An overview of the necessary thymic contributions to tolerance in transplantation

Clinical Immunology 173 (2016) 1–9

Contents lists available at ScienceDirect

Clinical Immunology journal homepage: www.elsevier.com/locate/yclim

Review Article

An overview of the necessary thymic contributions to tolerance in transplantation Joseph R. Scalea a,⁎,1, John B. Hickman b,1, Daniel J. Moore c, Kenneth L. Brayman b,d a

Division of Transplantation, Department of Surgery, University of Maryland, United States School of Medicine, University of Virginia, United States Division of Endocrinology, Department of Pediatrics, Department of Pathology, Microbiology and Immunology, Vanderbilt University, United States d Division of Transplantation, Department of Surgery, University of Virginia, United States b c

a r t i c l e

i n f o

Article history: Received 13 September 2016 Received in revised form 4 October 2016 accepted with revision 22 October 2016 Available online 27 October 2016

a b s t r a c t The thymus is important for the development of the immune system. However, aging leads to predictable involution of the thymus and immunodeficiency. These immunodeficiencies may be rectified with thymic rejuvenation. Atrophy of the thymus is governed by a complex interplay of molecular, cytokine and hormonal factors. Herein we review the interaction of these factors across age and how they may be targeted for thymic rejuvenation. We further discuss the growing pre-clinical evidence defining the necessary and sufficient contributions of the thymus to successful tolerance induction in transplantation. © 2016 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Review of thymic development, thymopoiesis, and thymic involution . . . . . . . . 2.1. Thymic embryology . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Thymopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Positive and negative selection . . . . . . . . . . . . . . . . . . . . . . 2.4. Thymic involution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The thymus and transplantation tolerance . . . . . . . . . . . . . . . . . . . . 3.1. Central tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Peripheral tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Convergence of central and peripheral tolerance mechanisms . . . . . . . . 4. The effect of thymic involution on tolerance induction: opportunities for intervention 4.1. Decrease in T regulatory cells may inhibit tolerance induction . . . . . . . . 4.2. Lymphostromal interactions may affect pathways of tolerance induction . . . 4.3. Degeneration of T cell signaling may inhibit the ability to induce tolerance . . 5. Thymic manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Chemical rejuvenation . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Androgenic rejuvenation . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Stem cell transplantation . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Thymic transplantation . . . . . . . . . . . . . . . . . . . . . . . . . 6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author at: University of Maryland, 29 S. Greene Street, 21201 Baltimore, MD, United States. E-mail address: [email protected] (J.R. Scalea). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.clim.2016.10.010 1521-6616/© 2016 Elsevier Inc. All rights reserved.

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1. Introduction The thymus is the central compartment for instruction of a safe and robust immune system. As such, it has long been an important target in

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the establishment of transplantation tolerance and in fact, recent data have confirmed that a functional thymus is essential for induction of transplantation tolerance. However, harnessing the thymus to produce clinical impact may be limited due to the dynamic nature of thymic function and its involution beginning at puberty. Indeed, the majority of the patients who would benefit from protocols of transplantation tolerance are older but these patients are the most likely to have the least thymic function available for tolerance induction. Aging impacts a number of innate and adaptive immune mechanisms, inclusive of thymopoiesis [1,2]. Aging is clinically relevant to transplantation because the number of elderly patients in the United States with end stage renal disease who require a renal transplant has more than doubled in the last two decades [3]. In addition, transplantation in the aged population is becoming increasingly common [4]. Therefore, thymic rejuvenation may be important for successful tolerance induction, particularly as the transplant population ages. Here, we review thymic involution, pathways of transplantation tolerance and novel methods of thymic rejuvenation. 2. Review of thymic development, thymopoiesis, and thymic involution In order to understand how thymic involution/rejuvenation meshes with transplantation tolerance induction, it is important to first briefly review thymic physiology. 2.1. Thymic embryology Paramount to its function is the thymus' ability to develop bonemarrow derived precursor cells into selected T cells capable of maintaining tolerance and immunity. The thymus develops primarily under the influence of the forkhead box protein N1 (FOXN1) transcription factor [5]. FOXN1 controls the differentiation and proliferation of cortical and medullary thymic epithelial cells (cTECs and mTECs) and is therefore required to establish the architecture upon which all T cells train [6]. Accordingly, FOXN1 expression has been used as a marker of thymic

function and rejuvenation (discussed below, see androgenic rejuvenation). The thymus produces γ/δ T cells, naïve CD4 and CD8 α/β T cells, natural killer T cells (NKT), T regulatory cells (Tregs), and intraepithelial lymphocytes (IEL) or IEL progenitors [7]. Mechanisms of transplantation tolerance largely focus on the balance of alloreactive populations (e.g. CD4 and CD8) and T regulatory cells, although non-T cell lymphocytes are important as well. Each of these cell populations is discussed below. 2.2. Thymopoiesis Immature thymocytes make up only 5% of total thymocytes, are derived from hematopoietic cells, and do not express T cell receptors (TCRs), CD4 or CD8 (Fig. 1) [2,8,9]. Entering through blood vessels located in the corticomedullary junction (CMJ), immature thymocytes mature as they transmigrate from the CMJ to the cortex and then to the medulla [7,8]. First, acquisition of both CD4 and CD8 as well as the rearrangement of the TCR genes and subsequent expression of the TCR occurs within the thymic cortex [8]. These CD4+ CD8+ thymocytes represent nearly 80% of total thymocytes [8]. From the cortex, maturing thymocytes then pass back through the CMJ and migrate toward the medulla: a process associated with the upregulation of CCR7 [7,10–12]. 2.3. Positive and negative selection In the non-involuted thymus, the testing of TCR avidity for antigens presented by cTECs is essential to the process of positive selection. While unsuccessful recognition results in apoptosis, successful binding triggers a signaling cascade that allows T cell survival and progression to single-positive (CD4+ CD8− or CD4− CD8+) lymphocytes [13]. The small proportions of cells that are positively selected then migrate to the medullary epithelium for negative selection [7,9,11,14]. In the medulla, mTECs express a comprehensive array of tissue-specific antigens (TSAs). To this end, the transcription factor autoimmune regulator (AIRE), in association with the protein deacetylase Sirtuin-1 (Sirt1), induces the promiscuous gene expression of nearly all of the body's selfantigens by mTECs [15]. As a result, single-positive thymocytes that

Fig. 1. Thymopoiesis. This is a cartoon of thymopoiesis based on the literature. Progenitor cells enter the gland at the corticomedullary junction, transmigrate to the cortex wherein they acquire CD4 and CD8 expression (double positive). Thereafter, thymocytes make their way to the medulla, where negative selection occurs.

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display an excessively high avidity for binding to self-tissue are negatively selected for clonal deletion by apoptosis or clonal anergy [10,14, 16–19]. Ultimately, the result of this thymic training is the release of naïve, single-positive T cells with auto/alloreactive and tolerogenic abilities [18]. Indeed, a balance of alloreactive T cells and T regulatory cells has been suggested as the underlying mechanism responsible for supporting long-term transplantation tolerance [20,21]. Important to the mechanisms of tolerance are FoxP3+/CD4+/C25+ T regulatory cells. These potently suppressive cells control both auto- and alloreactive responses in an antigen-specific manner through direct cell-to-cell contact [7,22].

unresponsiveness) in humans remains elusive [33,34]. It is possible that thymic involution plays a role in determining the outcome after attempted tolerance induction in human protocols. All three of the best-described human protocols of transplantation tolerance utilize lymphoid radiation, T cell depletion (or robust antiproliferative agents), in addition to bone marrow transplantation [34–37]. Human protocols were adapted from both small and large animal models. These animal models, particularly large animal models, have provided the framework upon which our understanding of tolerance induction has been derived [34].

2.4. Thymic involution

Tolerance mechanisms can be described as central or peripheral. Central tolerance refers to tolerance established through intrathymic mechanisms that take place during the maturation of T cells [38–40]. While the mechanisms remain elusive, it has been hypothesized that clonal deletion of highly-avid donor reactive thymocytes at least one mechanism by which central transplantation tolerance is induced [39, 41]. Indeed, in mouse models of mixed chimerism, chimerism mediates intrathymic deletion of anti-donor T cells [39]. Models of central tolerance utilizing bone marrow transplantation have also been extended to large animals [42–44]. The hypothesis that tolerance can be induced by means of clonal deletion following hematopoietic stem cell transplantation implies that the recipient thymus can delete donor-reactive T cell clones. Indeed, when mice rendered tolerant through mixed chimerism were thymectomized 7 weeks after tolerance induction [45]. The authors concluded that persistence of donor antigen in euthymic mice continued even after establishment of tolerance through mixed chimerism, and contributed to early maintenance of tolerance [45]. In addition, these data suggested intra-thymic clonal deletion, and not peripheral mechanisms, is the primary mechanism by which tolerance is maintained in mixed chimeras [45]. Taken together, these data provide evidence that a functional thymus is required for tolerance induction. However, clinically, most patients who require a renal transplant are older, and may have involuted thymi. Accordingly, rejuvenating the aged thymus may enhance, or even be required, for some tolerance protocol candidates. The requirement of the thymus for establishment and maintenance of tolerance in the mixed chimera model suggests that donor antigen is presented to the recipient in the context of the recipient thymus during the establishment of graft tolerance. On the other hand, there is convincing data that not all antigens prompting clonal deletion are expressed exclusively by the thymus. It has been suggested that certain cell types may migrate to the thymus and present donor antigen, which then leads to clonal deletion of the T cell with the cognate receptor for the presented antigen [46,47]. Investigators have shown that dendritic cells can migrate to the thymus. These dendritic cells then present donor antigens not natively expressed by the thymus. By presenting extrinsic antigens, clonal deletion of T cells with TCRs specific for these foreign antigens can occur [47]. In an animal model, when CD11+ MHC class-II-positive dendritic cells were labeled with CFSE and then adoptively transferred into syngeneic recipients, this sub-class of donor dendritic cells was identifiable within the thymus [47]. Not all antigens to which a recipient acquires tolerance are presented within the thymus. These data tell us that peripheral mechanisms of tolerance can affect mature T cells that have left the thymus [38]. Despite the above small animal data, it remains unclear as to whether or not mixed-chimerism induced tolerance in humans is mediated by clonal deletion [40]. In fact, there are some data that argue against this idea. For example, other strategies of tolerance induction in humans reliant also on bone-marrow transplantation, have demonstrated success with robust (rather than mixed), long-lasting chimerism. Indeed, those patients transplanted under protocols utilizing the mixed chimerism approach have not been completely free of rejection. While tolerance in those with robust (and in some cases full) chimerism appears stable,

Understanding the natural history of thymic involution is important for transplantation and for tolerance establishment. Thymic function begins to decline soon after birth [23]. As the histology of the thymus changes with time, so does the thymus' response to alloantigen. The primary events of involution involve epithelial atrophy, resulting in decreased production of the cells known as recent thymic emigrants (RTEs), that pool of T cells that have survived positive and negative selection and entered into the periphery [23]. While in many animals involution leads to a notable decrease in thymic size, in humans, adipocytes replace the atrophied epithelium, such that thymic volume is maintained. This alteration in thymic architecture, though, has important implications for thymopoiesis [24]. A progressive loss of thymopoietic function and a decrease in immune competence accompanies thymic involution [25,26]. While thymic involution has ramifications for all developing thymocytes, thymic production of new T regulatory cells appears to undergo the steepest decline [27]. This disproportionate decrease in T regulatory cell populations may support the finding that tolerance establishment is more difficult in aged animals [28,29]. It is likely that there are many triggers of thymic involution, but reduced FOXN1 expression with a subsequent, age-related loss of thymic epithelium is known to play a dominant role [30]. In addition, once an individual reaches puberty, the speed of thymic involution may increase suggesting an important role for the hormonal milieu in the involution process [2]. Ultimately, thymic involution results in a decreased T cell repertoire, impaired immune responses, and increased susceptibility to autoimmune diseases, cancer, and infections [18,30]. Why thymic involution occurs is not clear. It has been observed that thymic involution is an evolutionarily conserved process that may protect aging organisms from autoimmunity [2]. In addition, involution may conserve metabolic energy and redirect it toward more genetically useful pursuits. Given that the thymus has ostensibly produced a pool of T cells sufficiently large and diverse by adulthood to protect the host, it has been hypothesized that thymic persistence may be detrimental to the host [26]. Regardless of purpose, thymic involution is seemingly inevitable. Interestingly, investigators have shown that thymic stromal cells retain the capacity for regeneration after involution, giving hope to the idea of reconstituting a functional and active thymus and, potentially, reversing the age-related decline in immunity, termed immunosenescence [31]. 3. The thymus and transplantation tolerance The end result of thymocyte training is the ability of T cells to differentiate self from non-self. For this reason, the thymus is an attractive target for interventions aimed at transplantation tolerance induction. If the thymus can be adapted to generate RTE's that recognize a transplanted organ as self, it may be possible to safely withdraw pharmacologic immunosuppression from transplanted patients. While strides have been made toward achieving tolerance in humans [32], consistently achievable transplantation tolerance (immunologic

3.1. Central tolerance

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this improved rate of stable tolerance needs to be balanced by the potential risk of eventual graft versus host disease [35].

peripherally generated, antigen-specific T regulatory cells suppress alloreactive cellular responses, holding the anti-donor response at bay [20,21,62].

3.2. Peripheral tolerance 3.3. Convergence of central and peripheral tolerance mechanisms In contrast to central tolerance, acquisition of peripheral tolerance refers to educational mechanisms that take place outside the thymus. Pre-clinical models exploiting peripheral mechanisms of transplantation tolerance have yielded encouraging results. In an MHC class-I mismatched model of transplantation tolerance in euthymic miniature swine, a 12-day course of high-dose cyclosporine inhibition could lead to durable, long-term tolerance [48–51]. Bone marrow and chimerism were not required for establishment of chimerism in this model. In this pre-clinical model, infusion of IL-2 (allo-stimulatory), replacement of the transplanted kidney with a donor-matched graft, and a donormatched skin graft all failed to abrogate tolerance [20,50,52–54]. When transplantation across the same MHC barrier was attempted in aged animals (not euthymic), tolerance was not successfully induced. As such, investigators considered that involution of the aged thymus prohibited the development of tolerance [28,50,55–57]. Conversely, when euthymic animals were rendered tolerant and then subsequently thymectomized, tolerance was maintained. These findings suggested that the thymus was required for tolerance induction, but not for maintenance of immunologic unresponsiveness. This model of peripheral tolerance induction appears to be mediated by FoxP3+/CD4+/C25+ T regulatory cells circulating in the peripheral blood. While it is unclear how FoxP3+/CD4+/C25+ cells develop during the tolerance induction protocol, several facts point toward T regulatory cell involvement in tolerance induction and maintenance in this model. T regulatory cells within the peripheral blood and within the transplanted graft (graft infiltrating cells) have been correlated with donor specific tolerance [58–60]. To test the hypothesis that these thymic dependent T regulatory cells were required for tolerance induction and maintenance, investigators first attempted to remove regulatory cells through apheresis of circulating regulatory cells in addition to surgical nephrectomy of the transplanted kidney, and subsequent retransplantation of a donor-matched renal allograft [20,21,61]. Following removal of both of these T cell compartments, investigators observed a brisk rejection crisis which subsided [20]. This rigorous attempt to remove regulatory cells demonstrated the strength of tolerance in this model. In an experiment designed to mirror this removal of regulatory cells, investigators attempted the adoptive transfer of peripheral T regulatory cells by transfusing the apheresis product from a tolerant animal into a naïve euthymic MHC-matched recipient. Interestingly, the apheresed (and tolerant) cells alone did not render the naïve recipient animal tolerant in all cases [62,63]. However, when both the kidney and the apheresis product were administered together, tolerance in the MHC matched, naïve recipient was induced [20,62,63]. Taken together, these data suggest that the thymus is required for T tolerance induction and that T regulatory cells can support durable tolerance to MHC mismatched donors, even in the long term. Equally supportive of a thymic dependent T regulatory cell mechanism for tolerance induction in this model is evidence that transplantation of a juvenile thymus into an aged animal will render the aged animal tolerant [28,29,64]. Likewise, authors found in the same swine model that the aged thymus could be rejuvenated when transplanted into a juvenile recipient [29]. Additionally, tolerance induction was possible in animals with a rejuvenated, aged thymus [29,64]. The mechanisms underpinning thymic-dependent peripheral tolerance induction are unclear. Some authors have hypothesized that donor dendritic cells migrate to the recipient thymus and lead to clonal deletion of alloreactive clones [28,52,53]. What is known, however, is that the ongoing presentation of donor antigen, usually in the form a transplanted organ is required for tolerance maintenance [20,21,61, 62]. In light of the fact that T regulatory cells are thought to play a prominent role in peripheral tolerance, it may be that thymic or

There may be considerable overlap between central and peripheral mechanisms for development of immunologic unresponsiveness, mediated by an interchange of specialized peripheral cells traveling to and from the thymus [46,65,66]. While much of the data investigating mechanisms of peripheral tolerance has focused on T regulatory cells, newly emerging data have suggested that T regulatory cell populations are themselves upregulated following establishment of mixed chimerism through hematopoietic stem cell transplantation. Whether these T regulatory cells are responsible for tolerance or are simply a marker of a tolerant state is not clear. There is a building body of literature suggesting that regulatory myeloid cells (RMCs), of which there are 3 types, upstream to T regulatory cell development may be critical for tolerance induction [66–69]. In addition, there may be communication between these cells and the thymus during tolerance induction. The three populations of RMCs include: regulatory macrophages (M-regs), regulatory dendritic cells (DCregs), and myeloid derived suppressor cells (MDSCs). M-regs, which have been studied in humans, are produced ex-vivo from human peripheral blood mononuclear cells (PBMCs) and are not thought to have a naturally occurring counterpart. Mreg infusions have been shown to prolong graft survival in animal models [67, 70]. Accordingly, M-reg infusions are being investigated as a pathway to tolerance induction in humans. DCregs, through the action of IL-10 and TGF-beta are also derived from PBMCs [67,70]. Recipient derived DCregs may play a role in tolerance induction, but that role is presently unclear. There are exciting data from small and large animal models suggesting that MDSCs (CD11b+ Gr-1hiLyGloLy6Clo in mice; CD14+ CD33+ DR− in primates and humans) may induce not only peripheral (in contrast to intra-thymic) deletion of alloreactive T cell clones, but also the upregulation of donor-specific T regulatory cells [46,66–70]. MDSCs are highly immunosuppressive, largely through the action of arginase-1 [67,69]. Arginase-1 in MDSCs decreases levels of locally available L-arginine, which is required for lymphocyte function [67–69]. What is unclear is whether these MDSCs are donor-derived or recipient-derived [66,69]. In a small animal model, chimerism was inhibited when MDSCs were depleted using anti-Gr-1 (anti-MDSC) antibodies [66]. In addition, subsequent organ transplantation in animals treated with MDSC depletion leads to uniform graft rejection [66]. These RMCs may represent the point at which central and peripheral mechanisms of tolerance overlap. While data is limited, rigorous work has suggested that CD11b+ monocytes, which develop extrathymically, home to the thymus [71]. Blood-to-thymus migrating myeloid cells have also been associated with peripheral Treg development [67,72]. Whether this transmigration of CD11b+ myeloid cells to the thymus is responsible for Treg generation in central and/or peripheral tolerance mechanisms is not yet clear. While the complete mechanisms underlying tolerance induction remain elusive, evidence of intrathymic deletion, thymus-dependent Treg development, and MDSC-thymus interactions suggests that the thymus plays a key role [46]. Taken together, it appears that RMCs upstream to Tregs are important for both intrathymic and extrathymic mechanisms of tolerance induction. Further exploitation of RMCs may promote consistently achievable tolerance induction in humans. 4. The effect of thymic involution on tolerance induction: opportunities for intervention As the thymus supports multiple processes that promote immune tolerance and permit the generation of tolerogenic cells, the degree to which these processes fail during involution may compromise tolerance induction. Understanding the interaction of thymic

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involution and immune homeostasis indicates several opportunities to strengthen immune regulation for the establishment of transplantation tolerance.

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microenvironment are both critical to the impaired nature of T cells in the elderly [75,81]. Further, we can extrapolate that interventions aimed at rejuvenating the aged thymic stroma might enhance our ability to induce tolerance.

4.1. Decrease in T regulatory cells may inhibit tolerance induction As described above, T regulatory cells appear to be involved in both central and peripheral mechanisms of tolerance. At present it is unclear whether T regulatory cells are required for tolerance, or they are simply a marker for a different process (i.e. MDSC-mediated immunosuppression). Age-dependent decrement in T cell output from the thymus leads to decreases in all T cell counts, but at different rates. In a mouse model of thymic involution, investigators produced mice whose T cells expressed a transgene encoding green fluorescent protein (GFP) under the control of the recombination activating gene-2 (rag2) promoter [27,73,74]. Accordingly, the thymic output of various T cell subsets could be serially monitored with age. While both GFP + recent thymic emigrants, as well as thymocyte populations, decreased as thymi progressively involuted, GFP + T regulatory cells defined by CD4+ FoxP3+, decreased to a far greater degree (eight-fold decrease vs. three-fold decrease) in adult mice when compared to younger mice. Investigators found that in older animals, a negative feedback loop exists, whereby IL-2 is inhibited by recirculating Tregs [27]. The investigators hypothesized that the biologic basis for this negative feedback loop is to progressively reduce the number of newly produced Tregs, in order to avoid dilution of the existing Treg repertoire. Alternatively, authors have reasoned that recirculation of Tregs back to the thymus may provide feedback to the thymus with regard to peripheral regulation [27]. These data may provide a mechanistic basis for a) some of the immune deficiencies seen in aged patients and b) the basis by which tolerance is not readily established in aged, large animal models [27]. 4.2. Lymphostromal interactions may affect pathways of tolerance induction The age-dependent, functional decrement of T cells is likely a result of both changes in the bone marrow as well as the thymus. The interactions between the lymphocytes and non-lymphocytes within these spaces (bone marrow and thymus) are termed lymphostromal interactions [75]. Although both qualitative and quantitative changes occur in bone marrow progenitors with age, it is thought unlikely that the number of progenitor cells produced from the bone marrow alone is responsible for reduced T cell number and function [75–77]. Indeed, the number of bone marrow progenitors in the elderly is relatively similar to younger patients [75]. In older human subjects (N 70 years of age), investigators found that the percentage of multipotent CD34+ CD38− cells actually is increased compared with younger patients [76]. In contrast, the number of early B cell precursors decrease with age [76]. As such, a recent body of literature has suggested that stromal cells within the thymus may contribute significantly to age-related deficiencies of T cells. [77–80] When investigators transplanted young, CD45.1+ bone marrow progenitor cells into either young mice (approximately 2 months old) or older mice (approximately 22 months old), significantly fewer mature T cells were produced from older mice [75]. In addition, when aged bone marrow precursors were exposed to fetal thymi, the lymphohematopoietic cells were capable of generating similar numbers of thymocytes when compared with younger lymphohematopoietic cells [75]. In addition, the more time the aged bone marrow progenitors spent in contact with the younger thymic tissue, the less a distinction could be made between the CD4+ and CD8+ profiles generated by either younger or older cells. Furthermore, the degeneration of an aged stroma, which disallowed proper differentiation of younger bone marrow progenitors, occurred earlier when compared with the younger stroma. Taken together, these data demonstrated that non-lymphoid stromal cells in the bone marrow and the thymic

4.3. Degeneration of T cell signaling may inhibit the ability to induce tolerance Because defective adaptive immune responses are thought to be the driving force behind immunosenescence in the aged population, there has been significant interest in T cell signaling as a function of age [82, 83]. Efficacy of T cell signaling is reduced in the elderly [82]. In a study of elderly patients compared with younger patients, the expression of CD28, an important costimulatory molecule, was significantly reduced in the older population [82]. In addition, the decrease in CD28+ T cells correlated with a decrease in the CD4:CD8 T cell ratio. [82] The expression of CD25, a cell surface marker associated with activated T cells (and itself a component of the IL-2 receptor), is also decreased in the aged population [83]. Additionally, when both CD25 and CD28 expression levels are low, as is seen with the T cells of older patients, cells are more susceptible to apoptosis through the action of CD95 (also known as Fas, a molecule which is part of the tumor necrosis factor receptor super family) and CD95 ligand (FasL) [83]. These data, and others, have led to the hypothesis that decrements in T cell function which occur with age are also likely due to changes in lymphostromal interactions within the thymus and the bone marrow [75,79,84]. Synthesizing the above data, it would appear reasonable to conclude that returning the architecture of thymus to its youthful state would enhance the ability for transplant physicians to establish solid organ transplant tolerance in older patients. 5. Thymic manipulation At the core of the pursuit of allogeneic, immune unresponsiveness and restoration of immune function with aging are efforts to understand and reverse thymic involution. Thymopoiesis and the subsequent induction of tolerance are dependent on a complex exchange between the thymic microenvironment, developing thymocytes, and their progenitors [31]. This complexity is manifest by changes in cellularity, decreased rates of epithelial turnover and proliferation, alterations in thymic architecture, and changes in cytokine levels [31]. In addition, thymic involution compromises the body's ability to remove autoreactive T cells, resulting in chronic low-level inflammation [85]. While restoration of the aged thymus may occur through multiple routes, in general, they take one of three paths: a) chemical/androgenic rejuvenation, b) seeding of stem cells to regrow a juvenile and functional thymus, or c) direct transplantation of thymic tissue. 5.1. Chemical rejuvenation Multiple chemical and/or androgenic factors have been used to attempt thymic rejuvenation. One well-studied method involves the administration of keratinocyte growth factor (KGF). In murine models, KGF increases thymopoiesis, resulting in increased RTE output and increased thymic cellularity. [86] KGF affects the proliferation and differentiation of thymic epithelium, ultimately leading to an increase in mTECs and an apparent restoration of thymic architecture [86,87]. Alpdogan et al. demonstrated that while KGF-/- mice did not have an increased rate of involution, they were particularly susceptible to injury following irradiation, indicating an important role in maintenance of the thymic milieu [88]. Subsequently, the same group demonstrated that prophylactic administration of KGF enhanced thymopoiesis following irradiation and/or chemotherapeutic insult [88]. While this and other such discoveries has led to active interest in extending this protection to humans, others have recently reported that KGF was not effective in increasing thymopoiesis in immunocompromised patients with

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low CD4+ counts [89]. Further investigation of KGF may reveal a potential role for the agent in human trials of transplantation tolerance. The cytokines IL-7 and IL-22 have also shown to be thymogenic. Normally produced by thymic stromal cells, administration of exogenous IL-7 has been identified as necessary for the homeostatic proliferation of peripheral CD8+ T cells and IL-7 is essential for the establishment of immunologic memory [90–92]. Like KGF, IL-7 is likely unable to reverse thymic involution. However, exogenous IL-7 appears to improve the reconstitution of immune function following chemotherapy and bone marrow transplantation (BMT) [90,93]. Given this potential benefit, further applications of IL-7 are in development [94]. Damage to thymic epithelial tissue can also be ameliorated by IL-22 administration. [95] Dudakov et al. reported that IL-22 effectively regenerated the thymic microenvironment and enhanced proliferation of developing thymocytes [95]. The utility of IL-22 for human clinical trials requires further research [96]. 5.2. Androgenic rejuvenation Some of the most exciting work with thymic rejuvenation has surrounded the hormonal effects on thymic architecture. Given the relationship between involution and the onset of puberty, a great deal of work has been done determining the relationship between sex hormones and the thymus [24,97]. Underscoring the importance of this relationship is the observation that the thymus undergoes significant morphological changes, rejuvenation and subsequent involution during and after pregnancy as a result of hormonal interactions [98]. Indeed, steroid receptors are present in both stromal cells and thymocytes. One way that sex steroids affect T cell development is through inhibiting Delta-like 4 (Dll4) Notch ligand signaling, which is essential to the committed differentiation of T cell progenitors [99]. Given this evidence, sex steroid ablation (SSA), either surgically or chemically, has been used to rejuvenate the thymus. Luteinizing hormone-releasing hormone/gonadotropin-releasing hormone (LHRH/GnRH) receptor

a) Juvenile

b) Aged

c) Aged, 6 mo. after LI

f)

e)

Me a n T R E C v a lu e -T h y m ic Tis s u e

sjTRECs/100’000 cells

d)

analogues, in particular, have been found to restore thymic cellularity and the architecture of the thymic microenvironment while subsequently increasing the export of T cells to the periphery [24,100–102]. Indeed, chemical castration through suppression of testosterone by LHRH/GnRH agonists in small animal models has led to the rejuvenation of the aged thymus [103]. Conversely, pregnancy induced increases in circulating estrogens (and/or exogenous estrogens) have been shown to incite involution of the thymus through endocrine-induced immune modulations [104,105]. Mechanistically, it has been hypothesized that hormonally induced thymic involution results from a decrement in the thymic LHRH/GnRH binding sites, a reduction that occurs progressively with age [106,107]. Moreover, the recent work of Yamada et al. in large animal experiments has recapitulated these small animal data [24,64,108]. In fact, the use of LHRH/GnRH agonists in swine and baboons has led to rejuvenation of the aged thymus as detected by TREC analysis, histologic evaluation, and by MRI as well (Fig. 2). Furthermore, investigators have found that these surgically/chemically-rejuvenated thymi induced tolerance to allografts – a process that depended on the production of new Treg cells [102]. Griffith et al. warn, however, that the data produced from SSA studies reflecting increased T cell production does not necessarily “imply quality,” observing that regrown thymi still have decreased promiscuous gene expression and may therefore develop potentially self-reactive thymocytes [109]. Investigations of the thymorestorative or thymoprotective nature of leptin, ghrelin, growth factor, and corticotrophin-releasing factor are also promising [92,110–112]. These data tell us not only that thymic rejuvenation is possible, but secondarily that biomarkers such as TREC analysis may be helpful in determining the “health” of the thymus at the time of transplantation or during a period of attempted thymic rejuvenation. As mentioned above, FOXN1 expression is decreased with age [113]. In a mouse model utilizing inducible FOXN1 expression, investigators found that increased FOXN1 expression restored thymic function and size in animals with involuted thymi. Additionally, FOXN1 expression was associated with histologic regeneration of the thymic architecture

pre-injection

3 months post-injection

Fig. 2. Adapted from Scalea et al. (Transplant Immunology, 2014). Figs. 2a–c demonstrates the ability for leuprolide acetate (see Thymic Rejuvenation: androgenic rejuvenation) to rejuvenate the thymus, histologically, from an aged baboon. Fig. 2d–e show that this rejuvenation was also seen on imaging (MRI in this case). Fig. 2f shows an increase in T cell receptor excision circles measured in thymic tissue, after thymic rejuvenation with leuprolide acetate in a baboon.

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[114]. This suggests that FOXN1 is suppressed with age [114]. Further, authors concluded that FOXN1 expression reprogrammed fibroblasts in aged animals to become functional TECs [114]. Upon transplantation of treated animals, these induced TECs (iTECs) established a functional thymus, complete with recognizable thymic architecture capable of RTE production [114]. These iTECs, produced ex-vivo or through the upregulation of recipient FOXN1, potentially offer a novel avenue for improving preclinical and/or human tolerance protocols [114]. Controlling FOXN1 expression may have additional benefits beyond restoring thymic architecture. Counterintuitively, it has been shown that common thymic epithelial progenitors (TEPs)–those cells which are destined to become either cTECs or mTECs—do not depend on FOXN1 expression. [115] Early inhibition of FOXN1 expression may therefore be a useful strategy for preservation of TEPs [115]. In addition, TEPs can be produced ex-vivo [116]. When these ex-vivo produced TEPs were administered to animals, TEPs developed into functional TECs leading to rejuvenated thymic architecture and increased production of functional peripheral T cells once co-transplanted hematopoietic precursors were introduced [116]. Clearly, the upregulation and the timing of FOXN1 expression can both affect thymic architecture, depending on the cell type [117]. Harnessing FOXN1’s ability to generate TEPs ex-vivo may improve tolerance strategies by optimizing the thymic microenvironment. 5.3. Stem cell transplantation Intrathymic injection of stem cells has been investigated as method for thymic rejuvenation [99]. Adding KGF and IL-7 to the injection of stem cells enhanced the recipient responses after irradiation. Also, these two agents had a synergistic effect. When used in conjunction with one another, KGF and IL-7 led to increased thymic cellularity and increased immature T cell production. This finding provided an important reminder that maintaining, seeding, and, ultimately rejuvenating thymic tissue and function relies on the complex interplay of its disparate parts [99,116–118]. 5.4. Thymic transplantation For individuals born without a thymus, transplantation of allogeneic thymic tissue has been successful. FOXN1 deficient children, presenting with human severe combined immunodeficiency (SCID) and athymic infants with DiGeorge syndrome have both been successfully treated with thymic implants [119–122]. Thymic transplantation has resulted in diverse and functional T cell production with activation of B cells [119,121]. There does appear to be a potential risk for developing autoimmune disease, however, particularly with regard to the thyroid [119,121]. Nonetheless, removal of the thymus, as is common in cardiac transplantation in children, is clearly associated with increased autoimmune risk, which reinforces the need for a proper balance of thymic function to secure normal immune activity and optimal transplant outcomes [123]. Experimentally, transplantation of aged/involuted thymic tissue into juvenile, thymectomized swine resulted in the reversal of the involution process, suggesting that the underlying mechanism of involution relies more heavily on influences extrinsic, rather than intrinsic, to the thymus [64]. In addition, it has been shown that simultaneous transplantation of the thymus and renal allografts from a single donor to a thymectomized animal successfully induced tolerance in large animal models [29]. While euthymic swine rejected the grafts, thymectomized animals were able to accept both the thymi and kidneys, even across fully mismatched MHC barriers [29]. Beyond transplantation of thymic tissue, injection of allogeneic cells into recipient animal thymi was shown to successfully induce donorspecific tolerance, in a model of islet transplantation for diabetes mellitus [124]. In addition, direct implantation of pancreatic islet cells into canine thymi resulted in successful engraftment and survival.

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Notably, these cells were capable of insulin production [125]. As it stands to reason that the benefits of such intrathymic injections may be narrowed as the thymus involutes, the clinical efficacy of this technique requires further investigation. 6. Summary The natural involution of the thymus may partly explain findings of immunosenescence and, as such, its associated morbidity. On the other hand, the mechanisms hypothesized to support tolerance in the animal and human tolerance studies are thymic dependent. Specifically, intrathymic deletion of anti-donor T cells is thought to support tolerance induction even in adults. These findings imply that although atrophied and involuted, the thymus is still functional enough to facilitate tolerance induction. Clearly, our understanding of thymic biology is incomplete and requires further investigation. What we do know is that the thymus lies at the crossroads between these interlaced pathways. With additional knowledge of the mechanisms that drive the thymus' response to transplanted organs, improved immunosuppression, rejection avoidance, and possibly even transplantation tolerance can become a reality. References [1] D.B. Palmer, The effect of age on thymic function, Front. Immunol. 4 (2013) 316. [2] T. Boehm, J.B. Swann, Thymus involution and regeneration: two sides of the same coin? Nat. Rev. Immunol. 13 (2013) 831–838. [3] T. Heinbokel, A. Elkhal, G. Liu, K. Edtinger, S.G. Tullius, Immunosenescence and organ transplantation, Transplant. Rev. 27 (2013) 65–75. [4] A.J. Matas, J.M. Smith, M.A. Skeans, et al., OPTN/SRTR 2012 annual data report: kidney, Am. J. Transplant. 14 (Suppl 1) (2014) 11–44. [5] D.M. Su, S. Navarre, W.J. Oh, B.G. Condie, N.R. Manley, A domain of Foxn1 required for crosstalk-dependent thymic epithelial cell differentiation, Nat. Immunol. 4 (2003) 1128–1135. [6] M. Nehls, B. Kyewski, M. Messerle, et al., Two genetically separable steps in the differentiation of thymic epithelium, Science 272 (1996) 886–889. [7] M.A. Weinreich, K.A. Hogquist, Thymic emigration: when and how T cells leave home, J. Immunol. 181 (2008) 2265–2270. [8] W. Savino, The thymus is a common target organ in infectious diseases, PLoS Pathog. 2 (2006), e62. [9] P.E. Love, A. Bhandoola, Signal integration and crosstalk during thymocyte migration and emigration, Nat. Rev. Immunol. 11 (2011) 469–477. [10] F. Ramsdell, B.J. Fowlkes, Clonal deletion versus clonal anergy: the role of the thymus in inducing self tolerance, Science 248 (1990) 1342–1348. [11] Y. Takahama, Journey through the thymus: stromal guides for T-cell development and selection, Nat. Rev. Immunol. 6 (2006) 127–135. [12] M. Hauri-Hohl, S. Zuklys, G.A. Hollander, S.F. Ziegler, A regulatory role for TGF-beta signaling in the establishment and function of the thymic medulla, Nat. Immunol. 15 (2014) 554–561. [13] T.K. Starr, S.C. Jameson, K.A. Hogquist, Positive and negative selection of T cells, Annu. Rev. Immunol. 21 (2003) 139–176. [14] C.D. Surh, J. Sprent, T-cell apoptosis detected in situ during positive and negative selection in the thymus, Nature 372 (1994) 100–103. [15] A. Chuprin, A. Avin, Y. Goldfarb, et al., The deacetylase Sirt1 is an essential regulator of Aire-mediated induction of central immunological tolerance, Nat. Immunol. 16 (2015) 737–745. [16] K. Aschenbrenner, L.M. D'Cruz, E.H. Vollmann, et al., Selection of Foxp3+ regulatory T cells specific for self antigen expressed and presented by Aire+ medullary thymic epithelial cells, Nat. Immunol. 8 (2007) 351–358. [17] J. Derbinski, A. Schulte, B. Kyewski, L. Klein, Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self, Nat. Immunol. 2 (2001) 1032–1039. [18] T. Boehm, S. Scheu, K. Pfeffer, C.C. Bleul, Thymic medullary epithelial cell differentiation, thymocyte emigration, and the control of autoimmunity require lymphoepithelial cross talk via LTbetaR, J. Exp. Med. 198 (2003) 757–769. [19] P. Matzinger, S. Guerder, Does T-cell tolerance require a dedicated antigenpresenting cell? Nature 338 (1989) 74–76. [20] J.R. Scalea, M. Okumi, V. Villani, et al., Abrogation of renal allograft tolerance in MGH miniature swine: the role of intra-graft and peripheral factors in long-term tolerance, Am. J. Transplant. 14 (2014) 2001–2010. [21] J. Weiner, J. Scalea, Y. Ishikawa, et al., Tolerogenicity of donor major histocompatibility complex-matched skin grafts in previously tolerant Massachusetts general hospital miniature swine, Transplantation 94 (2012) 1192–1199. [22] A.D. Griesemer, J.C. Lamattina, M. Okumi, et al., Linked suppression across an MHCmismatched barrier in a miniature swine kidney transplantation model, J. Immunol. 181 (2008) 4027–4036. [23] G.G. Steinmann, B. Klaus, H.K. Muller-Hermelink, The involution of the ageing human thymic epithelium is independent of puberty. A morphometric study, Scand. J. Immunol. 22 (1985) 563–575.

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J.R. Scalea et al. / Clinical Immunology 173 (2016) 1–9

[24] J.R. Scalea, R. Torabi, A. Tena, et al., The rejuvenating effects of leuprolide acetate on the aged baboon's thymus, Transpl. Immunol. 31 (2014) 134–139. [25] H.E. Lynch, G.L. Goldberg, A. Chidgey, M.R. Van den Brink, R. Boyd, G.D. Sempowski, Thymic involution and immune reconstitution, Trends Immunol. 30 (2009) 366–373. [26] A.J. George, M.A. Ritter, Thymic involution with ageing: obsolescence or good housekeeping? Immunol. Today 17 (1996) 267–272. [27] N. Thiault, J. Darrigues, V. Adoue, et al., Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors, Nat. Immunol. 16 (2015) 628–634. [28] P.A. Vagefi, F.L. Ierino, P.R. Gianello, et al., Role of the thymus in transplantation tolerance in miniature swine: IV. The thymus is required during the induction phase, but not the maintenance phase, of renal allograft tolerance, Transplantation 77 (2004) 979–985. [29] C. Kamano, P.A. Vagefi, N. Kumagai, et al., Vascularized thymic lobe transplantation in miniature swine: thymopoiesis and tolerance induction across fully MHCmismatched barriers, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 3827–3832. [30] C.L. Ortman, K.A. Dittmar, P.L. Witte, P.T. Le, Molecular characterization of the mouse involuted thymus: aberrations in expression of transcription regulators in thymocyte and epithelial compartments, Int. Immunol. 14 (2002) 813–822. [31] D.H. Gray, N. Seach, T. Ueno, et al., Developmental kinetics, turnover, and stimulatory capacity of thymic epithelial cells, Blood 108 (2006) 3777–3785. [32] D.W. Scott, A. Azimzadeh, Transplantation tolerance: one - step towards the holy grail, Clin. Immunol. 136 (2010) 161. [33] A.D. Kirk, Clinical tolerance 2008, Transplantation 87 (2009) 953–955. [34] T. Kawai, D.H. Sachs, B. Sprangers, et al., Long-term results in recipients of combined HLA-mismatched kidney and bone marrow transplantation without maintenance immunosuppression, Am. J. Transplant. 14 (2014) 1599–1611. [35] E.S. Yolcu, J.R. Leventhal, S.T. Ildstad, Facilitating cells in tolerance induction for kidney transplantation, Curr. Opin. Organ Transplant. 20 (2015) 57–63. [36] T. Kawai, J. Leventhal, J.C. Madsen, S. Strober, L.A. Turka, K.J. Wood, Tolerance: one transplant for life, Transplantation 98 (2014) 117–121. [37] J.D. Scandling, S. Busque, J.A. Shizuru, et al., Chimerism, graft survival, and withdrawal of immunosuppressive drugs in HLA matched and mismatched patients after living donor kidney and hematopoietic cell transplantation, Am. J. Transplant. 15 (2015) 695–704. [38] Y. Xing, K.A. Hogquist, T-cell tolerance: central and peripheral, Cold Spring Harb. Perspect. Biol. 4 (2012). [39] T. Wekerle, P. Blaha, Z. Koporc, S. Bigenzahn, M. Pusch, F. Muehlbacher, Mechanisms of tolerance induction through the transplantation of donor hematopoietic stem cells: central versus peripheral tolerance, Transplantation 75 (2003) 21S–25S. [40] D.H. Sachs, Mixed chimerism as an approach to transplantation tolerance, Clin. Immunol. 95 (2000) S63–S68. [41] C. Tian, X. Yuan, P.T. Jindra, J. Bagley, M.H. Sayegh, J. Iacomini, Induction of transplantation tolerance to fully mismatched cardiac allografts by T cell mediated delivery of alloantigen, Clin. Immunol. 136 (2010) 174–187. [42] M. Sykes, D.H. Sachs, A.W. Nienhuis, D.A. Pearson, A.D. Moulton, D.M. Bodine, Specific prolongation of skin graft survival following retroviral transduction of bone marrow with an allogeneic major histocompatibility complex gene, Transplantation 55 (1993) 197–202. [43] G. Benichou, O. Nadazdin, A.B. Cosimi, T. Kawai, Successful tolerance to fully MHC disparate renal allografts via donor hematopoietic mixed chimerism in nonhuman primates, Am. J. Transplant. 13 (2013) 2500. [44] Y. Yamada, T. Ochiai, S. Boskovic, et al., Use of CTLA4Ig for induction of mixed chimerism and renal allograft tolerance in nonhuman primates, Am. J. Transplant. 14 (2014) 2704–2712. [45] A. Khan, Y. Tomita, M. Sykes, Thymic dependence of loss of tolerance in mixed allogeneic bone marrow chimeras after depletion of donor antigen. Peripheral mechanisms do not contribute to maintenance of tolerance, Transplantation 62 (1996) 380–387. [46] H. Morris, S. DeWolf, H. Robins, et al., Tracking donor-reactive T cells: Evidence for clonal deletion in tolerant kidney transplant patients, Sci. Transl. Med. 7 (2015) (272ra10). [47] R. Bonasio, M.L. Scimone, P. Schaerli, N. Grabie, A.H. Lichtman, U.H. von Andrian, Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus, Nat. Immunol. 7 (2006) 1092–1100. [48] C.V. Smith, K. Nakajima, A. Mixon, et al., Successful induction of long-term specific tolerance to fully allogeneic renal allografts in miniature swine, Transplantation 53 (1992) 438–444. [49] B.R. Rosengard, C.A. Ojikutu, P.C. Guzzetta, et al., Induction of specific tolerance to class I-disparate renal allografts in miniature swine with cyclosporine, Transplantation 54 (1992) 490–497. [50] P.R. Gianello, T. Lorf, K. Yamada, et al., Induction of tolerance to renal allografts across single-haplotype MHC disparities in miniature swine, Transplantation 59 (1995) 884–890. [51] P.R. Gianello, J.M. Fishbein, B.R. Rosengard, et al., Tolerance to class I-disparate renal allografts in miniature swine. Maintenance of tolerance despite induction of specific antidonor CTL responses, Transplantation 59 (1995) 772–777. [52] P.R. Gianello, G. Blancho, J.F. Fishbein, et al., Mechanism of cyclosporin-induced tolerance to primarily vascularized allografts in miniature swine. Effect of administration of exogenous IL-2, J. Immunol. 153 (1994) 4788–4797. [53] B.R. Rosengard, J.M. Fishbein, P. Gianello, et al., Retransplantation in miniature swine. Lack of a requirement for graft adaptation for maintenance of specific renal allograft tolerance, Transplantation 57 (1994) 794–799.

[54] B.R. Rosengard, E.O. Kortz, C.A. Ojikutu, et al., The failure of skin grafting to break tolerance to class I-disparate renal allografts in miniature swine despite inducing marked antidonor cellular immunity, Transplantation 52 (1991) 1044–1052. [55] K. Yamada, P.R. Gianello, F.L. Ierino, et al., Role of the thymus in transplantation tolerance in miniature swine. I. Requirement of the thymus for rapid and stable induction of tolerance to class I-mismatched renal allografts, J. Exp. Med. 186 (1997) 497–506. [56] F.L. Ierino, S. Gojo, P.T. Banerjee, et al., Transfer of swine major histocompatibility complex class II genes into autologous bone marrow cells of baboons for the induction of tolerance across xenogeneic barriers, Transplantation 67 (1999) 1119–1128. [57] K. Yamada, P.R. Gianello, F.L. Ierino, et al., Role of the thymus in transplantation tolerance in miniature swine: II. Effect of steroids and age on the induction of tolerance to class I mismatched renal allografts, Transplantation 67 (1999) 458–467. [58] O. Bestard, P. Nickel, J.M. Cruzado, et al., Circulating alloreactive T cells correlate with graft function in longstanding renal transplant recipients, J. Am. Soc. Nephrol. 19 (2008) 1419–1429. [59] O. Bestard, J.M. Cruzado, I. Rama, et al., Presence of FoxP3+ regulatory T cells predicts outcome of subclinical rejection of renal allografts, J. Am. Soc. Nephrol. 19 (2008) 2020–2026. [60] F. Veronese, S. Rotman, R.N. Smith, et al., Pathological and clinical correlates of FOXP3 + cells in renal allografts during acute rejection, Am. J. Transplant. 7 (2007) 914–922. [61] M. Okumi, J.M. Fishbein, A.D. Griesemer, et al., Role of persistence of antigen and indirect recognition in the maintenance of tolerance to renal allografts, Transplantation 85 (2008) 270–280. [62] M. Okumi, J.R. Scalea, B.C. Gillon, et al., The induction of tolerance of renal allografts by adoptive transfer in miniature swine, Am. J. Transplant. 13 (2013) 1193–1202. [63] V. Villani, K. Yamada, J.R. Scalea, et al., Adoptive transfer of renal allograft tolerance in a large animal model, Am. J. Transplant. (2015). [64] S. Nobori, A. Shimizu, M. Okumi, et al., Thymic rejuvenation and the induction of tolerance by adult thymic grafts, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 19081–19086. [65] T. Kawai, H. Sogawa, S. Boskovic, et al., CD154 blockade for induction of mixed chimerism and prolonged renal allograft survival in nonhuman primates, Am. J. Transplant. 4 (2004) 1391–1398. [66] D. Hongo, X. Tang, J. Baker, E.G. Engleman, S. Strober, Requirement for interactions of natural killer T cells and myeloid-derived suppressor cells for transplantation tolerance, Am. J. Transplant. 14 (2014) 2467–2477. [67] B.R. Rosborough, D. Raich-Regue, H.R. Turnquist, A.W. Thomson, Regulatory myeloid cells in transplantation, Transplantation 97 (2014) 367–379. [68] D.I. Gabrilovich, S. Ostrand-Rosenberg, V. Bronte, Coordinated regulation of myeloid cells by tumours, Nat. Rev. Immunol. 12 (2012) 253–268. [69] J.C. Ochando, H.R. Turnquist, Innate immune cell collaborations instigate transplant tolerance, Am. J. Transplant. 14 (2014) 2441–2443. [70] P. Conde, M. Rodriguez, W. van der Touw, et al., DC-SIGN(+) macrophages control the induction of transplantation tolerance, Immunity 42 (2015) 1143–1158. [71] J. Li, J. Park, D. Foss, I. Goldschneider, Thymus-homing peripheral dendritic cells constitute two of the three major subsets of dendritic cells in the steady-state thymus, J. Exp. Med. 206 (2009) 607–622. [72] K.J. Wood, A. Bushell, J. Hester, Regulatory immune cells in transplantation, Nat. Rev. Immunol. 12 (2012) 417–430. [73] R. Rezzani, L. Nardo, G. Favero, M. Peroni, L.F. Rodella, Thymus and aging: morphological, radiological, and functional overview, Age 36 (2014) 313–351. [74] N.M. Milicevic, Z. Milicevic, O. Piletic, S. Mujovic, V. Ninkov, Reactivity of thymic metallophilic cells during the regeneration after the application of cyclophosphamide, J. Reticuloendothel. Soc. 34 (1983) 501–507. [75] L. Sun, R. Brown, S. Chen, Q. Zhuge, D.M. Su, Aging induced decline in Tlymphopoiesis is primarily dependent on status of progenitor niches in the bone marrow and thymus, Aging 4 (2012) 606–619. [76] K. Kuranda, J. Vargaftig, P. de la Rochere, et al., Age-related changes in human hematopoietic stem/progenitor cells, Aging Cell 10 (2011) 542–546. [77] I.K. Chinn, C.C. Blackburn, N.R. Manley, G.D. Sempowski, Changes in primary lymphoid organs with aging, Semin. Immunol. 24 (2012) 309–320. [78] S.J. Morrison, A.M. Wandycz, K. Akashi, A. Globerson, I.L. Weissman, The aging of hematopoietic stem cells, Nat. Med. 2 (1996) 1011–1016. [79] E. Massasa, X.S. Costa, H.S. Taylor, Failure of the stem cell niche rather than loss of oocyte stem cells in the aging ovary, Aging 2 (2010) 1–2. [80] I.M. Conboy, M.J. Conboy, A.J. Wagers, E.R. Girma, I.L. Weissman, T.A. Rando, Rejuvenation of aged progenitor cells by exposure to a young systemic environment, Nature 433 (2005) 760–764. [81] V.P. Zediak, I. Maillard, A. Bhandoola, Multiple prethymic defects underlie agerelated loss of T progenitor competence, Blood 110 (2007) 1161–1167. [82] R.B. Effros, N. Boucher, V. Porter, et al., Decline in CD28+ T cells in centenarians and in long-term T cell cultures: a possible cause for both in vivo and in vitro immunosenescence, Exp. Gerontol. 29 (1994) 601–609. [83] N.S. Dennett, R.N. Barcia, J.D. McLeod, Age associated decline in CD25 and CD28 expression correlate with an increased susceptibility to CD95 mediated apoptosis in T cells, Exp. Gerontol. 37 (2002) 271–283. [84] S. Swain, K. Clise-Dwyer, L. Haynes, Homeostasis and the age-associated defect of CD4 T cells, Semin. Immunol. 17 (2005) 370–377. [85] B.D. Coder, H. Wang, L. Ruan, D.M. Su, Thymic involution perturbs negative selection leading to autoreactive T cells that induce chronic inflammation, J. Immunol. 194 (2015) 5825–5837. [86] D. Min, A. Panoskaltsis-Mortari, O.M. Kuro, G.A. Hollander, B.R. Blazar, K.I. Weinberg, Sustained thymopoiesis and improvement in functional immunity

J.R. Scalea et al. / Clinical Immunology 173 (2016) 1–9

[87] [88] [89]

[90]

[91]

[92] [93]

[94] [95] [96] [97]

[98]

[99] [100]

[101]

[102]

[103]

[104]

[105]

[106]

induced by exogenous KGF administration in murine models of aging, Blood 109 (2007) 2529–2537. M. Erickson, S. Morkowski, S. Lehar, et al., Regulation of thymic epithelium by keratinocyte growth factor, Blood 100 (2002) 3269–3278. O. Alpdogan, V.M. Hubbard, O.M. Smith, et al., Keratinocyte growth factor (KGF) is required for postnatal thymic regeneration, Blood 107 (2006) 2453–2460. J.M. Jacobson, H. Wang, R. Bordi, et al., A randomized controlled trial of palifermin (recombinant human keratinocyte growth factor) for the treatment of inadequate CD4+ T-lymphocyte recovery in patients with HIV-1 infection on antiretroviral therapy, J. Acquir. Immune Defic. Syndr. 66 (2014) 399–406. J.A. Phillips, T.I. Brondstetter, C.A. English, H.E. Lee, E.L. Virts, M.L. Thoman, IL-7 gene therapy in aging restores early thymopoiesis without reversing involution, J. Immunol. 173 (2004) 4867–4874. K.S. Schluns, W.C. Kieper, S.C. Jameson, L. Lefrancois, Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo, Nat. Immunol. 1 (2000) 426–432. M.S. Ventevogel, G.D. Sempowski, Thymic rejuvenation and aging, Curr. Opin. Immunol. 25 (2013) 516–522. C.L. Mackall, T.J. Fry, C. Bare, P. Morgan, A. Galbraith, R.E. Gress, IL-7 increases both thymic-dependent and thymic-independent T-cell regeneration after bone marrow transplantation, Blood 97 (2001) 1491–1497. C.L. Mackall, T.J. Fry, R.E. Gress, Harnessing the biology of IL-7 for therapeutic application, Nat. Rev. Immunol. 11 (2011) 330–342. J.A. Dudakov, A.M. Hanash, R.R. Jenq, et al., Interleukin-22 drives endogenous thymic regeneration in mice, Science 336 (2012) 91–95. J.A. Dudakov, A.M. Hanash, M.R. van den Brink, Interleukin-22: immunobiology and pathology, Annu. Rev. Immunol. 33 (2015) 747–785. E. Velardi, J.A. Dudakov, M.R. van den Brink, Sex steroid ablation: an immunoregenerative strategy for immunocompromised patients, Bone Marrow Transplant. 50 (2015) S77–S81. T.A. Tibbetts, F. DeMayo, S. Rich, O.M. Conneely, B.W. O'Malley, Progesterone receptors in the thymus are required for thymic involution during pregnancy and for normal fertility, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 12021–12026. E. Velardi, J.J. Tsai, A.M. Holland, et al., Sex steroid blockade enhances thymopoiesis by modulating notch signaling, J. Exp. Med. 211 (2014) 2341–2349. J.S. Sutherland, G.L. Goldberg, M.V. Hammett, et al., Activation of thymic regeneration in mice and humans following androgen blockade, J. Immunol. 175 (2005) 2741–2753. J.A. Dudakov, G.L. Goldberg, J.J. Reiseger, K. Vlahos, A.P. Chidgey, R.L. Boyd, Sex steroid ablation enhances hematopoietic recovery following cytotoxic antineoplastic therapy in aged mice, J. Immunol. 183 (2009) 7084–7094. G. Zhao, D.J. Moore, J.I. Kim, et al., Inhibition of transplantation tolerance by immune senescence is reversed by endocrine modulation, Sci. Transl. Med. 3 (2011) (87ra52). B.D. Greenstein, F.T. Fitzpatrick, M.D. Kendall, M.J. Wheeler, Regeneration of the thymus in old male rats treated with a stable analogue of LHRH, J. Endocrinol. 112 (1987) 345–350. V.D. Dixit, R. Sridaran, M.A. Edmonsond, D. Taub, W.E. Thompson, Gonadotropinreleasing hormone attenuates pregnancy-associated thymic involution and modulates the expression of antiproliferative gene product prohibitin, Endocrinology 144 (2003) 1496–1505. A.L. Zoller, G.J. Kersh, Estrogen induces thymic atrophy by eliminating early thymic progenitors and inhibiting proliferation of beta-selected thymocytes, J. Immunol. 176 (2006) 7371–7378. B. Marchetti, V. Guarcello, M.C. Morale, et al., Luteinizing hormone-releasing hormone (LHRH) agonist restoration of age-associated decline of thymus weight,

[107]

[108] [109]

[110]

[111]

[112] [113]

[114] [115]

[116] [117]

[118] [119]

[120] [121]

[122]

[123]

[124]

[125]

9

thymic LHRH receptors, and thymocyte proliferative capacity, Endocrinology 125 (1989) 1037–1045. B. Marchetti, V. Guarcello, M.C. Morale, et al., Luteinizing hormone-releasing hormone-binding sites in the rat thymus: characteristics and biological function, Endocrinology 125 (1989) 1025–1036. A. Hirakata, M. Okumi, A.D. Griesemer, et al., Reversal of age-related thymic involution by an LHRH agonist in miniature swine, Transpl. Immunol. 24 (2010) 76–81. A.V. Griffith, M. Fallahi, T. Venables, H.T. Petrie, Persistent degenerative changes in thymic organ function revealed by an inducible model of organ regrowth, Aging Cell 11 (2012) 169–177. J. Sreenivasan, S. Schlenner, D. Franckaert, J. Dooley, A. Liston, The thymoprotective function of leptin is indirectly mediated via suppression of obesity, Immunology 146 (2015) 122–129. M.P. Ullewar, S.N. Umathe, A possible role of endogenous central corticotrophin releasing factor in lipopolysaccharide induced thymic involution and cell apoptosis: effect of peripheral injection of corticotrophin releasing factor, J. Neuroimmunol. 280 (2015) 58–65. V.D. Dixit, H. Yang, Y. Sun, et al., Ghrelin promotes thymopoiesis during aging, J. Clin. Invest. 117 (2007) 2778–2790. E.C. Zook, P.A. Krishack, S. Zhang, et al., Overexpression of Foxn1 attenuates ageassociated thymic involution and prevents the expansion of peripheral CD4 memory T cells, Blood 118 (2011) 5723–5731. N. Bredenkamp, C.S. Nowell, C.C. Blackburn, Regeneration of the aged thymus by a single transcription factor, Development 141 (2014) 1627–1637. X. Jin, C.S. Nowell, S. Ulyanchenko, F.H. Stenhouse, C.C. Blackburn, Long-term persistence of functional thymic epithelial progenitor cells in vivo under conditions of low FOXN1 expression, PLoS One 9 (2014), e114842. M. Su, R. Hu, J. Jin, et al., Efficient in vitro generation of functional thymic epithelial progenitors from human embryonic stem cells, Sci. Rep. 5 (2015) 9882. G. Awong, J. Singh, M. Mohtashami, et al., Human proT-cells generated in vitro facilitate hematopoietic stem cell-derived T-lymphopoiesis in vivo and restore thymic architecture, Blood 122 (2013) 4210–4219. I. Politikos, V.A. Boussiotis, The role of the thymus in T-cell immune reconstitution after umbilical cord blood transplantation, Blood 124 (2014) 3201–3211. M.L. Markert, J.G. Marques, B. Neven, et al., First use of thymus transplantation therapy for FOXN1 deficiency (nude/SCID): a report of 2 cases, Blood 117 (2011) 688–696. M.L. Markert, A. Boeck, L.P. Hale, et al., Transplantation of thymus tissue in complete DiGeorge syndrome, N. Engl. J. Med. 341 (1999) 1180–1189. M.L. Markert, B.H. Devlin, M.J. Alexieff, et al., Review of 54 patients with complete DiGeorge anomaly enrolled in protocols for thymus transplantation: outcome of 44 consecutive transplants, Blood 109 (2007) 4539–4547. I.K. Chinn, B.H. Devlin, Y.J. Li, M.L. Markert, Long-term tolerance to allogeneic thymus transplants in complete DiGeorge anomaly, Clin. Immunol. 126 (2008) 277–281. J.H. Mouledoux, E.L. Albers, Z. Lu, B.R. Saville, D.J. Moore, D.A. Dodd, Clinical predictors of autoimmune and severe atopic disease in pediatric heart transplant recipients, Pediatr. Transplant. 18 (2014) 197–203. A.M. Posselt, C.F. Barker, J.E. Tomaszewski, J.F. Markmann, M.A. Choti, A. Naji, Induction of donor-specific unresponsiveness by intrathymic islet transplantation, Science 249 (1990) 1293–1295. M.M. Levy, R.J. Ketchum, J.E. Tomaszewski, A. Naji, C.F. Barker, K.L. Brayman, Intrathymic islet transplantation in the canine: I. Histological and functional evidence of autologous intrathymic islet engraftment and survival in pancreatectomized recipients, Transplantation 73 (2002) 842–852.