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1.  Ex Vivo Expanded Human Regulatory T Cells Can Prolong Survival of a Human Islet Allograft in a Humanized Mouse Model 
Transplantation  2013;96(8):707-716.
Background
Human regulatory T cells (Treg) offer an attractive adjunctive therapy to reduce current reliance on lifelong, nonspecific immunosuppression after transplantation. Here, we evaluated the ability of ex vivo expanded human Treg to prevent the rejection of islets of Langerhans in a humanized mouse model and examined the mechanisms involved.
Methods
We engrafted human pancreatic islets of Langerhans into the renal subcapsular space of immunodeficient BALB/c.rag2−/−.cγ−/− mice, previously rendered diabetic via injection of the β-cell toxin streptozocin. After the establishment of stable euglycemia, mice were reconstituted with allogeneic human peripheral blood mononuclear cells (PBMC) and the resultant alloreactive response studied. Ex vivo expanded CD25highCD4+ human Treg, which expressed FoxP3, CTLA-4, and CD62L and remained CD127low, were then cotransferred together with human PBMC and islet allografts and monitored for evidence of rejection.
Results
Human islets transplanted into diabetic immunodeficient mice reversed diabetes but were rejected rapidly after the mice were reconstituted with allogeneic human PBMC. Cotransfer of purified, ex vivo expanded human Treg prolonged islet allograft survival resulting in the accumulation of Treg in the peripheral lymphoid tissue and suppression of proliferation and interferon-γ production by T cells. In vitro, Treg suppressed activation of signal transducers and activators of transcription and inhibited the effector differentiation of responder T cells.
Conclusions
Ex vivo expanded Treg retain regulatory activity in vivo, can protect a human islet allograft from rejection by suppressing signal transducers and activators of transcription activation and inhibiting T-cell differentiation, and have clinical potential as an adjunctive cellular therapy.
Supplemental digital content is available in the article.
doi:10.1097/TP.0b013e31829fa271
PMCID: PMC3864182  PMID: 23917725
Regulatory T cells; Islet transplantation; Allograft; Humanized mouse model; Cellular therapy
2.  Regulatory T Cells and Human Myeloid Dendritic Cells Promote Tolerance via Programmed Death Ligand-1 
PLoS Biology  2010;8(2):e1000302.
Human regulatory T cells inhibit graft-versus-host disease that can occur after tissue transplantation, in part through expression of programmed death ligand 1 and modulation of antigen-presenting cells.
Immunotherapy using regulatory T cells (Treg) has been proposed, yet cellular and molecular mechanisms of human Tregs remain incompletely characterized. Here, we demonstrate that human Tregs promote the generation of myeloid dendritic cells (DC) with reduced capacity to stimulate effector T cell responses. In a model of xenogeneic graft-versus-host disease (GVHD), allogeneic human DC conditioned with Tregs suppressed human T cell activation and completely abrogated posttransplant lethality. Tregs induced programmed death ligand-1 (PD-L1) expression on Treg-conditioned DC; subsequently, Treg-conditioned DC induced PD-L1 expression in vivo on effector T cells. PD-L1 blockade reversed Treg-conditioned DC function in vitro and in vivo, thereby demonstrating that human Tregs can promote immune suppression via DC modulation through PD-L1 up-regulation. This identification of a human Treg downstream cellular effector (DC) and molecular mechanism (PD-L1) will facilitate the rational design of clinical trials to modulate alloreactivity.
Author Summary
Graft-versus-host disease (GVHD) is the most serious complication of bone marrow transplants between individuals (so-called allogenic transplants). The class of suppressor immune cells called regulatory T cells (Tregs) inhibit GVHD by dampening the effects of donor immune cells in the grafted tissue. The cellular and molecular mechanisms involved in this process have not been fully characterized, particularly for human cells. In this study, we report that human Tregs, which we generated from precursor cells ex vivo, express high levels of a cell surface protein called PD-L1 (programmed death ligand-1) that is known to mediate immune suppression. Coculture of these Tregs with allogeneic antigen-presenting cells (APCs), which are known to initiate GVHD, increased, in turn, the amount of PD-L1 on the APCs. The Treg-conditioned APCs were then less able than unconditioned APCs to provoke GVHD in a mouse model of the condition, preventing the death of the animals after transplantation. We found that an antibody against PD-L1 blocked the immunosuppressive effects of Tregs or Treg-conditioned APCs, indicating that this protein is an important part of the molecular mechanism. These findings are potentially important for attempts to modulate immune responses in disease by transplanting T cells into patients.
doi:10.1371/journal.pbio.1000302
PMCID: PMC2814822  PMID: 20126379
3.  Mechanisms of Cyclic Nucleotide Phosphodiesterases in Modulating T Cell Responses in Murine Graft-versus-Host Disease 
PLoS ONE  2013;8(3):e58110.
Graft-versus-host disease (GvHD) is a key contributor to the morbidity and mortality after allogeneic hematopoetic stem cell transplantation (HSCT). Regulatory Foxp3+ CD4+ T cells (Treg) suppress conventional T cell activation and can control GvHD. In our previous work, we demonstrate that a basic mechanism of Treg mediated suppression occurs by the transfer of cyclic adenosine monophosphate (cAMP) to responder cells. Whether this mechanism is relevant for Treg mediated suppression of GvHD is currently unknown. To address this question, bone marrow and T cells from C57BL/6 mice were transferred into lethally irradiated BALB/c recipients, and the course of GvHD and survival were monitored. Transplanted recipients developed severe GvHD that was strongly ameliorated by the transfer of donor Treg cells. Towards the underlying mechanisms, in vitro studies revealed that Treg communicated with DCs via gap junctions, resulting in functional inactivation of DC by a metabolic pathway involving cAMP that is modulated by the phosphodiesterase (PDE) 4 inhibitor rolipram. PDE2 or PDE3 inhibitors as well as rolipram suppressed allogeneic T cell activation, indirectly by enhancing Treg mediated suppression of DC activation and directly by inhibiting responder T cell proliferation. In line with this, we observed a cooperative suppression of GvHD upon Treg transfer and additional rolipram treatment. In conclusion, we propose that an important pathway of Treg mediated control of GvHD is based on a cAMP dependent mechanism. These data provide the basis for future concepts to manipulate allogeneic T cell responses to prevent GvHD.
doi:10.1371/journal.pone.0058110
PMCID: PMC3590136  PMID: 23483980
4.  Rapamycin-Conditioned Donor Dendritic Cells Differentiate CD4+CD25+Foxp3+ T Cells In Vitro with TGF-β 1 for Islet Transplantation 
Dendritic cells (DCs) conditioned with the mammalian target of rapamycin (mTOR) inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). This work addresses whether rapamycin-conditioned donor DCs could effectively induce CD4+CD25+Foxp3+ Tregs (iTregs) in cell cultures with alloantigen specificities, and whether such in vitro-differentiated CD4+CD25+Foxp3+ iTregs could effectively control acute rejection in allogeneic islet transplantation. We found that donor BALB/c bone marrow-derived DCs (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin had significantly enhanced ability to induce CD4+CD25+Foxp3+ iTregs of recipient origin (C57BL/6 (B6)) in vitro under Treg driving conditions compared to unmodified BMDCs. These in vitro-induced CD4+CD25+Foxp3+ iTregs exerted donor-specific suppression in vitro, and prolonged allogeneic islet graft survival in vivo in RAG−/− hosts upon coadoptive transfer with T-effector cells. The CD4+CD25+Foxp3+ iTregs expanded and preferentially maintained Foxp3 expression in the graft draining lymph nodes. Finally, the CD4+CD25+Foxp3+ iTregs were further able to induce endogenous naïve T cells to convert to CD4+CD25+Foxp3+ T cells. We conclude that rapamycin-conditioned donor BMDCs can be exploited for efficient in vitro differentiation of donor antigen-specific CD4+CD25+Foxp3+ iTregs. Such in vitro-generated donor-specific CD4+CD25+Foxp3+ iTregs are able to effectively control allogeneic islet graft rejection.
doi:10.1111/j.1600-6143.2010.03199.x
PMCID: PMC3995630  PMID: 20626386
Dendritic cells; graft rejection; induced regulatory T cells; islet transplantation; rapamycin; transforming growth factor-β 1
5.  Combined Administration of a Novel Mutant TGF-β1/Fc and Rapamycin Promotes Induction of Regulatory T Cells and Islet Allograft Tolerance1 
The critical roles of TGF-β in the reciprocal differentiation of tolerance-promoting CD4+Foxp3+ regulatory T cells (Treg) and pro-inflammatory Th17 effector cells impact alloimmune reactivity and transplant outcome. We reasoned that a strategy that harnessed TGF-β and blocked pro-inflammatory cytokines would inhibit the differentiation of Th17 cells and strengthen the cadre of Treg to promote tolerance induction and long-term allograft survival. Herein we report the development of a novel, long-lasting auto-active human mutant TGF-β1/Fc fusion protein that acts in conjunction with rapamycin to inhibit T cell proliferation and induce the de novo generation of Foxp3+ Treg in the periphery, while at the same time inhibiting IL-6-mediated Th17 cell differentiation. Short-term combined treatment with TGF-β1/Fc and rapamycin achieved long-term pancreatic islet allograft survival and donor-specific tolerance in a mouse model. This effect was accompanied by expansion of Foxp3+ Treg, enhanced alloantigen-specific Treg function, and modulation of transcript levels of Foxp3, IL-6 and IL-17. Our strategy of combined TGF-β1/Fc and rapamycin to target the IL-6-related Treg and Th17 signaling pathways provides a promising approach for inducing transplant tolerance and its clinical application.
doi:10.4049/jimmunol.1000769
PMCID: PMC3766970  PMID: 20844194
6.  Hepatic Stellate Cells Preferentially Expand Allogeneic CD4+CD25+FoxP3+ Regulatory T Cells in an IL-2 Dependent Manner 
Transplantation  2008;86(11):1492-1502.
Background
Organ transplantation has been successfully practice for decades, but the outcome of cell transplantation remains disappointing. This is the case in animal models; liver allografts in mice are spontaneous accepted without requirement of immunosuppression, whereas hepatocyte transplants in the same combination are acutely rejected, apparently resulting from immune attacks because syngeneic hepatocyte transplants survive indefinitely. This suggests that liver non-parenchymal cells play an important role in protecting parenchymal cell from rejection. We have shown that hepatic stellate cells (HpSC), well known to participate in liver repairing and fibrosis, mediate potent immunomodulatory functions via induction of activated T cell death.
Methods and Results
Here we report that HpSC acquired antigen presenting capacity following activated by IFN-γ. In contrast to professional APC dendritic cells (DC) that predominantly stimulated CD4+ T cells to generate CD25+Foxp3− effector cells, HpSC selectively expanded CD4+CD25+Foxp3+ cells in an IL-2 dependent manner. These expanded CD4+CD25+Foxp3+ cells showed regulatory T (Treg) cell activity in effectively inhibiting T cell proliferation in responses to anti-CD3 mAb or alloantigens in a MHC non-specific fashion. The Treg cells were expanded from the CD4+CD25+ population with the help of IL-2, independent of B7-H1 and TGF-β. Administration of HpSC into allogeneic recipients resulted in expansion of CD4+CD25+FoxP3+ cells in vivo.
Conclusion
Liver stromal HpSC acted as non-professional APC, and preferentially expanded CD25+FoxP3+ Treg cells, which may contribute to immune regulation in the liver.
doi:10.1097/TP.0b013e31818bfd13
PMCID: PMC2888269  PMID: 19077880
Hepatic stellate cell; Regulatory T cell; Dendritic cell; Antigen Presentation; IL-2; B7-H1; TGF-β; Immune regulation; T cell response
7.  Foxp3+ Treg Expanded from Patients with Established Diabetes Reduce Helios Expression while Retaining Normal Function Compared to Healthy Individuals 
PLoS ONE  2013;8(2):e56209.
Foxp3+ regulatory T cells (Treg) play a crucial role in regulating immune tolerance. The use of Treg to restore immune tolerance is considered an attractive novel approach to inhibit autoimmune disease, including type 1 diabetes (T1D), and to prevent rejection of organ transplants. In view of the goal of developing autologous Treg-based cell therapy for patients with long-term (>15 years) T1D, it will be necessary to expand a sufficient amount of functional Treg in vitro in order to study and compare Treg from T1D patients and healthy subjects. Our results have demonstrated that there is a comparable frequency of Treg in the peripheral blood lymphocytes (PBLs) of patients with long-term T1D relative to those in healthy subjects; however, Th1 cells, but not Th17 cells, were increased in the T1D patients. Further, more Treg in PBLs from T1D patients than from healthy subjects expressed the CD45RO+ memory cell phenotype, suggesting they were antigen-experienced cells. After isolation, Treg from both T1D patients and healthy subjects were successfully expanded with high purity. Although there was no difference in Helios expression on Treg in PBLs, in vitro expansion led to fewer Helios-expressing Treg from T1D patients than healthy subjects. While more Th1-like Treg expressing IFN-γ or TNF-α were found in the PBLs of T1D patients than healthy controls, there was no such difference in the expanded Treg. Importantly, expanded Treg from both subject groups were able to suppress autologous or allogeneic CD8+ effector T cells equally well. Our findings demonstrate that a large number of ex vivo expanded functional Treg can be obtained from long-term T1D patients, although fewer expanded Treg expressed a high level of Helios. Thus, based on the positive outcomes, these potent expanded Treg from diabetic human patients may be useful in treating T1D or preventing islet graft rejection.
doi:10.1371/journal.pone.0056209
PMCID: PMC3569420  PMID: 23409157
8.  Resistance of Foxp3+ Regulatory T Cells to Nur77-Induced Apoptosis Promotes Allograft Survival 
PLoS ONE  2008;3(5):e2321.
The NR4A nuclear receptor family member Nur77 (NR4A1) promotes thymocyte apoptosis during negative selection of autoreactive thymocytes, but may also function in mature extrathymic T cells. We studied the effects of over-expression of Nur77 on the apoptosis of murine peripheral T cells, including thymic-derived Foxp3+ regulatory (Treg) cells. Overexpression of Nur77 in the T cell lineage decreased numbers of peripheral CD4 and CD8 T cells by ∼80% compared to wild-type (WT) mice. However, the proportions of Treg cells were markedly increased in the thymus (61% of CD4+Foxp3+ singly positive thymocytes vs. 8% in WT) and secondary lymphoid organs (40–50% of CD4+Foxp3+ T cells vs. 7–8% in WT) of Nur77 transgenic (Nur77Tg) mice, and immunoprecipitation studies showed Nur77 was associated with a recently identified HDAC7/Foxp3 transcriptional complex. Upon activation through the T cell receptor in vitro or in vivo, Nur77Tg T cells showed only marginally decreased proliferation but significantly increased apoptosis. Fully allogeneic cardiac grafts transplanted to Nur77Tg mice survived long-term with well-preserved structure, and recipient splenocytes showed markedly enhanced apoptosis and greatly reduced anti-donor recall responses. Allografts in Nur77Tg recipients had significantly increased expression of multiple Treg-associated genes, including Foxp3, Foxp1, Tip60 and HDAC9. Allograft rejection was restored by CD25 monoclonal antibody therapy, indicating that allograft acceptance was dependent upon Treg function in Nur77Tg recipients. These data show that compared to conventional CD4 and CD8 T cells, Foxp3+ Tregs are relatively resistant to Nur77-mediated apoptosis, and that tipping the balance between the numbers of Tregs and responder T cells in the early period post-transplantation can determine the fate of the allograft. Hence, induced expression of Nur77 might be a novel means to achieve long-term allograft survival.
doi:10.1371/journal.pone.0002321
PMCID: PMC2386419  PMID: 18509529
9.  Low-Dose Rapamycin Treatment Increases the Ability of Human Regulatory T Cells to Inhibit Transplant Arteriosclerosis In Vivo 
American Journal of Transplantation  2012;12(8):2008-2016.
Regulatory T cells (Treg) are currently being tested in clinical trials as a potential therapy in cell and solid organ transplantation. The immunosuppressive drug rapamycin has been shown to preferentially promote Treg expansion. Here, we hypothesized that adjunctive rapamycin therapy might potentiate the ability of ex vivo expanded human Treg to inhibit vascular allograft rejection in a humanized mouse model of arterial transplantation. We studied the influence of combined treatment with low-dose rapamycin and subtherapeutic Treg numbers on the development of transplant arteriosclerosis (TA) in human arterial grafts transplanted into immunodeficient BALB/cRag2−/−Il2rg−/− mice reconstituted with allogeneic human peripheral blood mononuclear cell. In addition, we assessed the effects of the treatment on the proliferation and apoptosis of naïve/effector T cells. The combined therapy efficiently suppressed T-cell proliferation in vivo and in vitro. Neointima formation in the human arterial allografts was potently inhibited compared with each treatment alone. Interestingly, CD4+ but not CD8+ T lymphocytes were sensitive to Treg and rapamycin-induced apoptosis in vitro. Our data support the concept that rapamycin can be used as an adjunctive therapy to improve efficacy of Treg-based immunosuppressive protocols in clinical practice. By inhibiting TA, Treg and rapamycin may prevent chronic transplant dysfunction and improve long-term allograft survival
doi:10.1111/j.1600-6143.2012.04065.x
PMCID: PMC3440570  PMID: 22500984
Cell therapy; rejection; humanized mouse model; tolerance; Treg
10.  Rhesus Monkey Immature Monocyte-Derived Dendritic Cells Generate Alloantigen-Specific Regulatory T Cells From Circulating CD4+CD127−/lo T cells 
Transplantation  2009;88(9):1057-1064.
Background
Generation of non-human primate regulatory T cells (Treg) with alloantigen (alloAg) specificity would allow their testing in pre-clinical transplant models. Low recovery of Treg from peripheral blood limits their potential utility. In small animals and humans, conventional myeloid dendritic cells (DC) have been shown to select or induce alloAg-specific Treg.
Methods
We combined enrichment of rhesus macaque blood CD4+ Treg based on IL-7Rα (CD127) expression with their stimulation in mixed leukocyte cultures with immature, allogeneic, control or vitamin (Vit) D3/IL-10-conditioned monocyte-derived DC. Following co-culture in IL-2 and IL-15 for up to 14 days, the ability of the resulting T cells to suppress alloreactive effector T cell proliferation was assessed.
Results
CD4+CD127−/lo T cells represented approximately 7% of normal rhesus circulating CD4+ T cells, and were enriched for forkhead box P3 (Foxp3)+ cells. When stimulated with control allogeneic DC, they exhibited much inferior proliferative responses compared with bulk CD4+ or CD4+CD127+ cells. This anergic state was reversed by exogenous IL-2 and IL-15. Following 10–14 days culture of CD4+CD127−/lo T cells with immature allogeneic DC, particularly maturation-resistant VitD3/IL-10 DC, the frequency of Foxp3+ T cells was increased. The cultured cells markedly inhibited CD4+ effector T cell proliferation in a dose-related and donor alloAg-specific manner.
Conclusions
Stimulation of rhesus CD4+CD127−/lo T cells with immature and especially maturation-resistant allogeneic DC, generated highly-suppressive, alloAg-specific Treg. Without resorting to a more highly-purified starting population, this approach may have therapeutic utility in clinically-relevant transplant models.
doi:10.1097/TP.0b013e3181ba6b1f
PMCID: PMC2775499  PMID: 19898200
rhesus monkey; dendritic cells; vitamin D3; interleukin-10; regulatory T cells
11.  Generation of Adaptive Regulatory T Cells by Alloantigen Is Required for Some But Not All Transplant Tolerance Protocols 
Transplantation  2011;91(7):707-713.
Background
Because CD4+CD25+Foxp3+ regulatory T cells (Tregs) are essential for the maintenance of self-tolerance, significant interest surrounds the developmental cues for thymic-derived natural Tregs (nTregs) and periphery-generated adaptive Tregs (aTregs). In the transplant setting, the allograft may play a role in the generation of alloantigen-specific Tregs, but this role remains undefined. We examined whether the immune response to a transplant allograft results in the peripheral generation of aTregs.
Methods
To identify generation of aTregs, purified graft-reactive CD4+CD25− T cells were adoptively transferred to mice-bearing skin allograft. To demonstrate that aTregs are necessary for tolerance, DBA/2 skin was transplanted onto C57BL/6-RAG-1-deficient recipients adoptively transferred with purified sorted CD4+CD25− T cells; half of the recipients undergo tolerance induction treatment.
Results
By tracking adoptively transferred cells, we show that purified graft-reactive CD4+CD25− T lymphocytes up-regulate Foxp3 in mice receiving skin allografts in the absence of any treatment. Interestingly, cotransfer of antigen-specific nTregs suppresses the up-regulation of Foxp3 by inhibiting the proliferation of allograft-responsive T cells. In vitro data are consistent with our in vivo data—Foxp3+ cells are generated on antigen activation, and this generation is suppressed on coculture with antigen-specific nTregs. Finally, blocking aTreg generation in grafted, rapamycin-treated mice disrupts alloantigen-specific tolerance induction. In contrast, blocking aTreg generation in grafted mice treated with nondepleting anti-CD4 plus anti-CD40L antibodies does not disrupt graft tolerance.
Conclusions
We conclude that graft alloantigen stimulates the de novo generation of aTregs, and this generation may represent a necessary step in some but not all protocols of tolerance induction.
doi:10.1097/TP.0b013e31820e50b3
PMCID: PMC3727173  PMID: 21386770
Treg; Adaptive Tregs; Tolerance
12.  Induction of transplantation tolerance converts potential effector T cells into graft-protective regulatory T cells 
European Journal of Immunology  2010;41(3):726-738.
Naturally occurring FOXP3+CD4+ Treg have a crucial role in self-tolerance. The ability to generate similar populations against alloantigens offers the possibility of preventing transplant rejection without indefinite global immunosuppression. Exposure of mice to donor alloantigens combined with anti-CD4 antibody induces operational tolerance to cardiac allografts, and generates Treg that prevent skin and islet allograft rejection in adoptive transfer models. If protocols that generate Treg in vivo are to be developed in the clinical setting it will be important to know the origin of the Treg population and the mechanisms responsible for their generation. In this study, we demonstrate that graft-protective Treg arise in vivo both from naturally occurring FOXP3+CD4+ Treg and from non-regulatory FOXP3−CD4+ cells. Importantly, tolerance induction also inhibits CD4+ effector cell priming and T cells from tolerant mice have impaired effector function in vitro. Thus, adaptive tolerance induction shapes the immune response to alloantigen by converting potential effector cells into graft-protective Treg and by expanding alloreactive naturally occurring Treg. In relation to clinical tolerance induction, the data indicate that while the generation of alloreactive Treg may be critical for long-term allograft survival without chronic immunosuppression, successful protocols will also require strategies that target potential effector cells.
doi:10.1002/eji.201040509
PMCID: PMC3175037  PMID: 21243638
Transplantation tolerance; Treg
13.  Requirements for Prolongation of Allograft Survival with Regulatory T Cell Infusion in Lymphosufficient Hosts 
The Journal of surgical research  2011;169(1):e69-e75.
Background
For the clinical applicability of regulatory T cells (Tregs) in transplantation, it is critical to determine if donor antigen specificity is required for their immunosuppressive function. We developed an allospecific CD4+ T cell receptor transgenic (TCR-tg) mouse as a source for large numbers of Tregs with defined allospecificity and tested whether they are more effective than polyclonal Tregs at suppressing allograft rejection.
Materials and Methods
CD4+CD25+CD62Lhi T cells were sorted from the spleen and peripheral lymph nodes of wild-type (WT-Tregs) and TCR-tg (Allo-Tregs) mice, and expanded using IL-2 and anti-CD3/anti-CD28 conjugated magnetic beads. Tregs were tested for their ability to suppress the proliferation and cytokine production of alloreactive CD4+CD25- T cells in mixed leukocyte assays. Syngeneic WT hosts were adoptively transferred 5×106 Tregs and transplanted with allogeneic hearts.
Results
Using anti-CD3/anti-CD28 conjugated beads, Tregs were expanded in vitro 100-fold and maintained their suppressor phenotype and function. Allo-Tregs were 6-8 times more potent on a cell-for-cell basis than WT-Tregs in suppressing allospecific proliferation in vitro. Allo-Tregs were unable to suppress in the absence of allo-antigen. Adoptive transfer of expanded Allo-Tregs into WT recipients prolonged the graft survival in a F1 heart transplant model compared to WT-Treg or no treatment [20.0±4.4 d (n=6) vs. 10.4±1.2 (n=8) and 9.7±1.6 d (n=6)].
Conclusions
Unlike polyclonal Tregs, allospecific Tregs are able to prolong allograft survival. However, large numbers of Allo-Tregs were unable to induce tolerance, suggesting that Treg therapy in immunocompetent recipients will require conditioning and/or additional immunomodulation for the induction of tolerance.
doi:10.1016/j.jss.2011.03.021
PMCID: PMC3114634  PMID: 21571317
immunology; transplantation; alloimmunity; rejection; transgenic T cells; regulatory T cells
14.  Levels of Foxp3 in regulatory T cells reflect their functional status in transplantation1 
Foxp3 expressing CD4+CD25+ regulatory T cells (Tregs) have been shown to prevent allograft rejection in clinical and animal models of transplantation. However, the role of Foxp3 in regulating Treg function, and the kinetics and mechanism of action of Tregs in inducing allograft tolerance in transplantation, are still not fully understood. Thus, we investigated the kinetics and function of Tregs in a mouse model of orthotopic corneal transplantation, the most common form of tissue grafting worldwide. Here using in-vitro functional assays and in-vivo Treg adoptive transfer assays, we show that far more relevant than Treg frequency is their level of Foxp3 expression, which is directly associated with the potential of Tregs to prevent allograft rejection by producing regulatory cytokines and suppressing effector T cell activation. In addition, our data clearly demonstrate that Tregs primarily suppress the induction of alloimmunity in regional draining lymph nodes, rather than suppressing the effector phase of the immune response in the periphery. These findings provide new insights on Treg dynamics in transplantation which are crucial for designing therapeutic strategies to modulate Treg function, and to optimize Treg-based cell therapies for clinical translation.
PMCID: PMC2610348  PMID: 19109145
Regulatory T cells; Foxp3; Tolerance; Transplantation; Cornea
15.  Ex vivo-expanded human regulatory T cells prevent the rejection of skin allografts in a humanised mouse model1 
Transplantation  2010;90(12):1321-1327.
Background
Composite tissue transplantation effectively reconstructs the most complex defects, but its use is limited due to harmful immunosuppression and the high susceptibility of skin to rejection. Development of tolerance is an ideal solution, and protocols using regulatory T cells (Tregs) to achieve this have been promising in experimental animal models. The aim of this study was to investigate the ability of human Tregs to regulate immune responses to a human skin allograft in vivo.
Methods
We isolated and expanded naturally-occurring CD127loCD4+CD25+ human Tregs from peripheral blood mononuclear cells (PBMCs) ex vivo and examined their phenotype and suppressive activity in vitro. Using a clinically relevant chimeric humanised mouse system, we transplanted mice with human skin grafts followed by allogeneic populations of PBMCs with or without Tregs derived from the same PBMC donor.
Results
Ex vivo-expanded Tregs maintain the appropriate Treg markers and retain suppressive activity against allostimulated and polyclonally stimulated autologous PBMCs in vitro. Mice receiving allogeneic PBMCs alone consistently reject human skin grafts, whereas those also receiving Tregs display stable long-term human skin transplant survival along with a reduction in the CD8+ human cellular graft infiltrate.
Conclusions
We show for the first time the unique ability of human Tregs to prevent the rejection of a skin allograft in vivo, highlighting the therapeutic potential of these cells clinically.
doi:10.1097/TP.0b013e3181ff8772
PMCID: PMC3672995  PMID: 21048528
Regulatory T cell (Treg); tolerance; humanised mouse; rejection; skin transplantation
16.  Induction of Foxp3-Expressing Regulatory T-Cells by Donor Blood Transfusion Is Required for Tolerance to Rat Liver Allografts 
PLoS ONE  2009;4(11):e7840.
Background
Donor-specific blood transfusion (DST) prior to solid organ transplantation has been shown to induce long-term allograft survival in the absence of immunosuppressive therapy. Although the mechanisms underlying DST-induced allograft tolerance are not well defined, there is evidence to suggest DST induces one or more populations of antigen-specific regulatory cells that suppress allograft rejection. However, neither the identity nor the regulatory properties of these tolerogenic lymphocytes have been reported. Therefore, the objective of this study was to define the kinetics, phenotype and suppressive function of the regulatory cells induced by DST alone or in combination with liver allograft transplantation (LTx).
Methodology/Principal Findings
Tolerance to Dark Agouti (DA; RT1a) rat liver allografts was induced by injection (iv) of 1 ml of heparinized DA blood to naïve Lewis (LEW; RT1l) rats once per week for 4 weeks prior to LTx. We found that preoperative DST alone generates CD4+ T-cells that when transferred into naïve LEW recipients are capable of suppressing DA liver allograft rejection and promoting long-term survival of the graft and recipient. However, these DST-generated T-cells did not express the regulatory T-cell (Treg) transcription factor Foxp3 nor did they suppress alloantigen (DA)-induced activation of LEW T-cells in vitro suggesting that these lymphocytes are not fully functional regulatory Tregs. We did observe that DST+LTx (but not DST alone) induced the time-dependent formation of CD4+Foxp3+ Tregs that potently suppressed alloantigen-induced activation of naïve LEW T-cells in vitro and liver allograft rejection in vivo. Finally, we present data demonstrating that virtually all of the Foxp3-expressing Tregs reside within the CD4+CD45RC− population whereas in which approximately 50% of these Tregs express CD25.
Conclusions/Significance
We conclude that preoperative DST, in the absence of liver allograft transplantation, induces the formation of CD4+ T-cells that are not themselves Tregs but give rise directly or indirectly to fully functional CD4+CD45RC−Foxp3+Tregs when transferred into MHC mismatched recipients prior to LTx. These Tregs possess potent suppressive activity and are capable of suppressing acute liver allograft rejection. Understanding the mechanisms by which preoperative DST induces the generation of tolerogenic Tregs in the presence of alloantigens may lead to the development of novel antigen-specific immunological therapies for the treatment of solid organ rejection.
doi:10.1371/journal.pone.0007840
PMCID: PMC2776304  PMID: 19956764
17.  Suppression of Allograft Rejection by Tim-1-Fc through Cross-Linking with a Novel Tim-1 Binding Partner on T Cells 
PLoS ONE  2011;6(7):e21697.
Engagement of T-cell immunoglobulin mucin (Tim)-1 on T cells with its ligand, Tim-4, on antigen presenting cells delivers positive costimulatory signals to T cells. However, the molecular mechanisms for Tim-1-mediated regulation of T-cell activation and differentiation are relatively poorly understood. Here we investigated the role of Tim-1 in T-cell responses and allograft rejection using recombinant human Tim-1 extracellular domain and IgG1-Fc fusion proteins (Tim-1-Fc). In vitro assays confirmed that Tim-1-Fc selectively binds to CD4+ effector T cells, but not dendritic cells or natural regulatory T cells (nTregs). Tim-1-Fc was able to inhibit the responses of purified CD4+ T cells that do not express Tim-4 to stimulation by anti-CD3/CD28 mAbs, and this inhibition was associated with reduced AKT and ERK1/2 phosphorylation, but it had no influence on nTregs. Moreover, Tim-1-Fc inhibited the proliferation of CD4+ T cells stimulated by allogeneic dendritic cells. Treatment of recipient mice with Tim-1-Fc significantly prolonged cardiac allograft survival in a fully MHC-mismatched strain combination, which was associated with impaired Th1 response and preserved Th2 and nTregs function. Importantly, the frequency of Foxp3+ cells in splenic CD4+ T cells was increased, thus shifting the balance toward regulators, even though Tim-1-Fc did not induce Foxp3 expression in CD4+CD25− T cells directly. These results indicate that Tim-1-Fc can inhibit T-cell responses through an unknown Tim-1 binding partner on T cells, and it is a promising immunosuppressive agent for preventing allograft rejection.
doi:10.1371/journal.pone.0021697
PMCID: PMC3130052  PMID: 21750723
18.  Plasmodium falciparum–Mediated Induction of Human CD25hiFoxp3hi CD4 T Cells Is Independent of Direct TCR Stimulation and Requires IL-2, IL-10 and TGFβ 
PLoS Pathogens  2009;5(8):e1000543.
CD4+CD25+Foxp3+ regulatory T cells (Tregs) regulate disease-associated immunity and excessive inflammatory responses, and numbers of CD4+CD25+Foxp3+ Tregs are increased during malaria infection. The mechanisms governing their generation, however, remain to be elucidated. In this study we investigated the role of commonly accepted factors for Foxp3 induction, TCR stimulation and cytokines such as IL-2, TGFβ and IL-10, in the generation of human CD4+CD25+Foxp3+ T cells by the malaria parasite Plasmodium falciparum. Using a co-culture system of malaria-infected red blood cells (iRBCs) and peripheral blood mononuclear cells from healthy individuals, we found that two populations of Foxp3hi and Foxp3int CD4+CD25hi T cells with a typical Treg phenotype (CTLA-4+, CD127low, CD39+, ICOS+, TNFRII+) were induced. Pro-inflammatory cytokine production was confined to the Foxp3int subset (IFNγ, IL-4 and IL-17) and inversely correlated with high relative levels of Foxp3hi cells, consistent with Foxp3hi CD4 T cell–mediated inhibition of parasite-induced effector cytokine T cell responses. Both Foxp3hi and Foxp3int cells were derived primarily from proliferating CD4+CD25− T cells with a further significant contribution from CD25+Foxp3+ natural Treg cells to the generation of the Foxp3hi subset. Generation of Foxp3hi, but not Foxp3int, cells specifically required TGFβ1 and IL-10. Add-back experiments showed that monocytes expressing increased levels of co-stimulatory molecules were sufficient for iRBC-mediated induction of Foxp3 in CD4 T cells. Foxp3 induction was driven by IL-2 from CD4 T cells stimulated in an MHC class II–dependent manner. However, transwell separation experiments showed that direct contact of monocytes with the cells that acquire Foxp3 expression was not required. This novel TCR-independent and therefore antigen-non specific mechanism for by-stander CD4+CD25hiFoxp3+ cell induction is likely to reflect a process also occurring in vivo as a consequence of immune activation during malaria infection, and potentially a range of other infectious diseases.
Author Summary
Infection with the malaria parasite Plasmodium falciparum affects 300–600 million people each year. Regulatory T cells (Tregs) expressing the transcription factor Foxp3, which drives genes involved in immunosuppression, are specialized immune cells that can inhibit both protective and harmful inflammatory responses during malaria. While Treg numbers are increased during malaria infection, little is known about how they are induced by the parasite. We addressed this question using an in vitro culture system to model the interaction of the malaria parasite with human immune cells. We found that the parasite induced soluble immune mediators, including the T cell growth-factor IL-2 and the regulatory proteins IL-10 and TGFβ, which drive the induction and expansion of Tregs. These Tregs expressed high levels of Foxp3 and suppressed the production of inflammation and protective immunity-driving mediators by concurrently induced effector T cells. Importantly, we demonstrate that induction of Tregs by the malaria parasite did not necessarily require direct contact with antigen-presenting cells. Our findings suggest that the parasite induces Tregs in an antigen non-specific manner, which may explain why malarial immunosuppression is not confined to malaria-specific immune responses, and provide new insights into the mechanisms governing Treg induction during malaria infection, and potentially other infectious diseases.
doi:10.1371/journal.ppat.1000543
PMCID: PMC2718810  PMID: 19680449
19.  Recipient Myeloid-derived Immunomodulatory Cells Induce PD-1 Ligand-Dependent Donor CD4+Foxp3+ Treg Proliferation and Donor-Recipient Immune Tolerance After Murine Non-myeloablative Bone Marrow Transplantation§ 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(11):10.4049/jimmunol.1302191.
We have previously shown that non-myeloablative total lymphoid irradiation/rabbit anti-thymocyte serum (TLI/ATS) conditioning facilitates potent donor-recipient immune tolerance following bone marrow transplantation (BMT) across major histocompatibility complex (MHC) barriers via recipient invariant natural killer T cell (iNKT cell)-derived IL-4-dependent expansion of donor Foxp3+ naturally occurring Treg (nTreg). Here we report a more specific mechanism. Wild-type (WT) BALB/c (H-2d) hosts were administered TLI/ATS and BMT from WT or STAT6−/− C57BL/6 (H-2b) donors. Donor nTreg following STAT6−/− BMT demonstrated no loss of proliferation in vivo, indicating that an IL-4 responsive population in the recipient rather than the donor drives donor nTreg proliferation. In GVHD target organs, three recipient CD11b+ cell subsets (Gr-1highCD11cneg; Gr-1intCD11cneg; and Gr-1lowCD11c+) were enriched early after TLI/ATS + BMT versus TBI/ATS + BMT. Gr-1lowCD11c+ cells induced potent H-2Kb+CD4+Foxp3+ nTreg proliferation in vitro in 72-hr MLR. Gr-1lowCD11c+ cells were significantly reduced in STAT6−/− and iNKT cell-deficient Jα18−/− BALB/c recipients after TLI/ATS + BMT. Depletion of CD11b+ cells resulted in severe acute GVHD, and adoptive transfer of WT Gr-1lowCD11c+ cells to Jα18−/− BALB/c recipients of TLI/ATS + BMT restored day 6 donor Foxp3+ nTreg proliferation and protection from CD8 effector T cell-mediated GVHD. Blockade of PD-L1 or PD-L2, but not CD40, TGF-β, Arginase 1, or iNOS inhibited nTreg proliferation in co-cultures of recipient-derived Gr-1lowCD11c+ cells with donor nTreg. Through iNKT-dependent Th2 polarization, myeloid-derived immunomodulatory DCs are expanded after non-myeloablative TLI/ATS conditioning and allogeneic BMT, induce PD-1 ligand dependent donor nTreg proliferation, and maintain potent graft-versus-host immune tolerance.
doi:10.4049/jimmunol.1302191
PMCID: PMC3863612  PMID: 24190658
Graft-versus-host disease; transplantation; tolerance; T cells; monocytes
20.  CCR5 is Required for Regulation of Alloreactive T cell Responses to Single Class II MHC-Mismatched Murine Cardiac Grafts 
The effector CD4 T cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2bm12 cardiac allografts is restricted by CD4+CD25+ regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2bm12 cardiac allografts. In contrast to the long-term survival of B6.H-2bm12 allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5-/- recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-γ and IL-4 were markedly increased in spleens of B6.CCR5-/- vs. wild-type recipients. Wild-type and B6.CCR5-/- mice had equivalent numbers of splenic FoxP3+ Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro. However, diminished numbers of FoxP3+ Tregs infiltrated B6.H-2bm12 allografts in B6.CCR5-/- recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4+CD25+ Tregs to CCR5-/- recipients restored long-term survival of B6.H-2bm12 cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T cell responses to single class II MHC-mismatched cardiac allografts.
doi:10.1111/j.1600-6143.2009.02786.x
PMCID: PMC2760830  PMID: 19656127
21.  α-1-Antitrypsin Gene Delivery Reduces Inflammation, Increases T-Regulatory Cell Population Size and Prevents Islet Allograft Rejection 
Molecular Medicine  2011;17(9-10):1000-1011.
Antiinflammatory clinical-grade, plasma-derived human α-1 antitrypsin (hAAT) protects islets from allorejection as well as from autoimmune destruction. hAAT also interferes with disease progression in experimental autoimmune encephalomyelitis (EAE) and in collagen-induced arthritis (CIA) mouse models. hAAT increases IL-1 receptor antagonist expression in human mononuclear cells and T-regulatory (Treg) cell population size in animal models. Clinical-grade hAAT contains plasma impurities, multiple hAAT isoforms and various states of inactive hAAT. We thus wished to establish islet-protective activities and effect on Treg cells of plasmid-derived circulating hAAT in whole animals. Islet function was assessed in mice that received allogeneic islet transplants after mice were given hydrodynamic tail-vein injection with pEF-hAAT, a previously described Epstein-Barr virus (EBV) plasmid construct containing the EBV nuclear antigen 1 (EBNA1) and the family of repeat EBNA1 binding site components (designated “EF”) alongside the hAAT gene. Sera collected from hAAT-expressing mice were added to lipopolysaccharide (LPS)-stimulated macrophages to assess macrophage responsiveness. Also, maturation of peritoneal cells from hAAT-expressing mice was evaluated. hAAT-expressing mice accepted islet allografts (n = 11), whereas phosphate-buffered saline–injected animals (n = 11), as well as mice treated with truncated-hAAT-plasmid (n = 6) and untreated animals (n = 20) rapidly rejected islet allografts. In hAAT-expressing animals, local Treg cells were abundant at graft sites, and the IL-1 receptor antagonist was elevated in grafts and circulation. Sera from hAAT-expressing mice, but not control mice, inhibited macrophage responses. Finally, peritoneal cells from hAAT-expressing mice exhibited a semimature phenotype. We conclude that plasmid-derived circulating hAAT protects islet allografts from acute rejection, and human plasma impurities are unrelated to islet protection. Future studies may use this in vivo approach to examine the structure–function characteristics of the protective activities of AAT by manipulation of the hAAT plasmid.
doi:10.2119/molmed.2011.00145
PMCID: PMC3188864  PMID: 21670848
22.  Allograft rejection is restrained by short-lived TIM-3+PD-1+Foxp3+ Tregs  
The Journal of Clinical Investigation  2012;122(7):2395-2404.
Tregs play a pivotal role in inducing and maintaining donor-specific transplant tolerance. The T cell immunoglobulin and mucin domain-3 protein (TIM-3) is expressed on many fully activated effector T cells. Along with program death 1 (PD-1), TIM-3 is used as a marker for exhausted effector T cells, and interaction with its ligand, galectin-9, leads to selective death of TIM-3+ cells. We report herein the presence of a galectin-9–sensitive CD4+FoxP3+TIM-3+ population of T cells, which arose from CD4+FoxP3+TIM-3– proliferating T cells in vitro and in vivo and were often PD-1+. These cells became very prominent among graft-infiltrating Tregs during allograft response. The frequency and number of TIM-3+ Tregs peaked at the time of graft rejection and declined thereafter. Moreover, these cells also arise in a tolerance-promoting donor-specific transfusion model, representing a pool of proliferating, donor-specific Tregs. Compared with TIM-3– Tregs, TIM-3+ Tregs, which are often PD-1+ as well, exhibited higher in vitro effector function and more robust expression of CD25, CD39, CD73, CTLA-4, IL-10, and TGF-β but not galectin-9. However, these TIM-3+ Tregs did not flourish when passively transferred to newly transplanted hosts. These data suggest that a heretofore unrecognized graft-infiltrating, short-lived subset of Tregs can restrain rejection.
doi:10.1172/JCI45138
PMCID: PMC3386804  PMID: 22684103
23.  Characterization of Protective Human CD4+CD25+ FOXP3+ Regulatory T Cells Generated with IL-2, TGF-β and Retinoic Acid 
PLoS ONE  2010;5(12):e15150.
Background
Protective CD4+CD25+ regulatory T cells bearing the Forkhead Foxp3 transcription factor can now be divided into three subsets: Endogenous thymus-derived cells, those induced in the periphery, and another subset induced ex-vivo with pharmacological amounts of IL-2 and TGF-β. Unfortunately, endogenous CD4+CD25+ regulatory T cells are unstable and can be converted to effector cells by pro-inflammatory cytokines. Although protective Foxp3+CD4+CD25+ cells resistant to proinflammatory cytokines have been generated in mice, in humans this result has been elusive. Our objective, therefore, was to induce human naïve CD4+ cells to become stable, functional CD25+ Foxp3+ regulatory cells that were also resistant to the inhibitory effects of proinflammatory cytokines.
Methodology/Principal Findings
The addition of the vitamin A metabolite, all-trans retinoic acid (atRA) to human naïve CD4+ cells suboptimally activated with IL-2 and TGF-β enhanced and stabilized FOXP3 expression, and accelerated their maturation to protective regulatory T cells. AtRA, by itself, accelerated conversion of naïve to mature cells but did not induce FOXP3 or suppressive activity. The combination of atRA and TGF-β enabled CD4+CD45RA+ cells to express a phenotype and trafficking receptors similar to natural Tregs. AtRA/TGF-β-induced CD4+ regs were anergic and low producers of IL-2. They had potent in vitro suppressive activity and protected immunodeficient mice from a human-anti-mouse GVHD as well as expanded endogenous Tregs. However, treatment of endogenous Tregs with IL-1β and IL-6 decreased FOXP3 expression and diminished their protective effects in vivo while atRA-induced iTregs were resistant to these inhibitory effects.
Conclusions/Significance
We have developed a methodology that induces human CD4+ cells to rapidly become stable, fully functional suppressor cells that are also resistant to proinflammatory cytokines. This methodology offers a practical novel strategy to treat human autoimmune diseases and prevent allograft rejection without the use of agents that kill cells or interfere with signaling pathways.
doi:10.1371/journal.pone.0015150
PMCID: PMC3003689  PMID: 21179414
24.  Clinical Grade Manufacturing of Human Alloantigen-Reactive Regulatory T Cells for Use in Transplantation 
Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen-reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short-term cultures using good manufacturing practice-compliant reagents. This process uses CD40L-activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100- to 1600-fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen-expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model.
doi:10.1111/ajt.12433
PMCID: PMC4161737  PMID: 24102808
Cellular therapy; clinical application; regulatory T cells; tolerance induction
25.  Evidence that FoxP3+ Treg may play a role in promoting long-term acceptance of CTA1 
Transplantation  2011;91(8):908-915.
Background
FoxP3+/CD4+/CD25+ regulatory T cells (Treg) play an important role in maintaining peripheral tolerance and are potent suppressors of T cell activation. In the present studies we evaluated the role Treg might play in peripheral tolerance to composite tissue allotransplants (CTA).
Methods
Mixed allogeneic chimeric rats were prepared by pre-conditioning recipients with anti-αβ-TCR monoclonal antibody (mAb) followed by total body irradiation. Animals received T cell-depleted ACI bone marrow cells followed by anti-lymphocyte serum and FK-506. A modified osteomyocutaneous hind-limb flap composed of bone and all limb tissue components was placed at 29 days in animals with chimerism ≥ 1% on day 28. Recipients with CTA surviving ≥ 6 months were evaluated for Treg. Skin from tolerant long-term allogeneic transplanted, syngeneic transplanted, rejected and naïve animals were immunostained with fluorochrome conjugated anti-FoxP3 and anti-CD4 mAb and visualized under a laser confocal microscope.
Results
Significant CD4+/FoxP3+ Treg infiltrates were observed in tolerant donor-allograft skin samples. No graft infiltrating FoxP3+ cells were observed in rejector, naïve, or syngeneic CTA transplanted skin. In parallel experiments, mixed leukocyte reactions assays were performed to investigate the suppressor function of Treg cells. Splenocytes from tolerant, rejected, and naïve rats were sorted by flow cytometry for CD4+/CD25+ T cells. Treg demonstrated similar suppressive levels between the three groups.
Conclusions
These data suggest that Treg may play an important role in maintenance of tolerance and promoting graft acceptance in long-term CTA acceptors and may explain the favorable outcomes observed in clinical CTA recipients.
doi:10.1097/TP.0b013e31820fafb4
PMCID: PMC3592205  PMID: 21304439
composite tissue allotransplant; T regulatory cells; tolerance; bone marrow transplantation

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