The intrinsic role of endogenous IL-17A in spontaneous intestinal tumorigenesis has not been addressed previously to our knowledge. Ablation of IL-17A significantly reduced tumor development in mice bearing a heterozygote mutation in the adenomatous polyposis coli (APC) gene (Apc Min/+ mice). There was also a decrease in inflammatory cytokines and proinflammatory mediators, reduced infiltration of lymphocytes including T cells, and preservation of intestinal architecture and the presence of APC protein in intestinal epithelial cells. Interestingly, IL-17A ablation also corrected immunological abnormalities such as splenomegaly and thymic atrophy in Apc Min/+ mice. CD4 T cells from Apc Min/+ mice showed hyperproliferative potential in vitro and in vivo and increased levels of IL-17A and IL-10. The effector CD4 T cells from Apc Min/+ mice were more resistant to regulatory T cell-mediated suppression. Finally, these CD4 T cells induced colitis in immunodeficient mice upon adoptive transfer, whereas the ablation of IL-17A in CD4 T cells in Apc Min/+ mice completely abolished this pathogenic potential in vivo. Taken together, our results show that CD4 T cellderived IL-17A promotes spontaneous intestinal tumorigenesis with altered functions of CD4 T cells in Apc Min/+ mice.inflammation | T cells | colon cancer

Immunodeficient mice bearing components of a human immune system present a novel approach for studying human immune responses. We investigated the number, phenotype, developmental kinetics and function of developing human immune cells following transfer of CD34+ hematopoietic stem cell (HSC) preparations, originating from second trimester human fetal liver (HFL), umbilical cord blood (UCB), or granulocyte colony-stimulating factor-mobilized adult blood (G-CSF-AB) delivered via intrahepatic injection into sublethally irradiated neonatal NOD-scid/γc −/− , Balb/cRag1 −/− γc −/− , and C.B-17-scid/bg mice. HFL and UCB HSC provided the greatest number and breadth of developing cells. NOD-scid/γc −/− and Balb/c-Rag1 −/− γc −/− harbored human B and dendritic cells as well as human platelets in peripheral blood, whereas NOD-scid/γc −/− mice harbored higher levels of human T cells. NOD-scid/γc −/− mice engrafted with HFL CD34+ HSC demonstrated human immunological competence evidenced by white pulp expansion and increases in total human immunoglobulin following immunization with T-dependent antigens, and delayed type hypersensitivity-infiltrating leukocytes in response to antigenic challenge. In conclusion, we describe an encouraging base system for studying human hematopoietic lineage development and function utilizing human HFL or UCB HSC-engrafted NOD-scid/γc −/− mice that is well suited for future studies toward the development of a fully competent humanized mouse model.

CTLA-4 (CD152) negatively regulates T cell activation signaling, and the cytoplasmic domain of CTLA-4 (ctCTLA-4) itself has the capacity to inhibit T cell activation in vitro and in vivo. In this study, the inhibitory mechanisms of the cell-permeable recombinant protein Hph-1-ctCTLA-4 on T cell activation and its ability to prevent collageninduced arthritis were analyzed. Hph-1-ctCTLA-4 prevented human and mouse T cell activation and proliferation by inhibition of T cell receptor-proximal signaling and the arrest of the cell cycle. Furthermore, Hph-1-ctCTLA-4 protected human umbilical vein endothelial cells (HUVEC) from the human CTL allo-response. The incidence and severity of collagen-induced arthritis were significantly reduced and the erosion of cartilage and bone was effectively prevented by i.v. injection and transdermal administration of Hph-1-ctCTLA-4. Inflammatory cytokine production (IL-1␤, IL-6, TNF-␣, IL-17A) and collagenspecific antibody levels were significantly reduced, and the numbers of activated T cells and infiltrating granulocytes were substantially decreased. These results demonstrate that systemic or transdermal application of a cell-permeable form of the cytoplasmic domain of CTLA-4 offers an effective therapeutic approach for autoimmune diseases such as rheumatoid arthritis.autoimmune disease ͉ costimulatory molecule R heumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that leads to progressive joint destruction (1). Hallmarks of RA are joint inflammation, proliferation of synovial cells, and attachment and invasion of synovial fibroblasts into adjacent cartilage and bone (2, 3). Cumulative evidence has indicated that CD4 ϩ T cell-mediated autoimmune responses play a critical role in the pathogenesis of RA, in conjunction with activation of B cells and macrophages that infiltrate the synovium (4, 5). Recent biological therapies against IL-1␤, IL-6, or TNF-␣ have demonstrated promising effects against progressive joint destruction (6-8). However, the clinical use of these therapies has been limited because of several issues, including side effects and the increase of therapyinsensitive patients.Recently, several reports demonstrated that regulation of costimulatory signaling in the T cell is an important target for treatment of autoimmune diseases such as arthritis (9, 10). CTLA-4 is an activation-induced surface molecule on T cells and is essential for the negative regulation of T cell activation; its inhibitory effects can be accomplished either by competition with CD28 for their ligands B7-1 and -2 or by transmission of negative signals through its intracellular domain (11,12). The cytoplasmic domain of CTLA-4 (ctCTLA-4), which contains a tyrosine phosphorylation motif, has been found to be 100% conserved among different species, suggesting that this domain is critical for CTLA-4 functions (13,14). In addition, there are several splicing variants of CTLA-4, including full-length CTLA-4, soluble CTLA-4 that lacks the transmembrane domain, ligand-independent CTLA-4 (liC...

T cell interactions with vascular endothelial cells (EC) are of central importance for immune surveillance of microbes and for pathological processes such as atherosclerosis, allograft rejection, and vasculitis. Animal (especially rodent) models incompletely predict human immune responses, in particular with regard to the immunological functions of EC, and in vitro models may not accurately reflect in vivo findings. In this study, we describe the development of an immunodeficient SCID/bg murine model combining a transplanted human synthetic microvascular bed with adoptive transfer of human T lymphocytes allogeneic to the cells of the graft that more fully recapitulates T cell responses in natural tissues. Using this model, we demonstrate that transduced Bcl-2 protein in the engrafted EC effectively prevents injury even as it enhances T cell graft infiltration and replication.

It is thought that differentiation of beta-cell precursors into mature cells is largely autonomous, but under certain conditions differentiation can be modified by external factors. The factors that modify beta-cell differentiation have not been identified. In this study, we tested whether adult islet cells can affect the differentiation process in mouse and human pancreatic anlage cells. We assessed beta-cell proliferation and differentiation in mouse and human pancreatic anlage cells cocultured with adult islet cells or betaTC3 cells using cellular, molecular, and immunohistochemical methods. Differentiation of murine anlage cells into beta-cells was induced by mature islet cells. It was specific for beta-cells and not a general feature of endodermal derived cells. beta-Cell differentiation required cell-cell contact. The induced cells acquired features of mature beta-cells including increased expression of beta-cell transcription factors and surface expression of receptor for stromal cell-derived factor 1 and glucose transporter-2 (GLUT-2). They secreted insulin in response to glucose and could correct hyperglycemia in vivo when cotransplanted with vascular cells. Human pancreatic anlage cells responded in a similar manner and showed increased expression of pancreatic duodenal homeobox 1 and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A and increased production of proinsulin when cocultured with adult islets. We conclude that mature beta-cells can modify the differentiation of precursor cells and suggest a mechanism whereby changes in differentiation of beta-cells can be affected by other beta-cells.

BackgroundWidely accessible small animal models suitable for the study of hepatitis C virus (HCV) in vivo are lacking, primarily because rodent hepatocytes cannot be productively infected and because human hepatocytes are not easily engrafted in immunodeficient mice.Methodology/Principal FindingsWe report here on a novel approach for human hepatocyte engraftment that involves subcutaneous implantation of primary human fetal hepatoblasts (HFH) within a vascularized rat collagen type I/human fibronectin (rCI/hFN) gel containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) in severe combined immunodeficient X beige (SCID/bg) mice. Maturing hepatic epithelial cells in HFH/Bcl-2-HUVEC co-implants displayed endocytotic activity at the basolateral surface, canalicular microvilli and apical tight junctions between adjacent cells assessed by transmission electron microscopy. Some primary HFH, but not Huh-7.5 hepatoma cells, appeared to differentiate towards a cholangiocyte lineage within the gels, based on histological appearance and cytokeratin 7 (CK7) mRNA and protein expression. Levels of human albumin and hepatic nuclear factor 4α (HNF4α) mRNA expression in gel implants and plasma human albumin levels in mice engrafted with HFH and Bcl-2-HUVEC were somewhat enhanced by including murine liver-like basement membrane (mLBM) components and/or hepatocyte growth factor (HGF)-HUVEC within the gel matrix. Following ex vivo viral adsorption, both HFH/Bcl-2-HUVEC and Huh-7.5/Bcl-2-HUVEC co-implants sustained HCV Jc1 infection for at least 2 weeks in vivo, based on qRT-PCR and immunoelectron microscopic (IEM) analyses of gel tissue.Conclusion/SignificanceThe system described here thus provides the basis for a simple and robust small animal model of HFH engraftment that is applicable to the study of HCV infections in vivo.

The intestinal tract is subject to constant stimulation of immune cells and intestinal epithelial cells either by noninfectious antigens or infection. Recent studies identified the role of IL-17A in chronic inflammation in intestinal epithelium. Yet the intrinsic role of endogenous IL-17A in spontaneous intestinal tumorigenesis has not been addressed previously. Ablation of IL-17A significantly reduced tumor development in mice bearing a heterozygote mutation in the APC (adenomatous polyposis coli) gene (ApcMin/+ mice). There was a marked decrease in IL-6 and IL-23 (p19), as well as a decrease in IL-1β, suggesting that the inflammatory microenvironment was significantly changed by ablating IL-17A. We also found a significant decrease in CD44 and proinflammatory mediators such as KC (Keratinocyte Chemoattractant) and Cox-2. There was also a reduced infiltration of lymphocytes including T cells and preservation of intestinal architecture and the presence of APC protein in intestinal epithelial cells in IL-17AKO ApcMin/+ mice. Interestingly, IL-17A ablation also corrected immunological abnormalities such as splenomegaly and thymic atrophy in ApcMin/+ mice. We tested whether CD4 T cells from ApcMin/+ mice are more proliferative based on its role as a tumor suppressor gene, thereby regulating cell cycle. We observed that CD4 T cells from ApcMin/+ mice were hyperproliferative when compared with their wildtype littermate controls upon TcR (T cell receptor) stimulation. This hyperproliferation was more obvious following stronger stimulation through the TcR. Next, we tested whether the effector CD4 T cells are more refractory to regulatory T cell (Treg)-mediated suppression. Upon co-culture with wildtype Treg cells, ApcMin/+ CD4 effector T cells were more resistant to Treg-mediated suppression especially at a high ratio of effectors to Treg, suggesting that the suppression on ApcMin/+ CD4 effector T cells are not as efficient as for wildtype CD4 effector T cells. Finally, these CD4 T cells from ApcMin/+ mice induced colitis in immunodeficient mice upon adoptive transfer, while the ablation of IL-17A in CD4 T cells in ApcMin/+ mice (IL-17A KO ApcMin/+ mice) completely abolished this pathogenic potential in vivo, clearly showing that ablation of IL-17A abrogated hyperproliferation and colitogenic potential in immunodeficient mice. This was consistent with our observation of the phenotypic changes in intestinal tumorigenesis. Taken together, our results suggest an important proinflammatory role of IL-17A and its correction of immune abnormalities in spontaneous intestinal tumorigenesis. We demonstrate altered CD4 T cell functions from ApcMin/+ mice which favors intestinal tumorigenesis and the ablation of IL-17A can abolish these altered function, suggesting the importance of T-cell mediated IL-17A in spontaneous intestinal tumorigenesis Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5324.

No abstract