[Show abstract][Hide abstract] ABSTRACT: Regulatory T cells (Tregs) are a specialized subpopulation of T cells that control the immune response and thereby maintain immune system homeostasis and tolerance to self-antigens. Many subsets of CD4(+) Tregs have been identified, including Foxp3(+), Tr1, Th3, and Foxp3neg iT(R)35 cells. In this study, we identified a new subset of CD4(+)VEGFR1(high) Tregs that have immunosuppressive capacity. CD4(+)VEGFR1high T cells, which constitute approximately 1.0% of CD4(+) T cells, are hyporesponsive to T-cell antigen receptor stimulation. Surface marker and FoxP3 expression analysis revealed that CD4(+)VEGFR1(high) T cells are distinct from known Tregs. CD4(+)VEGFR1(high) T cells suppressed the proliferation of CD4(+)CD25(-) T cell as efficiently as CD4(+)CD25(high) natural Tregs in a contact-independent manner. Furthermore, adoptive transfer of CD4(+)VEGFR1(+) T cells from wild type to RAG-2-deficient C57BL/6 mice inhibited effector T-cell-mediated inflammatory bowel disease. Thus, we report CD4(+) VEGFR1(high) T cells as a novel subset of Tregs that regulate the inflammatory response in the intestinal tract.Cellular & Molecular Immunology advance online publication, 27 July 2015; doi:10.1038/cmi.2015.71.
[Show abstract][Hide abstract] ABSTRACT: Intraportal islet transplantation (ITx) causes instant blood-mediated inflammatory reaction (IBMIR), resulting in an early loss of transplanted islets. Porcine islets, transplanted intraportally into nonhuman primates (NHPs), induce complement activation, contributing to the development of IBMIR; however, the exact mechanism is not clear.
Complement activation were compared after incubation of purified adult porcine islets in 20% human serum with or without complement inhibitors, C1 esterase inhibitor (C1E-inh), anti-factor B, and purified human factor H. Intraportal porcine ITx was performed in diabetic NHPs to which cobra venom factor (CVF), factor H, or none of complement inhibitor was administered during the peritransplant period. The extent of complement activation and function of islet grafts were monitored after ITx.
The incubation of porcine islets with human serum resulted in generation of C3a, C4d, and factor Bb in the fluid phase. However, the generation of C3a after incubation was suppressed by anti-factor B or factor H, but not by C1E-inh. Moreover, in NHPs with porcine ITx, the administration of CVF or factor H ameliorated the increase in plasma C3a and factor Bb levels, as well as early release of porcine C-peptide after ITx. Furthermore, the functional survival of islet grafts was prolonged in the recipients of the CVF group compared to those in the control group.
The alternative complement pathway contributes to the development of IBMIR and the early loss of grafts in NHPs with porcine ITx. Complement inhibition during the peritransplant period may be beneficial for the survival of islet grafts.
[Show abstract][Hide abstract] ABSTRACT: Pancreatic islet transplantation is a promising treatment option for Type 1 diabetes, but organ supply shortage limits its wide adoption. Pig islets are the most promising alternative source and many important measures such as donor animal selection, pig islet production release criteria, preclinical data and zoonosis surveillance prior to human clinical trials have been put forward as a consensus through the efforts of the International Xenotransplantation Association. To bring pig islet transplantation to clinical reality, the development of clinically applicable immunosuppression regimens and methods to minimize immunosuppression to reduce side effects should be established. This review encompasses immune rejection mechanisms in islet xenotransplantation, immunosuppression regimens that have enabled long-term graft survival in pig-to-nonhuman primate experiments and strategies for minimizing immunosuppression in islet xenotransplantation. By thoroughly examining the drugs that are currently available and in development and their individual targets within the immune response, the best strategy for enabling clinical trials of pig islets for Type 1 diabetes will be proposed.
[Show abstract][Hide abstract] ABSTRACT: Non-human primate studies must be conducted prior to the clinical trial of xenotransplantation. In order to develop clinically applicable immune-modulatory regimen through non-human primate studies, close monitoring of xenogeneic immune responses is required. We adopted multiplex cytokine analysis in assessment of the immune status during the course of pig-to-non-human primate islet transplantation. This study aimed to assess the feasibility of this multiplex cytokine assay in the development of immune-modulatory regimen. Using this assay, we were able to detect different cytokines with a minimal usage of blood samples, and this allowed us to detect various immunological situations in the recipients. Detection of TNF-α surge (347.8 pg/mL) guided us to block TNF-α in the early phase of transplantation. Supportive information for in vivo efficacy of cytokine neutralizing antibody could be speculated by in vitro neutralization assay (1,250 pg/mL → 0 pg/mL). In addition, periodic monitoring of cytokines in peripheral blood allowed the detection of the infection episode prior to other routine assays. These benefits of multiplex cytokine assay may be generally applied to other pre-clinical research, which is a prerequisite for clinical trials.
Journal of Korean medical science 12/2013; 28(12):1729-33. DOI:10.3346/jkms.2013.28.12.1729 · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is a transmembrane protein that is structurally similar to CD28. As CTLA-4 has a much higher binding affinity to B7 than CD28, several approaches using soluble CTLA-4 have been tried to down-regulate T cell activity by blocking the interaction between CD28 and B7. We constructed soluble rhesus monkey CTLA-4 immunoglobulin (CTLA-4Ig) containing a critical binding site to B7 combined with a constant Ig heavy chain region in a mammalian system. Flow cytometry analyses indicated that soluble rhesus monkey CTLA-4Ig bound to rhesus monkey CD86 (B7.2). Moreover, soluble rhesus monkey CTLA-4Ig more effectively blocked the rhesus monkey-rhesus monkey allogeneic mixed lymphocyte reaction compared with that of humans. These results indicate that soluble rhesus monkey CTLA-4Ig may be useful in preclinical trials in a rhesus monkey model.
[Show abstract][Hide abstract] ABSTRACT: Maximum engraftment of transplanted islets is essential for the clinical application of a subcutaneous site. Significant barriers to the current approaches are associated with their low effectiveness, complexity and unproven biosafety. Here, we evaluated and optimized a fibrin-islet composite for effective glycemic control in a subcutaneous site whose environment is highly hypoxic due to low vascularization potential. In the setting of xenogeneic porcine islet transplantation into the subcutaneous space of a diabetic mouse, the in vivo islet functions were greatly affected by the concentrations of fibrinogen and thrombin. The optimized hydrogel-type fibrin remarkably reduced the marginal islet mass to approximately one tenth that of islets without fibrin. This marginal islet mass was comparable to that in the setting of the subcapsular space of the kidney, which is a highly vascularized organ. Highly vascularized structures were generated inside and on the outer surface of the grafts. A hydrogel-type fibrin-islet composite established early diabetic control within an average of 3.4days after the transplantation. In the mechanistic studies, fibrin promoted local angiogenesis, enhanced islet viability and prevented fragmentation of islets into single cells. In conclusion, in situ application of hydrogel-type fibrin-islet composite may be a promising modality in the clinical success of subcutaneous islet transplantation.
[Show abstract][Hide abstract] ABSTRACT: Human effector memory (EM) CD8(+) T cells include IL-7Rα(high) and IL-7Rα(low) cells with distinct cellular characteristics, including the expression of cytotoxic molecules. Both NK cells and the NK cell-associated molecule 2B4 that is expressed on CD8(+) T cells promote cytotoxicity. Here we analysed the expression of 2B4 on IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells and its contribution to cytotoxicity. We also analysed the frequency of IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells in patients with SLE or lupus and in healthy individuals given the potential role of cytotoxic CD8(+) T cells in the pathogenesis of lupus.
We used flow cytometry to measure the expression of 2B4 on IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells as well as the frequency of these cell populations in the peripheral blood of healthy individuals and patients with SLE. Also, 2B4-mediated cytotoxicity was quantitated in IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells using target cells with CD48 antigen.
We found that IL-7Rα(high) EM CD8(+) T cells had higher levels of 2B4 expression compared with IL-7Rα(low) EM CD8(+) T cells. Triggering 2B4 enhanced the cytotoxic function of IL-7Rα(low) EM CD8(+) T cells against target cells. We also noticed that patients with SLE had an increased frequency of IL-7Rα(low) EM CD8(+) T cells that correlated with disease manifestation.
Our findings show that SLE patients have increased IL-7Rα(low) EM CD8(+) T cells, possibly contributing to tissue damage through 2B4-mediated cytotoxicity.
[Show abstract][Hide abstract] ABSTRACT: The development of immunosuppressant treatments has enabled remarkable progress in the tissue and organ transplantation field by helping to prevent acute graft rejection. However, complications related to transplantation, such as infection by bacteria and viruses, and the occurrence of cancers resulting from prolonged immune suppression are major obstacles to overcome. Therefore, transplantation immunology research efforts should focus on the induction of donor-specific immune tolerance which preserves patient immune competence which promotes infection and cancer surveillance. Additionally, lifelong administration of immunosuppressants should be forgone in preference to short term therapies. In the 1990s, Dr. Shimon Sakaguchi identified the CD4+CD25+ regulatory T cells which develop in the thymus, and demonstrated that these cells play crucial roles in the maintenance of immune self tolerance. Studies which followed proved that these regulatory T cells are important to the control of autoimmune disease and prevention of graft rejection. Regulatory T cells have also been found to induce immune tolerance in rodent models. In this review, we discuss several considerations for the use of regulatory T cell therapy in the clinical transplantation field.
[Show abstract][Hide abstract] ABSTRACT: Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti-ICAM-1-induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection.
Journal of Experimental Medicine 11/2011; 208(12):2477-88. DOI:10.1084/jem.20111242 · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In clinical trials using adult porcine islet products, islets should be isolated from the designated pathogen-free (DPF) pigs under the current good manufacturing practice (GMP) regulations. Our previous studies suggested that male DPF pigs are better donors than retired breeder pigs and histomorphometrical parameters of donor pancreas predict the porcine islet quality. We aimed to investigate whether the use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy could improve the islet graft function in the context of islet isolation with DPF pigs.
Using 30 DPF pigs within a closed herd, we compared the islet yield of porcine islets isolated with Liberase PI (n = 11, as a historical control group), Liberase MTF C/T, which is a GMP-grade enzyme (n = 12), and CIzyme collagenase MA/BP protease (n = 7). We analyzed the relationship between the diabetes reversal rate of recipient NOD/SCID mice (n = 75) and histomorphometric parameters of each donor pancreas as well as donor characteristics.
Proportion of islets larger than 200 μm from the biopsied donor pancreas (P = 0.006) better predicted islet yield than age (P = 0.760) or body weight (P = 0.371) of donor. The proportion of islets larger than 200 μm from the biopsied donor pancreas was not related to the sex of the donor miniature pig (P = 0.358). The islet yield obtained with the three enzymes did not differ, even after stratification of the donor with the histomorphometric parameters of the biopsied donor pancreas and the sex of donor. The use of the newer bovine nervous tissue-free enzymes (P < 0.001), a higher proportion of large islets in donor pancreas (P = 0.006), and a male sex of the donor (P = 0.025) were independent predictors of earlier diabetes reversal.
Use of the newer bovine nervous tissue-free enzymes including a GMP-grade enzyme resulted in better islet quality than that of islet isolated using Liberase PI. To obtain high-quality islet from DPF pigs, the donor should be male pig and histomorphometrical parameters from donor pancreas should be considered.
[Show abstract][Hide abstract] ABSTRACT: 2477 Tolerance to specific antigens is the ultimate therapeutic goal in two major immunological fields, autoimmunity and transplantation rejec-tion. Over the past several decades, the genera-tion of a large array of immunosuppressive agents has increased the number of therapeutic tools available to address these two issues. The focus has now shifted to tackling the side effects of long-term immunosuppression. The final goal is to achieve T and B cell tolerance that is anti-gen specific without the need for long-term generalized immunosuppression. The mechanisms that underlie peripheral T cell tolerance have been explained, in part, al-though some points remain to be addressed. DCs have a key role in immune regulation (Steinman et al., 2003). Antigen presentation by immature and semimature DCs results in immune tolerance rather than effective T cell immunity because of the failure to provide sufficient co-stimulatory signals (Reis e Sousa, 2006; Morelli and Thomson, 2007; Shortman and Naik, 2007). These tolero-genic DCs are characterized by low-level expression of surface MHC molecules and sev-eral other co-stimulatory receptors and the pro-duction of low levels of Th1 cytokines, notably IL-12p70 (Morelli and Thomson, 2007). Among several mouse anti–human ICAM-1 (intercellular adhesion molecule 1) antibody clones we have developed thus far, we were able to select only one clone, MD-3, which CORRESPONDENCE Seong Hoe Park: email@example.com
Journal of Experimental Medicine 01/2011; 208(12):2477-2488. · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are multipotent, non-hematopoietic stem cells that exhibit the capacity to inhibit the proliferation of a variety of immune cells. However, the underlying mechanisms of the immunosuppressive effects of MSCs are still obscure. Therefore, we attempted to identify the mechanisms underlying immunosuppression toward the activated T lymphocytes by MSCs in a murine model. In particular, we aimed to find possible factors derived from MSCs that drive this phenomenon. We found that T lymphocytes incubated with conditioned media of MSCs (MSC CM) entered into apoptosis and were subjected to cell cycle arrest during the course of activation, and these phenomena were accompanied by the reduction of IL-2 production. Specifically, matrix metalloproteinases (MMPs) derived from MSCs caused cleavage of IL-2 receptor α (CD25) from the surface of activated T cells, and as a consequence, IL-2 signaling in response to engagement of the IL-2 receptor (IL-2R) was downregulated. The inhibition of MMP activity in the MSC CM by GM6001 abrogated CD25 cleavage and restored IL-2 production from the activated splenocytes. However, the blockade of MMP activity could not fully restore the proliferative response and apoptosis of T cells altered by MSC CM. In conclusion, MSC-derived MMPs have a significant role in the suppression of IL-2 production through induction of CD25 cleavage and have a partial role in the suppression of T cell proliferation.
[Show abstract][Hide abstract] ABSTRACT: Disease amelioration by mesenchymal stem cells (MSCs) has been shown to be closely related to their immunomodulatory functions on the host immune system in many disease models. However, the underlying mechanisms of how these cells affect the immune cells in vivo are not fully understood. In this study, we report findings that a small but significant number of MSCs accumulate in the secondary lymphoid organs and attenuate delayed-type hypersensitivity (DTH) response by inducing apoptotic cell death of surrounding immune cells in the draining lymph node (LN). In the migration study, i.v. infused GFP-MSCs preferentially accumulated at the boundary between the paracortical area and the germinal center in the LNs, in close proximity to various types of immune cells including T, B, and dendritic cells in a dose-dependent manner. As a result, accumulated MSCs markedly attenuated DTH response in proportion to the number of MSCs infused. During the DTH response, the infiltration of T cells in the challenged site was significantly decreased, whereas a number of apoptotic T cells were remarkably increased in the draining LN. Apoptosis was significantly induced in activated T cells (CD3(+) and BrdU(+)), but not in the resting T cells (CD3(+) and BrdU(-)). NO was associated with these apoptotic events. Taken together, we conclude that significant numbers of i.v. infused MSCs preferentially localize in the draining LN, where they induce apoptosis of the activated T cells by producing NO and thus attenuate the DTH response.
The Journal of Immunology 10/2010; 185(7):4022-9. DOI:10.4049/jimmunol.0902723 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Memory T cells specific for donor antigens are currently recognized as a significant barrier for maintaining a successful transplant. Furthermore, it has been shown that commonly used immunosuppressive drugs do not alleviate this memory response. Here, we report that rapamycin allows significant proliferation of memory T cells and bortezomib can abrogate the proliferation of rapamycin-resistant memory T cells when preserving the survival of regulatory T cells.
Peripheral blood mononuclear cells freshly isolated from non-human primates were stimulated with anti-CD3/CD28 antibodies, and inhibitory and apoptotic effects of rapamycin and bortezomib on memory T-cell proliferation were investigated. The CD95 marker in CD3+ T cells was used for the separate enrichment of memory T cells and naïve T cells.
Rapamycin at the level even higher than therapeutic concentration could not suppress the proliferation of a significant proportion of memory T cells. However, the combined administration of bortezomib and rapamycin abrogated the proliferation of rapamycin-resistant memory T cells. Furthermore, bortezomib preserved the survival of preexisting CD4+ FoxP3+ regulatory T cells, while inducing apoptosis of CD4+ FoxP3- conventional T cells. The combined administration of low doses of rapamycin and bortezomib also exerted an additive effect on suppressing T-cell proliferation. Cytokine analysis demonstrated that bortezomib could not only suppress rapamycin-permissive interleukin (IL)-6 production, but also production of interferon (IFN)-gamma, IL-4, and IL-10.
This article provides in vitro data from which immunosuppressive regimens for the effective control of memory T cells in non-human preclinical experiments and in clinical trials are selected.
[Show abstract][Hide abstract] ABSTRACT: Xenotransplantation using pigs as the transplant source holds great promise to resolve the severe shortage of human organ donors. Although stem-cell-derived organ and tissue regeneration have a potential to solve this as well for the future, it still remains as very early experimental phase. Likewise, artificial organs and mechanical devices have been simply used for bridge therapy to transplant. Therefore, xenotransplantation might provide the most imminent solution to the scarcity of human organ donors. In the last two decades, major progress has been made in understanding the mechanisms of xenografts rejection, zoonotic infections including porcine endogenous retrovirus (PERV) and production of genetically engineered pigs including α1,3-galactosyltransferase-deficient pigs. With these elaborations, it is now on the threshold of first clinical application. Particularly promising first target is porcine pancreatic islet xenotransplantation. Graft survival has been prolonged to almost one year in the non-human primate study and is waiting for the development of relatively non-toxic or clinically applicable immunosuppressive or tolerance-inducing regimens. This review highlights the currently known obstacles to translate xenotransplantation into clinical therapies and the possible strategies to overcome these hurdles, as well as current status and future perspective for clinical xenotransplantaion.
[Show abstract][Hide abstract] ABSTRACT: Human neural stem cells (hNSCs) can control inflammation in the central nervous system, although the underlying mechanisms are not understood fully. We investigated the immunomodulatory effect of hNSCs on human T cells and the underlying mechanisms. Culture supernatant from an immortalized hNSC cell line, HB1.F3, which has a therapeutic effect on acute stroke and intracerebral hemorrhage, suppressed the proliferation of allogeneically or mitogenically stimulated human peripheral T cells, including the CD3(+)CD103(+) subpopulation. CFSE labeling and flow cytometry showed that the suppression of proliferation was caused by cell cycle arrest and induction of apoptosis. The lack of significant change in caspase-8 levels and the significant reduction in Bcl-2 expression in the affected T cells suggest that the intrinsic pathway plays a major role in soluble-factor-mediated T-cell apoptosis. The addition of culture supernatant from hNSCs to activated T cells reduced the expression of the activation markers CD69 and CD25 at 24 hr after activation, but at 48 hr only CD69 was down-regulated. A cytometry bead assay showed that the secretion of interleukin (IL)-2 decreased significantly, whereas that of IL-4, IL-10, tumor necrosis factor-alpha, and interferon-gamma increased. These results show that hNSCs can negatively affect human peripheral T cells by suppressing their activation and proliferation through soluble mediators, suggesting that hNSCs have a bystander immunomodulatory effect on T cells.
Journal of Neuroscience Research 08/2009; 87(10):2264-72. DOI:10.1002/jnr.22050 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: N-glycan structures released from miniature pig endothelial and islet cells were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), negative ion electrospray ionization (ESI) MS/MS and normal-phase high performance liquid chromatography (NP-HPLC) combined with exoglycosidase digestion. Totally, the identified structures were 181 N-glycans including 129 sialylated and 18 alpha-galactosylated glycans from pig endothelial cells and 80 N-glycans including 41 sialylated and one alpha-galactosylated glycans from pig islet cells. The quantity of the alpha-galactosylated glycans from pig islet cells was certainly neglectable compared to pig endothelial cells. A number of NeuGc-terminated N-glycans (80 from pig endothelial cells and 13 from pig islet cells) are newly detected by our mass spectrometric strategies. The detailed structural information will be a matter of great interest in organ or cell xenotransplantation using alpha 1,3-galactosyltransferase gene-knockout (GalT-KO) pig.
Journal of Mass Spectrometry 07/2009; 44(7):1087-104. DOI:10.1002/jms.1587 · 2.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) is a proangiogenic mediator that promotes tumor growth. The role of VEGF in T lymphocytes is unknown. We found that T lymphocytes activated by either anti-CD3 monoclonal antibody (mAb) plus anti-CD28 mAb or by antigens on antigen-presenting cells transcribed mRNA for VEGF receptor 1 (VEGFR1) and VEGFR2. However, only VEGFR1 was expressed on the T cell surface. The addition of VEGF to either resting or activated T cells did not affect their proliferation, but VEGF increased IL-10 production and slightly decreased IFN-gamma production. A chemotaxis assay revealed that activated T lymphocytes migrate in response to VEGF. Our data suggest that VEGF has a direct immunomodulatory effect on T cells. Engagement of a high concentration of VEGF with VEGFR1 on T cells may cause T cells to migrate to tumor sites, and this interaction may play a role in IL-10-mediated immune evasion by tumor cells.
[Show abstract][Hide abstract] ABSTRACT: The ability to induce and maintain antigen-specific immunologic tolerance is the ultimate goal in allo-transplantation. Here, we report that the transplantation of unmanipulated murine pancreatic islets across the HY disparity induced transplantation tolerance that prevented HY-mismatched skin grafts being rejected.
Three hundred islet equivalent of freshly isolated islets from male C57BL/6 donor mice was transplanted underneath the kidney capsule of syngeneic female recipients rendered diabetic by streptozotocin. Nephrectomy was carried out to remove the islet graft and retransplantation was performed using the contralateral kidney. For skin transplantation, donor tail skin was transplanted onto the lateral thorax.
Islets from male C57BL/6 donors transplanted to syngeneic female recipients cured diabetes and the mice survived indefinitely. The acceptance of second grafts and rejection of third party islet grafts indicated antigen-specific transplantation tolerance. However, flow cytometry and ELISPOT analysis demonstrated that the HY-specific T cells were not deleted or anergized. A 2-fold increase of CD4+Foxp3+ regulatory T cells (Tregs) was observed in spleen and lymph nodes. Notably, CD25- Tregs increased threefold over levels in naïve mice. Adoptive transfer of CD4+ T cells to neonatal mice could transfer tolerance. At the graft site in long-term tolerant mice, CD4+ T cells, 40% of which were CD4+Foxp3+ Tregs (43% CD25-, 57% CD25+) infiltrated the peri-islet spaces.
Unmanipulated pancreatic islets can induce immunologic tolerance associated with peripherally induced CD4+Foxp3+ Tregs, a significant proportion of which notably are CD25-.