Protein cage architectures such as virus capsids and ferritins are versatile nanoscale platforms amenable to both genetic and chemical modification. Incorporation of multiple functionalities within these nanometer-sized protein architectures demonstrate their potential to serve as functional nanomaterials with applications in medical imaging and therapy. In the present study, we synthesized an iron oxide (magnetite) nanoparticle within the interior cavity of a genetically engineered human H-chain ferritin (HFn). A cell-specific targeting peptide, RGD-4C which binds alphavbeta3 integrins upregulated on tumor vasculature, was genetically incorporated on the exterior surface of HFn. Both magnetite-containing and fluorescently labeled RGD4C-Fn cages bound C32 melanoma cells in vitro. Together these results demonstrate the capability of a genetically modified protein cage architecture to serve as a multifunctional nanoscale container for simultaneous iron oxide loading and cell-specific targeting.

γδ T cells are innate immune cells that participate in host responses against many pathogens and cancers. Recently, phosphoantigen-based drugs, capable of expanding γδ T cells in vivo, entered clinical trials with the goal of enhancing innate immune system functions. Potential shortcomings of these drugs include the induction of nonresponsiveness upon repeated use and the expansion of only the Vδ2 subset of human γδ T cells. Vδ1 T cells, the major tissue subset, are unaffected by phosphoantigen agonists. Using FACS-based assays, we screened primary bovine cells for novel γδ T cell agonists with activities not encompassed by the current treatments in an effort to realize the full therapeutic potential of γδ T cells. We identified γδ T cell agonists derived from the condensed tannin fractions of Uncaria tomentosa (Cat’s Claw) and Malus domestica (apple). Based on superior potency, the apple extract was selected for detailed analyses on human cells. The apple extract was a potent agonist for both human Vδ1 and Vδ2 T cells and NK cells. Additionally, the extract greatly enhanced phosphoantigen-induced γδ T cell expansion. Our analyses suggest that a tannin-based drug may complement the phosphoantigen-based drugs, thereby enhancing the therapeutic potential of γδ T cells.

To elucidate the functions of circulating gammadelta T cells, in the absence of antigen stimulation, the differential gene expression of two circulating gammadelta T cell subsets was analyzed. The two subsets, with distinct trafficking phenotypes in young calves, were GD3.5(+), CD8(-), WC1(+) or GD3.5(-), CD2(+), WC1(-), and 90-100% CD8(+) and were sorted based on GD3.5 and gammadelta T cell receptor expression. Results from two different human arrays probed with cDNA from these gammadelta T cell subsets indicated that they have markedly different tissue-specific functions. The genes preferentially expressed by GD3.5(+) (CD8(-)) gammadelta T cells demonstrated that they were highly activated, proliferative, and inflammatory, whereas those expressed by GD3.5(-) (primarily CD8(+)) gammadelta T cells were involved in promoting quiescence, consistent with a role for gammadelta T cells as sentinel mucosal cells, and several were interferon-regulated genes. Gene expression and phenotypic assays indicated that CD8(+) gammadelta T cells were apoptotic, whereas CD8(-) gammadelta T cells were apoptosis-resistant. Differential expression of multiple genes was confirmed in both arrays: That of 14 genes was confirmed by quantitative reverse transcriptase-polymerase chain reaction and that of seven proteins was confirmed by flow cytometry. This novel, genomic analysis of circulating gammadelta T cell subsets, without confounding effects of the tissue microenvironment, offers new insight into the biology and development of neonatal gammadelta T cells.

Intracellular distribution of iron oxide nanoparticles incorporated within a ferritin mutant that displays genetically introduced cell‐targeted peptides (RGD‐4C) on its exterior surface are investigated using scanning transmission electron microscopy with a high‐angle annular dark‐field detector. The particles (indicated by arrows) internalized into macrophages much more effectively than those with noncell‐targeted ferritin.

Natural killer (NK) cells and dendritic cells (DCs) have been shown to link the innate and adaptive immune systems. Likewise, a new innate cell subset, interferon-producing killer DCs (IKDCs), shares phenotypic and functional characteristics with both DCs and NK cells. Here, we show IKDCs play an essential role in the resolution of experimental autoimmune encephalomyelitis (EAE) upon treatment with the tolerizing agent, myelin oligodendrocyte glycoprotein (MOG), genetically fused to reovirus protein σ1 (termed MOG-pσ1). Activated IKDCs were recruited subsequent MOG-pσ1 treatment of EAE, and disease resolution was abated upon NK1.1 cell depletion. These IKDCs were able to kill activated CD4+ T cells and mature dendritic DCs, thus, contributing to EAE remission. In addition, IKDCs were responsible for MOG-pσ1-mediated MOG-specific regulatory T cell recruitment to the CNS. The IKDCs induced by MOG-pσ1 expressed elevated levels of HVEM for interactions with cognate ligand-positive cells: LIGHT+ NK and Teff cells and BTLA+ B cells. Further characterization revealed these activated IKDCs being MHC class IIhigh, and upon their adoptive transfer (CD11c+NK1.1+MHC class IIhigh), IKDCs, but not CD11c+NK1.1+MHC class IIintermediate/low (unactivated) cells, conferred protection against EAE. These activated IKDCs showed enhanced CD107a, PD-L1, and granzyme B expression and could present OVA, unlike unactivated IKDCs. Thus, these results demonstrate the interventional potency induced HVEM+ IKDCs to resolve autoimmune disease.