Jamil Azzi

Congenic mesenchymal stem cell therapy reverses hyperglycemia in experimental type 1 diabetes

OBJECTIVE A number of clinical trials are underway to test whether mesenchymal stem cells (MSCs) are effective in treating various diseases, including type 1 diabetes. Although this cell therapy holds great promise, the optimal source of MSCs has yet to be determined with respect to major histocompatibility complex matching. Here, we examine this question by testing the ability of congenic MSCs, obtained from the NOR mouse strain, to reverse recent-onset type 1 diabetes in NOD mice, as well as determine the immunomodulatory effects of NOR MSCs in vivo. RESEARCH DESIGN AND METHODS NOR MSCs were evaluated with regard to their in vitro immunomodulatory function in the context of autoreactive T-cell proliferation and dendritic cell (DC) generation. The in vivo effect of NOR MSC therapy on reversal of recent-onset hyperglycemia and on immunogenic cell subsets in NOD mice was also examined. RESULTS NOR MSCs were shown to suppress diabetogenic T-cell proliferation via PD-L1 and to suppress generation of myeloid/inflammatory DCs predominantly through an IL-6-dependent mechanism. NOR MSC treatment of experimental type 1 diabetes resulted in long-term reversal of hyperglycemia, and therapy was shown to alter diabetogenic cytokine profile, to diminish T-cell effector frequency in the pancreatic lymph nodes, to alter antigen-presenting cell frequencies, and to augment the frequency of the plasmacytoid subset of DCs. CONCLUSIONS These studies demonstrate the inimitable benefit of congenic MSC therapy in reversing experimental type 1 diabetes. These data should benefit future clinical trials using MSCs as treatment for type 1 diabetes.

Calcineurin Inhibitors: 40 Years Later, Can’t Live Without …

Calcineurin inhibitors (CNIs) revolutionized the field of organ transplantation and remain the standard of care 40 years after the discovery of cyclosporine. The early impressive results of cyclosporine in kidney transplant recipients led to its subsequent use in other organ transplant recipients and for treatment of a variety of autoimmune diseases as well. In this review, we examine the discovery of CNIs, their mechanism of action, preclinical and clinical studies with CNIs, and the usage of CNIs in nontransplant recipients. We review the mechanisms of renal toxicity associated with CNIs and the recent efforts to avoid or reduce usage of these drugs. Although minimization strategies are possible, safe, and of potential long-term benefit, complete avoidance of CNIs has proven to be more challenging than initially thought.

Polylactide-cyclosporin A nanoparticles for targeted immunosuppression

Polymeric nanoparticles (NPs), prepared via coprecipitation of drugs and polymers, are promising drug delivery vehicles for treating diseases with improved efficacy and reduced toxicity. Here, we report an unprecedented strategy for preparing polylactide‐cyclosporine A (PLA‐CsA) NPs (termed CsA‐NPs) through CsA‐initiated ring‐opening polymerization of lactide (LA) followed by nanoprecipitation. The resulting CsA‐NPs have sub‐100 nm sizes and narrow particle size distributions, and release CsA in a sustained manner without a “burst”‐release effect. Both free CsA and CsA‐NPs displayed comparable suppression of T‐cell proliferation and production of inflammatory cytokines in various T‐cell assays in a dose‐dependent manner. The IC50 values for CsA and CsA‐NPs were 27.5 and 72.0 ng/ml, respectively. As lymph nodes are the main loci for T‐cell activation, we coupled dendritic cells (DCs) with CsA‐NPs and successfully delivered CsA selectively to the lymph nodes. Our studies indicated that CsA‐NPs could be internalized in the DCs with a sustained release of CsA to the culture medium, suppressing alloreactive T‐cell proliferation. Allogeneic DCs loaded with CsA‐NPs were able to migrate to the draining lymph nodes where the T‐cell priming was significantly reduced without any systemic release. This innovative nanoparticulate CsA delivery technology constitutes a strong basis for future targeted delivery of immunosuppressive drugs with improved efficiency and reduced toxicity.—Azzi, J., Tang, L., Tong, R., El Haddad, N., Akiyoshi, T., Mfarrej, B., Moore, R., Yang, S., Jurewicz, M., Ichimura, T., Lindeman, N., Cheng, J., Abdi, R. Polylactide‐cyclosporin A nanoparticles for targeted immunosuppression. FASEB J. 24, 3927–3938 (2010). www.fasebj.org

Mesenchymal stem cells express serine protease inhibitor to evade the host immune response.

Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow-derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6(-/-)) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6(-/-) MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell-mediated death in vitro. In addition, SPI6(-/-) MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases.

Long-term outcomes of kidney transplantation across a positive complement-dependent cytotoxicity crossmatch.

Background More than 30% of potential kidney transplant recipients have pre-existing anti–human leukocyte antigen antibodies. This subgroup has significantly lower transplant rates and increased mortality. Desensitization has become an important tool to overcome this immunological barrier. However, limited data is available regarding long-term outcomes, in particular for the highest risk group with a positive complement-dependent cytotoxicity crossmatch (CDC XM) before desensitization. Methods Between 2002 and 2010, 39 patients underwent living-kidney transplantation across a positive CDC XM against their donors at our center. The desensitization protocol involved pretransplant immunosuppression, plasmapheresis, and low-dose intravenous immunoglobulin±rituximab. Measured outcomes included patient survival, graft survival, renal function, rates of rejection, infection, and malignancy. Results The mean and median follow-up was 5.2 years. Patient survival was 95% at 1 year, 95% at 3 years, and 86% at 5 years. Death-censored graft survival was 94% at 1 year, 88% at 3 years, and 84% at 5 years. Uncensored graft survival was 87% at 1 year, 79% at 3 years, and 72% at 5 years. Twenty-four subjects (61%) developed acute antibody-mediated rejection of the allograft and one patient lost her graft because of hyperacute rejection. Infectious complications included pneumonia (17%), BK nephropathy (10%), and CMV disease (5%). Skin cancer was the most prevalent malignancy in 10% of patients. There were no cases of lymphoproliferative disorder. Mean serum creatinine was 1.7±1 mg/dL in functioning grafts at 5 years after transplantation. Conclusion Despite high rates of early rejection, desensitization in living-kidney transplantation results in acceptable 5-year patient and graft survival rates.

Immunological aspects of pancreatic islet cell transplantation.

Type 1 diabetes mellitus (T1DM) is one of the most common diseases of childhood. Insulin discovery changed the clinical course of T1DM from an acutely fatal disease to a chronic disease, but this discovery was later found to be inefficient to control its long-term complications. Whole-pancreas and islet cell transplantation seem to provide a potential solution by restoring the normal physiology of glucose–insulin homeostasis. Although islet transplantation is less invasive than whole-pancreas transplantation, the insulin-free state after islet transplantation remained low (10%) at 5 years after surgery. Here, we will present the specific immunologic challenges that are specific to islet cell transplantation, including instant blood-mediated inflammatory reaction and the recurrence of autoimmunity. We will also briefly discuss the immunosuppressive regimens used and the recent radiologic techniques in the detection of engraftment and early rejection of islet cells.

Essential role of PDL1 expression on nonhematopoietic donor cells in acquired tolerance to vascularized cardiac allografts.

The PD1:PDL1 pathway is an essential negative costimulatory pathway that plays a key role in regulating the alloimune response. PDL1 is expressed not only on antigen‐presenting cells (APCs) but also cardiac endothelium. In this study, we investigated the importance of PDL1 expression on donor cardiac allograft in acquired transplantation tolerance in a fully MHC‐mismatched model. We generated PDL1 chimeric mice on B6 background that expressed PDL1 on either hematopoietic cells or nonhematopoietic cells of the heart. Sham animals were used as controls. These hearts were then transplanted into BALB/c recipients and treated with CTLA4‐Ig to induce tolerance. Cardiac endothelium showed significant expression of PDL1, which was upregulated upon transplantation. While the absence of PDL1 on hematopoietic cells of the heart resulted in delayed rejection and prevented long‐term tolerance in most but not all recipients, we observed an accelerated and early graft rejection of all donor allografts that lacked PDL1 on the endothelium. Moreover, PDL1‐deficient endothelium hearts had significant higher frequency of IFN‐γ‐producing alloreactive cells as well as higher frequency of CD8+ effector T cells. These findings demonstrate that PDL1 expression mainly on donor endothelium is functionally important in a fully allogeneic mismatched model for the induction of cardiac allograft tolerance.

Immunosuppressive Activity of Size-Controlled PEG-PLGA Nanoparticles Containing Encapsulated Cyclosporine A

We encapsulated cyclosporine A (CsA) in poly(ethylene glycol)-b-poly(d,l-lactide-co-glycolide) (PEG-PLGA) nanoparticles (NPs) by nanoprecipitation of CsA and PEG-PLGA. The resulting CsA/PEG-PLGA-NPs were <100 nm in diameter with a narrow particle size distribution. The NP size could be controlled by tuning the polymer concentration, solvent, or water/solvent ratio during formulation. The PEGylated NPs maintained non-aggregated in salt solution. Solid NPs lyoprotected with bovine serum albumin were prepared for the convenience of storage and transportation. The release kinetics of CsA (55.6% released on Day 1) showed potential for maintaining therapeutic CsA concentrations in vivo. In T-cell assays, both free CsA and CsA/PEG-PLGA-NPs suppressed T-cell proliferation and production of inflammatory cytokines dose dependently. In a mixed lymphocyte reaction assay, the IC50 values for free CsA and CsA/PEG-PLGA-NPs were found to be 30 and 35 ng/mL, respectively. This nanoparticulate CsA delivery technology constitutes a strong basis for future targeted delivery of immunosuppressive drugs with improved efficiency and potentially reduced toxicity.

The renin-angiotensin system: an old, newly discovered player in immunoregulation

Clinical and experimental studies that discuss the different immune functions of the renin-angiotensin system (RAS) in kidney diseases were reviewed, with emphasis on studies of kidney transplantation. The RAS has been shown to affect both the innate and adaptive immune responses and has a well-established role in fibrinogenesis. Of special clinical interest is the ability of the RAS to activate the transforming growth factor beta(1) and the Smad pathways leading to fibrinogenesis. In addition to the RAS enhancing effect on the activity of T cells, several components of the RAS have also been shown to be chemotactic to macrophages, T cells, and natural killer cells. Experimental studies have found that RAS blockade decreases the histologic lesions of chronic allograft nephropathy but can enhance acute graft vasculopathy. Although the blockade of RAS has been commonly practiced to reduce posttransplantation hypertension, proteinuria, and erythrocytosis, however, its role in prolonging graft survival is not well established.

PI3kα and STAT1 Interplay Regulates Human Mesenchymal Stem Cell Immune Polarization

The immunomodulatory capacity of mesenchymal stem cells (MSCs) is critical for their use in therapeutic applications. MSC response to specific inflammatory cues allows them to switch between a proinflammatory (MSC1) or anti‐inflammatory (MSC2) phenotype. Regulatory mechanisms controlling this switch remain to be defined. One characteristic feature of MSC2 is their ability to respond to IFNγ with induction of indoleamine 2,3‐dioxygenase (IDO), representing the key immunoregulatory molecule released by human MSC. Here, we show that STAT1 and PI3Kα pathways interplay regulates IFNγ‐induced IDO production in MSC. Chemical phosphoinositide 3‐kinase (PI3K) pan‐inhibition, PI3Kα‐specific inhibition or shRNA knockdown diminished IFNγ‐induced IDO production. This effect involved PI3Kα‐mediated upregulation of STAT1 protein levels and phosphorylation at Ser727. Overexpression of STAT1 or of a constitutively active PI3Kα mutant failed to induce basal IDO production, but shifted MSC into an MSC2‐like phenotype by strongly enhancing IDO production in response to IFNγ as compared to controls. STAT1 overexpression strongly enhanced MSC‐mediated T‐cell suppression. The same effect could be induced using short‐term pretreatment of MSC with a chemical inhibitor of the counter player of PI3K, phosphatase and tensin homolog. Finally, downregulation of STAT1 abrogated the immunosuppressive capacity of MSC. Our results for the first time identify critical upstream signals for the induced production of IDO in MSCs that could be manipulated therapeutically to enhance their immunosuppressive phenotype. Stem Cells 2015;33:1892–1901