DR3 signaling modulates the function of Foxp3+ regulatory T cells and the severity of acute graft-versus-host disease.

CD4+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells, which regulate the immune system and enhance immune tolerance after transplantation. Donor-derived Treg prevent the development of lethal acute graft-versus-host disease (GVHD) in murine models of allogeneic hematopoietic stem cell transplantation. We recently demonstrated that a single treatment of the agonistic antibody to DR3 (death receptor 3, αDR3) to donor mice resulted in the expansion of donor-derived Treg and prevented acute GVHD, although the precise role of DR3 signaling in GVHD has not been elucidated. In this study, we comprehensively analyzed the immunophenotype of Treg after DR3 signal activation, demonstrating that DR3-activated Treg (DR3-Treg) had an activated/mature phenotype. Furthermore, the CD25+Foxp3+ subpopulation in DR3-Treg showed stronger suppressive effects in vivo. Prophylactic treatment of αDR3 to recipient mice expanded recipient-derived Treg and reduced the severity of GVHD, whereas DR3 activation in mice with ongoing GVHD further promoted donor T-cell activation/proliferation. These data suggest that the function of DR3 signaling was highly dependent on the activation status of the T cells. In conclusion, our data demonstrated that DR3 signaling affects the function of Treg and T-cell activation after alloantigen exposure in a time-dependent manner. These observations provide important information for future clinical testing using human DR3 signal modulation and highlight the critical effect of the state of T-cell activation on clinical outcomes after activation of DR3.

TNF-α priming enhances CD4+FoxP3+ regulatory T-cell suppressive function in murine GVHD prevention and treatment.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) have been shown to effectively prevent graft-versus-host disease (GVHD) when adoptively transferred in murine models of hematopoietic cell transplantation and in phase 1/2 clinical trials. Critical limitations to Treg clinical application are the paucity of cells and limited knowledge of the mechanisms of in vivo function. We hypothesized that inflammatory conditions in GVHD modify Treg characteristics and activity. We found that peripheral blood of recipient animals during acute GVHD (aGVHD) induces Treg activation and enhances their function. The serum contains high levels of tumor necrosis factor-α (TNF-α) that selectively activates Tregs without impacting CD4(+)FoxP3(-) T cells. TNF-α priming induces Treg in vivo proliferation, whereas it limits the ability of CD4 and CD8 conventional T cells (Tcons) to proliferate and induce GVHD. TNF-α-primed Tregs prolong animal survival as compared with unprimed Tregs when used at an unfavorable Treg:Tcon ratio, demonstrating enhanced in vivo efficacy of TNF-α-primed Tregs. Because TNF-α is produced by several immune cells during inflammation, our work elucidates aspects of the physiologic mechanisms of Treg function. Furthermore, TNF-α priming of Tregs provides a new tool to optimize Treg cellular therapies for GVHD prevention and treatment.

Treatment with agonistic DR3 antibody results in expansion of donor Tregs and reduced graft-versus-host disease.

The paucity of regulatory T cells (Tregs) limits clinical translation to control aberrant immune reactions including graft-versus-host disease (GVHD). Recent studies showed that the agonistic antibody to DR3 (αDR3) expanded CD4(+)FoxP3(+) Tregs in vivo. We investigated whether treating donor mice with a single dose of αDR3 could alleviate acute GVHD in a MHC-mismatched bone marrow transplantation model. αDR3 induced selective proliferation of functional Tregs. CD4(+) T cells isolated from αDR3-treated mice contained higher numbers of Tregs and were less proliferative to allogeneic stimuli. In vivo GVHD studies confirmed that Tregs from αDR3-treated donors expanded robustly and higher frequencies of Tregs within donor CD4(+) T cells were maintained, resulting in improved survival. Conventional T cells derived from αDR3-treated donors showed reduced activation and proliferation. Serum levels of proinflammatory cytokines (IFNγ, IL-1ß, and TNFα) and infiltration of donor T cells into GVHD target tissues (gastrointestinal tract and liver) were decreased. T cells from αDR3-treated donors retained graft-vs-tumor (GVT) effects. In conclusion, a single dose of αDR3 alleviates acute GVHD while preserving GVT effects by selectively expanding and maintaining donor Tregs. This novel strategy will facilitate the clinical application of Treg-based therapies.

Donor Requirements for Regulatory T Cell Suppression of Murine Graft-versus-Host Disease.

Adoptive transfer of freshly isolated natural occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) prevents graft-versus-host disease (GVHD) in several animal models and following hematopoietic cell transplantation (HCT) in clinical trials. Donor-derived Treg have been mainly used, as they share the same MHC with CD4(+) and CD8(+) conventional T cells (Tcon) that are primarily responsible for GVHD. Third party-derived Treg are a promising alternative for cellular therapy, as they can be prepared in advance, screened for pathogens and activity, and banked. We explored MHC disparities between Treg and Tcon in HCT to evaluate the impact of different Treg populations in GVHD prevention and survival. Third-party Treg and donor Treg are equally suppressive in ex vivo assays, whereas both donor and third-party but not host Treg protect from GVHD in allogeneic HCT, with donor Treg being the most effective. In an MHC minor mismatched transplantation model (C57BL/6 → BALB/b), donor and third-party Treg were equally effective in controlling GVHD. Furthermore, using an in vivo Treg depletion mouse model, we found that Treg exert their main suppressive activity in the first 2 d after transplantation. Third-party Treg survive for a shorter period of time after adoptive transfer, but despite the shorter survival, they control Tcon proliferation in the early phases of HCT. These studies provide relevant insights on the mechanisms of Treg-mediated protection from GVHD and support for the use of third-party Treg in clinical trials.

CD4+ invariant natural killer T cells protect from murine GVHD lethality through expansion of donor CD4+CD25+FoxP3+ regulatory T cells.

Dysregulated donor T cells lead to destruction of host tissues resulting in graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). We investigated the impact of highly purified (>95%) donor CD4(+) invariant natural killer T (iNKT) cells on GVHD in a murine model of allogeneic HCT. We found that low doses of adoptively transferred donor CD4(+) iNKT cells protect from GVHD morbidity and mortality through an expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). These Tregs express high levels of the Ikaros transcription factor Helios and expand from the Treg pool of the donor graft. Furthermore, CD4(+) iNKT cells preserve T-cell-mediated graft-versus-tumor effects. Our studies reveal new aspects of the cellular interplay between iNKT cells and Tregs in the context of tolerance induction after allogeneic HCT and set the stage for clinical translation.

Mast cells suppress murine GVHD in a mechanism independent of CD4+CD25+ regulatory T cells.

To investigate the role of mast cells in hematopoietic cell transplantation, we assessed graft-versus-host disease (GVHD) in C57BL/6-Kit(W-sh/W-sh) recipients, which virtually lack mast cells, compared with C57BL/6 WT recipients. GVHD was severely exacerbated in C57BL/6-Kit(W-sh/W-sh) mice (median survival time = 13 vs 60 days in wild-type [WT] mice; P < .0001). The increased mortality risk in C57BL/6-Kit(W-sh/W-sh) hosts correlated with increased T-cell numbers in lymph nodes, liver, and gastrointestinal tract sites, as indicated by bioluminescence imaging (P < .001). We did not detect any deficit in the number or function of CD4(+)CD25(+) regulatory T cells (Tregs) in C57BL/6-Kit(W-sh/W-sh) mice. Furthermore, Tregs were equally effective at reducing GVHD in C57BL/6-Kit(W-sh/W-sh) recipients compared with WT recipients containing mast cells. Furthermore, we found that survival of C57BL/6-Kit(W-sh/W-sh) mice during GVHD was significantly improved if the mice were engrafted with bone marrow-derived cultured mast cells from WT C57BL/6 mice but not from interleukin (IL)-10-deficient C57BL/6 mice. These data indicate that the presence of mast cells can significantly reduce GVHD independently of Tregs, by decreasing conventional T-cell proliferation in a mechanism involving IL-10. These experiments support the conclusion that mast cells can mediate a novel immunoregulatory role during hematopoietic cell transplantation.

Foxp3+ regulatory T cells maintain the bone marrow microenvironment for B cell lymphopoiesis.

Foxp3+ regulatory T cells (Treg cells) modulate the immune system and maintain self-tolerance, but whether they affect haematopoiesis or haematopoietic stem cell (HSC)-mediated reconstitution after transplantation is unclear. Here we show that B-cell lymphopoiesis is impaired in Treg-depleted mice, yet this reduced B-cell lymphopoiesis is rescued by adoptive transfer of affected HSCs or bone marrow cells into Treg-competent recipients. B-cell reconstitution is abrogated in both syngeneic and allogeneic transplantation using Treg-depleted mice as recipients. Treg cells can control physiological IL-7 production that is indispensable for normal B-cell lymphopoiesis and is mainly sustained by a subpopulation of ICAM1+ perivascular stromal cells. Our study demonstrates that Treg cells are important for B-cell differentiation from HSCs by maintaining immunological homoeostasis in the bone marrow microenvironment, both in physiological conditions and after bone marrow transplantation.

Generation of three-dimensional hepatocyte/gelatin structures with rapid prototyping system.

Using rapid prototyping technology, three-dimensional (3D) structures composed of hepatocytes and gelatin hydrogel have been formed. This technique employs a highly accurate 3D micropositioning system with a pressure-controlled syringe to deposit cell/biomaterial structures with a lateral resolution of 10 microm. The pressure-activated micro-syringe is equipped with a fine-bore exit needle for which a wide variety of 3D patterns with different arrays of channels (through-holes) were created. More than 30 layers of a hepatocyte/gelatin mixture were laminated into a high spacial structure using this method. The laminated hepatocytes remained viable and performed biological functions in the construct for more than 2 months. The rapid prototyping technology offers potential for eventual high-throughout production of artificial human tissues or organs.

Human Embryonic Stem Cell Lines with Lesions in FOXP3 and NF1.

Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of blastocyst staged embryos. Spare blastocyst staged embryos were obtained by in vitro fertilization (IVF) and donated for research purposes. hESCs carrying specific mutations can be used as a powerful cell system in modeling human genetic disorders. We obtained preimplantation genetic diagnosed (PGD) blastocyst staged embryos with genetic mutations that cause human disorders and derived hESCs from these embryos. We applied laser assisted micromanipulation to isolate the inner cell mass from the blastocysts and plated the ICM onto the mouse embryonic fibroblast cells. Two hESC lines with lesions in FOXP3 and NF1 were established. Both lines maintain a typical undifferentiated hESCs phenotype and present a normal karyotype. The two lines express a panel of pluripotency markers and have the potential to differentiate to the three germ layers in vitro and in vivo. The hESC lines with lesions in FOXP3 and NF1 are available for the scientific community and may serve as an important resource for research into these disease states.

Fabrication of viable tissue-engineered constructs with 3D cell-assembly technique.

We have recently developed an organ manufacturing technique that enables us to form cell/biomaterial complex three-dimensional (3D) architectures in designed patterns. This technique employs a highly accurate 3D micropositioning system with a pressue-controlled syringe to deposit cell/biomaterial structures with a lateral resolution of 10 microm. The pressure-activated micro-syringe is equipped with a fine-bore exit needle using which a wide variety of 3D patterns with different arrays of channels (through-holes) were created. The channels can supply living cells with nutrients and allow removing the cell metabolites. The embedded cells remain viable and perform biological functions as long as the 3D structures are retained. The new technology has the potential for eventual high-throughput production of artificial human tissues and organs.

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease.

The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.

Engraftment of embryonic stem cells and differentiated progeny by host conditioning with total lymphoid irradiation and regulatory T cells.

Embryonic stem cells (ESCs) hold promise for the treatment of many medical conditions; however, their utility is limited by immune rejection. The objective of our study is to establish tolerance or promote engraftment of transplanted ESCs as well as mature cell populations derived from ESCs. Luciferase (luc(+))-expressing ESCs were utilized to monitor the survival of the ESCs and differentiated progeny in living recipients. Allogeneic recipients conditioned with fractioned total lymphoid irradiation (TLI) and anti-thymocyte serum (ATS) or TLI plus regulatory T cells (T(reg)) promoted engraftment of ESC allografts after transplantation. Following these treatments, the engraftment of transplanted terminally differentiated endothelial cells derived from ESCs was also significantly enhanced. Our findings provide clinically translatable strategies of inducing tolerance to adoptively transferred ESCs for cell replacement therapy of medical disorders.

Characterization of stage-specific embryonic antigen-1 expression during early stages of human embryogenesis.

Extensive expression of stage-specific embryonic antigen-1 (SSEA-1) has been documented in some animal species, but not in human embryos. In this study, SSEA-1 was detected during human embryogenesis by whole-mount immunohistochemistry. Alkaline phosphatase (Ap) activity was detected to identify human primordial germ cells. SSEA-1 was expressed steadily and restrictedly in some cells/tissues, especially in the nephric duct and nephric tubule (including the pronephric duct and tubule, mesonephric duct and tubule, metanephric tissues) besides embryonic ectodermal cells and yolk sac from 3 to 7 weeks. High level of Ap activity was observed in vessels, part of the mesonephric duct, especially in embryonic primordial germ cells localized in the yolk sac, primitive gut, dorsal mesenteries and genital ridges. No colocalization of AP and SSEA-1 cells was observed. SSEA-1 was expressed in human embryos in a different pattern at early stages compared to that in mouse embryos. It was expressed in the nephric duct, nephric tubule, yolk sac and on the surface of embryonic ectodermal cells of the epidermis, but not in human primordial germ cells.

A new FACS approach isolates hESC derived endoderm using transcription factors.

We show that high quality microarray gene expression profiles can be obtained following FACS sorting of cells using combinations of transcription factors. We use this transcription factor FACS (tfFACS) methodology to perform a genomic analysis of hESC-derived endodermal lineages marked by combinations of SOX17, GATA4, and CXCR4, and find that triple positive cells have a much stronger definitive endoderm signature than other combinations of these markers. Additionally, SOX17(+) GATA4(+) cells can be obtained at a much earlier stage of differentiation, prior to expression of CXCR4(+) cells, providing an important new tool to isolate this earlier definitive endoderm subtype. Overall, tfFACS represents an advancement in FACS technology which broadly crosses multiple disciplines, most notably in regenerative medicine to redefine cellular populations.

In vitro neuronal differentiation of cultured human embryonic germ cells.

Human embryonic germ (hEG) cells, which have been advanced as one of the most important sources of pluripotent stem cells [the other one being human embryonic stem cells], can be propagated in vitro indefinitely in the primitive undifferentiated state while being capable of developing into all three germ layer derivatives, hence have become anticipated developing novel strategies of tissue regeneration and transplantation in the treatment of degenerative diseases. In the experiments here, we derived hEG cells from cultured human primordial germ cells (PGCs) of 6- to 9-week-post-fertilization embryos. They satisfied the criteria previously used to define hEG cells, including the expression of markers characteristic of pluripotent cells-abundant alkaline phosphatase (AP) activity, stage specific embryonic antigen (SSEA)-1(+), SSEA-3(-), SSEA-4(+), TRA-1-60(+), TRA-1-81(+), Oct-4(+), and hTERT(+), the retention of normal karyotypes, and possessing pluripotency by forming embryoid bodies (EBs) in vitro. Furthermore, these derived cells tended to neurally differentiate in vitro, especially under high-density culture conditions. We successfully isolated neural progenitor cells from differentiating hEG cultures and about 10% cells induced by 2microM all-trans-retinoic acid (RA) or 0.1mM dibutyryl cyclic AMP (dbcAMP)/1mM forskolin to mature neurons expressing microtubule-associated protein 2ab (MAP2ab), synaptophysin, beta-tubulin III, neuron-specific enolase (NSE), tyrosine hydroxylase (TH), but no glial fibrillary acid protein (GFAP) and choline acetyl transferase (ChAT). The data suggested that hEG cells may provide a potential source of cells for use in transplantation therapy for neurological degenerative diseases.

Cartilage-derived morphogenetic protein-1 promotes the differentiation of mesenchymal stem cells into chondrocytes.

Mesenchymal stem cells (MSCs) are able to differentiate into many types of cells including chondrocytes. Transforming growth factor beta1 (TGF-beta1) is very important in the regulation of chondrogenesis. Since cartilage-derived morphogenetic protein-1 (CDMP-1) belongs to the TGF-beta superfamily, we tested whether CDMP-1 plays any role in the regulation of the differentiation of MSCs into chondrocytes using a high density pellet culture system. Based on the histological staining of glycosaminoglycan using toluidine blue dye-binding method we found that CDMP-1 could initiate chondrogenic differentiation of MSCs as did TGF-beta1. However, CDMP-1 was less stimulatory than TGF-beta1. The combination of CDMP-1 and TGF-beta1 synergically induced chondrogenesis of MSCs. This synergic chondrogenic effect of CDMP-1 together with TGF-beta1 was further confirmed by quantification of GAG using dimethylmethylene blue dye-binding assay and immunohistochemical analysis of the expression of cartilage-specific protein collagen II. This study may provide an improved induction approach using MSCs for repairing damaged cartilage.

Human embryonic germ cells isolation from early stages of post-implantation embryos.

Human embryonic germ (hEG) cell is a very important alternative pluripotent stem cell resource. We describe the derivation of hEG cells from human embryonic fetal gonads over 6-8 weeks postconception. A large number of EG-like cell clumps were obtained at passage 1 and thus facilitated the following routine culture when the donor tissues were trypsinized with gentle pipetting and plated on feeder layer cells in the initial culture. Eight diploid hEG cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent stem cells. Human EG cells expressed transcription factor Oct4, a marker of pluripotency in mouse EG cells, at a high and steady level. Expression of markers indicative of differentiation along the three germ lineages was also observed in EBs. High level of alkaline phosphatase activity was shown in EG cells. These encouraging findings provide a starting point for potential applicability of hEG cells.