Hydrogen/deuterium exchange mass spectrometry and computational modeling reveal a discontinuous epitope of an antibody/TL1A Interaction.

TL1A, a tumor necrosis factor-like cytokine, is a ligand for the death domain receptor DR3. TL1A, upon binding to DR3, can stimulate lymphocytes and trigger secretion of proinflammatory cytokines. Therefore, blockade of TL1A/DR3 interaction may be a potential therapeutic strategy for autoimmune and inflammatory diseases.

A robust high throughput platform to generate functional recombinant monoclonal antibodies using rabbit B cells from peripheral blood.

We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies.

Selective inhibition of spleen tyrosine kinase (SYK) with a novel orally bioavailable small molecule inhibitor, RO9021, impinges on various innate and adaptive immune responses: implications for SYK inhibitors in autoimmune disease therapy.

Introduction: Spleen tyrosine kinase (SYK) is a key integrator of intracellular signals triggered by activated immunoreceptors, including Bcell receptors (BCR) and Fc receptors, which are important for the development and function of lymphoid cells. Given the clinical efficacy of Bcell depletion in the treatment of rheumatoid arthritis and multiple sclerosis, pharmacological modulation of Bcells using orally active small molecules that selectively target SYK presents an attractive alternative therapeutic strategy.

Methods: A SYK inhibitor was developed and assayed in various in vitro systems and in the mouse model of collagen-induced arthritis (mCIA).

Thymic stromal lymphopoietin receptor blockade reduces allergic inflammation in a cynomolgus monkey model of asthma.

Background: Thymic stromal lymphopoietin (TSLP) pathway blockade is a potential strategy for asthma treatment because the main activities of TSLP are activation of myeloid dendritic cells (mDCs) and modulation of cytokine production by mast cells. TSLP-activated mDCs prime the differentiation of naive T cells into inflammatory TH2 cells.

Objective: We sought to investigate mechanisms underlying the development of allergic lung inflammation in cynomolgus monkeys using gene expression profiling and to assess the effect of thymic stromal lymphopoietin receptor (TSLPR) blockade in this model.

RN486, a selective Bruton's tyrosine kinase inhibitor, abrogates immune hypersensitivity responses and arthritis in rodents.

Genetic mutation and pharmacological inhibition of Bruton's tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one (RN486), in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fcε receptor cross-linking-induced degranulation in mast cells (IC(50) = 2.

Human cytomegalovirus decreases constitutive transcription of MHC class II genes in mature Langerhans cells by reducing CIITA transcript levels.

Human cytomegalovirus (HCMV) productively infects CD34(+) progenitor-derived, mature Langerhans-type dendritic cells (matLC) and reduces surface expression of MHC class II complexes (MHC II) by increasing intracellular retention of these molecules. To determine whether HCMV also inhibits MHC II expression by other mechanisms, we assessed mRNA levels of the class II transcriptional regulator, CIITA, and several of its target genes in infected matLC. Levels of CIITA, HLA-DRA (DRA) and DRB transcripts, and new DR protein synthesis were compared in mock-infected and HCMV-infected cells by quantitative PCR and pulse-chase immunoprecipitation analyses, respectively.

DM influences the abundance of major histocompatibility complex class II alleles with low affinity for class II-associated invariant chain peptides via multiple mechanisms.

DM catalyses class II-associated invariant chain peptide (CLIP) release, edits the repertoire of peptides bound to major histocompatibility complex (MHC) class II molecules, affects class II structure, and thereby modulates binding of conformation-sensitive anti-class II antibodies. Here, we investigate the ability of DM to enhance the cell surface binding of monomorphic antibodies. We show that this enhancement reflects increases in cell surface class II expression and total cellular abundance, but notably these effects are selective for particular alleles.

Failed efficacy of soluble human CD83-Ig in allogeneic mixed lymphocyte reactions and experimental autoimmune encephalomyelitis: implications for a lack of therapeutic potential.

Soluble forms of CD83, a dendritic cell-specific surface glycoprotein, have been strongly proposed to be of therapeutic utility in inflammatory conditions such as multiple sclerosis and transplantation. We demonstrate here, however, that eukaryotically expressed, recombinant soluble human CD83-Ig molecules fail to achieve efficacy in model systems for those conditions: mouse experimental autoimmune encephalomyelitis models in vivo or in mixed lymphocyte reactions in vitro. These results raise concern as to the viability of a eukaryotically expressed soluble CD83 strategy for clinical therapeutic use.

WITHDRAWN: Failed efficacy of soluble human CD83-Ig in allogeneic mixed lymphocyte reactions and experimental autoimmune encephalomyelitis: implications for a lack of therapeutic potential?

This article has been withdrawn consistent with Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).

NKp46 and NKG2D recognition of infected dendritic cells is necessary for NK cell activation in the human response to influenza infection.

At an early phase of viral infection, contact and cooperation between dendritic cells (DCs) and NK cells activates innate immunity, and also influences recruitment, when needed, of adaptive immunity. Influenza, an adaptable fast-evolving virus, annually causes acute, widespread infections that challenge the innate and adaptive immunity of humanity. In this study, we dissect and define the molecular mechanisms by which influenza-infected, human DCs activate resting, autologous NK cells.

Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells.

Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC.

Human dendritic cell expression of HLA-DO is subset specific and regulated by maturation.

Expression of HLA-DO (DO) in cells that express HLA-DM (DM) results in an altered repertoire of MHC class II/peptide complexes, indicating that DO modulates DM function. Human and murine B cells and thymic epithelial cells express DO, while monocytes/macrophages do not. Monocyte-derived dendritic cells (DC) also have been found to be DO-negative, leading to the assumption that DC do not express DO.

Targeting the innate immune response with improved vaccine adjuvants.

Despite two centuries of vaccine use, only a few adjuvants and delivery systems are licensed for human use. This is partly because traditional vaccines based on attenuated live organisms already have them--their invasiveness provides efficient delivery to antigen-presenting cells and various naturally occurring components of the pathogens stimulate the innate immune system. But consideration of these immune potentiators and delivery systems has become important to the development of new subunit vaccines consisting of isolated antigens.

Interaction of HLA-DR with an acidic face of HLA-DM disrupts sequence-dependent interactions with peptides.

HLA-DM (DM) edits major histocompatibility complex class II (MHCII)-bound peptides in endocytic compartments and stabilizes empty MHCII molecules. Crystal structures of DM have revealed similarity to MHCII but not how DM and MHCII interact. We used mutagenesis to map a MHCII-interacting surface on DM.

Point mutations in or near the antigen-binding groove of HLA-DR3 implicate class II-associated invariant chain peptide affinity as a constraint on MHC class II polymorphism.

During maturation of MHC II molecules, newly synthesized and assembled complexes of MHC II alphabeta dimers with invariant chain (Ii) are targeted to endosomes, where Ii is proteolyzed, leaving remnant class II-associated Ii peptides (CLIP) in the MHC II peptide binding groove. CLIP must be released, usually with assistance from the endosomal MHC II peptide exchange factor, HLA-DM, before MHC II molecules can bind endosomal peptides. Structural factors that control rates of CLIP release remain poorly understood, although peptide side chain-MHC II specificity pocket interactions and MHC II polymorphism are important.

Role of IL-12-independent and IL-12-dependent pathways in regulating generation of the IFN-gamma component of T cell responses to Salmonella typhimurium.

Clearance of facultative intracellular pathogens such as Salmonella requires IFN-gamma from CD4 T cells. Mechanisms linking intracellular pathogen recognition with induction of IFN-gamma-producing T cells are still poorly understood. We show in this study that IL-12 is not required for commitment to the IFN-gamma-producing T cell response in infection with Salmonella typhimurium, but is needed for its maintenance.

Stabilization of soluble, low-affinity HLA-DM/HLA-DR1 complexes by leucine zippers.

The ectodomains of interacting membrane-bound proteins, when expressed as recombinant soluble molecules, often have low affinities for each other, hampering studies of their interaction. We reasoned that stabilization of unstable protein-protein complexes should aid our understanding of the structural and functional consequences of complex formation. Here, we have used fusion with leucine zipper (LZ) domains to stabilize a complex formed between the class II major histocompatibility complex (MHC-II) protein, HLA-DR1 (which binds peptides for presentation to CD4+ T cells) and HLA-DM (which catalyzes peptide exchange of MHC-II molecules).