Armenian Hamster Anti-Mouse CD40L (CD154) Monoclonal antibody, clone MR-1 Functional Grade

Interactions between different cell types are essential for multiple biological processes, including immunity, embryonic development and neuronal signalling. Although the dynamics of cell-cell interactions can be monitored in vivo by intravital microscopy, this approach does not provide any information on the receptors and ligands involved or enable the isolation of interacting cells for downstream analysis. Here we describe a complementary approach that uses bacterial sortase A-mediated cell labelling across synapses of immune cells to identify receptor-ligand interactions between cells in living mice, by generating a signal that can subsequently be detected ex vivo by flow cytometry. We call this approach for the labelling of ‘kiss-and-run’ interactions between immune cells ‘Labelling Immune Partnerships by SorTagging Intercellular Contacts’ (LIPSTIC). Using LIPSTIC, we show that interactions between dendritic cells and CD4(+) T cells during T-cell priming in vivo occur in two distinct modalities: an early, cognate stage, during which CD40-CD40L interactions occur specifically between T cells and antigen-loaded dendritic cells; and a later, non-cognate stage during which these interactions no longer require prior engagement of the T-cell receptor. Therefore, LIPSTIC enables the direct measurement of dynamic cell-cell interactions both in vitro and in vivo. Given its flexibility for use with different receptor-ligand pairs and a range of detectable labels, we expect that this approach will be of use to any field of biology requiring quantification of intercellular communication.

Current therapies to treat inflammatory bowel diseases have limited efficacy, significant side effects, and often wane over time. Little is known about the cellular and molecular mechanisms operative in the process of mucosal healing from colitis. To study such events, we developed a new model of reversible colitis in which adoptive transfer of CD4+CD45RBhi T cells into Helicobacter typhlonius-colonized lymphopenic mice resulted in a rapid onset of colonic inflammation that was reversible through depletion of colitogenic T cells. Remission was associated with an improved clinical and histopathological score, reduced immune cell infiltration to the intestinal mucosa, altered intestinal gene expression profiles, regeneration of the colonic mucus layer, and the restoration of epithelial barrier integrity. Notably, colitogenic T cells were not only critical for induction of colitis but also for maintenance of disease. Depletion of colitogenic T cells resulted in a rapid drop in tumor necrosis factor alpha (TNFalpha) levels associated with reduced infiltration of inflammatory immune cells to sites of inflammation. Although neutralization of TNFalpha prevented the onset of colitis, anti-TNFalpha treatment of mice with established disease failed to resolve colonic inflammation. Collectively, this new model of reversible colitis provides an important research tool to study the dynamics of mucosal healing in chronic intestinal remitting-relapsing disorders.

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.

Pancreatic islet transplantation is a promising strategy for diabetic patients. Unfortunately, host's immune cells rapidly reject transplanted islets. Cytoprotective gene therapy has been tried to protect them. However, the transduce yield of ex vivo gene delivery into islets is very rare due to the anatomical structure of them. Therefore, we newly designed an Arg-Gly-Asp (RGD) peptide-incorporated adenovirus vector expressing cytoprotective heme oxygenase-1 (HO1) gene (RGD-Adv-HO1) for effective gene therapy to islets via RGD integrin interaction between adenovirus and islet. After exposure to islets, the transduced RGD-Adv-HO1 did not affect the viability and insulin secretion of islets with higher transduction efficacy. Moreover, HO1 showed cytoprotective effect from the presence of paraquat-induced reactive oxygen species (ROS). When the transduced islets were xenotransplanted into streptozotocin-induced diabetic Balb/c mice, their survival time in vivo was significantly prolonged with curing blood glucose level, which was attributed to the stable expression of HO1. Additionally, we found that RGD-Adv-HO1 showed a good synergistic effect in xenotransplanted islets when accompanied with low dose of immunosuppressants tacrolimus and anti-CD154 monoclonal antibody. Collectively, this new combinatorial remedy of RGD-Adv-HO1 transduction with low dose of immunosuppressive agents could be an effective therapy for successful outcome of islet transplantation. (C) 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and T cell hyper-activation. Emerging evidence has shown that the stimulation of immunoglobulin D (IgD) induces T cell activation and may contribute to disease pathogenesis. In this study, the sIgD concentrations were positively associated with disease activity score in 28 joints (DAS28) and anti-cyclic citrullinated peptide (anti-CCP) in RA. We demonstrated that IgD-Fc-Ig (composed of human IgD Fc domain and IgG(1)Fc domain, obtained through prokaryotic protein expression and chromatography purification) effectively inhibited the activation and proliferation of T cells in healthy controls and PBMCs in RA patients stimulated by IgD, recovered the Th17/Treg cell subset balance, and downregulated p-Lck and p-ZAP70 expression. Moreover,in vivo, IgD-Fc-Ig decreased the swollen joint counts and arthritis indices in mice with collagen-induced arthritis (CIA), and ameliorated histopathological changes in joint and spleen tissue. It also downregulated thymocyte proliferation and reduced the percentage of helper T cells (Th) and CD154(+)T cells, reversed the imbalance of Th1/Th2 and Th17/Treg cell subsets, reduced cytokine and chemokine levels, and inhibited p-Lck and p-ZAP70 expression. Our data suggest that IgD-Fc-Ig fusion protein regulates T cell activity in RA. These findings have potential implications for IgD-targeted strategies to treat IgD-associated RA.