Kyle B. Woodward

Pancreatic islets engineered with SA-FasL protein establish robust localized tolerance by inducing regulatory T cells in mice.

Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL–engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL–engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4+CD25+Foxp3+ regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.

Local immunomodulation with Fas ligand-engineered biomaterials achieves allogeneic islet graft acceptance

Islet transplantation is a promising therapy for type 1 diabetes. However, chronic immunosuppression to control rejection of allogeneic islets induces morbidities and impairs islet function. T effector cells are responsible for islet allograft rejection and express Fas death receptors following activation, becoming sensitive to Fas-mediated apoptosis. Here, we report that localized immunomodulation using microgels presenting an apoptotic form of the Fas ligand with streptavidin (SA-FasL) results in prolonged survival of allogeneic islet grafts in diabetic mice. A short course of rapamycin treatment boosted the immunomodulatory efficacy of SA-FasL microgels, resulting in acceptance and function of allografts over 200 days. Survivors generated normal systemic responses to donor antigens, implying immune privilege of the graft, and had increased CD4+CD25+FoxP3+ T regulatory cells in the graft and draining lymph nodes. Deletion of T regulatory cells resulted in acute rejection of established islet allografts. This localized immunomodulatory biomaterial-enabled approach may provide an alternative to chronic immunosuppression for clinical islet transplantation.Islet transplantation for diabetes treatment requires immunosuppression to control rejection. A microgel presenting Fas ligand with immunomodulatory properties is now shown to prolong the survival of allogeneic islet grafts in vivo.

Novel technologies to engineer graft for tolerance induction.

Purpose of reviewConquering allograft rejection remains an elusive goal in spite of recent breakthroughs in the field of immunosuppression. Much of the problem lies in the toxicity and side-effects of long-term use of systemic immunosuppressant drugs, which are sometimes ineffective in controlling rejection, but also hinder establishment of transplant tolerance. In this review, we discuss novel technologies that use grafts engineered with immunomodulatory molecules as a means of inducing tolerance. Recent findingsSeveral recent studies have demonstrated the feasibility of engineering cells, tissues, or solid organ grafts with immunoregulatory biologics to achieve long termgraft survival without the use of chronic immunosuppression. This approach was shown to primarily change the ratio of T effector versus CD4+CD25+FoxP3+ T regulatory cells within the graft microenvironment in favor of attaining localized tolerance induction and maintenance. SummaryLocalized immunomodulation using biologic-engineered allografts represent a new paradigm for achieving long-term graft survival in the absence of chronic use of immunosuppression. The manipulation of the graft, rather than the recipient, not only ensures short- and long-term safety by minimizing the adverse effects of immunosuppression, but also allows retention of immune competency critical for the ability of the recipient to fight infections and cancer.

Posttransplantation systemic immunomodulation with SA-FasL-engineered donor splenocytes has robust efficacy in preventing cardiac allograft rejection in mice.

Apoptosis induced by the engagement of FasL with Fas receptor on the surface of lymphocytes is an important immune homeostatic mechanism that ensures tolerance to self-antigens under normal physiologic conditions. As such, FasL has been extensively tested as a tolerogenic molecule with the use of gene therapy in settings of autoimmunity and transplantation with conflicting outcomes. Although the mechanistic basis of these contradictory observations is largely unknown, the use of wild-type FasL and the means by which the gene was expressed may provide an explanation. To overcome these complications, we generated a chimeric FasL protein with streptavidin (SA-FasL) having potent apoptotic activity and displayed this molecule effectively and rapidly on biotinylated biologic membranes for immunomodulation. In the present study, we displayed SA-FasL on the surface of BALB/c splenocytes and injected 5 × 10(6) cells intraperitoneally into C57BL/6 recipients of BALB/c heart grafts on days 1, 3, and 5 after-transplantation. To control initial graft-reactive immune responses and facilitate FasL-mediated apoptosis, rapamycin was used as an immunosuppressant at 0.2 mg/kg daily for a total of 15 doses immediately after heart transplantation. All mice injected with SA-FasL-engineered donor splenocytes accepted their grafts during the 100-day observation period. In marked contrast, immunomodulation with control streptavidin protein-engineered BALB/c splenocytes had minimal effect on graft survival (mean survival, 21.4 ± 1.5 d). Taken together, these results establish posttransplantation systemic immunomodulation with SA-FasL-engineered donor splenocytes under transient cover of rapamycin as an effective regimen in preventing cardiac allograft rejection in rodents with important clinical implications.

The Transient Display of a Chimeric PD-L1 Protein on Pancreatic Islets Promotes Indefinite Survival in Allogeneic Recipients

Introduction and Objective Allogenic islet transplantation is an important therapeutic approach for the treatment of Type 1 Diabetes (T1D). However, graft rejection initiated and perpetuated by pathogenic T effector (Teff) cells presents a major barrier. One approach that has proven successful for promoting graft tolerance is shifting the T cell balance away from the induction of pathogenic Teff cells and towards the generation of protective T regulatory (Treg) cells. PD-1/PD-L1 immune checkpoint pathway plays an important role in the Teff and Treg balance with demonstrated clinical efficacy in cancer immunotherapy. The goal of this study was to assess the immunoregulatory function of PDL1 in controlling the rejection of allogeneic islets grafts. Materials and Methods A DNA construct encoding the extracellular portion of PD-L1 protein fused to a modified form of core streptavidin was generated. The chimeric SA-PDL1 protein was produced in insect cells, purified using immunoaffinity columns, and characterized for structure. The function of SA-PDL1 was assessed in vitro for the conversion of Teff into Treg cells, and its capacity to suppress Teff proliferation in response to allogeneic stimulation. For in vivo studies, BALB/c islets were modified with biotin followed by engineering with SA-PDL1 protein (~200 ng/1000 islets). Engineered islets (~500 islets/transplant) were then transplanted under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice under transient cover of rapamycin (0.2 mg/kg) administered daily for 15 days starting the day of transplantation. Groups with unmodified pancreatic islets and rapamycin treatment or SA-PDL1-engineered islets without rapamycin treatment served as controls. Results and Discussion SA-PDL1 was successfully expressed and purified. In vitro, the protein enhanced TGF-beta-induced conversion of Teff into Treg cells and effectively suppressed the proliferation of Teff cells in response to alloantigen stimulation. In vivo, SA-PDL1-engineered BALB/c islet grafts, with concurrent rapamycin treatment, remained functional for over a 100-day observation period. In marked contrast, unmodified islets, under the same rapamycin regimen, were acutely rejected within 20 days. SA-PDL1-engineered islets without rapamycin showed prolonged survival, although all grafts were eventually rejected within 40 days. Conclusion These results provide strong proof-of-efficacy and feasibility for the use of SA-PDL1 protein as an immunomodulator to promote allogeneic islet graft survival in the absence of continued immunosuppression. This study was funded in part by NIH (grants R21EB020107, R21AI113348, 185 R56AI121281, and F30AR069472).

Immunomodulation with SA-FasL-Engineered Microgels Achieves Long-Term Survival of Allogeneic Islet Grafts

Introduction and Objective Allogenic islet transplantation has shown efficacy in the clinic for the treatment of type 1 diabetes. However, sustained survival of allogeneic islet grafts requires chronic immunosuppression that has significant adverse effects. T effector (Teff) cells recognizing and responding to allograft antigens are the major culprit of graft rejection. Upon activation, Teff cells upregulate Fas death receptor on their surface and become sensitive to FasL-mediated apoptosis. Fas pathway, therefore, presents an important target for immunomodulation to block alloreactive responses with significant therapeutic potential. Herein, we assessed the efficacy of PEG microgels engineered with a novel form of FasL chimeric with a modified form of core streptavidin, SA-FasL, in achieving sustained survival of allogeneic islet grafts in the absence of chronic immunosuppression. Materials and Methods Microgels presenting biotin on their surface were produced by reacting biotin-PEG-thiol with a maleimide-terminated 4-arm poly(ethylene) glycol macromer, and generating 200 &mgr;m diameter microgels crosslinked with dithiothreitol. Biotinylated microgels were engineered with SA-FasL (1 &mgr;g/103 microgels) taking the advantage of the high affinity interaction between biotin and streptavidin. The apoptotic activity of SA-FasL-engineered microgels was tested on mouse A20 B cell lymphocytes in vitro. The immunomodulatory function of microgels for the prevention of graft rejection was tested by mixing ~500 BALB/c naïve islets with 1000 SA-FasL-engineered microgels and transplanting under the kidney capsule of streptozotocin-induced diabetic C57BL/6 recipients. A group of recipients were also treated transiently with rapamycin (0.2 mg/kg daily for 15 days starting the day of transplantation). Results and Discussion Biotin-PEG microgels were efficiently coupled with SA-FasL, and showed a dose-dependent apoptotic activity in A20 cells. Co-transplantation of SA-FasL-engineered microgels with naïve islets led to prolonged survival of grafts as compared with the control group (islets + unmodified microgels) with 20% surviving for a 200-day observation period. Transient use of low dose rapamycin, enhanced survival to > 90% of the grafts. In marked contrast, only 20% of islets co-transplanted with PEG microgels, and short-course rapamycin, survived long-term. Importantly, CD4+CD25+FoxP3+ Treg cells were required for graft survival as depletion of this cell population on day 50 post-transplantation resulted in prompt rejection. Conclusion These results provide strong proof-of-efficacy and feasibility for the use of SA-FasL-engineered microgels as an off-the-shelf product for the modulation of alloreactive responses with significant therapeutic potential. Funded in part by NIH (grants R21EB020107, R21AI113348, 185 R56AI121281, and F30AR069472) and the Juvenile Diabetes Research Foundation (2-SRA-186 2014-287-Q-R).