Immunology and Cell Biology (2015) 93, 43–50; doi:10.1038/icb.2014.95; published online 4 November 2014

Autophagy and haematopoietic stem cell transplantation

Lucie Leveque1, Laetitia Le Texier1, Katie E Lineburg1, Geoffrey R Hill1 and Kelli PA MacDonald1

1Department of Immunology, QIMR Berghofer Medical Research Institute, Brisbane, Australia

Correspondence: Dr KPA MacDonald, QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, Brisbane 4006, Australia. E-mail:

Received 15 September 2014; Revised 7 October 2014; Accepted 8 October 2014
Advance online publication 4 November 2014



Allogeneic haematopoietic stem cell transplantation (HSCT) represents the only curative therapy for the majority of bone marrow-derived cancers. Unfortunately, HSCT can result in serious complications such as graft-versus-host disease, graft failure and infection. In the last decade, there have been major advances in the understanding of the role of autophagy in many diseases and cellular processes. Recent findings have demonstrated a crucial role for autophagy in haematopoietic stem cell survival and function, antigen presentation, T-cell differentiation and response to cytokine stimulation. Given the critical requirement for each of these processes in HSCT and subsequent complications, it is surprising that the contribution of autophagy to HSCT per se is relatively unexplored. In addition, the increasing use of autophagy-modulating drugs in the clinic further highlights the need to understand the role of autophagy in allogeneic HSCT. This review will cover established and implicated roles of autophagy in HSCT, suggesting this pathway as an important therapeutic target for improving transplant outcomes.

Autophagy is a self-degradative process responsible for the elimination of cytosolic components including most long-lived proteins, aggregated proteins and also damaged organelles (for example, mitochondria, ribosomes, peroxysomes). Research over the last two decades has increased our understanding of the autophagic process, and has provided critical tools for dissecting the role of autophagy in various physiological settings. Autophagy is a multistep process regulated by protein complexes comprised of autophagy-related genes (Atg). Currently, more than 30 Atg genes have been described, and these proteins coordinately regulate the autophagy process. Initiation of autophagy through phosphoinositide 3-kinase class III-dependent activation of the complex ULK1/2-mTORC1/Ambra1 promotes the formation of a double membrane autophagosome, which encapsulates cytoplasmic components as it matures. The elongation and closure of the autophagosome are controlled by two ubiquitin-like conjugation systems: Atg12-Atg5-Atg16L1 and microtubule-associated light chain 3-phosphatidyl ethanolamine (LC3-PE). Subsequently, fusion of the autophagosome with a lysosome to form an autolysosome facilitates the degradation of luminal contents.1 These degraded products are recycled for the synthesis of new proteins and for energy production, and thus autophagy is a recognised pathway in cellular homoeostasis. In addition, autophagy can be activated as a cytoprotective response during cellular stress induced in the setting of nutrient starvation, cytokine stress, reactive oxygen species (ROS), hypoxia and accumulation of endoplasmic reticulum (ER).2 In addition to stress management, autophagy is also implicated in multiple physiological activities including development, differentiation and cellular remodelling. Autophagy is a critical mechanism in innate and adaptive immunity, playing important roles in pathogen clearance, antigen presentation and control of inflammation.3 Although autophagy primarily acts as a protective pathway, it is recognised that excessive autophagic activity may also lead to cell death.4 Therefore, depending on the different cellular contexts and stimuli, the outcome of autophagy can promote either cell survival or cell death. As such, autophagy has an intricate and vital role in the physiology of cells and organisms. Thus, it is not surprising that perturbations in this pathway have been linked to various human pathophysiologies, including cancer, infection, autoimmune disorders, neurodegeneration, myopathies, heart and liver diseases, and gastrointestinal disorders.5

Over the past decade, an increased understanding of the autophagic process has led to the development of new modulators of autophagy that enable us to monitor autophagic activity in vitro and in vivo. Approaches include both genetic, by knockout, knockdown or overexpression of key components of the pathway, or by pharmaceutical targeting to inhibit autophagy at different stages of the pathway. Deletion/reduction approaches for many autophagy-related genes, including Atg3, Atg5, Atg7, Atg9a, Atg16L1, Beclin-1,FIP200 and Ambra1, have been developed and these, function primarily to block autophagosome formation.6, 7, 8, 9, 10, 11, 12, 13, 14 Pharmaceutical reagents such as phosphoinositide 3-kinase class III inhibitors, including wortmannin or 3-methyladenine (3-MA), block the early step of autophagosome formation in vitro.15, 16 Later stages, including fusion between the autophagosome and lysosome or the degradation of the luminal components inside autolysosomes, can be inhibited by several reagents including chloroquine and bafilomycin A1 (BAF A1).17, 18 Conversely, the drug rapamycin activates autophagy by inhibiting the mammalian target of rapamycin (mTOR) pathway, which regulates signalling pathways involved in nutrient sensing.19 However, current pharmaceutical tools have poor specificity, as they also regulate diverse processes and pathways, thus the combination of both genetic and pharmaceutical approaches in study design is recommended.


Haematopoietic stem cell transplantation (HSCT)

HSCT is the preferred therapy for the majority of bone marrow-derived cancers (leukaemia, lymphoma and myeloma), oncologic malignancies as well as immunologic and metabolic disorders. The curative property of HSCT lies in the ability of the transferred haematopoietic stem cell (HSC) to engraft and reconstitute the patient’s immune system, which has been ablated as a consequence of irradiation/chemotherapy used to clear the underlying malignancy. In turn, the new donor-derived immune system is able to clear residual tumour by a process referred to as the graft-versus-leukaemia (GVL) effect. Unfortunately, the wider application of this procedure is limited by transplant-related complications, the most significant of which are graft-versus-host disease (GVHD) and graft failure. GVHD may be acute or chronic in nature and both forms contribute to significant mortality and morbidity post transplant. Acute GVHD occurs early after transplantation in the context of a T-helper type 1 (Th1)–dominant ‘cytokine storm’ that causes characteristic apoptosis in target tissues (gastrointestinal tract, the liver and the skin). The induction of GVHD depends on the presentation of host alloantigen by antigen-presenting cells (APCs) to naive donor T cells. Given that HSC repopulating function, inflammation, immune cell activation and differentiation are hallmarks of HSCT, there has been surprisingly little consideration of the role of autophagy in this process.

The success of HSCT relies on the capacity of the donor graft to reconstitute haematopoiesis and the immune system. Haematopoiesis is regulated by a rare pool of HSCs called long-term HSCs, which are characterised by their long life span, self-renewal, quiescence and differentiation abilities. In addition, in the context of a clinical stress such as HSCT, mobilisation or chemotherapy, HSCs are required to rapidly generate new mature blood cells. As such, it is suggested that HSC require autophagy, perhaps more than any other cells, in order to maintain their unique properties and survive long term. Indeed, recent studies using mice deficient in autophagy genes, such as FIP200,20 Atg721 and Atg12,22 which effectively disable the autophagy pathway, have demonstrated a critical role for autophagy in the development and function of HSCs including long-term HSCs20, 21, 22 and more mature progenitor cells.22 Following transplantation, HSCs specifically deficient in Atg7 or FIP200 failed to reconstitute lethally irradiated syngeneic recipients, suggesting a requirement for autophagy in HSC self-renewal. This was confirmed using an in vitro serial colony-forming unit assay in which Atg7-deficient HSCs exhibited reduced colony formation.21 Furthermore, blocking autophagy by culturing HSC in the presence of BAF A1 before transplantation was shown to reduce their self-renewal capacity following transplantation.22 Finally, a loss of the self-renewal capacity has been demonstrated in colony-forming unit assays within HSC from aged versus young mice following the inhibition of autophagy by BAF A1. This suggests that HSCs rely on high basal level of autophagy for their survival during the ageing process.22 Thus, it is possible that maintaining autophagy in HSC during ageing may help reduce the risk of blood diseases characterised by stem cell failure (for example, myelodysplasia). Following haematopoietic recovery, HSCs re-enter quiescence, a protective process critical for the maintenance of long-term self-renewal and again, autophagy is implicated in this process. In this regard, although the long-term HSCs were reduced in mice specifically lacking Atg7 in haematopoietic cells, there was a concomitant expansion in the HSC population in the bone marrow, suggestive of a loss of quiescent HSCs. The expanded HSCs exhibited an accumulation of mitochondrial mass and increased ROS production, with associated DNA damage, proliferation and apoptosis.21, 23 Thus, mitophagy, the selective removal of mitochondria by autophagy, appears to be critical for the promotion of quiescence in HSCs. Taken together, the data demonstrate that autophagy is indispensable for HSC long-term survival and function.

An additional complication following allogeneic HSCT is that of graft failure. Graft failure may occur as a consequence of either a lack of initial engraftment of donor cells (primary graft failure) or the loss of donor cells after initial engraftment (secondary graft failure).24 In addition to HSCs, HSC grafts also contain more mature committed progenitor cells, which contribute to initial engraftment and protection against opportunistic infection, a cause of post-HSCT morbidity and mortality. Definitive engraftment and expansion of HSC subsequently provide long-term haematopoietic and immune reconstitution. The established role for autophagy in HSC and progenitor cell function, particularly in the setting of syngeneic transplantation,22 permits speculation that pharmacological modulation of autophagy early post-transplant might impact on the quality of engraftment (Figure 1). In addition, the conditioning regimen (irradiation and/or chemotherapy) utilised before allogeneic HSCT, together with subsequent alloreactive T-cell responses result in a highly inflammatory milieu.25 In this scenario, there is likely to be a very high dependency on autophagy for HSC survival. Interestingly, secondary graft dysfunction is a common occurrence during GVHD and although the mechanism remains poorly defined, it is tempting to postulate that autophagy may be required to maintain HSC function in this setting. Taken together, therapeutic strategies, aimed at increasing autophagic activity in HSCs and their progenitors after transplantation, may thus promote their survival and differentiation leading to improved engraftment.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Role of autophagy in graft failure. Schematic view of the possible roles of autophagy in primary and secondary graft failure following allogeneic HSCT. Graft failure may be manifested as either lack of initial engraftment of donor cells (primary graft failure) or loss of donor cells after initial engraftment (secondary graft failure). Following irradiation, early reconstitution is primarily mediated by the progenitors, which require autophagy for their differentiation. The stress induced by preconditioning regimen (irradiation and chemotherapy) and also cytokines induce autophagy. Autophagy induction by pharmacological reagent may promote progenitor cell differentiation and improve early reconstitution. HSCs, which require autophagy for their survival and repopulating function, participate in the long-term reconstitution following HSCT. Under a stress situation such as GVHD, the release of cytokines, damage/pathogens-associated molecular patterns (DAMPS/PAMPS) and lipopolysaccharide (LPS) induce autophagy. Autophagy induction may help HSCs to overcome the stress induced by GVHD and allow their long-term reconstitution role. The absence of autophagy may promote the risk of primary and secondary graft failure. A full colour version of this figure is available at the Immunology and Cell Biology journal online.

Full figure and legend (113K)


Pre-transplant conditioning

Pre-transplant conditioning utilises intensive chemotherapy and/or total-body irradiation to debulk the underlying malignancy and ablate the host immune system to reduce graft rejection. This conditioning results in tissue damage, particularly in the gut where loss of epithelial barrier function permits the translocation of bacterial components, which in turn trigger the production of proinflammatory cytokines (cytokine storm). Current dogma supports the notion that autophagy is elicited in multiple cell types (both normal and cancerous) in response to radiation.26, 27 Indeed, total-body irradiation is reported to upregulate autophagy in radioresistant Paneth cells within the gut.28 Through their secretion of antimicrobial peptides (defensins) and proteins, Paneth cells are thought to protect the epithelium of the small intestine from invasion by the plethora of non-commensal lumenal microbes.29 In this manner, these cells contribute to the maintenance of epithelial barrier function and prevent inflammation. In addition, Paneth cells form part of the intestinal stem cell niche, where they secrete stem cell supportive factors. Notably, Paneth cells are recognised as a specific target of GVHD itself, which leads to reduced defensin production and outgrowth of septicaemia-inducing pathogens.30 Thus, irradiation-induced autophagy, in the setting of HSCT conditioning, likely has an important role by contributing to gut homeostasis. However, additional cellular stress induced by GVHD likely exceeds the threshold and compromises this capacity for autophagy to support Paneth cell survival and function. In line with this hypothesis, a recent study demonstrated that irradiation promoted the expression of autophagy-related genes such as Beclin-1, and an accumulation of autophagosome-associated LC3A and LC3B protein in endothelial cells, but repressed autophagic flux.31 Critically, in this setting, the promotion of autophagy following irradiation using SMER28, a small-molecule enhancer of autophagy via an mTOR-independent and Atg5-dependent pathway, provided protection against cell death. Taken together, these studies highlight the therapeutic potential for the pharmacological promotion of autophagy in the early transplant period following HSCT to protect the gut barrier and prevent inflammation.


Graft-versus-host disease

GVHD is the major cause of mortality and morbidity following HSCT and as such represents the most serious complication of this procedure. GVHD can manifest in either acute or chronic forms and both contribute to the significant mortality after transplant. Acute GVHD occurs early in the post-transplant period and culminates in extensive tissue destruction characterised by apoptosis. Its development is absolutely dependent on the presence and function of donor T cells in the donor inoculums and is closely tied to the curative GVL effect.32 Following HSCT, tissue injury and inflammation, defined by proinflammatory cytokine release, is initiated by the conditioning regimen.25 These cytokines, together with lipopolysaccharide (LPS) released as a result of conditioning-induced gut damage, promote downstream activation of host APC. Activated host APC then prime naive donor T cells and preferentially drive Th1 differentiation and expand effector CD8+ T cells, which mediate target tissue GVHD in the cytolytic effector pathway. Thus, the pathophysiology of acute GVHD involves multiple processes (inflammation mediated by cytokines, antigen presentation and T-cell activation and expansion), known to regulate, or be regulated by autophagy. The stepwise amplification of GVHD and the potential involvement of autophagy in this process is outlined below and illustrated in the Figure 2.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Role of autophagy in GVHD after allogeneic SCT. Schematic view of the possible roles of autophagy on the different parameters contributing to GVHD. The pathogenesis of GVHD is divided into four major phases: (1) pre-transplant conditioning (irradiation) induces gut damage allowing the release of lipopolysaccharide (LPS) that promotes (2) the production of proinflammatory cytokines (tumour necrosis factor (TNF), IL-6 and IL-1) and (3) contribute to host APC activation and subsequent priming of donor CD4+ T cells against host alloantigens presented via MHC class II. (4) Finally, target organ damage is mediated by both inflammatory cytokines and granzyme B/perforin-dependent cytotoxic NK and CD8+ T cells. The modulation of autophagy by pharmacological reagents could be used to prevent and/or attenuate GVHD. (1) Irradiation induces gut damage and cell death. It also upregulates autophagy. Blocking autophagy early post BMT could be a potential therapy to protect from the harmful effect of irradiation and also improve the early engraftment of myeloid progenitors. However, HSC requires autophagy for their survival and repopulating function. Induction of autophagy could therefore improve HSC engraftment and reduce GVHD development. (2) Proinflammatory cytokines (TNF, IL-1, IL-6) that are known to upregulate autophagy contributes to the inflammatory milieu leading to GVHD. Blocking autophagy has been shown to inhibit the secretion of IL-1α, TNF and IL-6 and induction of autophagy promotes the inflammasome degradation, which blocks IL-1β production. Therefore, modulating autophagy could potentially reduce the cytokine storm. (3) Modulating autophagy can also attenuate APC and donor T cells activation. Blocking autophagy may reduce CD4+ T-cell activation by inhibiting MHC class II antigen presentation by APCs. Moreover, blocking co-stimulation signal (CD40/CD40L) between APC and T cells may induce autophagy-mediated T-cell death. (4) Target organ damage induced by cytotoxic CD8+ T cells could be reduced by blocking autophagy with an aim to inhibit NK and T-cell cytotoxicity, T-cell activation and differentiation and the promotion of T-cell apoptosis. Alternately, the induction of autophagy may diminish cytotoxity by granzyme B degradation and also promote regulatory T-cell (Treg) expansion and stability, which will abrogate effector T-cell differentiation. A full colour version of this figure is available at the Immunology and Cell Biology journal online.

Full figure and legend (213K)


Acute GVHD: the cytokine storm

In the setting of compromised intestinal mucosal integrity, LPS and other damage/pathogens-associated molecular patterns are released systemically and lead to the secretion of the proinflammatory cytokines. The characteristic cytokines released by recipient cells at this early stage of GVHD pathogenesis are tumour necrosis factor (TNF), interleukin (IL)-1 and IL-6.33 Together, this inflammatory milieu promotes the activation of recipient APC to prime donor T cells, provides co-stimulatory signals to donor T cells and induces tissue inflammation/apoptosis, which facilitates donor T-cell access to GVHD target organs. The proinflammatory cytokines, IL-1, TNF and IL-6, like many cytokines, exhibit the capacity to enhance autophagy, whereas other cytokines (IL-4, IL-10 and IL-13) inhibit autophagy.34 Importantly, in addition to being regulated by cytokines, autophagy can regulate the production and secretion of cytokines. As such, autophagy can negatively regulate IL-1 expression, as blocking autophagy with 3-MA or wortmannin was shown to increase the secretion of IL-1α, IL-1β and IL-18, whereas induction of autophagy by rapamycin inhibited IL-1β secretion in response to LPS.10, 35 Indeed, autophagosomes containing IL-1β have been demonstrated in Toll-like receptor-activated macrophages, supporting the role for autophagy as an internal negative feedback loop limiting IL-1-induced inflammation. The Nlrp3 inflammasome is known to promote GVHD36 and autophagosomes can degrade inflammasome components37 that may in turn limit inflammation in GVHD. Therefore, the promotion of autophagy after transplant could be seen to provide a means to attenuate IL-1 secretion and associated mortality.38 However, given that autophagy can also promote secretion of other cytokines, the sum of these effects cannot easily be predicted. Indeed, autophagy can also inhibit IL-17 via the upstream suppression of IL-1 and IL-23.39 This complexity is further highlighted by the role of autophagy in the regulation of the nuclear factor κB (NF-κB) signalling pathway, which plays a complex role in inflammation and can contribute to both pro- and anti-inflammatory responses. Here, autophagy mediates the degradation of NF-κB components, however, depending on the component being targeted, autophagy functions to either terminate NF-κB signalling by inducing the clearance of inhibitor of NF-κB kinase (IKK) kinase complex or promote persistent activation of NF-κB through degradation of IkB kinase catalytic components alpha (IκBα).40 It should be noted that the role of autophagy in cytokine secretion during GVHD has yet to be directly addressed but data highlight the dichotomous contribution of autophagy to immune responses, which can act on similar targets yet have opposing effects depending on the cellular type and the immune context.



The priming and activation of donor T cells are a prerequisite for the development of acute GVHD. The process requires the interaction of the naive donor T cells with an APC presenting alloantigen, which promotes T-cell activation and differentiation. The proinflammatory milieu induced by conditioning and tissue damage results in the activation of host professional and non-professional APC within tissue and enhances their capacity to prime donor T cells.41, 42 As the most potent professional APC, recipient dendritic cells (DCs) have long been assumed critical for T-cell priming after transplant. However, recent studies from multiple groups have established that while recipient DCs can initiate GVHD, they are not required in isolation and may even regulate donor T-cell expansion via the induction of activation-induced donor T-cell death.42, 43 Notably, autophagy is required for monocyte differentiation into macrophages and DCs.44, 45 Autophagy provides a unique mechanism of major histocompatibility complex (MHC) class I-presentation by APC46 and also serves as a route for intracellular antigens to be processed and loaded in MHC class II molecules for presentation to CD4+ T cells.47, 48 The cross-presentation of exogenous antigens loaded on MHC class I molecules to CD8+ T cells and also MHC class II antigen presentation represent important pathways in GVHD and GVL.49 Therefore, studying the role of autophagy in antigen presentation in the context of HSCT will be of great interest. Moreover, several studies have reported the involvement of autophagy in DC activation. Induction of autophagy in DC is associated with increased MHC class II expression and antigen presentation after stimulation of the intracellular pattern-recognition receptor, NOD-2.50 Conversely, DCs deficient in Atg5 exhibit an impaired capacity to process and present phagocytosed antigens on MHC class II and drive Th1 responses.51 In addition, the loss of expression of autophagy-related molecules LC3A or Beclin-1, results in the alteration of bone marrow DC maturation in association with decreased TNF, IL-6 and IL-12p40 production in a murine model of respiratory syncytial viral infection.52 It is thus clear that autophagy is likely to play a role in antigen presentation and subsequent GVHD. Donor-derived DC can contribute to the maintenance of GVHD. This DC pool is critical for the generation of infectious immunity in transplant patients. Despite advances in the use of prophylactic antibiotic and antifungal therapies in patients with GVHD, infection remains a major cause of non-relapse mortality. We have reported that inflammation during GVHD alters DC development and specifically renders the MHC class II presentation pathway in donor DC incompetent. In contrast, both classical and cross-presentation within MHC class I remain largely intact.53 Intriguingly, a similar functional impairment in DC has been noted in Kaposis's sarcoma-associated herpesvirus infection in association with a block of autophagy.54 Thus, the contribution of autophagy to DC reconstitution and function after transplant warrants investigation.


Donor T-cell priming and differentiation

Donor T cells are critical effectors of GVHD, thus the regulation of their expansion, differentiation and function is crucial for the control of GVHD. Recent studies demonstrate the involvement of the autophagy process in T-cell biology,55, 56, 57 suggesting that targeting autophagy could be useful for the modulation of T-cell survival and function. Although there is a low basal level of autophagy in resting T cells, autophagy is induced after TCR engagement,55 chemokine receptor signalling58 and cytokine exposure.59 Notably, the requirement for autophagy differs between T-cell subsets, such that Th2 cells exhibit a greater dependency than Th1 cells.57 In addition, in Atg3fl/fl Lck-Cre mice in which autophagy deficiency is restricted to the T-cell compartment, memory CD44+CD62low T cells are increased relative to naive CD44CD62hi T cells, suggesting naive T cells have a greater dependency on autophagy for their survival.60 These results are interesting considering that naive T cells have a deleterious role in GVHD and memory T cells are unable to induce GVHD.61 Deficiency in Atg7, Atg3 or Atg5 in T cells is associated with increased mitochondria and ER mass and higher ROS production, which may explain the enhanced apoptosis and lower survival observed in autophagy disabled T cells.60, 62, 63, 64 Furthermore, pharmacological inhibition of autophagy has been shown to inhibit T-cell cytotoxicity, induce T-cell apoptosis and at high concentrations, block T-cell activation.65, 66, 67 Conversely, autophagy has also been associated with the induction of death in T cells. In this regard, siRNA knockdown of Beclin-1 or Atg7 in T cell lines increases their survival after the withdrawal of IL-2, which typically induces cell death.57 Thus, cell death induced by IL-2 deprivation may be mediated by autophagy. These findings are particularly relevant in HSCT where naive donor T cells are driven to differentiate into multiple lineages (Th1/Tc1, Th2 and Th17/Tc17) each of which contributes uniquely to GVHD pathology. In this regard, Th1 has a central role in gut injury, whereas Th17 and Tc17 have been demonstrated to contribute to lung and skin pathology.33 Although the skewing of Th1 to a Th2 phenotype has long been considered as a protective mechanism particularly associated with gut GVHD, the Th2 lineage can contribute to GVHD in the liver and skin.68 In addition, the cytotoxicity elicited by CD8+ T cells and natural killer (NK) cells is mediated in part by perforin/granzyme B.69 Induction of autophagy has been shown to allow the degradation of granzyme B (but not perforin),70 which could be beneficial to reduce NK cell cytotoxicity and therefore diminish GVHD pathogenesis. However, this could negatively impact on the GVL effect, which is also granzyme B dependent.71 Thus, carefully designed preclinical studies are required to determine the contribution of autophagy to the balance of effects on GVHD and GVL outcomes.


Autophagy and tolerance in HSCT

Regulatory T cells (Tregs) have a critical role in peripheral tolerance. Several studies in mouse and human have established an important role for Treg in tolerance after HSCT.72, 73, 74 In this regard, we and others have demonstrated that Treg suppresses both acute and chronic GVHD75, 76, 77 and that the number of Treg inversely correlates with the severity of disease.78 These findings have driven the development of Treg adoptive transfer strategies as a promising immunotherapy to temper both acute and chronic GVHD.79 The limited number of accessible natural Treg represents a significant limitation to the feasibility of these protocols, and is being addressed by natural Treg expansion protocols and the generation of induced Treg from conventional CD4 T cells.80, 81, 82 Although these in vitro derived Treg populations hold promise, FoxP3 stability and Treg survival following transfer in vivo remain a concern. Rapamycin, the mTOR inhibitor known to promote autophagy, is increasingly utilised as an immunosuppressant drug in clinical HSCT.83 Noteably, in natural Treg adoptive transfer studies, rapamycin was shown to suppress conventional T-cell differentiation and expansion while sparing the transferred Tregs.84 This was in marked contrast to treatment with cyclosporine A (CsA), which suppressed Treg expansion and enhanced GVHD severity relative to mice treated with rapamycin.84 Furthermore, FoxP3 expression is transient after adoptive transfer; however, co-administration of rapamycin with IL-2, but not IL-2 alone, promoted FoxP3 stability and maintained Treg numbers in vivo after allogeneic SCT.80, 85 Moreover, these rapamycin-stabilised induced Tregs were able to attenuate acute GVHD,80 although a direct effect of rapamycin induction of autophagy in the stabilisation of Treg remains to be demonstrated. Thus, further mechanistic studies are required to allow the development of strategies that culminate in improved Treg stability and survival in the clinic. In this regard, we have recently demonstrated that HSC mobilisation with granulocyte-colony-stimulating factor increases the expansion of Treg in both donors and in recipient mice after SCT, and this provides protection from GVHD. Moreover, microarray transcriptome analysis of Treg from saline and granulocyte-colony-stimulating factor-treated donors demonstrated the upregulation of several autophagy genes, suggesting a potential contribution of autophagy to this effect.86 As yet, there are no definitive reports of a functional role of autophagy in Treg, however, a recent study demonstrated an alterated Treg compartment in transgenic mice that do not express the autophagy-related phosphoinositide 3-kinase class III vacuolar protein sorting (Vps34) in T cells, which leads to an inflammatory wasting syndrome in aged animals.87 Notably, this phenotype bears similarity to the scurfy phenotype, which occurs as a result of FoxP3 and Treg deficiency.88 A new approach to expand Treg, using histone deacetylase inhibitors, has been shown to prevent GVHD in human clinical studies.89 Histone deacetylase inhibitors also induce autophagy by inhibiting mTOR, thus histone deacetylase inhibitors and rapamycin both promote Treg expansion and differentiation through a common pathway known to activate autophagy. Taken together, the data provide a compelling link between autophagy and Treg maintenance in vivo.


Modulating autophagy in clinical transplantation

Multiple autophagy-modulating drugs are already in use in the context of clinical transplantation (Table 1). Current therapeutic strategies to control GVHD involve the administration of immunosuppressive drugs, many of which impact on autophagy. Hence, calcineurin inhibitors such as CsA, which are commonly used after transplantation to control T-cell expansion, have been shown to induce autophagy by triggering ER stress.90 This effect appears to be a protective mechanism that counteracts the cell death induced by the drug’s cytotoxicity as seen on tubular cells after kidney transplantation.90 In accordance with these data, the induction of autophagy has been reported in glioma cells following CsA treatment. Silencing of ULK1, Atg5 or Atg7 contributes to increased apoptosis in response to CsA, further supporting a protective role of autophagy following CsA treatment.91 Similar to CsA, thiopurine can activate autophagy and also appears to serve as a compensatory protective mechanism to counter cytotoxicity.92 Thus, the strategic use of immunosuppressants that also promote autophagy might serve to decrease the intrinsic cytotoxic effects of these agents. Conversely, inhibitors of autophagic flux such as chloroquine and hydroxychloroquine have been shown to reduce the development of acute and chronic GVHD.65, 93, 94, 95 Furthermore, bortezomib, a proteasome inhibitor with known capacity to both increase and decrease autophagy dependent on the cell type has shown promising results in controlling GVHD in phase II trials.96, 97, 98 Based on these data, we speculate that modulating autophagy by pharmaceutical means will likely influence transplant outcomes. Blocking autophagy may in part maintain gut homoeostasis and inhibit systemic translocation of Toll-like receptor ligands, in turn reducing APC activation and donor T-cell priming. Conversely, inducing autophagy after transplantation may help limit the intrinsic cytotoxicity of many immunosuppressant drugs. The critical issue to now be determined is whether the beneficial effects of any of these compounds are mediated, at least in part, by autophagy.



Research in the last decade has enhanced our understanding of the role of autophagy in many diseases and cellular processes. However, the contribution of autophagy to HSCT and its complications such as graft failure and GVHD remain largely unexplored. Autophagy contributes to physiological stress responses in a multitude of ways, which are cell type and stimulus dependent. HSCT elicits multiple effects that are highly susceptible to autophagic regulation, starting with stress induced by conditioning pre-transplant, through to donor HSC reconstitution of haematopoiesis, antigen presentation, cytokine responses, and T-cell differentiation and survival thereafter. On balance, the majority of the literature supports the notion that the promotion of autophagy after HSCT could serve to improve transplant outcomes. However, GVHD pathophysiology is complex and autophagy can serve both to limit inflammation and promote cell survival, or conversely to promote cell death. Although, the sum of these effects cannot be predicted without further experimental analysis, future studies will likely allow the delivery of drugs that modulate autophagy in the context of pathway specificity, timing of delivery, cellular targets and concurrent therapies that may together improve HSCT outcome.



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