2,3,5,6-Tetrafluorophenyl N-(S-benzoylthioacetyl)glycylglycyl- p-aminobenzoate, a heterobifunctional (99m)Tc ligand for precomplexed antibody labeling.

Heterobifunctional (99m)Tc ligands are useful for antibody labeling using the precomplexation route. The aim of this work was to synthesize a ligand, which has sufficient chemical stability to be complexed with (99m)Tc without inactivating the reactive conjugation group. Using 2,3,5,6-tetrafluorophenyl N-(S-benzoylthioacetyl)glycylglycyl-p-aminobenzoate (OC2) >60% of the (99m)Tc complex was obtained at 80 degrees C in 20 min, which was separated from the free ligand and impurities by HPLC. After solvent evaporation, (99m)Tc-OC2 was conjugated with the monoclonal antibody mAb425 in 50% radiochemical yield. In all, the labeling method required about 1 h preparation time. The immunoreactive fraction of the (99m)Tc-OC2 mAb425 conjugate was 81%, indicating preserved binding capability after conjugation. Compared to recently described methods, which need in situ activation of the (99m)Tc complex, the application of OC2 saved time and reduced the number of manipulations with radioactive material.

Functional HLA-DM on the surface of B cells and immature dendritic cells.

HLA-DM (DM) plays a critical role in antigen presentation through major histocompatibility complex (MHC) class II molecules. DM functions as a molecular chaperone by keeping class II molecules competent for antigenic peptide loading and serves as an editor by favoring presentation of high-stability peptides. Until now, DM has been thought to exert these activities only in late endosomal/lysosomal compartments of antigen-presenting cells. Here we show that a subset of DM resides at the cell surface of B cells and immature dendritic cells. Surface DM engages in complexes with putatively empty class II molecules and controls presentation of those antigens that rely on loading on the cell surface or in early endosomal recycling compartments. For example, epitopes derived from myelin basic protein that are implicated in the autoimmune disease multiple sclerosis are down-modulated by DM, but are presented in the absence of DM. Thus, this novel concept of functional DM on the surface may be relevant to both protective immune responses and autoimmunity.

Quality control of MHC class II associated peptides by HLA-DM/H2-M.

For many years the crucial components involved in MHC class II mediated antigen presentation have been thought to be known: polymorphic MHC class II molecules, the monomorphic invariant chain (li) and a set of conventional proteases that cleave antigenic proteins thereby generating ligands able to associate with MHC class II molecules. However, in 1994 it was found that without an additional molecule, HLA-DM (DM), efficient presentation of protein antigens cannot be achieved. Biochemical studies showed that DM acts as a molecular chaperone protecting empty MHC class II molecules from functional inactivation. In addition, it serves as a peptide editor: DM catalyzes not only the release of the invariant chain remnant CLIP, but of all sorts of low-stability peptides, and simultaneously favors binding of high-stability peptides. Through this quality control of peptide loading, DM enables APCs to optimize MHC restriction and to display their antigenic peptide cargo on the surface for prolonged periods of time to be scrutinized by T cells.

HLA-DM acts as a molecular chaperone and rescues empty HLA-DR molecules at lysosomal pH.

HLA-DM (DM) is a nonclassical MHC class II molecule that interacts with classical MHC II molecules in acidic compartments. During this association DM is supposed to catalyze the release of invariant chain (Ii)-derived CLIP peptides, as well as other peptides bound with low kinetic stability. Here we provide evidence that in lysosomal compartments of B cells a considerable fraction of DM is stably associated with empty DR alphabeta dimers, thereby preventing their functional inactivation and aggregation. Upon encounter with cognate peptide, the DM-associated DR molecules can be rapidly loaded and no longer bind to DM. Thus, DM seems to act as a dedicated class II-specific chaperone. In view of the suggested shortage of DM-resistant self-peptides in the loading compartment, empty class II molecules that are chaperoned by DM may enable the antigen-processing system to respond promptly to the challenge by newly entering antigens.

Selection of the MHC class II-associated peptide repertoire by HLA-DM.

During the past five years considerable progress has been made in the field of major histocompatibility complex (MHC) class II-restricted antigen presentation. Several observations made in mutant cell lines with a presentation defect led to the identification of a novel protein, the nonclassic MHC class II molecule human leukocyte antigen (HLA)-DM. Cell biological and biochemical characterization of HLA-DM provided deeper insight into the molecular mechanism underlying the loading process: HLA-DM accumulates in acidic compartments where it binds to classic class II molecules as long as no high-stability ligand occupies the peptide-binding groove. Thus, HLA-DM prevents empty alpha beta dimers from functional inactivation in a chaperone-like fashion. At the same time HLA-DM acts as an editor by removing low-stability ligands, thereby skewing the class II peptide repertoire presentable to T-helper cells.