Using multivalent protein probes, an evolutionarily conserved endogenous ligand for EMR2, a human myeloid cell-restricted EGF-TM7 receptor, was identified on the surface of a number of adherent cell lines. In addition, in situ staining of the ligand has revealed specific in vivo patterns consistent with a connective tissue distribution. The interaction is conserved across species and mediated exclusively by the largest EMR2 isoform containing 5 epidermal growth factor (EGF)-like modules. Antibody-blocking studies subsequently revealed that the fourth EGF-like module constitutes the major ligandbinding site. The largest isoform of CD97, a related EGF-TM7 molecule containing an identical EGF-like module, also binds to the putative EMR2 ligand. Through the use of mutant Chinese hamster ovary (CHO) cell lines defective in glycosaminoglycans (GAGs) biosynthesis as well as the enzymatic removal of specific cell surface GAGs, the molecular identity of the EMR2 ligand was identified as chondroitin sulfate (CS). Thus, exogenous CS GAGs blocked the EMR2-ligand interaction in a dose-dependent manner. EMR2-CS interaction is Ca 2؉ -and sulphation-dependent and results in cell attachment. This is the first report of a GAG ligand for the TM7 receptors extending the already vast repertoire of stimuli of the GPCR superfamily.

The association between lymphopenia and autoimmunity is recognized, but the underlying mechanisms are poorly understood and have not been studied systematically in humans. People with multiple sclerosis treated with the lymphocyte-depleting monoclonal antibody alemtuzumab offer a unique opportunity to study this phenomenon; one in three people develops clinical autoimmunity, and one in three people develops asymptomatic autoantibodies after treatment. Here, we show that T-cell recovery after alemtuzumab is driven by homeostatic proliferation, leading to the generation of chronically activated (CD28) capable of producing proinflammatory cytokines. Individuals who develop autoimmunity after treatment are no more lymphopenic than their nonautoimmune counterparts, but they show reduced thymopoiesis and generate a more restricted T-cell repertoire. Taken together, these findings demonstrate that homeostatic proliferation drives lymphopeniaassociated autoimmunity in humans.T lymphocytes | reconstitution | immunotherapy T he anti-CD (cluster of differentiation molecule) 52 monoclonal antibody alemtuzumab has proven efficacy in relapsing remitting multiple sclerosis (RRMS) (1-3). Each cycle of alemtuzumab leads to profound panlymphopenia, but relatively infrequent dosing allows reconstitution to occur: B cells recovery rapidly, whereas CD4 and CD8 cells take 35 and 20 mo, respectively, to reach normal values (4). For 5 y after alemtuzumab and maximally, at 2 y, secondary autoimmune conditions may develop: 30% of individuals experience thyroid autoimmunity, and 1% of individuals have idiopathic thrombocytopenic purpura (ITP); there are rare cases of autoimmune hemolytic anemia, autoimmune neutropenia, and Goodpasture syndrome (1-3). One-third of patients develop asymptomatic autoantibodies.An association between lymphopenia and autoimmunity is recognized; in humans, T lymphopenia is a feature of systemic lupus erythematosus, rheumatoid arthritis, and Crohn and Sjogren syndromes (5), and animal models of autoimmunity often involve the induction of lymphopenia. The mechanism driving autoimmunity in these situations is unclear. In some cases it has been attributed to loss of regulatory cells; however, although treatment of lymphopenic hosts with CD4 +

Post-translational cleavage at the G protein-coupled receptor proteolytic site (GPS) has been demonstrated in many class B2 G protein-coupled receptors as well as other cell surface proteins such as polycystin-1. However, the mechanism of the GPS proteolysis has never been elucidated. Here we have characterized the cleavage of the human EMR2 receptor and identified the molecular mechanism of the proteolytic process at the GPS. Proteolysis at the highly conserved His-Leu2Ser 518 cleavage site can occur inside the endoplasmic reticulum compartment, resulting in two protein subunits that associate noncovalently as a heterodimer. Site-directed mutagenesis of the P ؉1 cleavage site (Ser 518) shows an absolute requirement of a Ser, Thr, or Cys residue for efficient proteolysis. Substitution of the P ؊2 His residue to other amino acids produces slow processing precursor proteins, which spontaneously hydrolyze in a defined cellfree system. Further biochemical characterization indicates that the GPS proteolysis is mediated by an autocatalytic intramolecular reaction similar to that employed by the N-terminal nucleophile hydrolases, which are known to activate themselves by self-catalyzed cisproteolysis. We propose here that the autoproteolytic cleavage of EMR2 represents a paradigm for the other GPS motif-containing proteins and suggest that these GPS proteins belong to a cell surface receptor subfamily of N-terminal nucleophile hydrolases.