Person:

Abdi, Reza

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 IC(_{50}) 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.

OBJECTIVES—To investigate a B-cell–depleting strategy to reverse diabetes in naïve NOD mice. RESEARCH DESIGN AND METHODS—We targeted the CD22 receptor on B-cells of naïve NOD mice to deplete and reprogram B-cells to effectively reverse autoimmune diabetes. RESULTS—Anti-CD22/cal monoclonal antibody (mAb) therapy resulted in early and prolonged B-cell depletion and delayed disease in pre-diabetic mice. Importantly, when new-onset hyperglycemic mice were treated with the anti-CD22/cal mAb, 100% of B-cell–depleted mice became normoglycemic by 2 days, and 70% of them maintained a state of long-term normoglycemia. Early therapy after onset of hyperglycemia and complete B-cell depletion are essential for optimal efficacy. Treated mice showed an increase in percentage of regulatory T-cells in islets and pancreatic lymph nodes and a diminished immune response to islet peptides in vitro. Transcriptome analysis of reemerging B-cells showed significant changes of a set of proinflammatory genes. Functionally, reemerging B-cells failed to present autoantigen and prevented diabetes when cotransferred with autoreactive (CD4^+) T-cells into NOD.SCID hosts. CONCLUSIONS—Targeting CD22 depletes and reprograms B-cells and reverses autoimmune diabetes, thereby providing a blueprint for development of novel therapies to cure autoimmune diabetes.

Type 1 diabetes (T1D) remains a major health problem worldwide, with a steadily rising incidence yet no cure. Phosphoinositide 3-kinase-γ (PI3Kγ), a member of a family of lipid kinases expressed primarily in leukocytes, has been the subject of substantial research for its role in inflammatory diseases. However, the role of PI3Kγ inhibition in suppressing autoimmune T1D remains to be explored. We tested the role of the PI3Kγ inhibitor AS605240 in preventing and reversing diabetes in NOD mice and assessed the mechanisms by which this inhibition abrogates T1D. Our data indicate that the PI3Kγ pathway is highly activated in T1D. In NOD mice, we found upregulated expression of phosphorylated Akt (PAkt) in splenocytes. Notably, T regulatory cells (Tregs) showed significantly lower expression of PAkt compared with effector T cells. Inhibition of the PI3Kγ pathway by AS605240 efficiently suppressed effector T cells and induced Treg expansion through the cAMP response element-binding pathway. AS605240 effectively prevented and reversed autoimmune diabetes in NOD mice and suppressed T-cell activation and the production of inflammatory cytokines by autoreactive T cells in vitro and in vivo. These studies demonstrate the key role of the PI3Kγ pathway in determining the balance of Tregs and autoreactive cells regulating autoimmune diabetes.

With a steady increase in its incidence and lack of curative treatment, type 1 diabetes (T1D) has emerged as a major health problem worldwide. To design novel effective therapies, there is a pressing need to identify regulatory targets controlling the balance of autoreactive to regulatory-T-cells (Tregs). We previously showed that the inhibition of the γ-subunit of the Phosphoinositide-3-kinase (PI3K), significantly suppress autoimmune-diabetes. To further delineate the mechanisms and the selectivity of specific immune modulation by PI3Kγ-inhibition, we developed a new NOD mouse model of T1D lacking the γ-subunit of PI3K. Strikingly, the loss of PI3Kγ protected 92% of the NOD-mice from developing spontaneous diabetes. The NOD.PI3Kγ-/- mice are protected from insulitis secondary to a defect in CD4 and CD8 autoreactive-T-cells activation and survival. In addition, PI3Kγ-deficiency promoted Treg generation in-vitro and in-vivo. Furthermore, PI3Kγ-inhibitor (AS605240) inhibited proliferation and cytokine production of a human CD4+ T-cell clone specific for GAD555-567 peptide that was isolated from a patient with T1D. These studies demonstrate the key role of the PI3Kγ pathway in regulating autoimmune-diabetes and provide rationales for future devise of anti- PI3Kγ therapy in T1D.

Mesenchymal stem cells (MSCs) are pluripotent stromal cells that have the potential to give rise to cells of diverse lineages. Interestingly, MSCs can be found in virtually all postnatal tissues. The main criteria currently used to characterize and identify these cells are the capacity for self-renewal and differentiation into tissues of mesodermal origin, combined with a lack in expression of certain hematopoietic molecules. Because of their developmental plasticity, the notion of MSC-based therapeutic intervention has become an emerging strategy for the replacement of injured tissues. MSCs have also been noted to possess the ability to impart profound immunomodulatory effects in vivo. Indeed, some of the initial observations regarding MSC protection from tissue injury once thought mediated by tissue regeneration may, in reality, result from immunomodulation. Whereas the exact mechanisms underlying the immunomodulatory functions of MSC remain largely unknown, these cells have been exploited in a variety of clinical trials aimed at reducing the burden of immune-mediated disease. This article focuses on recent advances that have broadened our understanding of the immunomodulatory properties of MSC and provides insight as to their potential for clinical use as a cell-based therapy for immune-mediated disorders and, in particular, type 1 diabetes.

Background: A number of studies have demonstrated the role of CX3CR1 in regulating the migration of monocytes into peripheral tissue and their transformation into dendritic cell (DC). No data are yet available on the importance of chemokine pathways in regulating homeostasis of DC in heart transplants. Recently, we showed that recipients of heart allografts from CX3CR1−/− donors show longer survival. To assess the trafficking of dDC, we have developed and tested a novel in vivo imaging tool in CX3CR1GFP/+ DC (B6 background) heart graft into BALB/c recipient model. Results: Majority of GFP+ cells were noted in the middle of cardiac myocyte. However few hours post transplant, they experienced morphological changes including stretching their extensions (3 and 24 h). However, images from 72 h at cardiac graft showed many of GFP+ cells moved to vessel areas. GFP+ cells were detected in near vessel wall. Only one GFP+ cell was observed in three lymph nodes (two mesenteric and one inguinal) (72 h). Conclusion: Our data indicate that immediately post transplant dDC undergo morphological changes and traffic out of the organs via systemic circulation. While, we still noted presence of dDC in the transplanted organs, their trafficking to lymphoid tissue remains to be fully explored.

SUMMARY Active-targeted delivery to lymph nodes represents a major advance toward more effective treatment of immune-mediated disease. The MECA79 antibody recognizes peripheral node address in molecules expressed by high endothelial venules of lymph nodes. By mimicking lymphocyte trafficking to the lymph nodes, we have engineered MECA79-coated microparticles containing an immunosuppressive medication, tacrolimus. Following intravenous administration, MECA79-bearing particles showed marked accumulation in the draining lymph nodes of transplanted animals. Using an allograft heart transplant model, we show that targeted lymph node delivery of microparticles containing tacrolimus can prolong heart allograft survival with negligible changes in tacrolimus serum level. Using MECA79 conjugation, we have demonstrated targeted delivery of tacrolimus to the lymph nodes following systemic administration, with the capacity for immune modulation in vivo.

Allogeneic hematopoietic cell transplantation (HCT) is the treatment of choice for a variety of hematologic malignancies and disorders. Unfortunately, acute graft-versus-host disease (GVHD) is a frequent complication of HCT. While substantial research has identified clinical, genetic and proteomic risk factors for acute GVHD, few studies have sought to develop risk prediction tools that quantify absolute risk. Such tools would be useful for: optimizing donor selection; guiding GVHD prophylaxis, post-transplant treatment and monitoring strategies; and, recruitment of patients into clinical trials. Using data on 9,651 patients who underwent first allogeneic HLA-identical sibling or unrelated donor HCT between 01/1999-12/2011 for treatment of a hematologic malignancy, we developed and evaluated a suite of risk prediction tools for: (i) acute GVHD within 100 days post-transplant and (ii) a composite endpoint of acute GVHD or death within 100 days post-transplant. We considered two sets of inputs: (i) clinical factors that are typically readily-available, included as main effects; and, (ii) main effects combined with a selection of a priori specified two-way interactions. To build the prediction tools we used the super learner, a recently developed ensemble learning statistical framework that combines results from multiple other algorithms/methods to construct a single, optimal prediction tool. Across the final super learner prediction tools, the area-under-the curve (AUC) ranged from 0.613–0.640. Improving the performance of risk prediction tools will likely require extension beyond clinical factors to include biological variables such as genetic and proteomic biomarkers, although the measurement of these factors may currently not be practical in standard clinical settings.

Ischemia reperfusion injuries (IRI) are unavoidable in solid organ transplantation. IRI augments alloimmunity but the mechanisms involved are poorly understood. Herein, we examined the effect of IRI on antigen specific alloimmunity. We demonstrate that ischemia promotes alloimmune activation, leading to more severe histological features of rejection, and increased CD4+ and CD8+ T cell graft infiltration, with a predominantly CD8+ IFNγ+ infiltrate. This process is dependent on the presence of alloreactive CD4+ T cells, where depletion prevented infiltration of ischemic grafts by CD8+ IFNγ+ T cells. IL-6 is a known driver of ischemia-induced rejection. Herein, depletion of donor antigen-presenting cells reduced ischemia-induced CD8+ IFNγ+ allograft infiltration, and improved allograft outcomes. Following prolonged ischemia, accelerated rejection was observed despite treatment with CTLA4Ig, indicating that T cell costimulatory blockade failed to overcome the immune activating effect of IRI. However, despite severe ischemic injury, treatment with anti-IL-6 and CTLA4Ig blocked IRI-induced alloimmune injury and markedly improved allograft survival. We describe a novel pathway where IRI activates innate immunity, leading to upregulation of antigen specific alloimmunity, resulting in chronic allograft injury. Based on these findings, we describe a clinically relevant treatment strategy to overcome the deleterious effect of IRI, and provide superior long-term allograft outcomes.

Phosphatidylinositol-3-kinases (PI3K) γ and δ are preferentially enriched in leukocytes, and defects in these signaling pathways have been shown to impair T cell activation. The effects of PI3Kγ and PI3Kδ on alloimmunity remain underexplored. Here, we show that both PI3Kγ −/− and PI3Kδ D910A/D910A mice receiving heart allografts have suppression of alloreactive T effector cells and delayed acute rejection. However, PI3Kδ mutation also dampens regulatory T cells (Treg). After treatment with low dose CTLA4-Ig, PI3Kγ −/−, but not PI3Κδ D910A/D910A, recipients exhibit indefinite prolongation of heart allograft survival. PI3Kδ D910A/D910A Tregs have increased apoptosis and impaired survival. Selective inhibition of PI3Kγ and PI3Kδ (using PI3Kδ and dual PI3Kγδ chemical inhibitors) shows that PI3Kγ inhibition compensates for the negative effect of PI3Kδ inhibition on long-term allograft survival. These data serve as a basis for future PI3K-based immune therapies for transplantation.