Gene expression analysis of dendritic cells that prevent diabetes in NOD mice: analysis of chemokines and costimulatory molecules.

We have demonstrated previously that BM-derived DCs can prevent diabetes development and halt progression of insulitis in NOD mice, the mouse model of type 1 diabetes. The DC population that was most effective in this therapy had a mature phenotype, expressed high levels of costimulatory molecules, and secreted low levels of IL-12p70. The protective DC therapy induced Treg and Th2 cells in vitro and in vivo. Microarray analysis of therapeutic and nontherapeutic DC populations revealed differences in the expression of OX40L, CD200, Ym-1, CCL2, and CCL5, which could play important roles in the observed DC-mediated therapy. The unique pattern of costimulatory molecules and chemokines expressed by the therapeutic DCs was confirmed by flow cytometry and ELISA. Using a novel cell-labeling and (19)F NMR, we observed that the chemokines secreted by the therapeutic DCs altered the migration of diabetogenic Th1 cells in vivo and attracted Th2 cells. These results suggest that the therapeutic function of DCs is mediated by a combination of costimulatory and chemokine properties that results in the attraction of diabetogenic Th1 and the induction of Th2 and/or Treg differentiation.

Marginal mass islet transplantation with autologous mesenchymal stem cells promotes long-term islet allograft survival and sustained normoglycemia.

Allogeneic islet transplantation is an option to treat diabetes however there are obstacles that are limiting its clinical use. We have examined whether mesenchymal stem cells (MSC) improve islet graft survival and whether such therapy allows for better graft acceptance with reduced requirement for immunosuppression. In vitro-expanded syngeneic bone marrow-derived MSC were co-transplanted with islets into omental pouch in a rat model of streptozotocin-induced diabetes. Marginal mass syngeneic islet transplantation into the omentum with MSC promoted sustained normoglycemia. Interestingly, allogeneic islets +MSC, but not islets alone, with short-term use of immunosuppression enhanced long-term islet graft survival, insulin expression in the grafts and induced normal serum insulin levels and normoglycemia. T cells from recipients transplanted with allogeneic islets +MSC produced low levels of IFN-gamma and TNF-alpha upon ex-vivo activation, and this transplantation protocol promoted the generation of IL-10-secreting CD4(+) T cells. These data encourage further preclinical and eventually, clinical MSC-based islet transplantation to improve the outcome of allogeneic islet transplantation in the treatment of diabetes.

Autologous bone marrow-derived rat mesenchymal stem cells promote PDX-1 and insulin expression in the islets, alter T cell cytokine pattern and preserve regulatory T cells in the periphery and induce sustained normoglycemia.

Cell-based therapies offer considerable promise for prevention or cure of diabetes. We explored the potential of autologous, self-renewing, mesenchymal stem cells (MSC) as a clinically-applicable approach to promote glucose homeostasis. In vitro-expanded syngeneic bone marrow-derived MSC were administered following or prior to diabetes induction into a rat model of streptozotocin-induced beta cell injury. MSC were CD45(-)/CD44(+)/CD54(+)/CD90(+)/CD106(+). MSC spontaneously secreted IL-6, HGF, TGF-beta1 and expressed high levels of SDF-1 and low levels of VEGF, IL-1beta and PGE(2), but no EGF, insulin or glucagon. MSC homed to the pancreas and this therapy allowed for enhanced insulin secretion and sustained normoglycemia. Interestingly, immunohistochemistry demonstrated that, the islets from MSC-treated rats expressed high levels of PDX-1 and that these cells were also positive for insulin staining. In addition, peripheral T cells from MSC-treated rats exhibited a shift toward IL-10/IL-13 production and higher frequencies of CD4(+)/CD8(+) Foxp3(+) T cells compared to the PBS-treated rats. These data suggest that the bioactive factors secreted by MSC establish a tissue microenvironment that supports beta cell activation/survival in the pancreas. In addition, because of anti-inflammatory and immunoregulatory effects of MSC on T cells, this work can lead to clinical trial of autologous MSC to prevent/cure type-1 diabetes.

Co-infusion of donor bone marrow with host mesenchymal stem cells treats GVHD and promotes vascularized skin allograft survival in rats.

We investigated the effect of autologous mesenchymal stem cells (MSC) on multiple unmodified donor bone marrow (BM) infusions and vascularized skin graft outcome. BM-derived rat MSC were examined for phenotype and function. MSC/MSC-conditioned-medium suppressed IFN-gamma production by T cells and modified DC function. Infusions of MSC with one-time BM improved vascularized skin graft survival, while with one-two-times BM reversed graft versus host disease (GVHD). Mixed chimerism was enhanced in recipients given two-four-times BM with MSC infusions. Interestingly, four-times BM infusions with MSC delayed GVHD onset, reduced host tissue damage and enhanced vascularized skin allograft survival compared to four-times BM alone. These data demonstrate that, the co-infusion of MSC with unmodified BM limit the toxicity of allogeneic BM transplantation, enhance mixed chimerism and improve vascularized skin graft survival. These findings provide insights for the development of autologous MSC-based BM transplantation and prevention of graft rejection or treatment of autoimmunity.

Long-term survival of limb allografts induced by pharmacologically conditioned, donor alloantigen-pulsed dendritic cells without maintenance immunosuppression.

BACKGROUND: We showed recently that limb allograft survival could be enhanced by administration of alloantigen (Ag)-pulsed immature dendritic cells (DC) after transplantation. Since indefinite graft survival was not achieved, we have further modified the DC by pharmacologic (rapamycin; Rapa) conditioning and ascertained their influence on graft survival, without continued immunosuppressive therapy. METHODS: We compared the ability of donor Ag-pulsed, Rapa-conditioned rat myeloid DC (Rapa DC) and control DC (CTR DC) to inhibit alloreactive T-cell responses after limb transplantation in antilymphocyte serum (ALS)-treated recipients given a short postoperative course of cyclosporine (CsA). RESULTS: Both DC populations expressed similar levels of major histocompatibility complex (MHC) II, CD40 and CD54, but Rapa DC expressed lower CD86. After toll-like receptor activation, both populations produced minimal interleukin (IL)-12p70, but Rapa DC secreted lower levels of IL-6 and IL-10. The capacity of DCs to stimulate T-cell proliferation in mixed leukocyte reactions was very low. Pulsing of the DC with donor Ag did not alter their phenotype or function. Interestingly, posttransplant administration of donor Ag-pulsed Rapa DC to rats given perioperative ALS and 21 days CsA significantly delayed graft rejection and promoted long-term (>125 days) graft survival. AlloAg-pulsed Rapa DC induced T-cell hyporesponsiveness and promoted the generation of IL-10-secreting CD4 T cells upon ex vivo challenge. CONCLUSIONS: Infusion of donor Ag-pulsed, Rapa-conditioned DC after composite tissue transplantation can prevent rejection of the grafts, including skin, across a full MHC mismatch and in the absence of continued immunosuppressive therapy.

Prolongation of composite tissue allograft survival by immature recipient dendritic cells pulsed with donor antigen and transient low-dose immunosuppression.

BACKGROUND: Composite tissue allograft transplantation is limited by risks of long-term immunosuppression. The authors investigated whether short-term immunosuppression combined with recipient immature dendritic cells pulsed with donor antigens promotes composite tissue allograft survival. METHODS: Orthotopic hind-limb transplants were performed (day 0) from Wistar-Furth (RT1) to Lewis (RT1(u)) rats. Recipient dendritic cells were propagated from bone marrow with granulocyte-macrophage colony-stimulating factor (bone marrow-derived dendritic cells) and pulsed with or without donor splenic cell lysate. Recipients were as follows: group I, control; group II, cyclosporine (10 mg/kg/day, days 0 through 6, intraperitoneally); group III, antilymphocyte serum plus cyclosporine (days -4 and +1, intraperitoneally); and groups IV and V, cyclosporine plus antilymphocyte serum, combined with 7 x 10(6) untreated or donor cell lysate-pulsed bone marrow-derived dendritic cells (days +7 and +14, intravenously), respectively. Epidermolysis/desquamation of donor skin defined rejection. Mixed leukocyte reaction determined recipient T-cell reactivity to donor. Tissue samples were obtained at 3 weeks and on the day of rejection. Groups comprised six or seven rats. RESULTS: Donor alloantigen-pulsed bone marrow-derived dendritic cells (group V) significantly prolonged median composite tissue allograft survival time (32.0 days) compared with groups II (18.0 days, p = 0.0012), III (22.5 days, p = 0.0043), and IV (26.5 days, p = 0.0043). Splenic T cells in group V exhibited hyporesponsiveness to donor alloantigen in mixed leukocyte reaction. Interestingly, the graft muscle component in the bone marrow-derived dendritic cell-treated group (group V) showed significant reduction in mononuclear cell infiltration relative to group II (p = 0.0317). CONCLUSIONS: Donor alloantigen-pulsed recipient bone marrow-derived dendritic cells combined with transient T-cell-directed immunosuppression significantly prolonged composite tissue allograft survival across a full major histocompatibility complex barrier. This may represent the basis for a novel, clinically applicable strategy to promote composite tissue allograft survival with reduced systemic immunosuppression.

Rapamycin-conditioned, alloantigen-pulsed dendritic cells promote indefinite survival of vascularized skin allografts in association with T regulatory cell expansion.

Clinically-applicable protocols that promote tolerance to vascularized skin grafts may contribute to more widespread use of composite tissue transplantation. We compared the properties of alloantigen (Ag)-pulsed, rapamycin (Rapa)-conditioned and control bone marrow-derived host myeloid dendritic cells (DCs) and their potential, together with transient immunosuppression (anti-lymphocyte serum+cyclosporine), to promote long-term, vascularized skin graft survival in Lewis rats across a full MHC barrier. Both types of DCs expressed low levels of CD86, but Rapa DC expressed lower levels of MHC II and CD40 and were less stimulatory in MLR. While both Rapa and control DCs produced low levels of IL-12p70 and moderate levels of IL-6 and IL-10 following TLR ligation, Rapa DC secreted significantly lower levels of IL-6 and IL-10 in response to LPS. Donor Ag-pulsed Rapa DC, but not control DC, induced long-term skin graft survival (median survival time >133 days) when administered 7 and 14 days post-transplant. Circulating T cells in hosts with long-surviving grafts were hyporesponsive to donor alloAg stimulation, but proliferated in response to third-party stimulation and produced IFN-gamma and IL-10. When recipients of long-surviving grafts were challenged with skin grafts, donor but not third-party grafts were prolonged, suggesting underlying regulatory mechanisms. Both flow cytometry and immunohistochemical analysis revealed that donor Ag-pulsed Rapa DC infusion expanded CD4+ Foxp3+ Treg in recipients' spleens, graft-associated lymph nodes and the graft. These data demonstrate for the first time that pharmacologically-modified, donor Ag-pulsed host DC administered post-transplant can promote indefinite vascularized skin graft survival, associated with Treg expansion.

Use of dendritic cells in drug selection, development and therapy.

BACKGROUND: Dendritic cells (DC) have the unique ability to induce immunity against tumors and various pathogens or to promote tolerance in autoimmunity and transplantation. Hence, they are central to the regulation of immune responses. OBJECTIVE/METHODS: Due to the unique tolerogenic ability of DC, understanding some of the key molecules that regulate DC function may help with targeting the relevant signals in DC as therapeutic options for many disease conditions. DC are also targets of drugs, and many of the anti-inflammatory and pharmaceutical agents used to prevent autoimmunity or inhibit graft rejection interfere with DC function. RESULTS/CONCLUSION: The drug-induced changes in DC may provide information for the selection of drugs and further drug discovery along with the use of DC as adjuvant in the treatment of autoimmunity and prevention of graft rejection in transplantation.

Strauch's technique for epigastric free flaps in rats revisited: a simple and effective method to increase patency rates.

Described by Strauch and Murray in 1967, the rodent epigastric free flap remains a versatile tool for microsurgery research and training. We report herein three sequential phases of our quest to improve efficiency and effectiveness of the original technique, making it more accessible to more microsurgeons. Ninety-six allotransplants were performed. Surgical technique, complication rates, clinical findings, and histopathologic correlation of each phase are reported. In phase I, two experienced microsurgeons employed the original technique and succeeded in 77% of the procedures. In phase II, two junior microsurgeons achieved a patency rate of 16.6% using the same technique, as opposed to 100% in phase III, utilizing the not-yet-described simplified flow-thru technique. Although patency rate using the original method varies from 9 to 78% (according to other reports), this technical modification can increase even the less experienced microsurgeons' success rates, perpetuating the use of Strauch's epigastric flap in experimental microsurgery.

Allopeptide-pulsed dendritic cells and composite tissue allograft survival.

Composite tissue allografts (CTAs) contain their own reservoir of vascularized bone marrow, offering novel aspects for the induction of donor-specific tolerance. Additionally, the manipulation of recipient dendritic cells, pulsed with donor allopeptide, has been shown to engender solid organ allograft survival. To exploit these modalities, we have developed a protocol utilizing injection of recipient bone marrow-derived dendritic cells (BMDCs) pulsed with a donor-derived peptide for use in CTA transplantation. Six days prior to orthotopic hind-limb transplantation, Lewis rats received IV injection of donor allopeptide-pulsed, recipient BMDCs, in conjunction with a single dose of anti-lymphocyte serum. Control groups displayed signs of allograft rejection within 5 days postoperatively. Animals within the primary experimental cohort demonstrated prolongation of graft survival to an average of 8 days, and exhibited low numbers of donor T cells. The use of BMDCs in conjunction with transient immunosuppression has potential therapeutic application for induction of donor-antigen-specific tolerance to hind limb allografts.

Intrinsic ability of GM+IL-4 but not Flt3L-induced rat dendritic cells to promote allogeneic T cell hyporesponsiveness.

The influence of GM+IL-4 and Flt3 ligand (FL) on phenotype and function of BM-derived DC from Lewis rats was investigated. GM+IL-4-induced DC, despite expression of CD80/CD86, were less stimulatory than FL-induced DC that expressed low CD80/CD86 and were efficient stimulators of allogeneic T cells. GM+IL-4 DC were CD11b+ OX62lo, whereas FL DC were CD11blo OX62+. Following activation, GM+IL-4 DC produced IL-10 and IL-6, but no IL-12p70, and were resistant to further maturation. FL DC produced IL-12p70, IFN-alpha/beta, IL-10 and IL-6 and underwent maturation. Repeated stimulation of T cells with GM+IL-4 DC inhibited proliferation, cytokine production and induced early T cell apoptosis. FL DC-activated T cells produced large amounts of IFN-gamma/IL-10 and exhibited late T cell apoptosis/necrosis. In vivo, GM+IL-4 DC induced alloAg-specific hyporesponsiveness following T cell restimulation. These results demonstrate that GM+IL-4 DC display intrinsic regulatory properties, inducing passive-cell-death in T cells with potential for inactivation/regulation of alloreactive T cells in transplantation.

Dendritic cell immunotherapy for autoimmune diabetes.

Dendritic cells (DC) play important roles in the initiation of immune responses and maintenance of self-tolerance. We have been studying the role of DC in the pathogenesis of type 1 diabetes and exploring the ability of specific DC subsets to prevent diabetes in non-obese diabetic (NOD) mice. DC subsets that prevent diabetes in this model have a mature phenotype and induce the production of regulatory Th2 cells. We review here recent advances in this area and highlight the importance of optimizing culture conditions and purification methods in the isolation of therapeutic DC.

Polarization of naive T cells into Th1 or Th2 by distinct cytokine-driven murine dendritic cell populations: implications for immunotherapy.

Dendritic cells (DCs) activate T cells and regulate their differentiation into T helper cell type 1 (Th1) and/or Th2 cells. To identify DCs with differing abilities to direct Th1/Th2 cell differentiation, we cultured mouse bone marrow progenitors in granulocyte macrophage-colony stimulating factor (GM), GM + interleukin (IL)-4, or GM + IL-15 and generated three distinct DC populations. The GM + IL-4 DCs expressed high levels of CD80/CD86 and major histocompatibility complex (MHC) class II and produced low levels of IL-12p70. GM and GM + IL-15 DCs expressed low levels of CD80/CD86 and MHC class II. The GM + IL-15 DCs produced high levels of IL-12p70 and interferon (IFN)-gamma, whereas GM DCs produced only high levels of IL-12p70. Naive T cells stimulated with GM + IL-4 DCs secreted high levels of IL-4 and IL-5 in addition to IFN-gamma. In contrast, the GM + IL-15 DCs induced higher IFN-gamma production by T cells with little or no Th2 cytokines. GM DCs did not induce T cell polarization, despite producing large amounts of IL-12p70 following activation. A similar pattern of T cell activation was observed after in vivo administration of DCs. These data suggest that IL-12p70 production alone, although necessary for Th1 differentiation, is not sufficient to induce Th1 responses. These studies have implications for the use of DC-based vaccines in immunotherapy of cancer and other clinical conditions.

Dendritic cells transduced to express interleukin-4 prevent diabetes in nonobese diabetic mice with advanced insulitis.

Our previous studies demonstrated that adoptive transfer of dendritic cells (DC) prevents diabetes in young nonobese diabetic (NOD) mice by inducing regulatory T(H)2 cells. In this report, as a means of treating NOD mice with more advanced insulitis, we infected DC with adenoviral vectors expressing interleukin (IL)-4 (Ad.IL-4), eGFP (Ad.eGFP), or empty vector (Ad psi 5). DC infected with any of the Ad vectors expressed higher levels of CD40, CD80, and CD86 molecules than uninfected DC and Ad.IL-4 DC produced IL-4 after lipopolysaccharide (LPS) and interferon (IFN)-gamma stimulation. Ad-infected DC efficiently stimulated allogeneic T cells, and cultures of T cells with Ad.IL-4 DC produced lower levels of IFN-gamma and marginally higher levels of IL-4. In vivo studies demonstrated that the Ad.eGFP DC trafficked to the pancreatic lymph nodes within 24 hr of intravenous administration, and could be visualized in the T cell areas of the spleen. The intrapancreatic IFN-gamma:IL-4 or IFN-gamma:IL-10 cytokine ratios were lower in 10-week-old mice treated with Ad.IL-4 DC, and these mice were significantly protected from disease. These results demonstrate, for the first time, that genetically modified DC can prevent diabetes in the context of advanced insulitis.

Regulatory Th2 response induced following adoptive transfer of dendritic cells in prediabetic NOD mice.

We previously demonstrated that immunotherapy with dendritic cells (DC) prevented diabetes development in prediabetic NOD mice and that this effect was optimal when using a stimulatory DC population generated from bone marrow cells cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. In this study, we have investigated the mechanism by which GM-CSF- and IL-4-cultured DC prevent diabetes in prediabetic NOD mice. Histological analysis of pancreatic tissue from DC-treated mice revealed a reduction in the severity of insulitis compared to controls. Analysisof the T cell response in DC-treated mice suggested a general shift towards a Th2-dominated response, as determined by cytokine production following either concanavalin A or anti-TCR stimulation. Furthermore, sorted CD45RB(lo) CD25+ CD4+ T cells from the spleen of DC-treated mice produced high amounts of Th2 cytokines following anti-TCR stimulation, suggesting that these cells are responsible for the apparent Th2 shift. We conclude that DC therapy may have corrected the immunoregulatory defect in the NOD mouse, thus restoring a balance between pathogenic Th1 cells and protective Th2 cells.