Transglutaminase 2 interacts with syndecan-4 and CD44 at the surface of human macrophages to promote removal of apoptotic cells

Tissue transglutaminase (TG2) is a multifunctional protein cross-linking enzyme that has been implicated in apoptotic cell clearance but is also important in many other cell functions including cell adhesion, migration and monocyte to macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4 and β1 and β3 integrins. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise role of TG2 in apoptotic cell clearance remains ill-defined. Our work addresses the role of macrophage extracellular TG2 in apoptotic cell corpse clearance. Here we reveal TG2 expression and activity (cytosolic and cell surface) in human macrophages and demonstrate that inhibitors of protein crosslinking activity reduce macrophage clearance of dying cells. We show also that cell-impermeable TG2 inhibitors significantly inhibit the ability of macrophages to migrate and clear apoptotic cells through reduced macrophage recruitment to, and binding of, apoptotic cells. Association studies reveal TG2-syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus our data define a role for TG2 activity at the surface of human macrophages in multiple stages of AC clearance and we propose that TG2, in association with heparan sulphates, may exert its effect on AC clearance via a mechanism involving the crosslinking of CD44.

Physical and functional characterisation of apoptotic cell-derived extracellular vesicles

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in the exposure of 'flags' at the dying cell surface and the release of attractive signals to recruit phagocytes. Together these changes ensure efficient phagocytic removal of dying cells and prevention of inflammatory and autoimmune disorders. Extracellular vesicles (EV) are released from a variety of cells (both viable and apoptotic) and they serve as a novel means of intercellular communication. They range in size: 70-100nm ('exosomes') through 100-1000nm ('microparticles') to large vesicles released from dying cells ('apoptotic bodies'). Release of apoptotic cell-derived extracellular vesicles (acdEV) of less than 1000nm is an important mechanism by which phagocytes are attracted to sites of cell death. Using a variety of approaches we characterize the release, physical characteristics and function of acdEV. Using fluorescence microscopy we demonstrate release of ICAM-3 on acdEV from dying leukocytes and, through the use of resistive pulse technology (qNano, IZON Science), we accurately size and quantitate acdEV release. The function of acdEV is revealed through the use of both horizontal chemotaxis assays (Dunn chambers) and vertical transwell migration assays (Cell-IQ, CM Technologies). These assays reveal potent chemoattractive capacity of acdEV and associated ICAM-3. Additionally we demonstrate an additional novel function of acdEV as anti-inflammatory immune-modulators. These data support an integrated approach to the physical and functional analyses of EV.

EpiJen:a server for multistep T cell epitope prediction

Background - The main processing pathway for MHC class I ligands involves degradation of proteins by the proteasome, followed by transport of products by the transporter associated with antigen processing (TAP) to the endoplasmic reticulum (ER), where peptides are bound by MHC class I molecules, and then presented on the cell surface by MHCs. The whole process is modeled here using an integrated approach, which we call EpiJen. EpiJen is based on quantitative matrices, derived by the additive method, and applied successively to select epitopes. EpiJen is available free online. Results - To identify epitopes, a source protein is passed through four steps: proteasome cleavage, TAP transport, MHC binding and epitope selection. At each stage, different proportions of non-epitopes are eliminated. The final set of peptides represents no more than 5% of the whole protein sequence and will contain 85% of the true epitopes, as indicated by external validation. Compared to other integrated methods (NetCTL, WAPP and SMM), EpiJen performs best, predicting 61 of the 99 HIV epitopes used in this study. Conclusion - EpiJen is a reliable multi-step algorithm for T cell epitope prediction, which belongs to the next generation of in silico T cell epitope identification methods. These methods aim to reduce subsequent experimental work by improving the success rate of epitope prediction.

Transporter associated with antigen processing preselection of peptides binding to the MHC:a bioinformatic evaluation

TAP is responsible for the transit of peptides from the cytosol to the lumen of the endoplasmic reticulum. In an immunological context, this event is followed by the binding of peptides to MHC molecules before export to the cell surface and recognition by T cells. Because TAP transport precedes MHC binding, TAP preferences may make a significant contribution to epitope selection. To assess the impact of this preselection, we have developed a scoring function for TAP affinity prediction using the additive method, have used it to analyze and extend the TAP binding motif, and have evaluated how well this model acts as a preselection step in predicting MHC binding peptides. To distinguish between MHC alleles that are exclusively dependent on TAP and those exhibiting only a partial dependence on TAP, two sets of MHC binding peptides were examined: HLA-A*0201 was selected as a representative of partially TAP-dependent HLA alleles, and HLA-A*0301 represented fully TAP-dependent HLA alleles. TAP preselection has a greater impact on TAP-dependent alleles than on TAP-independent alleles. The reduction in the number of nonbinders varied from 10% (TAP-independent) to 33% (TAP-dependent), suggesting that TAP preselection is an important component in the successful in silico prediction of T cell epitopes.