In vivo imaging of type 1 diabetes immunopathology using eye-transplanted islets in NOD mice.

AIMS/HYPOTHESIS: Autoimmune attack against the insulin-producing beta cells in the pancreatic islets results in type 1 diabetes. However, despite considerable research, details of the type 1 diabetes immunopathology in situ are not fully understood mainly because of difficult access to the pancreatic islets in vivo. METHODS: Here, we used direct non-invasive confocal imaging of islets transplanted in the anterior chamber of the eye (ACE) to investigate the anti-islet autoimmunity in NOD mice before, during and after diabetes onset. ACE-transplanted islets allowed longitudinal studies of the autoimmune attack against islets and revealed the infiltration kinetics and in situ motility dynamics of fluorescence-labelled autoreactive T cells during diabetes development. Ex vivo immunostaining was also used to compare immune cell infiltrations into islet grafts in the eye and kidney as well as in pancreatic islets of the same diabetic NOD mice. RESULTS: We found similar immune infiltration in native pancreatic and ACE-transplanted islets, which established the ACE-transplanted islets as reliable reporters of the autoimmune response. Longitudinal studies in ACE-transplanted islets identified in vivo hallmarks of islet inflammation that concurred with early immune infiltration of the islets and preceded their collapse and hyperglycaemia onset. A model incorporating data on ACE-transplanted islet degranulation and swelling allowed early prediction of the autoimmune attack in the pancreas and prompted treatments to intercept type 1 diabetes. CONCLUSIONS/INTERPRETATION: The current findings highlight the value of ACE-transplanted islets in studying early type 1 diabetes pathogenesis in vivo and underscore the need for timely intervention to halt disease progression.

Correction to: In vivo imaging of type 1 diabetes immunopathology using eye-transplanted islets in NOD mice.

Unfortunately there is a mistake in the presentation of the affiliations in this paper.

High-resolution, noninvasive longitudinal live imaging of immune responses.

Intravital imaging emerged as an indispensible tool in biological research, and a variety of imaging techniques have been developed to noninvasively monitor tissues in vivo. However, most of the current techniques lack the resolution to study events at the single-cell level. Although intravital multiphoton microscopy has addressed this limitation, the need for repeated noninvasive access to the same tissue in longitudinal in vivo studies remains largely unmet. We now report on a previously unexplored approach to study immune responses after transplantation of pancreatic islets into the anterior chamber of the mouse eye. This approach enabled (i) longitudinal, noninvasive imaging of transplanted tissues in vivo; (ii) in vivo cytolabeling to assess cellular phenotype and viability in situ; (iii) local intervention by topical application or intraocular injection; and (iv) real-time tracking of infiltrating immune cells in the target tissue.

Replenishable prevascularized cell encapsulation devices increase graft survival and function in the subcutaneous space.

Beta cell replacement therapy (BCRT) for patients with type 1 diabetes (T1D) improves blood glucose regulation by replenishing the endogenous beta cells destroyed by autoimmune attack. Several limitations, including immune isolation, prevent this therapy from reaching its full potential. Cell encapsulation devices used for BCRT provide a protective physical barrier for insulin-producing beta cells, thereby protecting transplanted cells from immune attack. However, poor device engraftment posttransplantation leads to nutrient deprivation and hypoxia, causing metabolic strain on transplanted beta cells. Prevascularization of encapsulation devices at the transplantation site can help establish a host vascular network around the implant, increasing solute transport to the encapsulated cells. Here, we present a replenishable prevascularized implantation methodology (RPVIM) that allows for the vascular integration of replenishable encapsulation devices in the subcutaneous space. Empty encapsulation devices were vascularized for 14 days, after which insulin-producing cells were inserted without disrupting the surrounding vasculature. The RPVIM devices were compared with nonprevascularized devices (Standard Implantation Methodology [SIM]) and previously established prevascularized devices (Standard Prevascularization Implantation Methodology [SPVIM]). Results show that over 75% of RPVIM devices containing stem cell-derived insulin-producing beta cell clusters showed a signal after 28 days of implantation in subcutaneous space. Notably, not only was the percent of RPVIM devices showing signal significantly greater than SIM and SPVIM devices, but the intraperitoneal glucose tolerance tests and histological analyses showed that encapsulated stem-cell derived insulin-producing beta cell clusters retained their function in the RPVIM devices, which is crucial for the successful management of T1D.

Selective deletion of human leukocyte antigens protects stem cell-derived islets from immune rejection.

Stem cell-based replacement therapies hold the promise to restore function of damaged or degenerated tissue such as the pancreatic islets in people with type 1 diabetes. Wide application of these therapies requires overcoming the fundamental roadblock of immune rejection. To address this issue, we use genetic engineering to create human pluripotent stem cells (hPSCs) in which the majority of the polymorphic human leukocyte antigens (HLAs), the main drivers of allogeneic rejection, are deleted. We retain the common HLA class I allele HLA-A2 and less polymorphic HLA-E/F/G to allow immune surveillance and inhibition of natural killer (NK) cells. We employ a combination of in vitro assays and humanized mouse models to demonstrate that these gene manipulations significantly reduce NK cell activity and T-cell-mediated alloimmune response against hPSC-derived islet cells. In summary, our approach produces hypoimmunogenic hPSCs that can be readily matched with recipients to avoid alloimmune rejection.

Precision Engineering of an Anti-HLA-A2 Chimeric Antigen Receptor in Regulatory T Cells for Transplant Immune Tolerance.

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-ζ signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

Low-dose carbon monoxide inhibits progressive chronic allograft nephropathy and restores renal allograft function.

Chronic allograft nephropathy (CAN) represents progressive deterioration of renal allograft function with fibroinflammatory changes. CAN, recently reclassified as interstitial fibrosis (IF) and tubular atrophy (TA) with no known specific etiology, is a major cause of late renal allograft loss and remains a significant deleterious factor of successful renal transplantation. Carbon monoxide (CO), an effector byproduct of heme oxygenase pathway, is known to have potent anti-inflammatory and antifibrotic functions. We hypothesized that inhaled CO would inhibit fibroinflammatory process of CAN and restore renal allograft function, even when the treatment was initiated after CAN was established. Lewis rat kidney grafts were orthotopically transplanted into binephrectomized allogenic Brown Norway rats under brief tacrolimus (0.5 mg/kg im, days 0-6). At day 60, CO (20 ppm) inhalation was initiated to recipients and continued until day 150 or animal death. Development of CAN was confirmed at day 60 with decreased creatinine clearance (CCr), significant proteinuria, and histopathological findings of TA, IF, and intimal arteritis. Air-treated control recipients continued to deteriorate with further declines of CCr and increases of urinary protein excretion and died with a median survival of 82 days. In contrast, progression of CAN was decelerated when recipients received CO on days 60-150, showing markedly improved graft histopathology, restored renal function, and improved recipient survival to a median of >150 days. CO significantly reduced intragraft mRNA levels for IFN-gamma and TNF-alpha at day 90. Expression of profibrotic TGF-beta/Smad was significantly suppressed with CO, together with downregulation of ERK-MAPK pathways. Continuous CO (20 ppm) treatment for days 0-30, days 30-60, or days 0-90, or daily 1-h CO (250 ppm) treatment for days 0-90, also showed efficacy in inhibiting CAN. The study demonstrates that CO is able to inhibit progression of fibroinflammatory process of CAN, restore renal allograft function, and improve survival even when the treatment is started after CAN is diagnosed.

Supporting Survival of Transplanted Stem-Cell-Derived Insulin-Producing Cells in an Encapsulation Device Augmented with Controlled Release of Amino Acids.

Pancreatic islet transplantation is a promising treatment for type I diabetes, which is a chronic autoimmune disease in which the host immune cells attack insulin-producing beta cells. The impact of this therapy is limited due to tissue availability and dependence on immunosuppressive drugs that prevent immune rejection of the transplanted cells. These issues can be solved by encapsulating stem cell-derived insulin-producing cells in an immunoprotective device. However, encapsulation exacerbates ischemia, and the lack of vasculature at the implantation site post-transplantation worsens graft survival. Here, an encapsulation device that supplements nutrients to the cells is developed to improve the survival of encapsulated stem cell-derived insulin-producing cells in the poorly vascularized subcutaneous space. An internal compartment in the device is fabricated to provide zero-order release of alanine and glutamine for several weeks. The amino acid reservoir sustains viability of insulin-producing cells in nutrient limiting conditions in vitro. Moreover, the reservoir also increases cell survival by 30% after transplanting the graft in the subcutaneous space.

Ex vivo carbon monoxide prevents cytochrome P450 degradation and ischemia/reperfusion injury of kidney grafts.

Renal ischemia/reperfusion injury is a major complication of kidney transplantation. We tested if ex vivo delivery of carbon monoxide (CO) to the kidney would ameliorate the renal injury of cold storage that can complicate renal transplantation. Orthotopic syngeneic kidney transplantation was performed in Lewis rats following 24 h of cold preservation in University of Wisconsin solution equilibrated without or with CO (soluble CO levels about 40 microM). Ischemia/reperfusion injury in control grafts resulted in an early upregulation of inflammatory mediator mRNAs and progressive deterioration of graft function. In contrast, the grafts preserved with CO had significantly less oxidative injury and this was associated with improved recipient survival compared to the control group. Renal injury in the control group showed considerable degradation of cytochrome P450 heme proteins, active heme metabolism and increased detrimental intracellular free heme levels. Kidney grafts preserved in CO-equilibrated solution maintained their cytochrome P450 protein levels, had normal intracellular heme levels and had less lipid peroxidation. Our results show that CO-mediated suppression of injurious heme-derived redox reactions offers protection of kidney grafts from cold ischemia/reperfusion injury.

Galantamine and carbon monoxide protect brain microvascular endothelial cells by heme oxygenase-1 induction.

Galantamine, a reversible inhibitor of acetylcholine esterase (AChE), is a novel drug treatment for mild to moderate Alzheimer's disease and vascular dementia. Interestingly, it has been suggested that galantamine treatment is associated with more clinical benefit in patients with mild-to-moderate Alzheimer disease compared to other AChE inhibitors. We hypothesized that the protective effects of galantamine would involve induction of the protective gene, heme oxygenase-1 (HO-1), in addition to enhancement of the cholinergic system. Brain microvascular endothelial cells (mvECs) were isolated from spontaneous hypertensive rats. Galantamine significantly reduced H(2)O(2)-induced cell death of mvECs in association with HO-1 induction. These protective effects were completely reversed by nuclear factor-kappaB (NF-kappaB) inhibition or HO inhibition. Furthermore, galantamine failed to induce HO-1 in mvECs which lack inducible nitric oxide synthase (iNOS), supplementation of a nitric oxide (NO) donor or iNOS gene transfection on iNOS-deficient mvECs resulted in HO-1 induction with galantamine. These data suggest that the protective effects of galantamine require NF-kappaB activation and iNOS expression, in addition to HO-1. Likewise, carbon monoxide (CO), one of the byproducts of HO, up-regulated HO-1 and protected mvECs from oxidative stress in a similar manner. Our data demonstrate that galantamine mediates cytoprotective effects on mvECs through induction HO-1. This pharmacological action of galantamine may, at least in part, account for the superior clinical efficacy of galantamine in vascular dementia and Alzheimer disease.

Carbon monoxide ameliorates renal cold ischemia-reperfusion injury with an upregulation of vascular endothelial growth factor by activation of hypoxia-inducible factor.

BACKGROUND: We have previously shown that carbon monoxide (CO) inhalation at a low concentration provides protection against cold ischemia-reperfusion (I/R) injury after kidney transplantation. As vascular endothelial growth factor (VEGF) may promote the recovery process of impaired vascular endothelial cells during I/R injury, we examined whether protective effects of CO involved VEGF induction and its upstream hypoxia-inducible factor (HIF)-1 activation. METHODS: Lewis rat kidney graft, preserved in University of Wisconsin at 4 degrees C for 24 hr, was orthotopically transplanted into syngeneic recipient. Recipients were continuously maintained in air or exposed to CO (250 ppm) for 1 hr before and 24 hr after transplant. RESULTS: Prolonged cold preservation resulted in progressive impairment of kidney graft function with early inflammatory responses. Carbon monoxide significantly protected kidney grafts from cold I/R injury, improved renal function and enhanced recipient survival. Real-time reverse transcriptase-polymerase chain reaction revealed upregulation of HIF-1alpha and VEGF in the CO-treated kidney grafts as early as 1 hr after reperfusion. Western blot showed CO significantly upregulated VEGF expression 1 to 3 hr after kidney transplantation. Considerably more VEGF-positive cells were observed mainly in tubular epithelial cells in CO-treated, but not air-exposed, kidney grafts at 3 hr after reperfusion. YC-1, HIF-1alpha inhibitor, completely abrogated the actions of CO on VEGF induction and reversed the protective effects afforded by CO. Nitric oxide production in the grafts was increased by CO, however, abolished by YC-1. CONCLUSION: These results demonstrate that the protective effect of CO against renal cold I/R injury may involve VEGF upregulation through its upstream signal, HIF-1 activation.

Nanoporous Immunoprotective Device for Stem-Cell-Derived ß-Cell Replacement Therapy.

Encapsulation of human embryonic stem-cell-differentiated beta cell clusters (hES-ßC) holds great promise for cell replacement therapy for the treatment of diabetics without the need for chronic systemic immune suppression. Here, we demonstrate a nanoporous immunoprotective polymer thin film cell encapsulation device that can exclude immune molecules while allowing exchange of oxygen and nutrients necessary for in vitro and in vivo stem cell viability and function. Biocompatibility studies show the device promotes neovascular formation with limited foreign body response in vivo. The device also successfully prevented teratoma escape into the peritoneal cavity of mice. Long-term animal studies demonstrate evidence of engraftment, viability, and function of cells encapsulated in the device after 6 months. Finally, in vivo study confirms that the device was able to effectively immuno-isolate cells from the host immune system.

Mitigating Ischemic Injury of Stem Cell-Derived Insulin-Producing Cells after Transplant.

The advent of large-scale in vitro differentiation of human stem cell-derived insulin-producing cells (SCIPC) has brought us closer to treating diabetes using stem cell technology. However, decades of experiences from islet transplantation show that ischemia-induced islet cell death after transplant severely limits the efficacy of the therapy. It is unclear to what extent human SCIPC are susceptible to ischemia. In this study, we show that more than half of SCIPC die shortly after transplantation. Nutrient deprivation and hypoxia acted synergistically to kill SCIPC in vitro. Amino acid supplementation rescued SCIPC from nutrient deprivation, likely by providing cellular energy. Generating SCIPC under physiological oxygen tension of 5% conferred hypoxia resistance without affecting their differentiation or function. A two-pronged strategy of physiological oxygen acclimatization during differentiation and amino acid supplementation during transplantation significantly improved SCIPC survival after transplant.

Assessment of Immune Isolation of Allogeneic Mouse Pancreatic Progenitor Cells by a Macroencapsulation Device.

BACKGROUND: Embryonic stem cell (ESC)-derived ß cells hold the promise of providing a renewable source of tissue for the treatment of insulin-dependent diabetes. Encapsulation may allow ESC-derived ß cells to be transplanted without immunosuppression, thus enabling wider application of this therapy. METHODS: In this study, we investigated the immunogenicity of mouse pancreatic progenitor cells and efficacy of a new macroencapsulation device in protecting these cells against alloimmune and autoimmune responses in mouse models. RESULTS: Mouse pancreatic progenitor cells activated the indirect but not the direct pathway of alloimmune response and were promptly rejected in immune competent hosts. The new macroencapsulation device abolished T cell activation induced by allogeneic splenocytes and protected allogeneic MIN6 ß cells and pancreatic progenitors from rejection even in presensitized recipients. In addition, the device was effective in protecting MIN6 cells in spontaneously diabetic nonobese diabetic recipients against both alloimmune and recurring autoimmune responses. CONCLUSIONS: Our results demonstrate that macroencapsulation can effectively prevent immune sensing and rejection of allogeneic pancreatic progenitor cells in fully sensitized and autoimmune hosts.

Silicon nanopore membrane (SNM) for islet encapsulation and immunoisolation under convective transport.

Problems associated with islet transplantation for Type 1 Diabetes (T1D) such as shortage of donor cells, use of immunosuppressive drugs remain as major challenges. Immune isolation using encapsulation may circumvent the use of immunosuppressants and prolong the longevity of transplanted islets. The encapsulating membrane must block the passage of host's immune components while providing sufficient exchange of glucose, insulin and other small molecules. We report the development and characterization of a new generation of semipermeable ultrafiltration membrane, the silicon nanopore membrane (SNM), designed with approximately 7 nm-wide slit-pores to provide middle molecule selectivity by limiting passage of pro-inflammatory cytokines. Moreover, the use of convective transport with a pressure differential across the SNM overcomes the mass transfer limitations associated with diffusion through nanometer-scale pores. The SNM exhibited a hydraulic permeability of 130 ml/hr/m(2)/mmHg, which is more than 3 fold greater than existing polymer membranes. Analysis of sieving coefficients revealed 80% reduction in cytokines passage through SNM under convective transport. SNM protected encapsulated islets from infiltrating cytokines and retained islet viability over 6 hours and remained responsive to changes in glucose levels unlike non-encapsulated controls. Together, these data demonstrate the novel membrane exhibiting unprecedented hydraulic permeability and immune-protection for islet transplantation therapy.

Polycaprolactone Thin-Film Micro- and Nanoporous Cell-Encapsulation Devices.

Cell-encapsulating devices can play an important role in advancing the types of tissue available for transplantation and further improving transplant success rates. To have an effective device, encapsulated cells must remain viable, respond to external stimulus, and be protected from immune responses, and the device itself must elicit a minimal foreign body response. To address these challenges, we developed a micro- and a nanoporous thin-film cell encapsulation device from polycaprolactone (PCL), a material previously used in FDA-approved biomedical devices. The thin-film device construct allows long-term bioluminescent transfer imaging, which can be used for monitoring cell viability and device tracking. The ability to tune the microporous and nanoporous membrane allows selective protection from immune cell invasion and cytokine-mediated cell death in vitro, all while maintaining typical cell function, as demonstrated by encapsulated cells' insulin production in response to glucose stimulation. To demonstrate the ability to track, visualize, and monitor the viability of cells encapsulated in implanted thin-film devices, we encapsulated and implanted luciferase-positive MIN6 cells in allogeneic mouse models for up to 90 days. Lack of foreign body response in combination with rapid neovascularization around the device shows promise in using this technology for cell encapsulation. These devices can help elucidate the metrics required for cell encapsulation success and direct future immune-isolation therapies.

Intravital imaging of cytotoxic T lymphocytes.

Intravital imaging approaches are proving to be essential to address new questions to better understand how the immune system operates. These approaches are especially valuable to characterize the complex organization of immune responses in vivo. Here, we examine how to take advantage of the cornea as a natural body window to apply noninvasive imaging techniques to assess cytotoxic T lymphocyte involvement in the immune rejection process in a model of intraocular allogeneic islet transplantation.

Prevention of autoimmune diabetes and induction of ß-cell proliferation in NOD mice by hyperbaric oxygen therapy.

We evaluated the effects of hyperbaric oxygen therapy (HOT) on autoimmune diabetes development in nonobese diabetic (NOD) mice. Animals received no treatment or daily 60-min HOT 100% oxygen (HOT-100%) at 2.0 atmospheres absolute and were monitored for diabetes onset, insulitis, infiltrating cells, immune cell function, and ß-cell apoptosis and proliferation. Cyclophosphamide-induced diabetes onset was reduced from 85.3% in controls to 48% after HOT-100% (P < 0.005) and paralleled by lower insulitis. Spontaneous diabetes incidence reduced from 85% in controls to 65% in HOT-100% (P = 0.01). Prediabetic mice receiving HOT-100% showed lower insulitis scores, reduced T-cell proliferation upon stimulation in vitro (P < 0.03), increased CD62L expression in T cells (P < 0.04), reduced costimulation markers (CD40, DC80, and CD86), and reduced major histocompatibility complex class II expression in dendritic cells (DCs) (P < 0.025), compared with controls. After autoimmunity was established, HOT was less effective. HOT-100% yielded reduced apoptosis (transferase-mediated dUTP nick-end labeling-positive insulin-positive cells; P < 0.01) and increased proliferation (bromodeoxyuridine incorporation; P < 0.001) of insulin-positive cells compared with controls. HOT reduces autoimmune diabetes incidence in NOD mice via increased resting T cells and reduced activation of DCs with preservation of ß-cell mass resulting from decreased apoptosis and increased proliferation. The safety profile and noninvasiveness makes HOT an appealing adjuvant therapy for diabetes prevention and intervention trials.