Natural killer (NK) cells attack tumor and infected cells, but the receptors and ligands that stimulate them are poorly understood. Here we report the expression cloning of two murine ligands for the lectin-like receptor NKG2D. The two ligands, H-60 and Rae1 beta, are distant relatives of major histocompatibility complex class I molecules. NKG2D ligands are not expressed by most normal cells but are up-regulated on numerous tumor cells. We show that mouse NKG2D is expressed by NK cells, activated CD8+ T cells and activated macrophages. Expression of either NKG2D ligand by target cells triggers NK cell cytotoxicity and interferon-gamma secretion by NK cells, as well as nitric oxide release and tumor necrosis factor alpha transcription by macrophages. Thus, through their interaction with NKG2D, H-60 and Rae1 beta are newly identified potent stimulators of innate immunity.

The ability of killer T cells carrying the CD8 antigen to detect tumours or intracellular pathogens requires an extensive display of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of potential target cells. These peptides are derived from almost all intracellular proteins and reveal the presence of foreign pathogens and mutations. How cells produce thousands of distinct peptides cleaved to the precise lengths required for binding different MHC class I molecules remains unknown. The peptides are cleaved from endogenously synthesized proteins by the proteasome in the cytoplasm and then trimmed by an unknown aminopeptidase in the endoplasmic reticulum (ER). Here we identify ERAAP, the aminopeptidase associated with antigen processing in the ER. ERAAP has a broad substrate specificity, and its expression is strongly upregulated by interferon-gamma. Reducing the expression of ERAAP through RNA interference prevents the trimming of peptides for MHC class I molecules in the ER and greatly reduces the expression of MHC class I molecules on the cell surface. Thus, ERAAP is the missing link between the products of cytosolic processing and the final peptides presented by MHC class I molecules on the cell surface.

The a/fl T-cell receptor recognies a complex ligand formed by the association ofantigenic peptides with molecules ofthe major histocompatibility complex (MHC). The inherent limitations of the conventional T-cell activation assays used to detect these peptide/MHC ligands have, until now, hampered the development of expression cloning systems for T-cell antigens. To overcome these limitations, we have recently introduced a method for detecting ligand-induced activation of individual T cells. This assay, which makes use of a lacZ reporter construct, differs from conventional ligadinduced activation assays in that it allows the detection of single, activated T cells in large pools of resting cells. We applied the IacZ assay to the problem of screening expression libraries, which requires the ability to detect ligand-bearing antigen-presenting cells when they are present at very low frequency. We show here that ligand-expressing antigenpresenting cells can be detected at frequencies of 1:103-104, a level of sensitivity compatible with the screening of cDNA libraries. Furthermore, by using as antigen-presenting cells COS-7 cells stably transfected with the murine Kb class I MHC molecule, we demonstrate that transiently expressed ovalbumin is efficiently processed and presented to an ovalbumin/Kb_ specific T-cell hybridoma. IacZ expression is induced in a detectable number of cocultured T cells, even when the ovalbumin cDNA consists of only 1:104 of the total DNA used to transfect the COS cells. These results suggest that unknown T-cell antigens may be identified by screening cDNA libraries in MHC-expressing COS cells using lacZ-inducible T cells as indicators of peptide antigen expression.The a/X3 T-cell receptor (TCR) recognizes small antigenic peptides bound to major histocompatibility complex (MHC)

The development of protein-based vaccines remains a major challenge in the fields of immunology and drug delivery. Although numerous protein antigens have been identified that can generate immunity to infectious pathogens, the development of vaccines based on protein antigens has had limited success because of delivery issues. In this article, an acid-sensitive microgel material is synthesized for the development of protein-based vaccines. The chemical design of these microgels is such that they degrade under the mildly acidic conditions found in the phagosomes of antigenpresenting cells (APCs). The rapid cleavage of the microgels leads to phagosomal disruption through a colloid osmotic mechanism, releasing protein antigens into the APC cytoplasm for class I antigen presentation. Ovalbumin was encapsulated in microgel particles, 200 -500 nm in diameter, prepared by inverse emulsion polymerization with a synthesized acid-degradable crosslinker. Ovalbumin is released from the acid-degradable microgels in a pH-dependent manner; for example, microgels containing ovalbumin release 80% of their encapsulated proteins after 5 h at pH 5.0, but release only 10% at pH 7.4. APCs that phagocytosed the acid-degradable microgels containing ovalbumin were capable of activating ovalbumin-specific cytoxic T lymphocytes. The aciddegradable microgels developed in this article should therefore find applications as delivery vehicles for vaccines targeted against viruses and tumors, where the activation of cytoxic T lymphocytes is required for the development of immunity.polymer ͉ crosslinker ͉ encapsulation ͉ vaccination ͉ cytotoxic T lymphocyte