Dendritic cells (DCs) in the peripheral tissues act as sentinels of the immune system. They detect and capture pathogens entering the body and present their antigens to T cells to trigger responses directed towards elimination of the pathogen. The induction of peripheral tolerance against self and certain foreign antigens is also believed to be mediated through DCs. The outcome of any immune response is largely controlled by the microenvironment of antigen capture, processing and presentation by DCs. The "context" of antigen delivery to DCs will directly influence the microenvironment of antigen presentation and hence the regulation of immune responses. We report here preliminary investigations describing the formulation of a pharmaceutically acceptable, biodegradable, and strategic nanoparticulate delivery system, and its application for efficient antigen loading of DCs to achieve antigen specific T cell activation. "Pathogen-mimicking" nanoparticles capable of interacting with DCs were fabricated by incorporating monophosphoryl lipid A (MPLA; toll-like receptor (TLR) 4 ligand) or CpG ODN (seq #2006; TLR9 ligand) in biodegradable copolymer, poly(D,L,-lactic-co-glycolic acid) (PLGA). The uptake of PLGA nanoparticles by human umbilical cord blood derived DCs (DCs propagated from CD34 progenitors) was conclusively demonstrated by scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Cell phenotype at day 12 of cultures was determined as immature DC using specific cell surface markers, i.e. CD11c (approximately 90%), MHC-II (approximately 70%), CD86 (approximately 20%), CD83 (approximately 5%), CD80 (approximately 40%), CD40 (approximately 40%), and CCR7 (approximately 5%). Tetanus toxoid (TT), a model antigen, was encapsulated in nanoparticles along with an immunomodulator, i.e. either MPLA or CpG ODN. DCs pulsed with various antigen formulations were co-cultured with autologous naïve T cells at various cell ratios (DC: T cells were 1:5-20). The DCs pulsed with TT and MPLA together in nanoparticles induced significantly higher T cell proliferation (P<0.05) as compared to when DCs pulsed with TT and MPLA in solution were employed. A similar trend was observed when CpG ODN was used instead of MPLA in the TT nanoparticles. This strategy of antigen delivery to DCs was then tested with a cancer vaccine candidate, a MUC1 lipopeptide. The T cell proliferation observed in the presence of nanoparticulate MUC1 and MPLA pulsed-DCs was much higher than DCs pulsed with soluble antigen (P<0.0005). These results indicate that PLGA nanoparticles mimicking certain features of pathogens are efficient delivery systems for targeting vaccine antigens to DCs and activation of potent T cell responses.

Transfection of allogeneic mouse islets with human CTLA4-Ig results in prolonged allograft survival. Although on histology mononuclear cells are present in the area of the transfected graft, they do not appear to infiltrate or destroy the islet graft.

Our results indicate that local production of human CTLA4-Ig or soluble human Fas ligand by biolistically transfected islets can promote allograft survival. This approach should be valuable as a potential immunoprotective therapeutic strategy in tissue transplantation.

No abstract

To utilize gene therapy, we required an efficient method to transfect intact islets before their use in transplantation. The biolistic method transforms cells by bombarding them with microprojectiles coated with DNA. Once internalized, the DNA is solubilized and expressed. We used the firefly luciferase gene driven by the human cytomegalovirus immediate early promoter as a reporter construct in freshly isolated BALB/c mouse islets to compare the transfection efficiency using either the biolistic method, lipofection, or recombinant adenoviral infection (n=4 in each case). The biolistic method achieved, on average, a 35-fold higher level of luciferase activity than the lipofection method (mean +/- SEM: 42.6 +/- 14.2 vs. 1.1 +/- 0.2 relative light units (RLU)/islet). Adenoviral infection achieved, on average, a further 25-fold higher level of luciferase activity than the biolistic method (1136.0 +/- 542.0 RLU/islet). The average proportion of islets recovered 48 hr after the biolistic blast was 53% (n=20). The average number of dissociated cells found to express the foreign gene product using beta-galactosidase as a reporter construct was 3% (n=6). Furthermore, nontransformed and biolistically transformed islets responded similarly to an in vitro glucose challenge (stimulation index of insulin release at 20.0 mM glucose/insulin release at 2.8 mM glucose = 2.8 and 3.0, respectively, P=0.9). Syngeneic, biolistically transfected islets functioned to reverse the diabetic state when transplanted (500 islets) beneath the renal capsule of alloxan-induced diabetic BALB/c recipients (n=7). This methodology can achieve efficient transfection of pancreatic islets while preserving their function and thus holds promise for ex vivo gene therapy of isolated islets prior to transplantation.