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.

A series of T cell lines transfected to stably express HIV-1 envelope (env) glycoproteins were analyzed for viability and for T cell signaling. One transfectant was distinguished by its stable expression of gp120 and gp41, whereas the remainder of the T cell lines were similar to previously reported env-expressing T cells in synthesizing predominantly unprocessed env glycoprotein gp160. All of the transfectants were additionally constructed to express tat and rev proteins. None of these cell lines displayed growth abnormalities or spontaneous cell fusion, although the cell line synthesizing env gp120/gp41 could be induced to fuse and die when cocultured with a second cell expressing surface CD4. A cell line expressing only gp160 and the transfectant expressing gp160, gp120, and gp41 could be triggered normally via CD3-cross-linking as measured by protein tyrosine phosphorylation and by the induction of the CD69 activation marker. At levels of env protein expression sufficient to mediate syncytium formation and to kill cells, these HIV-1 env transfectants displayed no intrinsic T cell signaling abnormalities, suggesting that mechanisms other than a direct intracellular action of the tat or env proteins may be contributing to the deficit in Ag-specific T cell activation described subsequent to HIV infection in vivo and in vitro.