Il-Hee Yoon

Soluble mediators from mesenchymal stem cells suppress T cell proliferation by inducing IL-10

Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either naïve or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naïve and pre-activated T cells in which IL-10 and IDO play important roles.

Long-term control of diabetes in immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets.

Pig islets are an alternative source for islet transplantation to treat type 1 diabetes (T1D), but reproducible curative potential in the pig‐to‐nonhuman primate (NHP) model has not been demonstrated. Here, we report that pig islet grafts survived and maintained normoglycemia for >6 months in four of five consecutive immunosuppressed NHPs. Pig islets were isolated from designated pathogen‐free (DPF) miniature pigs and infused intraportally into streptozotocin‐induced diabetic rhesus monkeys under pretreatment with cobra venom factor (CVF), anti‐thymocyte globulin (ATG) induction and maintenance with anti‐CD154 monoclonal antibody and low‐dose sirolimus. Ex vivo expanded autologous regulatory T cells were adoptively transferred in three recipients. Blood glucose levels were promptly normalized in all five monkeys and normoglycemia (90–110 mg/dL) was maintained for >6 months in four cases, the longest currently up to 603 days. Intravenous glucose tolerance tests during the follow‐up period showed excellent glucose disposal capacity and porcine C‐peptide responses. Adoptive transfer of autologous regulatory T cells was likely to be associated with more stable and durable normoglycemia. Importantly, the recipients showed no serious adverse effects. Taken together, our results confirm the clinical feasibility of pig islet transplantation to treat T1D patients without the need for excessive immunosuppressive therapy.

Vascular endothelial growth factor-induced chemotaxis and IL-10 from T cells

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.

Immunomodulation of Delayed-Type Hypersensitivity Responses by Mesenchymal Stem Cells Is Associated with Bystander T Cell Apoptosis in the Draining Lymph Node

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.

Bortezomib can suppress activation of rapamycin-resistant memory T cells without affecting regulatory T-cell viability in non-human primates.

Background. 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. Methods. 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. Results. 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)-γ, IL-4, and IL-10. Conclusions. 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.

Pancreatic Islets Induce Cd4+cd25−foxp3+ T-cell Regulated Tolerance to Hy-mismatched Skin Grafts

Background. 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. Methods. 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. Results. 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. Conclusions. Unmanipulated pancreatic islets can induce immunologic tolerance associated with peripherally induced CD4+Foxp3+ Tregs, a significant proportion of which notably are CD25−.

High-dose cyclophosphamide-mediated anti-tumor effects by the superior expansion of CD44high cells after their selective depletion

As alkylating agents, cyclophosphamides (CTX) are used to treat various cancers and, ironically, to boost immune responses. In the present study, we attempted to elucidate the mechanism responsible for the immunomodulatory effect of high-dose CTX in an established tumor model. A single injection of high-dose CTX increased the survival rate of immunocompetent, but not immunodeficient, mice. Notably, 10 days after CTX injection, the number of CD44(high) memory T cells significantly increased, without a selective decrease in the actual number and percentage of CD4+CD25+Foxp3+ regulatory T cells (Tregs). However, the proportion of Tregs among CD4+ T cells decreased due to expansion of memory and other CD4+ T cell subtypes. This outcome was accompanied by an increase in IL-15 mRNA and up-regulation of IL-15 receptors in the CD44+CD8+ T cell compartment. We postulate that the CTX-induced change in T cell balance may increase anti-tumor immunity.

Porcine antigen-specific IFN-γ ELISpot as a potentially valuable tool for monitoring cellular immune responses in pig-to-non-human primate islet xenotransplantation.

Recent progress in xenotransplantation of porcine islets to non‐human primates (NHPs) gives hope for human clinical trials in the near future. Thus, implementation of an appropriate monitoring method to detect the development of detrimental porcine antigen‐specific cellular immune responses is necessary. The enzyme‐linked immunospot (ELISpot) assay has been widely used to monitor antigen‐specific alloreactive T‐cell responses in humans; however, the utility of porcine islet‐specific ELISpot assay has not yet been thoroughly evaluated for pig‐to‐NHPs intraportal islet xenotransplantation.

Induction, management, and complications of streptozotocin-induced diabetes mellitus in rhesus monkeys.

Diabetes mellitus (DM) model using streptozotocin (STZ) which induces chemical ablation of β cell in the pancreas has been widely used for various research purposes in non‐human primates. However, STZ has been known to have a variety of adverse effects such as nephrotoxicity, hepatotoxicity, and even mortality. The purpose of this study is to report DM induction by STZ, toxicity associated with STZ and procedure and complication of exogenous insulin treatment for DM management in rhesus monkeys (Macaca mulatta) that are expected to be transplanted with porcine islets within 2 months.

CD4＋VEGFR1HIGH T cell as a novel Treg subset regulates inflammatory bowel disease in lymphopenic mice

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+VEGFR1high 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+VEGFR1high T cells are distinct from known Tregs. CD4+VEGFR1high T cells suppressed the proliferation of CD4+CD25− T cell as efficiently as CD4+CD25high 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+ VEGFR1high T cells as a novel subset of Tregs that regulate the inflammatory response in the intestinal tract.