SummaryTo investigate the development of HLA-DR-associated autoimmune diseases, we generated transgenic (Tg) mice with HLA-DRA-IEcx and HLA-DRBI*0401-IE[3 chimeric genes. The transgene-encoded proteins consisted of antigen-binding domains from HLA-DRA and HLA-DRBI*0401 molecules and the remaining domains from the IEd-ot and IEd-[3 chains. The chimeric molecules showed the same antigen-binding specificity as HLA-DRBI*0401 molecules, and were functional in presenting antigens to T cells. The Tg mice were backcrossed to MHC class II-deficient (IAl3-,IEoe-) mice to eliminate any effect of endogenous MHC class II genes on the development of autoimmune diseases. As expected, IA~x[3 or IEot[3 molecules were not expressed in Tg mice. Moreover, cell-surface expression of endogenous IE[3 associated with HLA-DRA-IEci was not detectable in several Tg mouse lines by flow cytometric analysis. The HLA-DRA-IEo~/HLA-DRBI*0401-IE[3 molecules rescued the development ofCD4 + T cells in MHC class II-deficient mice, but T cells expressing VI35, V1311, and VI312 were specifically deleted.Tg mice were immunized with peptides, myelin basic protein (MBP) 87-106 and proteolipid protein (PLP) [175][176][177][178][179][180][181][182][183][184][185][186][187][188][189][190][191][192], that are considered to be immunodominant epitopes in HLA-DR4 individuals. PLP175-192 provoked a strong proliferative response of lymph node T cells from Tg mice, and caused inflammatory lesions in white matter of the CNS and symptoms of experimental allergic encephalomyelitis (EAE). Immunization with MBP87-106 elicited a very weak proliferative T cell response and caused mild EAE. Non-Tg mice immunized with either PLP175-192 or MBP87-106 did not develop EAE. These results demonstrated that a human MHC class II binding site alone can confer susceptibility to an experimentally induced murine autoimmune disease.

The phosphatase of regenerating liver (PRL) family, a unique class of oncogenic phosphatases, consists of three members: PRL-1, PRL-2, and PRL-3. Aberrant overexpression of PRL-3 has been found in multiple solid tumor types. Ectopic expression of PRLs in cells induces transformation, increases mobility and invasiveness, and forms experimental metastases in mice. We have now shown that small interfering RNAmediated depletion of PRL expression in cancer cells results in the down-regulation of p130Cas phosphorylation and expression and prevents tumor cell anchorage-independent growth in soft agar. We have also identified a small molecule, 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (thienopyridone), which potently and selectively inhibits all three PRLs but not other phosphatases in vitro. The thienopyridone showed significant inhibition of tumor cell anchorage-independent growth in soft agar, induction of the p130Cas cleavage, and anoikis, a type of apoptosis that can be induced by anticancer agents via disruption of cell-matrix interaction. Unlike etoposide, thienopyridone-induced p130Cas cleavage and apoptosis were not associated with increased levels of p53 and phospho-p53 (Ser 15 ), a hallmark of genotoxic druginduced p53 pathway activation. This is the first report of a potent selective PRL inhibitor that suppresses tumor cell three-dimensional growth by a novel mechanism involving p130Cas cleavage. This study reveals a new insight into the role of PRL-3 in priming tumor progression and shows that PRL may represent an attractive target for therapeutic intervention in cancer. [Cancer Res 2008;68(4):1162-9]

Dendritic cells (DC) are central regulators of immunity. Signal-induced maturation of DCs is assumed to be the starting point for specific immune responses. To further understand this process, we analyzed the alteration of transcript profiles along the time course of CD40 ligand-induced maturation of human myeloid DCs by Affymetrix GeneChip microarrays covering >6800 genes. Besides rediscovery of genes already described as associated with DC maturation proving reliability of the methods used, we identified clusterin as novel maturation marker. Looking across the time course, we observed synchronized kinetics of distinct functional groups of molecules whose temporal coregulation underscores known cellular events during dendritic cell maturation. For example, an early-peaking wave of inflammatory chemokines was followed by a sustained increase of constitutive chemokines and accompanied by slow but continuous induction of survival proteins. After an immediate but transient induction of cytokine-responsive transcripts, there was an increased expression of a group of genes involved in not only the regulation of cytokine effects, but also of transcription in general. Our results demonstrate that microarray studies along time courses combined with real-time PCR not only discover new marker molecules with functional implications, but also dissect the molecular kinetics of biological processes identifying complex pathways of regulation.

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