Adoptive cell transfer in autoimmune hepatitis.

Review

Expert Review of Gastroenterology & Hepatology Downloaded from informahealthcare.com by Nanyang Technological University on 04/27/15 For personal use only.

Adoptive cell transfer in autoimmune hepatitis Expert Rev. Gastroenterol. Hepatol. Early online, 1–16 (2015)

Albert J Czaja Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, 200 First Street S.W, Rochester, MN 55905, USA Tel.: +1 507 284 2691 Fax: +1 507 284 0538 [email protected]

Adoptive cell transfer is an intervention in which autologous immune cells that have been expanded ex vivo are re-introduced to mitigate a pathological process. Tregs, mesenchymal stromal cells, dendritic cells, macrophages and myeloid-derived suppressor cells have been transferred in diverse immune-mediated diseases, and Tregs have been the focus of investigations in autoimmune hepatitis. Transferred Tregs have improved histological findings in animal models of autoimmune hepatitis and autoimmune cholangitis. Key challenges relate to discrepant findings among studies, phenotypic instability of the transferred population, uncertain side effects and possible need for staged therapy involving anti-inflammatory drugs. Future investigations must resolve issues about the purification, durability and safety of these cells and consider alternative populations if necessary. KEYWORDS: adoptive transfer . autoimmune hepatitis . Tregs . treatment

Adoptive cell transfer is a therapeutic intervention in which autologous immune cells that have been modified, expanded or induced ex vivo are re-introduced to mitigate a pathological process. Recipient-derived cells that are modified ex vivo and restored to the same individual minimize graft-versus-host reactions [1], and the adoptive transfer of functional autologous immune cells that have been genetically modified, antigensensitized, expanded or pre-treated with stabilizers have been used in animal models and humans to treat various forms of cancer (melanoma, leukemia, lymphomas and nasopharyngeal cancer) [2–5] and autoimmune diseases (diabetes, collagen-induced arthritis, myasthenia gravis, inflammatory bowel disease, experimental autoimmune encephalitis and autoimmune liver disease) [6–13]. Autoimmune hepatitis is a consequence of an immunological intolerance of self-antigens and the failure to modulate a promiscuous immune response to these antigens and their homologues within the liver [14]. Immunosuppressive drugs have been successful in ameliorating the disease, but their actions have been undirected and results have been variable and often unsustainable [15]. Adoptive transfer is a treatment option that might improve outcomes in autoimmune hepatitis by correcting a crucial regulatory defect, restoring immune homeostasis and inducing a durable tolerance of self-antigens. informahealthcare.com

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The goals of this review are to describe the principal cell populations that have been adoptively transferred in autoimmune disease, indicate the methods that have been applied to improve results, examine the potential value of the adoptive transfer of Tregs in autoimmune hepatitis and outline the challenges that must be overcome to advance this intervention. Principal cell populations for adoptive transfer in autoimmune diseases

Tregs, mesenchymal stromal cells (MSCs), dendritic cells, macrophages and myeloid-derived suppressor cells have been the principal populations considered for adoptive cell therapy in autoimmune diseases, and Tregs have been the focus of investigations in autoimmune hepatitis. Regulatory T cells

Tregs are characterized by the expression of CD4, CD25 and the transcription factor, Forkhead box protein 3 (Foxp3) (TABLE 1) [16,17]. Other activated lymphocytes may express Foxp3 [18], and the low or absent expression of CD127 (IL-7 receptor) [19], the presence of CD39 [20] and the demonstration of sitespecific demethylation of the Foxp3 gene are other features that typify this population [21]. Natural Tregs are derived from the thymus, and they comprise 5–10% of the circulating CD4+ lymphocytes [17]. Induced Tregs are derived from T cells after antigen exposure,

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Table 1. Candidate cell populations for adoptive transfer in autoimmune hepatitis.


Cell population

Features +

Investigational experiences +

+

Tregs

CD4 CD25 Foxp3 phenotype [16,17] Low or no expression of CD127 [19] Thymic-derived (natural) and induced types [22] Broad immunosuppressive effects [24] Induced cells more potent than natural cells [24] Strengthened by multiple drugs (steroids) [33–35] Antigen-specificity improves targeting [24,39,44] Convert CD4+ cells to Tregs [46] Generate memory cells [6] Suppress maturation of dendritic cells [24]

Natural cells in animal models: Prevents autoimmune gastritis [39] Ineffective in established disease [24] Induced cells in animal models: Effective in GVHD, EAE, diabetes, autoimmune gastritis [6,7,42] Expanded autologous cells effective in colitis, PBC and AIH [11,13,26] Prevents allograft rejection [48,49]

Mesenchymal stromal cells

Multipotent, self-renewing cells [50,51] No class II MHC molecules [50,55] Low immunogenicity [56,57] Polarize macrophages to anti-inflammatory type [58] Inhibit natural killer cells by tryptophan depletion [65] Limit dendritic cell differentiation and function [68] Inhibit B and T lymphocytes by NO and HLA-G [51] Expand Tregs [79,80]

Human trials in immune diseases: Steroid refractory Crohn’s disease [86] Fistulizing Crohn’s disease [54,87] Refractory GVHD [88–90] Kidney and liver transplantation [51] Multiple sclerosis [92] Effective in murine model of RA [80] Prevent radiation-induced injury [85]

Dendritic cells

Antigen-presenting cells [10,118,119] Modulate innate and adaptive responses [119] Actions directed by inflammatory milieu [120] Immature or semi-mature cells tolerogenic [121] Expand Tregs [121] Block expansion of activated T cells [126,129] Induce apoptosis of effector T cells [10]

Animal studies: Effective in RA [10,122] Human studies: Effective in diabetes, RA [123,124]

Macrophages

Innate immune response to tissue injury [132] Proinflammatory M1 type, TNF-a secretion [133] Anti-inflammatory M2 type, IL-10 secretion [132]

Animal studies: Improved diabetes [132] Protected pancreatic islets [132]

Myeloid-derived suppressor cells

Characterized by CD11b and Gr-1 [130,134] Inhibit proliferation and function of T cells [130] Activated by cannabidiol [130,134]

Effective in models of AIH, acute hepatitis, MG [12,130,136]

AIH: Autoimmune hepatitis; EAE: Experimental autoimmune encephalitis; GVHD: Graft-versus-host disease; MG: Myasthenia gravis; NO: Nitric oxide; PBC: Primary biliary cirrhosis; RA: Rheumatoid arthritis.

stimulation with TGF-b and upregulation of Foxp3 [22]. Cytotoxic T lymphocyte antigen-4 also modulates the differentiation of induced Tregs in vitro, and cytotoxic T lymphocyte antigen-4 and TGF-b are important for the induction of Foxp3 in conventional T lymphocytes [23]. Induced Tregs vary in number in accordance with the inflammatory stimulus, and they have greater immunosuppressive effects than natural Tregs [24]. Tregs have broad immunosuppressive actions (TABLE 1). They express PPAR-g which is a nuclear receptor that suppresses signaldependent proinflammatory genes and limits the proliferation and survival of inflammatory and immune cells by inducing their apoptosis [25]. PPAR-g also decreases the production of IFN-g by CD4+ lymphocytes [25], dampens the inflammatory response [26] and inhibits the proliferation and chemotaxis of hepatic stellate cells [27]. Tregs produce the anti-inflammatory cytokine, IL-10, inhibit the production of IL-17 and decrease the expansion of Th17 lymphocytes [28]. Tregs are critical in modulating inflammatory and immune responses, and their numerical and functional deficiencies in

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diverse autoimmune diseases, including autoimmune hepatitis, have justified efforts to strengthen their activity [29–31]. Pharmacological agents (corticosteroids [32,33], mycophenolate mofetil [34], rapamycin [35], 1-a,25-dihydroxyvitamin D3 [34], all trans-retinoic acid [36] and proteasome inhibitors [37]) and cellular interventions (adoptive transfer [38]) have been used in this effort. The prospect of applying a disease-specific, individualized therapy with durable results has been the principal allure of adoptive transfer. Adoptive transfer of Tregs has had variable effects in the treatment of autoimmune diseases depending in part on the transfer of natural or induced populations [24,38]. The transfer of natural, thymic-derived Tregs has been able to prevent autoimmune gastritis in animal models [39], but it has had little or no effect on established diseases (autoimmune gastritis [39], collagen-induced arthritis [24,38] and lupus glomerulonephritis [40]). In contrast, the adoptive transfer of induced Tregs has improved the laboratory features and survival of animals with chronic graft-versus-host disease [41], experimental autoimmune

Expert Rev. Gastroenterol. Hepatol.


Adoptive cell transfer in autoimmune hepatitis

encephalitis [7], type 1 diabetes [6] and autoimmune gastritis [42]. Whereas IL-6 can convert natural Tregs to IL-17-secreting T cells, induced Tregs are resistant to this proinflammatory cytokine, and they are more functionally stable in an inflammatory milieu [43]. Outcomes can also be affected by the antigen-specificity of the Tregs (TABLE 1) [42]. Antigen-specificity can direct the adoptively transferred Tregs to the target organ and results in a stronger immunosuppressive effect than that observed after the adoptive transfer of polyclonal Tregs [44]. Antigen-specificity directs the migration of the modified cells to the intended target, selectively enhances their accumulation in the chosen organ and focuses their immunosuppressive actions on the local pathogenic elements [45]. Organ-specific Tregs can be effective without knowledge of the pathogenic antigen that actually triggers the disease because the immunosuppressive actions of Tregs are not disease-specific [45]. Adoptive transfer of induced Tregs can also have a longlasting immunosuppressive effect. The transferred CD4+CD25+ T cells can stimulate the generation of secondary Tregs from CD4+CD25– T cells by direct cell-to-cell contact and by the secretion of TGF-b [46]. These newly generated functional Tregs express Foxp3 [46] and independently suppress disease activity [7]. They are a possible mechanism by which immune tolerance can be sustained and the immunosuppressive effect expanded (‘infectious tolerance’) [46,47]. Induced Tregs can persist for at least 1 year as memory cells characterized as CD25–Foxp3+ cells. These cells can regenerate as functional Tregs after repeat antigen exposure [6]. Induced tolerance of the triggering antigen is another mechanism by which adoptively transferred Tregs can sustain immunological homeostasis. Transferred Tregs suppress the accumulation and maturation of dendritic cells [24], and they may thereby impart a durable antigen tolerance during steadystate intervals that are free of inflammatory activity. This property is being evaluated to prevent allograft rejection in organ transplantation [48,49]. The demonstration of correctable deficiencies in the number and function of Tregs in patients with autoimmune hepatitis, clarification of the preferred Treg subset for transfer and improved culture methods to render antigenspecific Tregs with stable phenotypes have moved the Treg population forward as the prime candidate for adoptive transfer in autoimmune hepatitis. Mesenchymal stromal cells

MSCs are multipotent, self-renewing cells that have fibroblasticlike features, wide distribution throughout the body and the ability to differentiate into mesodermal, endodermal, and ectodermal cells [50,51]. They also have diverse effects on the innate and adaptive immune systems, which have justified their evaluation in the treatment of immune-mediated diseases [52–54] and graft rejection after solid organ transplantation (TABLE 1) [51]. MSCs are characterized by the expression of the surface markers, CD105, CD73, CD44, CD9, CD90 and CD80. They are also defined by their inability to express CD45, CD34, CD11b, CD11c, CD14, informahealthcare.com

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CD19, CD79a, CD86 and class II molecules of the MHC [50,55]. Their low expression of class I MHC molecules, inability to express class II MHC molecules and limited expression of co-stimulatory molecules decrease their immunogenicity and support their candidacy for cell transfer [56,57]. MSCs influence the actions of almost all components of the innate and adaptive immune systems (TABLE 1) [50,51,58]. Their effects on the macrophages, natural killer (NK) cells and dendritic cells are pivotal in modulating the innate immune response and suppressing tissue damage. Their inhibitory effects on B and T lymphocytes can dampen the adaptive immune response and limit the loss of self-tolerance. MSCs polarize macrophages in the presence of IL-4 and IL-13 from an M1 phenotype, which produces the proinflammatory cytokines, IL-12 and TNF-a, to an M2 phenotype, which produces the anti-inflammatory cytokine, IL-10 and reduced amounts of IL-12 and TNF-a [58–60]. Furthermore, human MSCs produce prostaglandin E2 which can inhibit macrophage production of the proinflammatory cytokines, IL-6, TNF-a and IFN-g. The net effect is to promote a ‘regulatory’ phenotype that avidly phagocytizes apoptotic cells and stabilizes the microenvironment [61]. MSCs are upregulated by exposure to the proinflammatory cytokines, IFN-g, TNF-a and IL-1, to produce indoleamine 2,3-dioxygenase, which catalyzes the degradation of tryptophan [62–64]. Tryptophan depletion and the local accumulation of metabolites in turn inhibits the actions of human NK cells as well as the functions of activated B and T lymphocytes (TABLE 1) [63,65]. Dendritic cells are also inhibited by MSCs [50]. MSCs prevent the generation of monocyte-derived dendritic cells [66,67], limit differentiation and function of dendritic cells [68] and impair T-cell priming by inhibiting dendritic cells from secreting proinflammatory cytokines, migrating to draining lymph nodes and presenting antigens to CD4+ T lymphocytes [69]. MSCs mediate the conversion of mature dendritic cells to a ‘regulatory’ immunosuppressive subset [70]. Critical components of the adaptive immune system are also inhibited by MSCs (TABLE 1) [50]. B lymphocytes are unable to proliferate or differentiate because MSCs arrest their cell cycle and limit their viability [71,72], possibly by activating the programmed death 1 pathway [50,73]. IgG production in blood and spleen lymphocytes can be stimulated or inhibited by MSCs in co-culture depending on the level of stimulation by viral antigens or lipopolysaccharide [74]. T lymphocytes are inhibited by MSCs that interrupt their cell cycle [75], increase their exposure to nitric oxide and secrete HLA-G [51]. The expression of inducible nitric oxide synthase is upregulated in MSCs after exposure to IFN-g, TNF-a and IL-1, and high local concentrations of nitric oxide in turn inhibit T-cell proliferation [76,77]. HLA-G is a non-classical class I MHC molecule produced by MSCs that can interact with the receptors of NK cells, dendritic cells and T lymphocytes and inhibit their functions [51,78,79]. HLA-G can also induce the expansion of the Treg population [79], and MSCs derived from bone marrow or human gingival tissue have expanded Tregs and suppressed markers of inflammation in a murine model of collagendoi: 10.1586/17474124.2015.1019470


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induced arthritis [80]. Other molecules produced by the MSCs, such as prostaglandin E2, TGF-b and hepatocyte growth factor, also contribute to the suppression of T-cell activity [50,81]. MSCs act by direct contact with the effector cells [51,79], and in some instances, under the mediation of IL-10 [79,82]. They must be primed by exposure to the cytokines, IFN-g, TNF-a or IL-1b [77]. Adoptive transfer of MSCs has been effective in animal models of diverse immune-mediated diseases, including rheumatoid arthritis [80,83], diabetes [84] and radiation-induced liver injury [85]. Two hundred clinical trial sites, including 22 sites for autoimmune diseases, have been registered to assess this intervention in the treatment of human diseases [52,54]. Preliminary human studies in immune-mediated diseases have suggested their value in the management of steroid-refractory Crohn’s disease [86], fistulizing Crohn’s disease [52,54,87], steroidresistant graft-versus-host disease [88–90], tissue toxicity after allogeneic hematopoietic stem cell transplantation [91], advanced multiple sclerosis [92] and allograft rejection after kidney transplantation [93,94]. Human studies are also ongoing to evaluate the prevention of graft rejection after liver transplantation [51]. The therapeutic promise of MSC infusion has extended beyond immune-mediated diseases. Preliminary studies in murine models of myocardial infarction [95] and musculoskeletal disorders [96] and in humans with acute respiratory distress syndrome [97] have been encouraging. Furthermore, the differentiation of adipose-derived MSCs into mature hepatocytes that incorporate into the liver parenchyma heralds a possible alternative therapy for liver failure [98]. MSCs of autologous and allogeneic origins have been expanded ex vivo and used in human studies, and the immunogenicity of MSCs has been low and the actions comparable regardless of cell origin (allogeneic or autologous) or cell source (bone marrow, adipose tissue or umbilical cord) [52,54,99]. Serious side effects have not been observed in mid-term clinical trials [51,100]. Therapy with MSCs has not been assessed in animal models or humans with autoimmune hepatitis, and several unresolved issues may hamper investigations in this disease [51,100,101]. The effects of cell transfer cannot be easily reversed and the longterm consequences of this intervention are still uncertain [51,101]. Methods to expand the MSC population ex vivo have not been standardized, and variations in cell preparation may have contributed to discrepant findings in some studies [50,51]. Chromosomal aberrations [102] and malignant transformation [103] have been described in short-term and long-term cultures of murine MSCs, and transient aneuploidy has also been described in MSCs derived from human bone marrow [104] and adipose tissue [105]. Rare occurrences of immunogenicity, manifested by the induction of memory T-cell responses and rejection, have followed the infusion of allogeneic MSCs in some models [50,51,106,107], and the rapid clearance of MSCs by the lungs after peripheral vein infusion may limit its effectiveness [108]. Immunosuppressive drugs used in the treatment of refractory autoimmune hepatitis (mycophenolate mofetil, tacrolimus and rapamycin) [109,110] have decreased the actions of MSCs doi: 10.1586/17474124.2015.1019470

in vitro, and the MSCs have in turn limited the actions of these immunosuppressive drugs [51,111,112]. Under such circumstances, the net benefit of MSC transfer may be limited. Laboratory and clinical experiences are providing reassurances about the safety of infusion therapy with MSCs, and barriers to its assessment in autoimmune hepatitis are diminishing. Human MSCs have not undergone malignant transformation in limited clinical studies [101,113,114], and the one investigation describing a high frequency of malignant transformation in culture [115] may have been cross-contaminated with cancerous cells [116]. Allogeneic and autologous MSCs have had similar efficacy and safety in clinical trials [52,54], and the frequency of opportunistic infections following the administration of MSCs in a randomized trial of patients transplanted with livingrelated kidneys has been low and actually better than that in untreated patients [117]. The need, rationale, techniques, safety and clinical precedents are aligning to encourage investigations of cell therapy with MSCs in autoimmune hepatitis. Dendritic cells

Dendritic cells are powerful antigen-presenting cells, and they modulate the innate and adaptive immune responses in tissuespecific autoimmune diseases [10,118,119]. Dendritic cells process and present foreign and self-antigens, and they can promote an adaptive immune response or induce tolerance to an antigen based on the accompanying inflammatory conditions [120]. Dendritic cells dampen the immune response by expanding Tregs and inducing tolerance to foreign and self-antigens (TABLE 1) [121]. The adoptive transfer of autologous dendritic cells has suppressed inflammatory activity and induced the expansion of Tregs in animal models of rheumatoid arthritis [122]. Clinical trials involving the transfer of tolerogenic dendritic cells have been instituted in patients with type 1 diabetes [123] and rheumatoid arthritis [124], and tolerogenic dendritic cells generated from patients with multiple sclerosis have maintained a stable semi-mature phenotype and ability to induce an antigen-specific hypo-responsiveness [125]. Characterization and isolation of tolerogenic dendritic cells are requisites for adoptive transfer [10,126]. The salient features of this subset are low surface densities of antigen-presenting molecules of the MHC, limited expression of the co-stimulatory molecules that activate CD4+ T-helper lymphocytes and reduced secretion of the proinflammatory cytokine, IL-12 [10,127]. They can be induced by the anti-inflammatory cytokines, IL-10 and TGF-b, and they are resistant to maturation [128]. Tolerogenic dendritic cells are typically immature or semimature cells that have limited function as antigen-presenting cells (TABLE 1) [128]. Dendritic cells express Fas ligand, which can induce the apoptosis of activated CD4+ T cells [10], indoleamine 2,3 dioxygenase, a tryptophan-degrading enzyme, which can block clonal T-cell expansion [129] and arginase, which can deplete activated T lymphocytes of L-arginine and limit their proliferation and function [130]. Tolerogenic dendritic cells can also promote the differentiation and proliferation of Tregs, and in this fashion, they can generate a self-amplification loop that Expert Rev. Gastroenterol. Hepatol.


prolongs their immunosuppressive effects. The expanded Tregs secrete the anti-inflammatory cytokine, IL-10, which in turn limits the maturation of the dendritic cells and preserves their tolerogenic phenotype [10,131]. The adoptive transfer of dendritic cells has not been evaluated in autoimmune hepatitis.


Macrophages

Macrophages are part of the innate immune response to tissue injury, and they can have proinflammatory and anti-inflammatory actions depending on their state of activation (TABLE 1) [132]. Proinflammatory M1 macrophages express nitric oxide synthase and increase oxidative stress within the microenvironment. They are induced by lipopolysaccharide, which is a Toll-like receptor agonist, and by IFN-g [132]. The proinflammatory cytokines, TNF-a and IL-12, are secreted as a consequence of these signaling pathways [132,133]. Anti-inflammatory M2 macrophages secrete IL-10 and little or no proinflammatory cytokines. The adoptive transfer of M2 macrophages has reduced the onset of diabetes in nonobese diabetic mice and protected pancreatic islets [132]. M2 macrophages are candidates for adoptive cell therapy in other autoimmune diseases, but their preference over Tregs, MSCs and tolerogenic dendritic cells is uncertain, especially since they lack antigen-specificity and broad immunosuppressive actions. Myeloid-derived suppressor cells

Myeloid-derived suppressor cells have a lineage shared by dendritic cells and macrophages, and they limit the proliferation and function of activated T cells by expressing arginase [130]. Myeloidderived suppressor cells are characterized by the surface markers, CD11b and Gr-1, and they can be activated by cannabidiol, a non-psychoactive component of marijuana (TABLE 1) [134]. Stimulation of this activation pathway by cannabidiol has reduced the markers of liver inflammation in experimental autoimmune hepatitis [135], and the adoptive transfer of myeloid-derived suppressor cells has been associated with potent anti-inflammatory effects in murine models of autoimmune hepatitis [130], acute hepatitis associated with polymicrobial sepsis [136] and experimental myasthenia gravis [12].

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Antigen-specificity can be obtained by pulsing cell cultures with the desired antigen or by introducing genes for specific T-cell antigen receptors (TCRs). Retroviral gene transfer in established Tregs or retroviral transfer of TCRs and the Foxp3 gene in naı¨ve CD4+CD25– T cells ensure the generation of Tregs that will accumulate in the desired organ [45,143]. Engineered fusion molecules (chimeric antigen receptors) [144,145] and the insertion of multiple antigenic specificities in a single cell population [146] are other advances that may improve targeting and effectiveness. Genetic engineering also promises to increase yields of the desired antigen-specific population [145,147]. Optimizing dendritic cells for adoptive transfer

The key to preserving tolerogenic dendritic cells is to prevent their maturation [10]. Epigenetic manipulations of the transcription pathway responsible for the maturation of dendritic cells make this objective possible [148]. Myeloid-derived dendritic cells mature along an activation pathway dependent on the family of transcription factors associated with NF-kB [124,149]. The component designated, RelB, is essential for antigen detection and immune responsiveness in dendritic cells. The production or nuclear translocation of RelB can be blocked by soluble inhibitors or small molecules of interfering RNA [124,149,150]. The modified dendritic cells are rendered tolerogenic, antigenspecificity can be induced and the normal immune response to other antigens can be preserved [124]. Advantages of using Tregs for adoptive transfer in autoimmune hepatitis

Investigational efforts to develop adoptive transfer for autoimmune hepatitis have focused on the Tregs. Deficiencies in this population have been identified that are correctable by adoptive transfer; preferred features of the transferred subset have been described; successful animal studies have been performed and controversial areas have been discovered that will ultimately refine the knowledge base and treatment strategy. These advantages have made Tregs the leading candidate for adoptive transfer in autoimmune hepatitis. Correctable Treg deficiencies

Optimizing the Treg & dendritic populations for adoptive transfer

Tregs, especially natural or thymic-derived cells, can stop expressing Foxp3 in response to inflammatory cytokines [137], and the transformed cells can lose immunosuppressive activity [138], secrete proinflammatory cytokines (IL-17 and IFN-g) [139] and become immunogenic [140]. Similarly, dendritic cells can have deleterious, proinflammatory actions [118]. Genetic and epigenetic manipulations can modify, intensify and re-direct these populations to optimize their performance after adoptive transfer. Optimizing Tregs for adoptive transfer

Phenotypic stability can be strengthened by epigenetic manipulations of the Foxp3 gene using inhibitors of DNA methyltransferase or histone protein deacetylase [38,141,142]. informahealthcare.com

Patients with autoimmune hepatitis are deficient in the number and function of Tregs [31,32], and these deficiencies can decrease tolerance of liver-related antigens and promote the development of autoimmune hepatitis (TABLE 2) [11]. The reduced number of circulating Tregs in autoimmune disease may reflect impaired induction or increased apoptosis rather than re-distribution [30], whereas the deficient function may reflect intrinsic defects in the Tregs [151]. Galectin-9 is a b-galactosidase-binding protein expressed by Tregs, and it is required to inhibit the immune responses of CD4+ effector cells [151,152]. Galectin-9 binds to the T-cell immunoglobulin and mucin domain-3 receptor expressed on Th1 lymphocytes and dendritic cells, and this ligation induces apoptosis and downregulates the immune response [152]. Reduced expression of galectin-9 on Tregs and impaired doi: 10.1586/17474124.2015.1019470

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Table 2. Adoptive transfer using Tregs for autoimmune hepatitis. Advantages

Disadvantages

Correctable Treg deficiencies: Deficient number and function [31,32,162] Reduced expression of galectin-9 [151] Impaired ligation with TIM-3 on Th1 cells [151] Decreased number and function of CD39+ cells [154] Reversible defects with corticosteroids [32]

Uncertain pathological deficiencies in Tregs: Discrepant findings [159] Uncertain distinction from normal response [159] Unclear contributions to autoimmunity [38]

Characterization of the optimal subset: Induced cells preferred over natural cells [24] Antigen-specificity improves tissue targeting [24,157] Retroviral transfer of TCR, Foxp3 gene possible [45] Effective without knowledge of antigenic trigger [145] Genetic engineering improves yields and function [145] Phenotypic stability by Foxp3 protectors [38,141,142]

Disease-related factors impair performance: Deficiencies may recur after transfer [137,138] Suppressor phenotype may become pathogenic [138] Poor results in established diseases [24,40] Optimal timing for transfer uncertain [31,154] Pre-treatment with drugs may be needed [32,38] Drug response may supersede need for transfer [38]

Effective in murine models of autoimmune liver disease: Prevents fatal AIH in neonatally thymectomized mice [8] Liver accumulation in AIH [11] Immune tolerance to hepatic autoantigen in AIH [11] Decreased histological activity in AIH [11] Less portal inflammation, bile duct damage in PBC [13]

Unknown safety profile: Suppressor phenotype may become pathogenic [138] Detrimental cell defect may be expanded [24] Healthy tissues may be targeted [145] Malignancy and infection risk unclear [145]

Controversial areas identified: Discrepancies about Treg deficiencies in AIH [159,162] Inconsistent definition of Treg phenotype [159,162] Varied methods of Treg purification [160,161] Varied assessments of Treg function [160,161] Non-standardized tissue assessments for Tregs [159,165]

Methodological and Procedural uncertainties: Adequacies of cell yields for transfer [24] Identity, purity, potency, and sterility [38] Differentiating natural from induced subsets [38] Establishing phenotypic stability [38] Developing preservation techniques [38] Ensuring availability and delivery [1,49]

AIH: Autoimmune hepatitis; Foxp3: Forkhead box protein 3; PBC: Primary biliary cirrhosis; TCR: T-cell antigen receptor; TIM-3: T-cell immunoglobulin and mucin domain-3.

expression of T-cell immunoglobulin and mucin domain-3 on Th1 cells have been described in autoimmune hepatitis, and these deficiencies may prevent the ligation necessary for effective immunosuppression [151]. CD39 is an ectonucleotidase that is expressed on Tregs and involved in the conversion of ATP and ADP to AMP [20]. AMP is converted to adenosine, and this immune-regulatory nucleoside in turn contributes to the immunosuppressive actions of Tregs [153]. In autoimmune hepatitis, the number and function of CD39+ Tregs are reduced, and the failure to hydrolyze proinflammatory nucleotides, generate adenosine and suppress IL-17 production may promote autoimmune hepatitis [154]. These defects are associated with inflammatory activity, and they are reversible by treatments that suppress liver inflammation and restore or replace the impaired Tregs [32]. Characterization of the preferred Treg subset

Freshly generated Tregs induced from autologous undifferentiated CD4+CD25– T cells have properties that justify their preference over naturally occurring Tregs (TABLE 2). The undifferentiated CD4+CD25– T cells from patients with autoimmune hepatitis can be expanded ex vivo with high-dose IL-2 and TGF-b, and some will acquire Foxp3, CD25 and regulatory properties [155,156]. Induced Tregs have greater phenotypic stability than natural Tregs in inflammatory conditions, doi: 10.1586/17474124.2015.1019470

greater immunosuppressive activity and more resistance to apoptosis [24,155]. Antigen-specificity is also preferred in the transferred population as it improves their accumulation in the target organ and localizes their immunosuppressive actions [24]. Both the induced and natural Tregs can be rendered antigen-specific by antigen pulsing or gene transfer of TCRs [24,44,45,145]. In type 2 autoimmune hepatitis, Tregs directed against the triggering antigen (cytochrome P450 2D6) have greater immunosuppressive actions than polyclonal Tregs [157]. Disease suppression in animal models

Two studies in murine models of autoimmune hepatitis have demonstrated improvement after the adoptive transfer of Tregs. In BALB/c mice with disruption of the programmed cell death-1 (PD-1–/–) signaling pathway, neonatal thymectomy depletes naturally occurring CD4+CD25+Foxp3+ Tregs, accelerates antibody responses, increases the frequency of pathogenic self-reactive T cells and precipitates a fatal autoimmune hepatitis. The adoptive transfer of Tregs has suppressed the induction of fatal autoimmune hepatitis in this model (TABLE 2) [8]. Furthermore, splenectomy in these thymectomized mice has prevented the occurrence of fatal autoimmune hepatitis and prolonged the salutary effects of dexamethasone, presumably by preventing the migration of splenic follicular T-helper cells to the liver and counterbalancing the Treg deficiency [158]. Expert Rev. Gastroenterol. Hepatol.



In another murine model of autoimmune hepatitis, the adoptive transfer of autologous Tregs accumulated in the inflamed liver, restored peripheral tolerance to a liver autoantigen (formiminotransferase cyclodeaminase), and significantly reduced the histological activity indices (TABLE 2) [11]. The expanded Tregs expressed high levels of the cytokine receptor, CXCR3, and migration to the liver was probably directed by increased hepatic production of the cytokine ligands, CXCL9 and CXCL10 [11]. Immunological tolerance was maintained for at least 1 month despite reduction in the number of intrahepatic Tregs to control levels. These observations indicated that the adoptive transfer of Tregs could be effective in preventing and treating autoimmune hepatitis in two different animal models [8,11] and that an inactive state could be maintained in the absence of a triggering factor or persistently high levels of intrahepatic Tregs [11]. Experiences in a murine model of autoimmune cholangitis in which the adoptive transfer of Tregs reduced portal inflammation, bile duct damage and the inflammatory response have supported these findings [13] and underscored the therapeutic potential of Treg transfer in immune-mediated liver diseases. Identification of controversial areas

The concept that Tregs have numerical and functional deficiencies in autoimmune hepatitis has been challenged [159], and the bases for these discrepancies may relate to differences in the definition of Tregs and the methods for their assessment (TABLE 2) [160,161]. Early studies defined Tregs by the surface markers, CD4+CD25+ [31,32,162], and subsets with different functions were subsequently described based on the expression of Foxp3 [155], CD127 [163] and CD39 [154]. Cell cultures could be contaminated with CD127+ cells [163], IL-17-producing cells [164], effector cells transiently expressing Foxp3 [18] and Tregs that converted to effector phenotypes [154]. The cumulative experience with Tregs in autoimmune hepatitis has probably been compromised by the inclusion of invalid cell populations in the early studies [159]. Later studies that defined Tregs by the surface markers, CD4+CD25+ (high)C127(low)Foxp3+, found Tregs to be normal in number and function in autoimmune hepatitis [159]. Furthermore, the frequency of circulating Tregs was higher in patients with active inflammation than in patients with quiescent disease, and Tregs were more frequent in the liver tissue of patients with autoimmune hepatitis than in patients with non-alcoholic steatohepatitis [159]. These findings varied in every aspect to earlier studies in autoimmune hepatitis [31,162] and to histological studies using different methods of cell purification and staining [165]. The discrepant observations underscore the importance of codifying the essential phenotype of Tregs and standardizing the methods for assessing their function and tissue infiltration (TABLE 2) [159]. Furthermore, the circumstances under which the assessments are performed must be carefully documented as findings can vary with the degree of inflammatory activity and the use of immunosuppressive drugs [32]. The identification of informahealthcare.com

Review

controversial areas affords an opportunity to improve the rigor of future investigations and strengthen the knowledge base. Disadvantages of using Tregs for adoptive transfer in autoimmune hepatitis

The emergence of adoptive cell therapy using Tregs in autoimmune hepatitis requires the reassurance that a crucial cellular defect will be corrected without compromising other protective homeostatic mechanisms. Observations in humans with autoimmune hepatitis have not clearly separated the normal protective functions of the Tregs from a pathological response. These uncertainties have been generated by discrepant findings in patients with the disease [159], and they must be resolved before the strategy can be fully justified (TABLE 2). Animal studies have supported the adoptive transfer of Tregs in experimental models of autoimmune hepatitis [8,11], and studies in animals and humans have indicated that the ex vivo expansion or induction of functionally abnormal Tregs can be restored to normal [11,155]. This finding suggests that the deficiencies in Tregs are induced by factors associated with the disease (possibly proinflammatory cytokines). Recurrence of the original abnormalities in the transferred Tregs is a justifiable concern. The disease-related factors that limit Treg function must be defined, and the optimal circumstances for cell transfer must be determined. The prospect of a staged therapy comprising initial anti-inflammatory medications (corticosteroids) followed by adoptive transfer must be considered. If staged therapy is preferred, the indications for subsequent adoptive transfer and its advantages must be clarified (TABLE 2). The safety profile of adoptive transfer in autoimmune hepatitis is unknown. Major concerns are that an unsuspected irreversible intrinsic defect within the autologous Tregs will be inadvertently expanded to the detriment of the patient [24] and that the transferred immunosuppressive phenotype will convert in vivo to an immunogenic phenotype [137,138]. Other theoretical concerns are that misdirected tissue targeting will injure healthy tissues and that the risk of infection or malignancy will be increased by compromised immune surveillance [145]. Better understanding of the cell defects contributing to the disease, refined methods for selecting healthy cell populations for expansion or induction, manipulations that stabilize the preferred phenotype in diverse conditions and a complete catalogue of side effects associated with the intervention are warranted (TABLE 2). Other challenges in developing adoptive cell transfer for autoimmune hepatitis are methodological and procedural. Improving yields, establishing the identity, purity, potency and sterility of cell preparations, differentiating naturally occurring from induced subsets, generating phenotypic stability and developing reliable preservation techniques are methodological challenges [38]. The delivery of a therapy that by nature is highly individualized, labor-intensive, expensive and restricted to specialized medical centers is the daunting procedural challenge [1,49]. The promise of adoptive cell therapy in autoimmune hepatitis is sufficiently compelling to address these challenges and encourage the next step forward. doi: 10.1586/17474124.2015.1019470

Review

Czaja


Expert commentary

The ideal therapeutic intervention for autoimmune hepatitis would correct the critical immunological defect that sustains the disease, have liver-specific actions that do not imbalance other homeostatic mechanisms, maintain a treatment-free quiescent state long-term and have few or no side effects. Multiple pharmacological, molecular and cellular interventions have emerged that have a rationale in autoimmune hepatitis, and the continued pursuit of therapies that promise to enhance or replace current treatments must be encouraged and supported [110,166]. The ideal treatment for autoimmune hepatitis is yet to be discovered. Multiple defects in the regulatory mechanisms that control the activation, differentiation and proliferation of Th1, Th2 and Th17 lymphocytes have been described in autoimmune hepatitis, and genetic factors that may contribute to the occurrence and behavior of the disease have been identified [14,167]. Adoptive cell therapy has the prospect of correcting a critical immuneregulatory defect, maintaining organ-specificity and achieving a durable response [24,38]. Investigations are ongoing to develop an evidence-based foundation that supports or rejects this therapeutic option in autoimmune hepatitis. Autologous Tregs are appropriate candidates for adoptive cell therapy in autoimmune hepatitis. The intervention has already been shown to reduce inflammatory activity in experimental models of autoimmune hepatitis [8,11] and other immune-mediated diseases [6,7,13,24,39,41]. The demonstrated immune-regulatory actions of Tregs are comprehensive and pertinent to autoimmune hepatitis [38]. The preferred Tregs for adoptive transfer (antigenspecific, freshly generated Tregs) have been characterized [155,157], and improvements in the engineering, purification and yield of the preferred Treg population are ongoing [38]. Furthermore, studies of other cell populations for adoptive transfer therapy (MSCs, dendritic cells, macrophages and myeloid-derived suppressor cells) are preliminary [80,118,130,132], or the candidate alternative population (MSCs or dendritic cells) is effective in part through a Treg mechanism [79,80,121]. The clinical need, putative actions and investigational evidence support further study of Tregs in adoptive cell therapy for autoimmune hepatitis [31,32,162]. Progress is impeded mainly by uncertainties in the Treg abnormalities that perpetuate the disease [159]. Studies in diverse immune-mediated diseases have indicated that the number and function of Tregs are variable between diseases and inconsistent in the same disease [31,38,159,162]. The discrepant findings of different laboratories evaluating Treg performance in autoimmune hepatitis must be resolved before full commitment to the development of this intervention [160,161]. The discrepancies in the studies of Tregs in autoimmune hepatitis highlight the difficulties in studying homogenous patient populations under comparable conditions, applying uniform and strict designations for Tregs in an evolving science and using similar laboratory methods for assessing function and tissue infiltration [160,161]. The markedly discrepant findings in studies of Tregs in autoimmune hepatitis support the likelihood doi: 10.1586/17474124.2015.1019470

that the differences reflect patient selection, testing conditions and laboratory assessments rather than invalid conclusions. They also suggest opportunities to standardize designations, study conditions and laboratory methods. Other impediments to the emergence of adoptive cell therapy with Tregs for autoimmune hepatitis are the need to develop antigen-specificities that concentrate the immunosuppressive actions within the liver [45,145], minimize systemic homeostatic imbalance [145], stabilize the suppressor phenotype in inflammatory conditions [24], ensure purity, potency and sterility of preparations [38] and demonstrate an excellent safety profile [145]. These advances will follow as investigational commitment to this intervention strengthens among the diverse disciplines already evaluating this strategy and a network of laboratories committed to assessing this intervention in autoimmune hepatitis is established. Five-year view

The controversies regarding the role of Tregs in the pathogenesis of autoimmune hepatitis will be resolved. Designations for the Treg population will be codified and uniformly applied in all studies. Treg subsets will be fully characterized and thymicderived (natural) Tregs will be reliably distinguished from induced Tregs by assays that detect subset-specific transcription factors such as Helios [168]. Investigational testing protocols will evaluate homogeneous patient populations at similar stages of inflammatory activity and treatment; laboratory methods for the detection and functional assessment of Tregs will be standardized and correctable deficiencies in the Treg population in certain patients with autoimmune hepatitis will be substantiated. The disease-related factors that reversibly affect Treg performance in autoimmune hepatitis will be defined, probably as interactions between proinflammatory cytokines and chemokines that maintain self-amplification loops impairing Treg performance. Tregs will become a focus of investigational activity in several centers committed to the study of autoimmune hepatitis, and management strategies based on pharmacological agents (corticosteroids, mycophenolate mofetil, rapamycin and proteasome inhibitors) and adoptive cell transfer will emerge as individual, combined or sequenced regimens in experimental animal models. The results of these studies will direct the nature of preliminary clinical trials and foster a multi-centered network of clinical collaborators. Tregs will be a central topic of interest in autoimmune hepatitis, but adoptive cell therapy will still have much to prove. Studies based on the adoptive transfer of MSCs or dendritic cells may emerge if progress in the development of Treg therapy falters. Financial & competing interests disclosure

AJ Czaja researched, designed and wrote this article. The tables are original, constructed by Czaja, fully referenced and developed solely for this review. The review article is original, current and comprehensive, and it has not been published previously. This review did not receive financial support from a funding agency or institution, and AJ Czaja has no conflict of interests to declare. The author has no relevant affiliations or Expert Rev. Gastroenterol. Hepatol.


financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies,

Review

honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.

Key issues Expert Review of Gastroenterology & Hepatology Downloaded from informahealthcare.com by Nanyang Technological University on 04/27/15 For personal use only.

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Adoptive transfer of autologous immune cells (Tregs, mesenchymal stromal cells, dendritic cells, macrophages and myeloid-derived suppressor cells) that have been modified, expanded or induced ex vivo have been used in animal models and humans to treat various immune-mediated diseases, including autoimmune hepatitis.

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The principal candidates for adoptive cell therapy in autoimmune hepatitis are Tregs because they have broad immunosuppressive actions and correctable defects in autoimmune hepatitis.

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Tregs can be rendered antigen-specific, induce antigen tolerance and yield a durable protection by generating secondary Tregs (‘infectious tolerance’) and suppressing the accumulation and maturation of dendritic cells.

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Tregs that have been induced from CD4+CD25– T lymphocytes after stimulation with TGF-b have greater immunosuppressive effects and functional stability in an inflammatory milieu than natural, thymic-derived Tregs, and they are the preferred Treg subset for adoptive cell therapy in autoimmune hepatitis.

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Adoptive transfer of Tregs in murine models of autoimmune hepatitis have prevented the occurrence of fatal disease, reduced inflammatory activity in liver tissue, restored peripheral tolerance to a liver autoantigen (formiminotransferase cyclodeaminase) and maintained immunological tolerance for at least 1 month.

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Uncertainties exist in the occurrence and nature of Treg abnormalities in autoimmune hepatitis, and studies that have indicated abnormalities in the number and function of Tregs have been challenged.

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Key difficulties in developing the adoptive transfer of Tregs for autoimmune hepatitis are the phenotypic instability of these cells in an inflammatory milieu, their uncertain safety profile and the lack of standardized methodologies to ensure identity, purity, potency and sterility of cell preparations.

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Combination or sequential therapies using pharmacological agents (corticosteroids, mycophenolate mofetil, rapamycin or proteasome inhibitors) to suppress inflammatory activity, expand the Treg population and protect the phenotype of the induced Tregs may be necessary to optimize adoptive cell therapy.

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Prime alternative cell populations to be considered for adoptive transfer in autoimmune hepatitis are mesenchymal stromal cells and dendritic cells.

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Multi-center collaboration will be necessary to develop, refine, evaluate, establish and distribute a treatment that is highly individualized, labor intensive, expensive and restricted to specialized centers.

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Francois S, Mouiseddine M, Allenet-Lepage B, et al. Human mesenchymal stem cells provide protection against radiation-induced liver injury by antioxidative process, vasculature protection, hepatocyte differentiation, and trophic effects. Biomed Res Int 2013;2013:151679 Duijvestein M, Vos AC, Roelofs H, et al. Autologous bone marrow-derived mesenchymal stromal cell treatment for refractory luminal Crohn’s disease: results of a phase I study. Gut 2010;59:1662-9 Ciccocioppo R, Bernardo ME, Sgarella A, et al. Autologous bone marrow-derived mesenchymal stromal cells in the treatment of fistulising Crohn’s disease. Gut 2011;60: 788-98 Ringden O, Uzunel M, Rasmusson I, et al. Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplantation 2006;81:1390-7

94.

Perico N, Casiraghi F, Gotti E, et al. Mesenchymal stromal cells and kidney transplantation: pretransplant infusion protects from graft dysfunction while fostering immunoregulation. Transpl Int 2013;26:867-78

95.

Pasha Z, Wang Y, Sheikh R, et al. Preconditioning enhances cell survival and differentiation of stem cells during transplantation in infarcted myocardium. Cardiovasc Res 2008;77:134-42

96.

97.

98.

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First human trial of MSCs in eight patients with steroid-refractory graft-versus-host disease demonstrating resolution in seven patients and improved survival compared with untreated patients during the same period. Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 2008;371:1579-86

99.

Prasad VK, Lucas KG, Kleiner GI, et al. Efficacy and safety of ex vivo cultured adult human mesenchymal stem cells (Prochymal) in pediatric patients with severe refractory acute graft-versus-host disease in a compassionate use study. Biol Blood Marrow Transplant 2011;17:534-41

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Ringden O, Uzunel M, Sundberg B, et al. Tissue repair using allogeneic mesenchymal stem cells for hemorrhagic cystitis, pneumomediastinum and perforated colon. Leukemia 2007;21:2271-6 Yamout B, Hourani R, Salti H, et al. Bone marrow mesenchymal stem cell transplantation in patients with multiple sclerosis: a pilot study. J Neuroimmunol 2010;227:185-9 Peng Y, Ke M, Xu L, et al. Donor-derived mesenchymal stem cells combined with low-dose tacrolimus prevent acute rejection after renal transplantation: a clinical pilot study. Transplantation 2013;95:161-8


103.

Jeong JO, Han JW, Kim JM, et al. Malignant tumor formation after transplantation of short-term cultured bone marrow mesenchymal stem cells in experimental myocardial infarction and diabetic neuropathy. Circ Res 2011;108: 1340-7

104.

Tarte K, Gaillard J, Lataillade JJ, et al. Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation. Blood 2010;115:1549-53

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Human MSCs can develop aneuploidy in culture but the chromosomal aberrations are transient, growth arrest is progressive and all cultures enter senescence without malignant transformation.

105.

Meza-Zepeda LA, Noer A, Dahl JA, et al. High-resolution analysis of genetic stability of human adipose tissue stem cells cultured to senescence. J Cell Mol Med 2008;12: 553-63

106.

Nauta AJ, Westerhuis G, Kruisselbrink AB, et al. Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood 2006;108: 2114-20

107.

Kern S, Eichler H, Stoeve J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006;24:1294-301

Eliopoulos N, Stagg J, Lejeune L, et al. Allogeneic marrow stromal cells are immune rejected by MHC class I- and class II-mismatched recipient mice. Blood 2005;106:4057-65

108.

Morphology and immune phenotype are similar in MSCs obtained from bone marrow, adipose tissue and umbilical cord, but successful isolation of cells was lowest from umbilical cord (63 vs 100%).

Eggenhofer E, Benseler V, Kroemer A, et al. Mesenchymal stem cells are short-lived and do not migrate beyond the lungs after intravenous infusion. Front Immunol 2012;3:297

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Important study demonstrating that MSCs infused in the peripheral vein are cleared rapidly from the peripheral circulation and are unlikely to get beyond the lungs, raising questions about how their immunosuppressive actions are delivered and the most effective route of cell infusion (peripheral vein vs portal vein).

109.

Czaja AJ. Drug choices in autoimmune hepatitis: part B - nonsteroids. Expert Rev Gastroenterol Hepatol 2012;6:617-35

110.

Czaja AJ. Current and prospective pharmacotherapy for autoimmune hepatitis. Expert Opin Pharmacother 2014;15: 1715-36

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Update of emerging pharmacotherapies for autoimmune hepatitis including agents that may expand Treg populations.

Gao J, Caplan AI. Mesenchymal stem cells and tissue engineering for orthopaedic surgery. Chir Organi Mov 2003;88:305-16 Zheng G, Huang L, Tong H, et al. Treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebo-controlled pilot study. Respir Res 2014;15:39 Banas A, Teratani T, Yamamoto Y, et al. Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hepatology 2007;46:219-28 Demonstration that MSCs derived from adipose tissue can be transformed into liver cells that express liver-specific markers, exhibit liver-related functions, incorporate into the liver parenchyma when transplanted and may prove useful in the treatment of liver failure.

100.

Otto WR, Wright NA. Mesenchymal stem cells: from experiment to clinic. Fibrogenesis Tissue Repair 2011;4:20

101.

Haarer J, Johnson CL, Soeder Y, Dahlke MH. Caveats of mesenchymal stem cell therapy in solid organ transplantation. Transpl Int 2015;28:1-9

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102.

Review

Cautionary review of cell therapy with MSCs emphasizing that unlike pharmacological therapy the effects of cell therapy cannot be easily reversed. Miura M, Miura Y, Padilla-Nash HM, et al. Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells 2006;24:1095-103

doi: 10.1586/17474124.2015.1019470

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Hoogduijn MJ, Crop MJ, Korevaar SS, et al. Susceptibility of human mesenchymal stem cells to tacrolimus, mycophenolic acid, and rapamycin. Transplantation 2008;86: 1283-91 Buron F, Perrin H, Malcus C, et al. Human mesenchymal stem cells and immunosuppressive drug interactions in allogeneic responses: an in vitro study using human cells. Transplant Proc 2009;41: 3347-52

113.

Casiraghi F, Remuzzi G, Abbate M, Perico N. Multipotent mesenchymal stromal cell therapy and risk of malignancies. Stem Cell Rev 2013;9:65-79

114.

Prockop DJ, Brenner M, Fibbe WE, et al. Defining the risks of mesenchymal stromal cell therapy. Cytotherapy 2010;12:576-8

115.

Rosland GV, Svendsen A, Torsvik A, et al. Long-term cultures of bone marrow-derived human mesenchymal stem cells frequently undergo spontaneous malignant transformation. Cancer Res 2009;69:5331-9

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116.

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117.

118.

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119.

Concerns about the emergence of chromosomal aberrations and malignant transformation of MSCs in culture are heightened by findings that 46% of cultures maintained for 5–106 weeks undergo spontaneous malignant change. Torsvik A, Rosland GV, Svendsen A, et al. Spontaneous malignant transformation of human mesenchymal stem cells reflects cross-contamination: putting the research field on track - letter. Cancer Res 2010;70: 6393-6 Cross-contamination of cultures of MSCs with cancerous cells is an explanation for the high frequency of malignant transformation.

120.

Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol 2003;21:685-711

121.

Moseman EA, Liang X, Dawson AJ, et al. Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4+CD25+ regulatory T cells. J Immunol 2004;173: 4433-42

122.

Jaen O, Rulle S, Bessis N, et al. Dendritic cells modulated by innate immunity improve collagen-induced arthritis and induce regulatory T cells in vivo. Immunology 2009;126:35-44

123.

Giannoukakis N, Phillips B, Finegold D, et al. Phase I (safety) study of autologous tolerogenic dendritic cells in type 1 diabetic patients. Diabetes Care 2011;34:2026-32

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Early clinical experience with adoptive transfer of dendritic cells in the treatment of human immune-mediated disease.

124.

Thomas R. Dendritic cells and the promise of antigen-specific therapy in rheumatoid arthritis. Arthritis Res Ther 2013;15:204

125.

Raich-Regue D, Grau-Lopez L, Naranjo-Gomez M, et al. Stable antigen-specific T-cell hyporesponsiveness induced by tolerogenic dendritic cells from multiple sclerosis patients. Eur J Immunol 2012;42:771-82

126.

127.

Manicassamy S, Pulendran B. Dendritic cell control of tolerogenic responses. Immunol Rev 2011;241:206-27

128.

Torres-Aguilar H, Aguilar-Ruiz SR, Gonzalez-Perez G, et al. Tolerogenic dendritic cells generated with different immunosuppressive cytokines induce antigen-specific anergy and regulatory properties in memory CD4+ T cells. J Immunol 2010;184:1765-75

Tan J, Wu W, Xu X, et al. Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial. JAMA 2012;307:1169-77 Mbongue J, Nicholas D, Firek A, Langridge W. The role of dendritic cells in tissue-specific autoimmunity. J Immunol Res 2014;2014:857143 Excellent review of the nature, subsets and diverse functions of dendritic cells including their ability under steady-state conditions to induce antigen tolerance. McKenna K, Beignon AS, Bhardwaj N. Plasmacytoid dendritic cells: linking innate and adaptive immunity. J Virol 2005;79: 17-27

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Hilkens CM, Isaacs JD. Tolerogenic dendritic cell therapy for rheumatoid arthritis: where are we now? Clin Exp Immunol 2013;172:148-57

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Mellor AL, Baban B, Chandler P, et al. Cutting edge: induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion. J Immunol 2003;171:1652-5 Hegde VL, Nagarkatti PS, Nagarkatti M. Role of myeloid-derived suppressor cells in amelioration of experimental autoimmune hepatitis following activation of TRPV1 receptors by cannabidiol. PLoS One 2011;6:e18281 Ito T, Yang M, Wang YH, et al. Plasmacytoid dendritic cells prime IL-10-producing T regulatory cells by

inducible costimulator ligand. J Exp Med 2007;204:105-15 132.

Parsa R, Andresen P, Gillett A, et al. Adoptive transfer of immunomodulatory M2 macrophages prevents type 1 diabetes in NOD mice. Diabetes 2012;61:2881-92

133.

Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol 2008;8:958-69

134.

Hegde VL, Nagarkatti M, Nagarkatti PS. Cannabinoid receptor activation leads to massive mobilization of myeloid-derived suppressor cells with potent immunosuppressive properties. Eur J Immunol 2010;40:3358-71

135.

Hegde VL, Hegde S, Cravatt BF, et al. Attenuation of experimental autoimmune hepatitis by exogenous and endogenous cannabinoids: involvement of regulatory T cells. Mol Pharmacol 2008;74:20-33

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Demonstration that natural cannabinoids can modulate inflammatory activity in experimental autoimmune hepatitis by suppressing the production of inflammatory cytokines and increasing the absolute number of intrahepatic Tregs.

136.

Sander LE, Sackett SD, Dierssen U, et al. Hepatic acute-phase proteins control innate immune responses during infection by promoting myeloid-derived suppressor cell function. J Exp Med 2010;207:1453-64

137.

Komatsu N, Okamoto K, Sawa S, et al. Pathogenic conversion of Foxp3+ T cells into TH17 cells in autoimmune arthritis. Nat Med 2014;20:62-8

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Important caveat regarding the use of Tregs in inflammatory immune-mediated diseases as they can transform into pathogenic cells and augment the proinflammatory response.

138.

Zhou X, Bailey-Bucktrout SL, Jeker LT, et al. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat Immunol 2009;10:1000-7

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Phenotype instability of Tregs in inflammatory conditions can contribute to the disease process, and it is a distressing limitation that must be overcome to advance the therapy.

139.

Ayyoub M, Deknuydt F, Raimbaud I, et al. Human memory FOXP3+ Tregs secrete IL-17 ex vivo and constitutively express the T(H)17 lineage-specific transcription factor RORgamma t. Proc Natl Acad Sci USA 2009;106:8635-40




140.

Duarte JH, Zelenay S, Bergman ML, et al. Natural Treg cells spontaneously differentiate into pathogenic helper cells in lymphopenic conditions. Eur J Immunol 2009;39:948-55

151.

Liberal R, Grant CR, Holder BS, et al. The impaired immune regulation of autoimmune hepatitis is linked to a defective galectin-9/tim-3 pathway. Hepatology 2012;56:677-86

141.

Moon C, Kim SH, Park KS, et al. Use of epigenetic modification to induce FOXP3 expression in naive T cells. Transplant Proc 2009;41:1848-54

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142.

Floess S, Freyer J, Siewert C, et al. Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol 2007;5:e38

143.

Murphy A, Westwood JA, Teng MW, et al. Gene modification strategies to induce tumor immunity. Immunity 2005;22: 403-14

Demonstration of a defective potentially correctable signaling pathway in autoimmune hepatitis by which Tregs with reduced levels of Gal9 are less capable of immunosuppression and CD4+CD25– effectors cells with reduced levels of T-cell immunoglobulin and mucin domain-3 are less responsive to Treg control.

144.

145.

..

146.

147.

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Maher J. Immunotherapy of malignant disease using chimeric antigen receptor engrafted T cells. ISRN Oncol 2012;2012:278093 Jethwa H, Adami AA, Maher J. Use of gene-modified regulatory T-cells to control autoimmune and alloimmune pathology: is now the right time? Clin Immunol 2014;150:51-63 Excellent review of methods to improve target specificity of Tregs by genetic modifications that allow expression of desired T-cell antigen receptors. Joffre O, Santolaria T, Calise D, et al. Prevention of acute and chronic allograft rejection with CD4+CD25+Foxp3+ regulatory T lymphocytes. Nat Med 2008;14:88-92 Wright GP, Ehrenstein MR, Stauss HJ. Regulatory T-cell adoptive immunotherapy: potential for treatment of autoimmunity. Expert Rev Clin Immunol 2011;7:213-25

Maruoka R, Aoki N, Kido M, et al. Splenectomy prolongs the effects of corticosteroids in mouse models of autoimmune hepatitis. Gastroenterology 2013;145:209-20

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Interesting study implicating the chemokine-directed migration of splenic follicular cells to the liver as a basis for fatal autoimmune hepatitis in neonatally thymectomized mice and demonstrating that splenectomy prevents this occurrence and enhances the salutary effects of dexamethasone.

159.

Peiseler M, Sebode M, Franke B, et al. FOXP3+ regulatory T cells in autoimmune hepatitis are fully functional and not reduced in frequency. J Hepatol 2012;57: 125-32

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Critical study using rigid criteria for Tregs that demonstrates normal numbers and function of Tregs in patients with autoimmune hepatitis and challenges the results of studies demonstrating Treg deficiencies in this disease.

153.

Ernst PB, Garrison JC, Thompson LF. Much ado about adenosine: adenosine synthesis and function in regulatory T cell biology. J Immunol 2010;185:1993-8

154.

Grant CR, Liberal R, Holder BS, et al. Dysfunctional CD39(POS) regulatory T cells and aberrant control of T-helper type 17 cells in autoimmune hepatitis. Hepatology 2014;59:1007-15

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Additional support that functional abnormalities of Tregs are present in autoimmune hepatitis.

160.

Longhi MS, Meda F, Wang P, et al. Expansion and de novo generation of potentially therapeutic regulatory T cells in patients with autoimmune hepatitis. Hepatology 2008;47:581-91

Longhi MS, Ma Y, Mieli-Vergani G, Vergani D. Regulatory T cells in autoimmune hepatitis. J Hepatol 2012;57: 932-3

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Criticism of study demonstrating normal Treg numbers and function in autoimmune hepatitis based on differences in methodologies for assessing function and sampling conditions.

161.

Peiseler M, Sebode M, Schramm C, Herkel J. Reply to: “Regulatory T cells in autoimmune hepatitis”. J Hepatol 2012;57: 933-4

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Defense of normal findings of Treg numbers and function in autoimmune hepatitis by emphasizing the rigidity of the definition for Tregs (CD4+CD25+(high)C127(low)Foxp3+) and the validity of the methodology applied in the discrepant study.

162.

Longhi MS, Ma Y, Bogdanos DP, et al. Impairment of CD4(+)CD25(+) regulatory T-cells in autoimmune liver disease. J Hepatol 2004;41:31-7

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Key paper justifying subsequent efforts to improve the immunosuppressive actions of Tregs in autoimmune hepatitis by demonstrating deficient numbers and function in this population.

155.

..

Demonstration that autologous Tregs from patients with autoimmune hepatitis can be expanded ex vivo as natural Tregs and conventional T lymphocytes can be transformed into Tregs for possible use in adoptive transfer.

148.

Martin E, Capini C, Duggan E, et al. Antigen-specific suppression of established arthritis in mice by dendritic cells deficient in NF-kappaB. Arthritis Rheum 2007;56: 2255-66

Chen W, Jin W, Hardegen N, et al. Conversion of peripheral CD4+CD25naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 2003;198:1875-86

157.

149.

O’Sullivan BJ, MacDonald KP, Pettit AR, Thomas R. RelB nuclear translocation regulates B cell MHC molecule, CD40 expression, and antigen-presenting cell function. Proc Natl Acad Sci USA 2000;97:11421-6

Longhi MS, Hussain MJ, Kwok WW, et al. Autoantigen-specific regulatory T cells, a potential tool for immune-tolerance reconstitution in type-2 autoimmune hepatitis. Hepatology 2011;53:536-47


158.

Rodriguez-Manzanet R, DeKruyff R, Kuchroo VK, Umetsu DT. The costimulatory role of TIM molecules. Immunol Rev 2009;229:259-70

156.

Li M, Zhang X, Zheng X, et al. Immune modulation and tolerance induction by RelB-silenced dendritic cells through RNA interference. J Immunol 2007;178: 5480-7

inhibiting the release of inflammatory cytokines and the preferred subset for adoptive cell therapy in type 2 autoimmune hepatitis.

152.

Excellent perspective of the evolving role of adoptive cell therapy using Tregs in the treatment of immune-mediated diseases.

150.

Review

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Demonstration that the co-culturing of Tregs with semi-mature dendritic cells loaded with cytochrome P450 2D6 under conditions of appropriate HLA-DR restriction generated antigen-specific Tregs that were highly effective in suppressing autoreactive T lymphocytes,

doi: 10.1586/17474124.2015.1019470

Review 163.


164.

.

165.

Czaja

Longhi MS, Mitry RR, Samyn M, et al. Vigorous activation of monocytes in juvenile autoimmune liver disease escapes the control of regulatory T-cells. Hepatology 2009;50: 130-42 Longhi MS, Liberal R, Holder B, et al. Inhibition of interleukin-17 promotes differentiation of CD25(-) cells into stable T regulatory cells in patients with autoimmune hepatitis. Gastroenterology 2012;142:1526-35 Demonstration of phenotypic instability in Tregs and need to purify Treg population of IL-17-producing proinflammatory T lymphocytes. Ferri S, Longhi MS, De Molo C, et al. A multifaceted imbalance of T cells with regulatory function characterizes

doi: 10.1586/17474124.2015.1019470

DRB1*0301 and DRB1*0401 as the principal susceptibility alleles and suggesting that two polymorphisms outside the MHC (SH2B3 and Card10) are contributory to a genetic predisposition that may overlap with other immune-mediated liver diseases such as primary sclerosing cholangitis and primary biliary cirrhosis.

type 1 autoimmune hepatitis. Hepatology 2010;52:999-1007 .

Demonstration of reduced numbers of CD4+CD25+ T lymphocytes during active autoimmune hepatitis with reduced levels of Foxp3 expression and decreased ability to inhibit target cell proliferation compared with normal subjects.

166.

Czaja AJ, Manns MP. Advances in the diagnosis, pathogenesis and management of autoimmune hepatitis. Gastroenterology 2010;139:58-72

167.

de Boer YS, van Gerven NM, Zwiers A, et al. Genome-wide association study identifies variants associated with autoimmune hepatitis type 1. Gastroenterology 2014;147:443-52

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First genome-wide association study in type 1 autoimmune hepatitis confirming

168.

Thornton AM, Korty PE, Tran DQ, et al. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J Immunol 2010;184:3433-41


Antibodies to neurofascin exacerbate adoptive transfer experimental autoimmune neuritis.

Adoptive transfer of autoimmune diabetes and thyroiditis to athymic rats.

Antigen-specific inhibition of the adoptive transfer of experimental autoimmune encephalomyelitis in Lewis rats.

Exogenous administration of IL-1 alpha inhibits active and adoptive transfer autoimmune diabetes in NOD mice.

Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3(+) regulatory T cells.

Trial Watch: Adoptive cell transfer for oncological indications.

Adoptive transfer of murine autoimmune orchitis with sperm-specific T lymphoblasts.

Adoptive Transfer of Lung Antigen Presenting Cells.

Adoptive Transfer of Isolated Bone Marrow Neutrophils.

Selective bispecific T cell recruiting antibody and antitumor activity of adoptive T cell transfer.

Autoimmune hepatitis.

Autoimmune Hepatitis in Hawai'i.

Adoptive Transfer of Memory B Cells.

[Autoimmune hepatitis].

T-cell-directed hepatocyte damage in autoimmune chronic active hepatitis.

Adoptive transfer of autoimmune splenic dendritic cells to lupus-prone mice triggers a B lymphocyte humoral response.

Autoimmune hepatitis and immunoglobulin G4-associated autoimmune hepatitis.

Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones.

Adoptive transfer of immune cells from glaucomatous mice provokes retinal ganglion cell loss in recipients.

A new hope in immunotherapy for malignant gliomas: adoptive T cell transfer therapy.

Is there a future for adoptive cell transfer in melanoma patients?

Autoimmune hepatitis: trust in transaminases.

Transfer of adoptive immunity to tuberculosis in mice.

Adoptive T-Cell Immunotherapy.