 38. These observations do not negate that creating additional space promotes bone marrow engraftment further. Mobilizing recipient bone marrow with a CXCR4 antagonist (AMD3100) enhanced mixed chimerism in a congenic mouse model 52. In the allogeneic setting AMD3100 increased donor chimerism shortly after BMT, but the effect vanished gradually over time without promoting skin graft tolerance 53. Depriving CXCR4 from its ligand CD26 by Diprotin A or sitagliptin did not improve bone marrow engraftment in non‐myeloblative irradiated recipients 54. In contrast, depleting haematopoietic stem cells from their niches by an antibody directed against the stem cell factor c‐Kit was effective in establishing donor bone marrow engraftment in immunodeficient mice. In combination with blockade of CD47, α‐c‐Kit antibodies even extinguished > 99% of host haematopoietic stem cells, thus promoting bone marrow engraftment in immunocompetent mice 55. Accordingly, creating space itself enhances bone marrow engraftment, but needs to be combined with strategies to prevent rejection of allogeneic cells to be most effective.

Innate immunity rises from the shadow

Adaptive immunity has long been regarded as the driving force of organ transplant rejection. Nonetheless, innate immunity emerged as crucial additional contributor to alloresponses, especially in the absence of co‐stimulatory signalling 56, 57. Innate immune cells infiltrate the graft in response to the inflammation elicited by the surgical trauma and ischaemia/reperfusion injury, become activated and augment adaptive immunity through the release of proinflammatory cytokines. However, growing evidence suggests that innate cells themselves also possess the ability to recognize foreign tissues despite the lack of somatically rearranged receptors 58. In contrast to organ transplants, NK cells have long been recognized as major mediators of cell transplant rejection, as they readily kill allogeneic bone marrow through their ability to recognize ‘missing‐self’59. NK cells are resistant to co‐stimulation blockade 60 and their depletion or blockade [through lymphocyte function‐associated antigen 1 (LFA1)], which is necessary for NK–target cell interactions 61), promoted engraftment of conventional numbers of allogeneic bone marrow under co‐stimulation blockade and busulfan 62. Apart from NK cells, monocytes have been reported to augment allograft rejection by non‐self‐recognition which is driven by CD47/signal regulatory protein alpha (SIRPα) interactions 63. CD47 is expressed on a wide range of cells and delivers a ‘don't eat me’ signal to macrophages via ligation of SIRPα. The highly polymorphic binding site of SIRPα presumably explains the ability of monocytes/macrophages to discriminate between self and non‐self 64. With regard to bone marrow transplantation, it is noteworthy that CD47 is also present on haematopoietic stem cells and progenitors. CD47^–/– BM cannot engraft in lethally irradiated syngeneic WT mice 65, implying that macrophages engulf stem cells if their SIRPα receptor cannot bind CD47 66, 67. In line with this, the macrophage depleting agent clodronate promoted the induction of haematopoietic chimerism and donor‐specific skin graft tolerance 68.

Mechanisms preserving tolerance

Depending on the specifics of the regimen used to induce mixed chimerism, tolerance is maintained by different mechanisms which can be divided broadly into deletional and regulatory. The specific elimination of alloreactive T cell clones (clonal deletion) can occur either in the thymus (central deletion) or the periphery (peripheral deletion). In chimeras, newly developing T cells directed against alloantigens are deleted specifically in the thymus by donor APCs arising from the BM graft 69, 70. Dendritic cells have long been implicated as the primary source of donor APCs in the thymus, although it is now acknowledged widely that there is an intensive cross‐talk between the thymus and the periphery 71. In particular, activated T cells, T_regs and B cells have been shown to re‐enter the thymus and to participate in negative selection 72, 73, 74. So far, however, the influence of these cell populations for clonal deletion in mixed chimeras remains undefined. Sparing the pre‐existing T cell pool by avoiding T cell depletion through co‐stimulation blockade revealed that alloreactive T cells can also be purged in the periphery. Peripheral deletion is a rapid process that is initiated shortly after BMT 24, 75. Nevertheless, it has been recognized for a long period of time that some grafts were still rejected in mixed chimeras even despite seemingly complete clonal deletion. This phenomenon was more pronounced in the presence of minor antigen disparities, implying a role for indirect alloreactivity 76, 77. So far, the available methodologies have only allowed assessing the deletion of ‘direct’ alloreactive T cells, while the fate of ‘indirect’ alloreactive T cells remains an unresolved issue. Several studies, however, indicated that regulatory mechanisms can control indirect alloreactivity 77. In an irradiation‐free protocol deploying T_reg transfer, active regulatory mechanisms preserved tolerance to minor antigen disparities in the absence of complete clonal deletion 39. Endogenous thymus‐derived T_regs were recruited to the graft, where they kept alloreactive T effector cells at bay. Depleting T_regs (α‐CD25) or blocking their effector molecules (α‐PD1, α‐CTLA4) suspended the cover of regulation leading to rapid rejection of the tolerized grafts. The infused T_regs seemed to pass on their suppressive behaviour as they vanished over time 77. This observation was also made in a non‐myeloablative model based on T_reg therapy in which the injected cells were required for the induction of tolerance, but not its maintenance 78. The significance for intragraft regulation probably lies in its ability to control alloimunity directed against tissue‐specific donor antigens.

The balanced interplay of clonal deletion and regulation seems to be the most solid foundation for a durable and stable state of tolerance. Deletional mechanisms are more robust, as they physically eliminate alloreactive T cells which can then no longer be reactivated subsequently during the course of an inflammation. In line with this, viral infection of stable mixed chimeras exhibiting complete clonal deletion neither broke tolerance nor abrogated chimerism 79. Moreover, clonal deletion is necessary to reduce the clone size of MHC‐reactive T cells. Conversely, T cells recognizing antigens expressed on donor bone marrow can undergo clonal deletion while T cells reactive towards tissue‐specific antigens (e.g. skin) possibly escape. This might explain why some protocols induce tolerance to certain tissues (e.g. haematopoietic cells) while others (e.g. skin) are rejected, a phenomenon known as split tolerance 80. Therefore, regulatory mechanisms are critical, as they are capable of tolerizing tissue‐specific antigens expressed by the transplanted organ. Regulatory mechanisms are, however, more susceptible to environmental influences. In this regard, TLR agonists abrogated co‐stimulation blockade‐induced prolongation of skin 81, and tolerance towards heart allografts induced by α‐CD40L and donor‐specific transfusion could be broken easily upon infection with Listeria monocytogenes. Intriguingly, the donor‐specific tolerance re‐emerged after clearance of the pathogen, as shown by the acceptance of a donor‐matched second transplant 82.

It has been described recently that cells of the innate immune system can also exhibit features of adaptive immunity 83, raising the question of whether innate cells can adapt to donor antigens. NK cells readily reject allogeneic bone marrow cells through missing‐self recognition, but are apparently tolerant towards donor and recipient cells in stable mixed chimeras. In non‐myeloablative models, NK cell tolerance could be broken by high amounts of IL‐2 in vitro but not in vivo 84, 85. Restoration of NK cell alloreactivity implies that NK cells adjust their activation threshold towards donor cells rather than being deleted. Studies with MHC class I‐deficient bone marrow have already suggested that NK cells adapt readily to the surrounding supply of MHC molecules 86. Recently, we found that NK cells of mixed chimeras reshape their receptor repertoire in favour of donor MHC to attenuate alloreactivity 87. However, the detailed mechanisms how NK cells are tolerized towards donor antigens in mixed chimeras currently remains incompletely understood (Fig. 1).

Figure 1.

Mechanisms maintaining tolerance in mixed chimeras: the following mechanisms contribute to tolerance after donor stem cells have engrafted in a recipient's bone marrow niches, leading to hematopoietic mixed chimerism. Donor‐derived antigen‐presenting cells (APCs) (dendritic cells and B cells) invade the thymus to eliminate developing donor‐reactive T cells (central clonal deletion). Pre‐existing alloreactive T cells and those escaping central deletion are either eliminated in the periphery (peripheral clonal deletion) or suppressed by regulatory T cells (T_regs) (peripheral regulation). Peripheral regulation can either occur in secondary lymphoid organs or in the allograft itself (intragraft regulation). Natural killer (NK) cells adapt to donor cells by reshaping their receptor repertoire (NK cell education).

Roadblocks limiting mouse models

Recently, the usability of young and healthy mice to predict reliably the behaviour of the experienced immune system of human patients has been increasingly questioned 88. A major cause for this discrepancy is the high frequency of alloreactive memory T cells found in adult humans in contrast to laboratory mice 89. Memory T cells are less susceptible to regulatory mechanisms of the immune system and are less affected by immunosuppressive drugs than naive T cells 90. Due to the advanced age of most transplant recipients, their immune system is highly experienced and compromises an increased pool of memory T cells, as well as other alterations of their immune system, which may hamper the potential to induce tolerance 91, 92. When a model of mixed chimerism established in young mice was tested in old mice it was found that advanced age by itself does not impair bone marrow engraftment or tolerance induction. The overall frequency of memory T cells increased with age, but without a detectable concomitant increase in alloreactive memory T cells 93. In an alternative approach, BMT recipients enriched with donor‐reactive memory T cells were exploited as a model to identify strategies that overcome the barrier of T cell sensitization without concomitant humoral sensitization. Adjunctive short‐term treatment with rapamycin or α‐LFA‐1 was effective in counteracting donor‐reactive memory T cells in this model, leading to long‐term mixed chimerism and donor‐specific tolerance 94, suggesting that chimerism protocols developed in young laboratory mice can be translated to clinically more relevant settings with moderate adaptations. Another circumstance that aggravates the interpretation of several mouse studies is the use of clinically unrealistic mismatch combinations. Several studies of mixed chimerism deployed strain combinations without minor antigen disparities which, however, exist universally in the human setting, and have been shown recently in murine models to impede long‐lasting chimerism and tolerance 76, 95.

Skin grafts are used commonly to monitor tolerance as they are technically not challenging, considered to be very immunogenic and are easy to monitor 96. However, the mechanisms of skin rejection differ greatly from that of primarily vascularized grafts. Kidney allografts would be most appropriate as they are also used in human tolerance studies, but are accepted spontaneously in several mouse strain combinations 97 and murine kidney transplant models are performed rarely due to technical challenges. Cardiac allografts are currently the most appreciated model of vascularized transplants, but also suffer from some disadvantages. In mice, heart allografts are regarded typically as rejected if no heartbeat is palpable, but substantial immunological damage can occur in the heterotopic, non‐life‐sustaining heart graft long before it stops beating. Therefore, a combination of donor graft types should be evaluated ideally in murine studies.

In order to allow translation of a protocol from mouse to humans, pharmaceuticals which are currently available, or will be in the foreseeable future, should be used. In most murine protocols a blocking antibody against CD40L is used to induce tolerance. In the human setting α‐CD40L antibodies were withdrawn from the market due to thromboembolic complications 98. Although several encouraging alternatives are currently tested, it is still unclear if and when they will reach clinical maturity 99. Blocking CD40 may not be equivalent to blocking CD40L, as CD40L can also ligate CD11b (Mac‐1) 100. Recently, it was shown that CTLA4‐Ig and rapamycin synergizes in a non‐myleoablative BMT model to replace α‐CD40L 101, at least to some degree, suggesting that CD40L blockade is not indispensable in chimerism‐based tolerance approaches. Similarly, bone marrow doses should be used that are clinically feasible. Typically, 15–20 × 10⁶ unseparated bone marrow cells are used to induce mixed chimerism, although this already constitutes the upper limit of clinical feasibility. Furthermore, in the clinic, mobilized peripheral blood stem cells are often preferred over unfractioned bone marrow 102. Notably, in mice, murine‐mobilized peripheral blood stem cells were less potent than bone marrow to induce mixed chimerism due to a higher amount of T cells 103, highlighting the importance of the donor cell source (Fig. 2).

Figure 2.

Factors influencing the translational relevance of mouse studies for chimerism‐based tolerance: Several factors make mouse protocols more relevant for potential clinical translation. With regard to patient safety, conditioning should ideally be non‐cytotoxic and drugs tested should be clinically available or at least under development. The donor and recipient pairs should be mismatched in major (MHC) and minor antigens (miHag), as is typically the case in clinical transplantation. A clinically realistic dose of donor bone marrow should be deployed and recipients of different ages should be tested. Different types of donor grafts (e.g. heart and skin) should be evaluated to test for tolerance in order to allow assessment of acute and chronic immunological damage and to include a range of tissue‐specific antigens. Mice not kept under specific pathogen‐free conditions should be evaluated as recipients, as heterologous immunity constitutes a considerable barrier to tolerance induction.

Summary and outlook

Chimerism‐based tolerance remains an attractive strategy for clinical translation and has recently proved its feasibility in a set of pilot trials. Three centres in the United States have elaborated distinct approaches to induce tolerance in recipients of living donor kidney transplants via donor haematopoietic stem cell transplantation. Tolerance was achieved in approximately 50% of human leucocyte antigen (HLA)‐mismatched patients, but safety issues were also noted. These trials are discussed in detail in a separate paper within this review series 104. Despite varying degrees of success, these trials constitute an important step towards clinical translation. Efforts are now being made to improve both efficacy and safety of current conditioning regimens in order to allow broader application. Those trials are based on protocols whose principles have been generated in mouse models, demonstrating the important role of mouse studies as a basis for the development of clinical tolerance strategies. Recent advances have allowed to reduce and to almost eliminate the remaining toxicity of chimerism protocols in mice and might pave the way for a more widespread clinical translation of this approach.

Disclosure

The authors of this manuscript have no conflicts of interest to disclose.