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Immunoregulatory properties of rapamycin-conditioned monocyte-derived dendritic cells and their role in transplantation

Immunoregulatory properties of rapamycin-conditioned monocyte-derived dendritic cells and their... In efforts to minimize the chronic administration of immunosuppression (IS) drugs in transplantation and autoimmune disease, various cell-based tolerogenic therapies, including the use of regulatory or tolerogenic dendritic cells (tolDC) have been developed. These DC-based therapies aim to harness the inherent immunoregulatory potential of these professional antigen-presenting cells. In this short review, we describe both the demonstrated tolerogenic properties, and current limitations of rapamycin-conditioned DC (RAPA-DC). RAPA-DC are generated through inhibition of the integrative kinase mammalian target of rapamycin (mTOR) by the immunosuppressive macrolide rapamycin during propagation of monocyte-derived DC. Consistent with the characteristics of tolDC, murine RAPA-DC display resistance to phenotypic maturation induced by pro-inflammatory stimuli; exhibit the ability to migrate to secondary lymphoid tissue (important for ‘cross-presentation’ of antigen to T cells), and enrich for naturally-occurring CD4 regulatory T cells. In rodent models, delivery of recipient-derived RAPA-DC pulsed with donor antigen prior to organ transplantation can prolong allogeneic heart-graft survival indefinitely, especially when combined with a short course of IS. These encouraging data support ongoing efforts to develop RAPA-DC for clinical testing. When compared to murine RAPA-DC however, human RAPA-DC have proven only partially resistant to maturation triggered by pro-inflammatory cytokines, and display heterogeneity in their impact on effector T-cell expansion and function. In total, the evidence suggests the need for more in-depth studies to better understand the mechanisms by which mTOR controls human DC function. These studies may facilitate the development of RAPA-DC therapy alone or together with agents that preserve/enhance their tolerogenic properties as clinical immunoregulatory vectors. Keywords: Dendritic cells, Antigen presentation, Rapamycin, T cells, Regulatory T cells, Tolerance, Transplantation Introduction protocols, including cell-based therapies, such as the use Given their capacity to safely prevent and/or reverse of tolerogenic dendritic cells (tolDC), is a dynamic area acute allograft rejection, immunosuppressive agents have of investigation and may provide a means to minimize proven crucial to the successful clinical development of or even replace use of IS drugs [1]. In addition to redu- organ transplantation. However, there are major limita- cing the toxic burden of chronic IS, it is hoped that tions associated with drug-based immunosuppression these innovative approaches will prevent/reduce chronic (IS), including lack of antigen (Ag) specificity, failure to rejection, given the strong immunological involvement support tolerance induction, deficiencies in the preven- in its etiology [2,3]. tion of late graft failure (chronic rejection), and signifi- DC play critical roles in Ag presentation to naïve and cant morbidity. Evaluation of novel, tolerance-promoting memory T cells and can either promote T-cell immunity or support the induction of tolerance [4-6]. Experimen- * Correspondence: [email protected] tal protocols are currently being developed with the goal Thomas E. Starzl Transplantation Institute, Department of Surgery, University of harnessing the inherent tolerogenicity of DC to act as of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15261, ‘negative cellular vaccines’, which can inhibit immune USA Department of Immunology, University of Pittsburgh School of Medicine, responses in an alloAg-specific manner and promote 200 Lothrop Street, Pittsburgh, PA 15261, USA © 2012 Macedo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Macedo et al. Transplantation Research 2012, 1:16 Page 2 of 7 http://www.transplantationresearch.com/content/1/1/16 Table 1 Mouse vs. human immature RAPA-DC tolerance to transplanted cells and organs. TolDC are characteristically immature, express low surface MHC Mouse Human molecules, a low ratio of co-stimulatory to co-inhibitory Phenotype [19,23,26,27] signals, and an impaired ability to secrete T-helper-1 MHC Class II # - (Th1) cell-driving or inflammatory cytokines [2]. CD86/ CD40 ## # In particular, administration of tolDC presenting allo- B7-H1 ## Ag promotes transplant survival through the induction of Migration [10,23,24] T-cell hyporesponsiveness to allo-Ag, deletion of allor- CCR7 - " eactive T cells, or increased Treg incidence or functions Treg [21-23] [7-10]. This knowledge has driven efforts to identify enrichment/induction/expansion "" mechanisms that underline the tolerogenic properties of T cell apoptosis [10] ↑ ND DC to promote transplant tolerance. Various anti-inflammatory and immunosuppressive Function [15,19,20,23,27-29] agents have been used to generate tolDC in vitro, includ- Endocytosis ## ND ing interleukin (IL)-10 and transforming growth factor-β MLR ## # (TGF-β), cytotoxic T lymphocyte Ag-4 Ig (CTLA4-Ig), IL-12p70 # - prostaglandin E2 (PGE2), dexamethasone, and vitamin IL-10 # - D3 (vitD3), among others [11-13]. Of special interest to ND, not done; -, not changed. our lab has been defining the molecular and functional impact of the immunosuppressive pro-drug rapamycin (RAPA) on DC. recipient-derived RAPA-DC pulsed with alloAg (day −7) followed by a short-term course of low-dose RAPA [25]. When rodent and human DC are generated in Phenotypic and functional characteristics of RAPA-DC clinically-relevant concentrations of RAPA, they are RAPA is a macrocyclic triene antibiotic with immuno- phenotypically immature, with low levels of cell surface suppressant properties, that was discovered in 1975 as a T-cell co-stimulatory molecules (CD86, CD40); however, product of the bacterium Streptomyces hygroscopicus in only murine RAPA-DC maintain their immature pheno- a soil sample from Rapa Nui (Easter Island) [14]. This type when exposed to inflammatory stimuli, such as bac- immunosuppressant inhibits the mammalian target of terial lipopolysaccharide (LPS) [11,25]. Also, mouse and rapamycin (mTOR), a highly conserved serine/threonine human RAPA-DC exhibit a paradoxical decrease in cell kinase that controls cellular responses to environmental surface expression of B7-H1 (also known as pro- cues [15-17]. In mouse models, RAPA has a profound grammed death ligand-1; PD-L1), a PD-1 ligand, which impact on DC in vitro, impairing their maturation fol- contributes to the negative regulation of T lymphocyte lowing exposure to Toll-like receptor (TLR) ligands and activation and promotes peripheral tolerance [13,28] suppressing their T-cell allostimulatory function [11,18-22] (Table 1). (Table 1). RAPA has been reported to have unique Murine RAPA-DC induce hyporesponsiveness and/or tolerance-promoting and Treg facilitating/sparing prop- apoptosis of alloreactive T cells [10,11,18,22,25]. Like- erties in small animal models of organ transplantation wise, human RAPA-DC are poorly stimulatory and in- [23,24]. We [10,25] and others [7,21,26,27] have shown duce T-cell hyporesponsiveness [11]. Furthermore, that when donor-derived, RAPA-conditioned DC murine RAPA-DC retain the capacity to stimulate (RAPA-DC) or recipient-derived RAPA-DC pulsed with mouse naturally-occurring Foxp3 Treg, resulting in donor allo-Ag are administered to recipients prior to an overall enrichment of this population relative to T transplantation, donor graft survival is prolonged indef- effector cells [25]. A similar capacity for promotion of initely, especially when combined with a short course of Foxp3 cells in T-cell cultures has been reported for low dose IS, such as RAPA, cyclosporine, or FK506 human RAPA-DC [12] (Table 1). (Table 2). Taner et al. have shown, in the mouse model, Another important feature of murine RAPA-DC is prolongation of heart allograft survival when recipient- their unaltered chemokine receptor (CCR7) expression derived RAPA-DC pulsed with allo-Ag were given i.v. and capacity for migration to CCL19/CCL21, and thus prior transplantation. Such results were improved with to secondary lymphoid tissues [10,25,26]. Human short-term administration of subtherapeutic dose FK506, RAPA-DC have been reported to upregulate CCR7 ex- which alone did not prolong graft survival or repeated pression and to display significantly augmented migra- infusion of RAPA-DC pulsed with allo-Ag (x3; days −10, tion to CCL21 compared to control DC or other -3, and 0) [10]. Turnquist et al. have also shown long- ex vivo-generated human tolDC, such as those condi- term heart allograft survival, after a single i.v. dose of tioned with IL-10, dexamethasone, TGF-β, or vitamin Macedo et al. Transplantation Research 2012, 1:16 Page 3 of 7 http://www.transplantationresearch.com/content/1/1/16 Table 2 Prolongation of allograft survival by RAPA-DC Species Graft Source of DC IS agent in vivo Reference Mouse Heart Bone marrow (BM) recipient-derived None 10 FK506 10 RAPA 23 Rat Limb BM recipient-derived ALS, CSA 18 Rat Vascularized skin BM recipient-derived ALS, CSA 7 Mouse Hematopoietic cells Autologous BM-derived (unpulsed) None 24 Mouse Pancreatic islets BM donor-derived None 25 ALS, anti-lymphocyte serum; CSA, cyclosporine A; IS, immunosuppression. D3 [13,30]. The ability of RAPA-DC to retain CCR7 cell therapy in organ transplantation involves the use of expression/regulation and to migrate in vivo to second- donor-derived tolDC in an effort to improve graft sur- ary lymphoid tissue, while maintaining low expression of vival; however, such protocols can only be applied in a CD86 and diminished T-cell allostimulatory capacity, has live-donor setting since the in-vitro generation of tolDC important implications for their function as cellular ther- takes 5 to 7 days, precluding use of tolDC generated apy (that is, ‘negative’ vaccines) for prevention of trans- from deceased donors. The generation of recipient- plant rejection [31] (Table 1). derived DC loaded with donor allo-Ag (donor cell lysate, RAPA-DC are also characterized by their unique cyto- apoptotic cells, or exosomes) is more advantageous, kine production profile upon LPS or pro-inflammatory since the generation of autologous RAPA-DC can be cocktail (IL-1β, tumor necrosis factor (TNF)-α, IL-6, performed at any time before transplantation and host IFN-γ) stimulation. While IL-10 production is consist- peripheral mononuclear cells (PBMC) can be cryopre- ently reduced in RAPA-DC [12,28], their production of served until time of tolDC generation/infusion. In IL-12p70 may be affected differently. DC exposed to addition, Ag presentation via the indirect pathway is RAPA in vivo exhibit decreased IL-12p70 production in thought to play an important role in the development response to IL-4 stimulation; likewise, when DC are gen- of chronic rejection, making recipient-derived DC, if erated in culture with long exposure to RAPA, followed successful in regulating indirectly-alloreactive T cells, a by stimulation with agonistic anti-CD40 mAb, these potentially ground-breaking tolerogenic cell therapy in RAPA-DC display reduced IL-12p40 [18,25]. However, transplantation [37]. Immature DC such as RAPA-DC we have described increased IL-12p70 production by can also regulate the expansion and differentiation of human monocyte-derived RAPA-DC after stimulation Treg in vitro and in vivo, resulting in a ‘feedback’ regula- with LPS [11,29] or pro-inflammatory cytokines tory loop [38,39]. On the current evidence, we cannot (Macedo et al., manuscript in preparation). Increased say whether pre- or post-transplant administration of production of IL-12p70 by RAPA-DC upon maturation tolDC, or whether autologous or donor-derived tolDCs, (LPS stimulation) has been associated with augmented will prove to be a superior treatment; however, it is our Th1/Th2-polarization of alloreactive CD4 T cells [32] personal opinion that alloantigen-pulsed recipient- and with Th1 responses upon pro-inflammatory cytokine derived DCs represent a pragmatic approach and offer stimulation, towards IFN-γ production (Macedo et al., certain theoretical advantages because of their indirect manuscript in preparation). Further definition of the presentation of alloantigen. It is an exciting prospect precise mechanisms by which mTOR controls and coor- that The ONE Study consortium will directly compare dinates cytokine production and expression of B7-H1 by different approaches to tolerogenic APC therapy in a DC upon exposure to pro-inflammatory stimuli will be coherent clinical trial. important to fundamental understanding of DC immu- A means to obtain large numbers of monocytes is nobiology and aid efforts to harness these promising through their enrichment from peripheral blood leuka- TM immunoregulatory vectors in transplant medicine and pheresis products. The Elutra cell separation system autoimmune disease. enriches monocytes untouched by antibodies or microbeads within a closed system on the basis of size RAPA-DC from the bench to the clinic and density [35,36]. Although not yet approved for clin- The use of immunogenic or tolDC-based cell therapy in ical use in many countries, as an alternative, the Clini- the clinic has been reported by groups working in differ- MACS cell separation system isolates monocytes by ent medical fields (cancer, HIV infection, and auto- positive selection using CD14 microbeads within a immune diseases) with positive outcomes in terms of its closed system with a good purity [40,41]. Monocytes feasibility and safety [33-36]. One aspect of tolDC-based generated using these techniques can be cultured in Macedo et al. Transplantation Research 2012, 1:16 Page 4 of 7 http://www.transplantationresearch.com/content/1/1/16 medium containing cGMP-grade GM-CSF and IL-4, or free and prolongation of graft survival. However, in the using an Aastrom Replicell system [12,35]. After 5 to following year, Kawai et al. [51] documented a high inci- 7 days of incubation, DC can be loaded with allo-Ag, dence of thromboembolic complications after the use of then evaluated for sterility, viability, recovery, and monoclonal antibody against CD40L in monkeys. Never- phenotype; and either aliquoted for cryopreservation or theless, further studies and pre-clinical evaluation of infused into the patient. We envisaged that the addition CD40-CD40L pathway blockade in conjunction with of RAPA during human DC culture/expansion would tolDC should be explored, including use of anti-CD40 promote the tolerogenic features described above for prior to its application in patients. murine RAPA-DC (Table 3). However, exposure of A further clinically applicable question concerning human RAPA-DC to maturation-inducing factors, such tolDC therapy is the route of DC administration, since it as pro-inflammatory cytokines or TLR4 ligands in vitro could promote different outcomes. Giannoukakis et al. increased their production of IL-12p70, a Th1-inducing [36] have reported injection of autologous NF-κB- cytokine that could augment pathogen-specific CD8 T inhibited DC intradermally in the abdominal wall cell responses and/ or promote alloimmunity [11,42] overlying the anatomic location of the pancreas in type- and (Macedo et al., manuscript in preparation). As such, 1 diabetic patients. As previously described by our methods to limit IL-12p70 production should be exam- group, in the mouse model, systemic (intravenous) ad- ined as part of any protocol for RAPA-DC generation ministration of RAPA-DC was successful in significantly [11,43-45]. Recently, we have shown that increased IL- prolonging alloAg-specific heart graft survival [10]. 12p70 production, by both mouse and human RAPA- Macatangay et al. found no difference between subcuta- DC, following TLR4 ligation results from lost regulation neous and intravenous administration of autologous of glycogen synthase kinase 3 (GSK-3) [11]. As treat- monocyte-derived DC loaded with HIV-1 peptides deliv- ment of RAPA-DC with GSK-3 inhibitors, such as lith- ered to subjects with chronic HIV-1 infection on anti- ium chloride, ablated IL-12p70 production, RAPA-DC retroviral therapy [52]. Since CCR7 and CD62L treatment with GSK-3 inhibitors may be useful in limit- expression on RAPA-DC is not affected by mTOR inhib- ing any potential danger of increase Th1 immunity fol- ition, this may allow the cells to traffic normally to sec- lowing RAPA-DC administration [11]. ondary lymphoid tissues, where their immunoregulatory Another way to maintain RAPA-DC tolerogenicity fol- function is mediated [10,26,30]. lowing infusion of donor-derived or allo-Ag-pulsed recipient-derived tolDC is the concomitant use of How studies of human RAPA-DC relate to others’ work costimulation-blocking agents, such as abatacept (CTLA4- In recent years, tolDC protocols have offered a potential Ig) or belatacept (Lea 29Y), a first and second-generation therapeutic tool in solid organ transplantation [1,2,53]. CTLA4-Ag, respectively, that block the B7-CD28 costi- In order to compare different tolDC protocols, specific mulatory pathway [46,47]. Lu et al. [48] showed an characteristics of the tolDC need to be analyzed. These increase in experimental organ graft survival when anti- include the phenotype, migration capability, cytokine CD40L mAb was administered in conjunction with production (in both immature and mature states), the donor-derived myeloid DC to block the CD40/CD40L ability to induce allogeneic T-cell proliferation, and the pathway, which plays an important role in allogeneic expansion/induction of Treg. For the purpose of generat- DC-T cell interactions in vivo. Later, Kirk et al. [49] and ing tolDC, these can be manipulated in vitro with differ- Kenyon et al. [50] showed promising results in renal and ent immune modulators such as RAPA, dexamethasone, pancreatic islet transplantation, respectively, following IL-10, TGF-β, or vitD3 [1,2,11-13]. the administration of humanized CD154-specific mono- In humans, the majority of the tolDC generated using clonal antibody in rhesus monkeys with acute rejection- the protocols mentioned above exhibit an immature to Table 3 Generation of immature RAPA-DC Research Clinical W W W Monocyte isolation CD14 microbeads (AutoMACS or CliniMACS ) Elutriation or CD14 microbeads (CliniMACS ) Culture conditions 10% fetal calf serum 10% human serum (7 days) AIM-V media AIM-V media (c-GMP grade) 1000 U/mL rhIL-4 1000 U/mL rhIL-4 (c-GMP grade) 1000 U/mL GM-CSF 1000 U/mL GM-CSF (c-GMP grade) 10 ng/mL Rapamycin 10 ng/mL Rapamycin (c-GMP grade) Cell culture plates Cell culture plates or Aastrom Replicell system Macedo et al. Transplantation Research 2012, 1:16 Page 5 of 7 http://www.transplantationresearch.com/content/1/1/16 semi-mature cell surface phenotype, with low to inter- and IL-10. However, methods to prevent increased IL- mediate expression of MHC II, CD86, CD83, and B7- 12p70 production by RAPA-DC (such as use of lithium H1. RAPA-DC and TGF-β-DC have a higher migration chloride [11] or sanglifehrin A [60]) have been identified. capability in response to CCL19 and CCL21 in vitro when Likewise, IL-10 could be delivered with vaccination to compared to IL-10- and vitD3-DC, with higher expression offset the reduced capacity of RAPA-DC to make IL-10. of CCR7. Interestingly, all tolDC (dexamethasone-, IL-10-, Further insights into how mTOR regulates DC cytokine RAPA-, TGF-β-, and VitD3-DC) exhibit diminished pro- production are critical for development of improved duction of IL-23 when compared to mature untreated- ‘negative’ and ‘positive’ cellular vaccines in general, and DC, whereas IL-10-DC and dexamethasone-DC were the to begin to translate these technologies to the bedside. only population to show increased production of IL-10 Abbreviations [11-13]. However, the ability of tolDC to suppress T-cell Ag: Antigen; CTLA4-Ig: Cytotoxic T lymphocyte Ag-4 immunoglobulin; proliferation in humans is variable. IL-10-, TGF-β-, and DC: Dendritic cells; GM-CSF: Granulocyte macrophage colony stimulating VitD3-DC can each suppress T-cell proliferation [11-13]. factor; GSK3: Glycogen synthase kinase 3; IL: Interleukin; IS: Immunosuppression; LPS: Lypopolysaccharide; MLR: Mixed leukocyte Contradictory effects of RAPA on DC in culture have been reaction; mTOR: Mammalian target of rapamycin; PBMC: Peripheral blood reported. We have shown allo-PBMC hyporesponsiviness mononuclear cells; PGE2: Prostaglandin E2; RAPA: Rapamycin; in MLR when stimulated with RAPA-DC [11]. Naranjo- TGFβ1: Transforming growth factor β1; TLR: Toll-like receptors; TolDC: Tolerogenic dendritic cells; Treg: Regulatory T cells; VEGF: Vascular Gomez et al. [12] have also shown RAPA-DC suppression endothelial growth factor. of T-cell proliferation in CFSE-MLR on the other hand, Boks et al. [13] did not find RAPA-DC to be suppressive Competing interests in MLR and Haidinger et al. found an allostimulatory The authors declare that they have no competing interests. effect of RAPA on DC (augmentation of IL-12, CD86, Authors’ contributions IL-1β, and Ag presentation) [28]. Interestingly, Naranjo- CM wrote the manuscript. HT, DM, and AWT participated in editing the final Gomez et al. have shown, in humans, as we reported pre- manuscript. All authors read and approved the final manuscript. viously in mice, that RAPA-DC are capable of significantly sparing/expanding Treg, which suppress effector T-cell Acknowledgements This work was supported by grant NIH-RO1 AI 067541-NAID (PI: AW allo-reactivity [11,12]. Thomson). 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Hackstein H, Steinschulte C, Fiedel S, Eisele A, Rathke V, Stadlbauer T, Taner T, Thomson AW, Tillmanns H, Bein G, Holschermann H: Sanglifehrin a blocks key dendritic cell functions in vivo and promotes long-term allograft survival together with low-dose CsA. Am J Transplant 2007, 7:789–798. doi:10.1186/2047-1440-1-16 Cite this article as: Macedo et al.: Immunoregulatory properties of rapamycin-conditioned monocyte-derived dendritic cells and their role in transplantation. Transplantation Research 2012 1:16. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Transplantation Research Springer Journals

Immunoregulatory properties of rapamycin-conditioned monocyte-derived dendritic cells and their role in transplantation

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Springer Journals
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Copyright © 2012 by Macedo et al.; licensee BioMed Central Ltd.
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Medicine & Public Health; Transplant Surgery; Internal Medicine; Immunology
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10.1186/2047-1440-1-16
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Abstract

In efforts to minimize the chronic administration of immunosuppression (IS) drugs in transplantation and autoimmune disease, various cell-based tolerogenic therapies, including the use of regulatory or tolerogenic dendritic cells (tolDC) have been developed. These DC-based therapies aim to harness the inherent immunoregulatory potential of these professional antigen-presenting cells. In this short review, we describe both the demonstrated tolerogenic properties, and current limitations of rapamycin-conditioned DC (RAPA-DC). RAPA-DC are generated through inhibition of the integrative kinase mammalian target of rapamycin (mTOR) by the immunosuppressive macrolide rapamycin during propagation of monocyte-derived DC. Consistent with the characteristics of tolDC, murine RAPA-DC display resistance to phenotypic maturation induced by pro-inflammatory stimuli; exhibit the ability to migrate to secondary lymphoid tissue (important for ‘cross-presentation’ of antigen to T cells), and enrich for naturally-occurring CD4 regulatory T cells. In rodent models, delivery of recipient-derived RAPA-DC pulsed with donor antigen prior to organ transplantation can prolong allogeneic heart-graft survival indefinitely, especially when combined with a short course of IS. These encouraging data support ongoing efforts to develop RAPA-DC for clinical testing. When compared to murine RAPA-DC however, human RAPA-DC have proven only partially resistant to maturation triggered by pro-inflammatory cytokines, and display heterogeneity in their impact on effector T-cell expansion and function. In total, the evidence suggests the need for more in-depth studies to better understand the mechanisms by which mTOR controls human DC function. These studies may facilitate the development of RAPA-DC therapy alone or together with agents that preserve/enhance their tolerogenic properties as clinical immunoregulatory vectors. Keywords: Dendritic cells, Antigen presentation, Rapamycin, T cells, Regulatory T cells, Tolerance, Transplantation Introduction protocols, including cell-based therapies, such as the use Given their capacity to safely prevent and/or reverse of tolerogenic dendritic cells (tolDC), is a dynamic area acute allograft rejection, immunosuppressive agents have of investigation and may provide a means to minimize proven crucial to the successful clinical development of or even replace use of IS drugs [1]. In addition to redu- organ transplantation. However, there are major limita- cing the toxic burden of chronic IS, it is hoped that tions associated with drug-based immunosuppression these innovative approaches will prevent/reduce chronic (IS), including lack of antigen (Ag) specificity, failure to rejection, given the strong immunological involvement support tolerance induction, deficiencies in the preven- in its etiology [2,3]. tion of late graft failure (chronic rejection), and signifi- DC play critical roles in Ag presentation to naïve and cant morbidity. Evaluation of novel, tolerance-promoting memory T cells and can either promote T-cell immunity or support the induction of tolerance [4-6]. Experimen- * Correspondence: [email protected] tal protocols are currently being developed with the goal Thomas E. Starzl Transplantation Institute, Department of Surgery, University of harnessing the inherent tolerogenicity of DC to act as of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15261, ‘negative cellular vaccines’, which can inhibit immune USA Department of Immunology, University of Pittsburgh School of Medicine, responses in an alloAg-specific manner and promote 200 Lothrop Street, Pittsburgh, PA 15261, USA © 2012 Macedo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Macedo et al. Transplantation Research 2012, 1:16 Page 2 of 7 http://www.transplantationresearch.com/content/1/1/16 Table 1 Mouse vs. human immature RAPA-DC tolerance to transplanted cells and organs. TolDC are characteristically immature, express low surface MHC Mouse Human molecules, a low ratio of co-stimulatory to co-inhibitory Phenotype [19,23,26,27] signals, and an impaired ability to secrete T-helper-1 MHC Class II # - (Th1) cell-driving or inflammatory cytokines [2]. CD86/ CD40 ## # In particular, administration of tolDC presenting allo- B7-H1 ## Ag promotes transplant survival through the induction of Migration [10,23,24] T-cell hyporesponsiveness to allo-Ag, deletion of allor- CCR7 - " eactive T cells, or increased Treg incidence or functions Treg [21-23] [7-10]. This knowledge has driven efforts to identify enrichment/induction/expansion "" mechanisms that underline the tolerogenic properties of T cell apoptosis [10] ↑ ND DC to promote transplant tolerance. Various anti-inflammatory and immunosuppressive Function [15,19,20,23,27-29] agents have been used to generate tolDC in vitro, includ- Endocytosis ## ND ing interleukin (IL)-10 and transforming growth factor-β MLR ## # (TGF-β), cytotoxic T lymphocyte Ag-4 Ig (CTLA4-Ig), IL-12p70 # - prostaglandin E2 (PGE2), dexamethasone, and vitamin IL-10 # - D3 (vitD3), among others [11-13]. Of special interest to ND, not done; -, not changed. our lab has been defining the molecular and functional impact of the immunosuppressive pro-drug rapamycin (RAPA) on DC. recipient-derived RAPA-DC pulsed with alloAg (day −7) followed by a short-term course of low-dose RAPA [25]. When rodent and human DC are generated in Phenotypic and functional characteristics of RAPA-DC clinically-relevant concentrations of RAPA, they are RAPA is a macrocyclic triene antibiotic with immuno- phenotypically immature, with low levels of cell surface suppressant properties, that was discovered in 1975 as a T-cell co-stimulatory molecules (CD86, CD40); however, product of the bacterium Streptomyces hygroscopicus in only murine RAPA-DC maintain their immature pheno- a soil sample from Rapa Nui (Easter Island) [14]. This type when exposed to inflammatory stimuli, such as bac- immunosuppressant inhibits the mammalian target of terial lipopolysaccharide (LPS) [11,25]. Also, mouse and rapamycin (mTOR), a highly conserved serine/threonine human RAPA-DC exhibit a paradoxical decrease in cell kinase that controls cellular responses to environmental surface expression of B7-H1 (also known as pro- cues [15-17]. In mouse models, RAPA has a profound grammed death ligand-1; PD-L1), a PD-1 ligand, which impact on DC in vitro, impairing their maturation fol- contributes to the negative regulation of T lymphocyte lowing exposure to Toll-like receptor (TLR) ligands and activation and promotes peripheral tolerance [13,28] suppressing their T-cell allostimulatory function [11,18-22] (Table 1). (Table 1). RAPA has been reported to have unique Murine RAPA-DC induce hyporesponsiveness and/or tolerance-promoting and Treg facilitating/sparing prop- apoptosis of alloreactive T cells [10,11,18,22,25]. Like- erties in small animal models of organ transplantation wise, human RAPA-DC are poorly stimulatory and in- [23,24]. We [10,25] and others [7,21,26,27] have shown duce T-cell hyporesponsiveness [11]. Furthermore, that when donor-derived, RAPA-conditioned DC murine RAPA-DC retain the capacity to stimulate (RAPA-DC) or recipient-derived RAPA-DC pulsed with mouse naturally-occurring Foxp3 Treg, resulting in donor allo-Ag are administered to recipients prior to an overall enrichment of this population relative to T transplantation, donor graft survival is prolonged indef- effector cells [25]. A similar capacity for promotion of initely, especially when combined with a short course of Foxp3 cells in T-cell cultures has been reported for low dose IS, such as RAPA, cyclosporine, or FK506 human RAPA-DC [12] (Table 1). (Table 2). Taner et al. have shown, in the mouse model, Another important feature of murine RAPA-DC is prolongation of heart allograft survival when recipient- their unaltered chemokine receptor (CCR7) expression derived RAPA-DC pulsed with allo-Ag were given i.v. and capacity for migration to CCL19/CCL21, and thus prior transplantation. Such results were improved with to secondary lymphoid tissues [10,25,26]. Human short-term administration of subtherapeutic dose FK506, RAPA-DC have been reported to upregulate CCR7 ex- which alone did not prolong graft survival or repeated pression and to display significantly augmented migra- infusion of RAPA-DC pulsed with allo-Ag (x3; days −10, tion to CCL21 compared to control DC or other -3, and 0) [10]. Turnquist et al. have also shown long- ex vivo-generated human tolDC, such as those condi- term heart allograft survival, after a single i.v. dose of tioned with IL-10, dexamethasone, TGF-β, or vitamin Macedo et al. Transplantation Research 2012, 1:16 Page 3 of 7 http://www.transplantationresearch.com/content/1/1/16 Table 2 Prolongation of allograft survival by RAPA-DC Species Graft Source of DC IS agent in vivo Reference Mouse Heart Bone marrow (BM) recipient-derived None 10 FK506 10 RAPA 23 Rat Limb BM recipient-derived ALS, CSA 18 Rat Vascularized skin BM recipient-derived ALS, CSA 7 Mouse Hematopoietic cells Autologous BM-derived (unpulsed) None 24 Mouse Pancreatic islets BM donor-derived None 25 ALS, anti-lymphocyte serum; CSA, cyclosporine A; IS, immunosuppression. D3 [13,30]. The ability of RAPA-DC to retain CCR7 cell therapy in organ transplantation involves the use of expression/regulation and to migrate in vivo to second- donor-derived tolDC in an effort to improve graft sur- ary lymphoid tissue, while maintaining low expression of vival; however, such protocols can only be applied in a CD86 and diminished T-cell allostimulatory capacity, has live-donor setting since the in-vitro generation of tolDC important implications for their function as cellular ther- takes 5 to 7 days, precluding use of tolDC generated apy (that is, ‘negative’ vaccines) for prevention of trans- from deceased donors. The generation of recipient- plant rejection [31] (Table 1). derived DC loaded with donor allo-Ag (donor cell lysate, RAPA-DC are also characterized by their unique cyto- apoptotic cells, or exosomes) is more advantageous, kine production profile upon LPS or pro-inflammatory since the generation of autologous RAPA-DC can be cocktail (IL-1β, tumor necrosis factor (TNF)-α, IL-6, performed at any time before transplantation and host IFN-γ) stimulation. While IL-10 production is consist- peripheral mononuclear cells (PBMC) can be cryopre- ently reduced in RAPA-DC [12,28], their production of served until time of tolDC generation/infusion. In IL-12p70 may be affected differently. DC exposed to addition, Ag presentation via the indirect pathway is RAPA in vivo exhibit decreased IL-12p70 production in thought to play an important role in the development response to IL-4 stimulation; likewise, when DC are gen- of chronic rejection, making recipient-derived DC, if erated in culture with long exposure to RAPA, followed successful in regulating indirectly-alloreactive T cells, a by stimulation with agonistic anti-CD40 mAb, these potentially ground-breaking tolerogenic cell therapy in RAPA-DC display reduced IL-12p40 [18,25]. However, transplantation [37]. Immature DC such as RAPA-DC we have described increased IL-12p70 production by can also regulate the expansion and differentiation of human monocyte-derived RAPA-DC after stimulation Treg in vitro and in vivo, resulting in a ‘feedback’ regula- with LPS [11,29] or pro-inflammatory cytokines tory loop [38,39]. On the current evidence, we cannot (Macedo et al., manuscript in preparation). Increased say whether pre- or post-transplant administration of production of IL-12p70 by RAPA-DC upon maturation tolDC, or whether autologous or donor-derived tolDCs, (LPS stimulation) has been associated with augmented will prove to be a superior treatment; however, it is our Th1/Th2-polarization of alloreactive CD4 T cells [32] personal opinion that alloantigen-pulsed recipient- and with Th1 responses upon pro-inflammatory cytokine derived DCs represent a pragmatic approach and offer stimulation, towards IFN-γ production (Macedo et al., certain theoretical advantages because of their indirect manuscript in preparation). Further definition of the presentation of alloantigen. It is an exciting prospect precise mechanisms by which mTOR controls and coor- that The ONE Study consortium will directly compare dinates cytokine production and expression of B7-H1 by different approaches to tolerogenic APC therapy in a DC upon exposure to pro-inflammatory stimuli will be coherent clinical trial. important to fundamental understanding of DC immu- A means to obtain large numbers of monocytes is nobiology and aid efforts to harness these promising through their enrichment from peripheral blood leuka- TM immunoregulatory vectors in transplant medicine and pheresis products. The Elutra cell separation system autoimmune disease. enriches monocytes untouched by antibodies or microbeads within a closed system on the basis of size RAPA-DC from the bench to the clinic and density [35,36]. Although not yet approved for clin- The use of immunogenic or tolDC-based cell therapy in ical use in many countries, as an alternative, the Clini- the clinic has been reported by groups working in differ- MACS cell separation system isolates monocytes by ent medical fields (cancer, HIV infection, and auto- positive selection using CD14 microbeads within a immune diseases) with positive outcomes in terms of its closed system with a good purity [40,41]. Monocytes feasibility and safety [33-36]. One aspect of tolDC-based generated using these techniques can be cultured in Macedo et al. Transplantation Research 2012, 1:16 Page 4 of 7 http://www.transplantationresearch.com/content/1/1/16 medium containing cGMP-grade GM-CSF and IL-4, or free and prolongation of graft survival. However, in the using an Aastrom Replicell system [12,35]. After 5 to following year, Kawai et al. [51] documented a high inci- 7 days of incubation, DC can be loaded with allo-Ag, dence of thromboembolic complications after the use of then evaluated for sterility, viability, recovery, and monoclonal antibody against CD40L in monkeys. Never- phenotype; and either aliquoted for cryopreservation or theless, further studies and pre-clinical evaluation of infused into the patient. We envisaged that the addition CD40-CD40L pathway blockade in conjunction with of RAPA during human DC culture/expansion would tolDC should be explored, including use of anti-CD40 promote the tolerogenic features described above for prior to its application in patients. murine RAPA-DC (Table 3). However, exposure of A further clinically applicable question concerning human RAPA-DC to maturation-inducing factors, such tolDC therapy is the route of DC administration, since it as pro-inflammatory cytokines or TLR4 ligands in vitro could promote different outcomes. Giannoukakis et al. increased their production of IL-12p70, a Th1-inducing [36] have reported injection of autologous NF-κB- cytokine that could augment pathogen-specific CD8 T inhibited DC intradermally in the abdominal wall cell responses and/ or promote alloimmunity [11,42] overlying the anatomic location of the pancreas in type- and (Macedo et al., manuscript in preparation). As such, 1 diabetic patients. As previously described by our methods to limit IL-12p70 production should be exam- group, in the mouse model, systemic (intravenous) ad- ined as part of any protocol for RAPA-DC generation ministration of RAPA-DC was successful in significantly [11,43-45]. Recently, we have shown that increased IL- prolonging alloAg-specific heart graft survival [10]. 12p70 production, by both mouse and human RAPA- Macatangay et al. found no difference between subcuta- DC, following TLR4 ligation results from lost regulation neous and intravenous administration of autologous of glycogen synthase kinase 3 (GSK-3) [11]. As treat- monocyte-derived DC loaded with HIV-1 peptides deliv- ment of RAPA-DC with GSK-3 inhibitors, such as lith- ered to subjects with chronic HIV-1 infection on anti- ium chloride, ablated IL-12p70 production, RAPA-DC retroviral therapy [52]. Since CCR7 and CD62L treatment with GSK-3 inhibitors may be useful in limit- expression on RAPA-DC is not affected by mTOR inhib- ing any potential danger of increase Th1 immunity fol- ition, this may allow the cells to traffic normally to sec- lowing RAPA-DC administration [11]. ondary lymphoid tissues, where their immunoregulatory Another way to maintain RAPA-DC tolerogenicity fol- function is mediated [10,26,30]. lowing infusion of donor-derived or allo-Ag-pulsed recipient-derived tolDC is the concomitant use of How studies of human RAPA-DC relate to others’ work costimulation-blocking agents, such as abatacept (CTLA4- In recent years, tolDC protocols have offered a potential Ig) or belatacept (Lea 29Y), a first and second-generation therapeutic tool in solid organ transplantation [1,2,53]. CTLA4-Ag, respectively, that block the B7-CD28 costi- In order to compare different tolDC protocols, specific mulatory pathway [46,47]. Lu et al. [48] showed an characteristics of the tolDC need to be analyzed. These increase in experimental organ graft survival when anti- include the phenotype, migration capability, cytokine CD40L mAb was administered in conjunction with production (in both immature and mature states), the donor-derived myeloid DC to block the CD40/CD40L ability to induce allogeneic T-cell proliferation, and the pathway, which plays an important role in allogeneic expansion/induction of Treg. For the purpose of generat- DC-T cell interactions in vivo. Later, Kirk et al. [49] and ing tolDC, these can be manipulated in vitro with differ- Kenyon et al. [50] showed promising results in renal and ent immune modulators such as RAPA, dexamethasone, pancreatic islet transplantation, respectively, following IL-10, TGF-β, or vitD3 [1,2,11-13]. the administration of humanized CD154-specific mono- In humans, the majority of the tolDC generated using clonal antibody in rhesus monkeys with acute rejection- the protocols mentioned above exhibit an immature to Table 3 Generation of immature RAPA-DC Research Clinical W W W Monocyte isolation CD14 microbeads (AutoMACS or CliniMACS ) Elutriation or CD14 microbeads (CliniMACS ) Culture conditions 10% fetal calf serum 10% human serum (7 days) AIM-V media AIM-V media (c-GMP grade) 1000 U/mL rhIL-4 1000 U/mL rhIL-4 (c-GMP grade) 1000 U/mL GM-CSF 1000 U/mL GM-CSF (c-GMP grade) 10 ng/mL Rapamycin 10 ng/mL Rapamycin (c-GMP grade) Cell culture plates Cell culture plates or Aastrom Replicell system Macedo et al. Transplantation Research 2012, 1:16 Page 5 of 7 http://www.transplantationresearch.com/content/1/1/16 semi-mature cell surface phenotype, with low to inter- and IL-10. However, methods to prevent increased IL- mediate expression of MHC II, CD86, CD83, and B7- 12p70 production by RAPA-DC (such as use of lithium H1. RAPA-DC and TGF-β-DC have a higher migration chloride [11] or sanglifehrin A [60]) have been identified. capability in response to CCL19 and CCL21 in vitro when Likewise, IL-10 could be delivered with vaccination to compared to IL-10- and vitD3-DC, with higher expression offset the reduced capacity of RAPA-DC to make IL-10. of CCR7. Interestingly, all tolDC (dexamethasone-, IL-10-, Further insights into how mTOR regulates DC cytokine RAPA-, TGF-β-, and VitD3-DC) exhibit diminished pro- production are critical for development of improved duction of IL-23 when compared to mature untreated- ‘negative’ and ‘positive’ cellular vaccines in general, and DC, whereas IL-10-DC and dexamethasone-DC were the to begin to translate these technologies to the bedside. only population to show increased production of IL-10 Abbreviations [11-13]. However, the ability of tolDC to suppress T-cell Ag: Antigen; CTLA4-Ig: Cytotoxic T lymphocyte Ag-4 immunoglobulin; proliferation in humans is variable. IL-10-, TGF-β-, and DC: Dendritic cells; GM-CSF: Granulocyte macrophage colony stimulating VitD3-DC can each suppress T-cell proliferation [11-13]. factor; GSK3: Glycogen synthase kinase 3; IL: Interleukin; IS: Immunosuppression; LPS: Lypopolysaccharide; MLR: Mixed leukocyte Contradictory effects of RAPA on DC in culture have been reaction; mTOR: Mammalian target of rapamycin; PBMC: Peripheral blood reported. We have shown allo-PBMC hyporesponsiviness mononuclear cells; PGE2: Prostaglandin E2; RAPA: Rapamycin; in MLR when stimulated with RAPA-DC [11]. Naranjo- TGFβ1: Transforming growth factor β1; TLR: Toll-like receptors; TolDC: Tolerogenic dendritic cells; Treg: Regulatory T cells; VEGF: Vascular Gomez et al. [12] have also shown RAPA-DC suppression endothelial growth factor. of T-cell proliferation in CFSE-MLR on the other hand, Boks et al. [13] did not find RAPA-DC to be suppressive Competing interests in MLR and Haidinger et al. found an allostimulatory The authors declare that they have no competing interests. effect of RAPA on DC (augmentation of IL-12, CD86, Authors’ contributions IL-1β, and Ag presentation) [28]. Interestingly, Naranjo- CM wrote the manuscript. HT, DM, and AWT participated in editing the final Gomez et al. have shown, in humans, as we reported pre- manuscript. All authors read and approved the final manuscript. viously in mice, that RAPA-DC are capable of significantly sparing/expanding Treg, which suppress effector T-cell Acknowledgements This work was supported by grant NIH-RO1 AI 067541-NAID (PI: AW allo-reactivity [11,12]. Thomson). Other immunosuppressive cell types of myeloid origin, such as macrophages [54], myeloid-derived suppressor Received: 6 June 2012 Accepted: 4 September 2012 Published: 28 September 2012 cells generated in the presence of PGE2 [55], and mes- enchymal stem cells [56], have been introduced recently References to the transplant field, with suppressive properties that 1. Ezzelarab M, Thomson AW: Tolerogenic dendritic cells and their role in may be suitable for clinical use. DC treated with differ- transplantation. Semin Immunol 2011, 23:252–263. ent cytokines and/or IS agents, macrophages, and mes- 2. Morelli AE, Thomson AW: Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol 2007, 7:610–621. enchymal stem cells mentioned above are being studied 3. Libby P, Pober JS: Chronic rejection. Immunity 2001, 14:387–397. currently by The ONE Study, a multinational clinical 4. 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Hackstein H, Steinschulte C, Fiedel S, Eisele A, Rathke V, Stadlbauer T, Taner T, Thomson AW, Tillmanns H, Bein G, Holschermann H: Sanglifehrin a blocks key dendritic cell functions in vivo and promotes long-term allograft survival together with low-dose CsA. Am J Transplant 2007, 7:789–798. doi:10.1186/2047-1440-1-16 Cite this article as: Macedo et al.: Immunoregulatory properties of rapamycin-conditioned monocyte-derived dendritic cells and their role in transplantation. Transplantation Research 2012 1:16. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

Journal

Transplantation Research – Springer Journals

Published: Sep 28, 2012

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