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The ability to leverage antibodies to agonize disease relevant biological pathways has tremendous potential for clinical investigation. Yet while antibodies have been successful as antagonists, immune mediators, and targeting agents, they are not readily effective at recapitulating the biology of natural ligands. Among the important determinants of antibody agonist activity is the geometry of target receptor engagement. Here, we describe an engineering approach inspired by a naturally occurring Fab-Fab homotypic interaction that constrains IgG in a unique i-shaped conformation. i-shaped antibody (iAb) engineering enables potent intrinsic agonism of five tumor necrosis factor receptor superfamily (TNFRSF) targets. When applied to bispecific antibodies against the heterodimeric IL-2 receptor pair, constrained bispecific IgG formats recapitulate IL-2 agonist activity. iAb engineering provides a tool to tune agonist antibody function and this work provides a framework for the development of intrinsic antibody agonists with the potential for generalization across broad receptor classes. In contrast to their clinical success as inhibitors and targeting agents, antibodies have generally been ineffective as receptor agonists. Here, Romei et al. leverage a natural homotypic interface to tune antibody geometry, enabling optimization of agonist activity for multiple therapeutic targets. [ABSTRACT FROM AUTHOR]

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W. UBE2W catalyzes the ubiquitination of protein N-termini but its substrate spectrum is largely unknown. Here, the authors discover mAbs selective for peptides derived from N-terminally ubiquitinated proteins, solve the structure of a peptide-bound mAb and apply the mAbs to map endogenous UBE2W substrates by proteomics. [ABSTRACT FROM AUTHOR]

Macrophage colony-stimulating factor (M-CSF) influences the proliferation and survival of mononuclear phagocytes through the receptor CSF-1R. The adaptor protein DAP12 is critical for the function of mononuclear phagocytes. DAP12-mutant mice and humans have defects in osteoclasts and microglia, as well as brain and bone abnormalities. Here we show DAP12 deficiency impaired the M-CSF-induced proliferation and survival of macrophages in vitro. DAP12-deficient mice had fewer microglia in defined central nervous system areas, and DAP12-deficient progenitors regenerated myeloid cells inefficiently after bone marrow transplantation. Signaling by M-CSF through CSF-1R induced the stabilization and nuclear translocation of β-catenin, which activated genes involved in the cell cycle. DAP12 was essential for phosphorylation and nuclear accumulation of β-catenin. Our results provide a mechanistic explanation for the many defects of DAP12-deficient mononuclear phagocytes. [ABSTRACT FROM AUTHOR]

Over the past 20 years great progress has been made in defining most of the key signalling pathways that functionally regulate immune cells. Recently, it has become clear that scaffold proteins have a crucial role in regulating many of these signalling cascades. By binding two or more components of a signalling pathway, scaffold proteins can help to localize signalling molecules to a specific part of the cell or to enhance the efficacy of a signalling pathway. Scaffold proteins can also affect the thresholds and the dynamics of signalling reactions by coordinating positive and negative feedback signals. In this Review, we focus on recent progress in the understanding of the function of scaffold proteins in immune cells. [ABSTRACT FROM AUTHOR]

The 'lipid raft' hypothesis has been a contentious topic over the past 5 years, with much of the immunology community divided into 'believers' and 'nonbelievers'. The disagreement is due mainly to the inability to observe these membrane domains directly and to the widespread use of experimental approaches of dubious utility. As a lipid raft 'dilettante' who has dabbled in the area over the years, I view the lipid raft model with some skepticism and disinterest because of that confusion. Although progress in the field has helped clarify some of the issues, more work is still needed to formally confirm the lipid raft hypothesis and to reestablish the scientific credibility of this area. [ABSTRACT FROM AUTHOR]

Mice lacking CD2-associated protein (CD2AP−/−) develop glomerular lesions resembling human focal segmental glomerulosclerosis (FSGS) between 3–4 weeks of age and die approximately 2 weeks later from massive proteinuria and renal failure. The mechanisms involved in the glomerular injury in this model are unclear. In this study, we used laser capture microdissection (LCM) and real-time PCR, and examined expression of TGF-ß isoforms in CD2AP−/− mice at the level of isolated glomeruli. Total RNA yield from cryosections of 30 glomeruli was 10.71 ng (SD, 5.45) in CD2AP+/+ group ( n =7), and 4.20 ng (SD, 2.04) in CD2AP−/− group ( n =8), p =0.008. Expression of TGF-ß1 mRNA was increased 1.5-fold in the whole kidney ( p =0.030), and twofold in isolated CD2AP−/− glomeruli ( p =0.026). Whole kidney mRNA of TGF-ß receptor I (RI) and II (RII) was not different in CD2AP−/− and CD2AP+/+ animals, but it was increased in CD2AP−/− glomerular samples by 4.38-fold ( p =0.001) and 11.37-fold ( p =0.0163), respectively. By using LCM we confirmed increased glomerular expression levels of TGF-ß isoforms previously described by our group in glomeruli isolated by sieving in CD2AP KO mice and underscored the importance of local factors in the development of glomerulosclerosis. [ABSTRACT FROM AUTHOR]

As with all scientific disciplines, advances in immunology are driven by experimental observations. Observations yield facts that can be integrated into hypotheses and theoretical models that are amenable to further experimental tests. The ultimate product of this iterative process of making observations and theoretical model building is mechanistic understanding. The creation of models has been essential in many important advances in biology; the most famous example is Watson and Crick's model for the structure of DNA. Exploration of ideas with conceptual models before the availability of experimental data has also led to the development of some of the basic paradigms of immunology. In the past three decades, advances in cell culture techniques, immunochemistry, recombinant DNA methodology, X-ray crystallography and the use of transgenic mice have enabled the explanation of diverse immunological phenomena in terms of protein structure and specific biochemical events. However, there are some recent indications that quantitative theoretical models can partner fruitfully with cellular experiments to substantially advance the understanding of molecular mechanisms that underlie key events such as T cell activation, T cell migration and thymocyte selection.

Although the Src family tyrosine kinase Lck is essential for T cell receptor (TCR) signaling, whether or how Lck is activated is unknown. Using a phosphospecific antiserum to Lck, we show here that Lck becomes autophosphorylated when T cells are stimulated by antigen-presenting cells (APCs). We found that TCR cross-linking alone could not stimulate Lck autophosphorylation and CD45 was not required for this process. Instead, the T cell accessory molecules CD4 and CD28 cooperated to induce autophosphorylation of Lck. CD4 recruited Lck to the T cell?APC interface, whereas CD28 sustained Lck activation. These data show how the multiple interactions afforded by the immunological synapse drive efficient and highly specific signaling. [ABSTRACT FROM AUTHOR]

According to the two-signal model of T cell activation, costimulatory molecules augment T cell receptor (TCR) signaling, whereas adhesion molecules enhance TCR?MHC-peptide recognition. The structure and binding properties of CD28 imply that it may perform both functions, blurring the distinction between adhesion and costimulatory molecules. Our results show that CD28 on naïve T cells does not support adhesion and has little or no capacity for directly enhancing TCR?MHC-peptide interactions. Instead of being dependent on costimulatory signaling, we propose that a key function of the immunological synapse is to generate a cellular microenvironment that favors the interactions of potent secondary signaling molecules, such as CD28. [ABSTRACT FROM AUTHOR]

Looks at two studies that show the spatial-temporal organization of lipid second messengers or phosphoinositols (PI) during immunological synapse formation. Role of phosphatidylinositol-3 kinase during T cell antigen-presenting cell interactions; Adaptation of technology to study PI metabolism in T cell activation.

Reports that immunological and neurological synapses use a common molecule called agrin. T cell activation; Recruitment of lipid rafts; Deglycosylation of extracellular ligands.

Mesangial cells are stromal cells that are important for kidney glomerular homeostasis and the glomerular response to injury. A growing body of evidence demonstrates that mesenchymal stromal cells, such as stromal fibroblasts, pericytes and vascular smooth muscle cells, not only specify the architecture of tissues but also regulate developmental processes, vascularization and cell fate specification. In addition, through crosstalk with neighbouring cells and indirectly through the remodelling of the matrix, stromal cells can regulate a variety of processes such as immunity, inflammation, regeneration and in the context of maladaptive responses - fibrosis. Insights into the molecular phenotype of kidney mesangial cells suggest that they are a specialized stromal cell of the glomerulus. Here, we review our current understanding of mesenchymal stromal cells and discuss how it informs the function of mesangial cells and their role in disease. These new insights could lead to a better understanding of kidney disease pathogenesis and the development of new therapies for chronic kidney disease. [ABSTRACT FROM AUTHOR]