Maryam Feili-Hariri

Receptor-mediated endocytosis of iron-oxide particles provides efficient labeling of dendritic cells for in vivo MR imaging

Dendritic cells (DCs) function as antigen presenting cells in vivo and play a fundamental role in numerous diseases. New methods are described for high‐efficiency intracellular labeling of DCs with superparamagnetic iron‐oxide (SPIO) utilizing a receptor‐mediated endocytosis (RME) mechanism. Bone marrow‐derived DCs or a fetal skin‐derived DC line were incubated with SPIO conjugated to anti‐CD11c monoclonal antibody (mAb) under conditions favoring RME. These cells exhibited approximately a 50‐fold increase in uptake relative to DCs incubated with SPIO without the mAb. Flow cytometry studies assaying cell surface markers showed a down‐modulation of CD11c, but no other changes in phenotype. Immunological function of the DCs was unmodified by the labeling, as determined by cytokine secretion assays. The RME mechanism was confirmed using electron microscopy, endocytosis inhibition assays, and incubation experiments with SPIO conjugated to mAbs against accessory molecules that are not expressed on DCs. Labeled DCs were injected into murine quadriceps and monitored in vivo for several days using MR microimaging at 11.7 T. DCs were observed to remain within the muscle for >24 hr. The use of RME is an efficient way to label immune cells for in vivo MRI and can be applied to a wide variety of cell types. Magn Reson Med 49:1006–1013, 2003.

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.

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.

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 CD45RBlo 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.

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 ow 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)‐γ, 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‐γ. In contrast, the GM + IL‐15 DCs induced higher IFN‐γ 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.

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.

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.

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 (RT1u) to Lewis (RT1l) 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 × 106 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.

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.

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 19F 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.