CD167 Acts As A Novel Costimulatory Receptor In T Cell Activation

It is well accepted that activation of resting T cells requires two distinct signals. Signal one is derived from recognition of MHC-peptide complexes by the TCR and signal two is provided by the binding of the T cell-expressed costimulatory molecule to its ligands on antigen-presenting cell (APC). Most T cells activated in the absence of appropriate costimulatory events appear to cause peripheral tolerance or die prematurely. Intensive research has characterized the T cell molecule CD28 as the principal receptor that generates costimulatory signals in resting T cells for their activation. In fact, multiple molecules on the T cell have also been shown to costimulate resting T cells, including CD2, CD5, CD9 and CD99. In most studies costimulation has been evaluated as the capacity of each mAb against these molecules to enhance proliferation of resting T cells stimulated with suboptimal doses of anti-CD3. These co-signaling pathways play overlapping and distinct regulatory roles at various stages of T-cell response, so that the immune responses process in a proper manner and intensity. In recent years, the number of cell surface molecules known to possess costimulatory activity or to function as their receptors has grown rapidly. Costimulation has become an area of intense investigation because a mechanistic understanding of costimulatory signals may enable rational therapeutic approaches for manipulating immune responses.CD167 (previously also known as discoidin domain receptor, DDR) is a tyrosine receptor kinase and contains two members, CD167a (DDR1) and CD167b (DDR2), which are characterised by the presence of an extracellular discoidin homology (DS) domain. Both CD167a and CD167b are widely expressed during development and in adult tissues. CD167a is mostly found in epithelial cells and leukocytes, while CD167b is confined to mesenchymal cells. Both receptors regulate cell proliferation, adhesion and motility, and control remodelling of the extracellular matrix by regulating the expression and activity of matrix metalloproteinases. With respect to human disease, CD167a and CD167b are associated with fibrotic diseases of the lung and osteoarthritis.Structurally, each CD167 consists of a discoidin (DS) domain and a stalk region in its extracellular portion, and an intracellular tyrosine kinase, connected by a transmembrane region. In addition, CD167a and CD167b share 88.5% homology within their discoidin domains and 89.5% homology within their tyrosine kinase domains. The direct interaction of collagen type I with the DS domain of CD167 trigger the activation of its intracellular tyrosine kinase, leading to downstream intracellular signaling. Current evidence indicates that CD167 signaling is independent ofβ1 integrins, the other main class of collagen type I -binding receptors. Interestingly, theα4β1-integrin (also known as very late antigen-4, VLA-4) accumulates at the IS and can function as a co-stimulatory molecule for T-cell activation. Therefore, we hypothesized whether CD167 might be expressed on T cells and involved in T cell activation. The research program was focused on this question.In this report, we first analyzed the expression profile of CD167 on resting and activated human T cells. Real-time quantitative polymerase chain reaction (PCR) analysis and triple-color stained and analyzed by flow cytometry showed that CD167 expression is up-regulated in activated T cells. Furthermore, in the presence of anti-CD167 mAb or collagen type I plus anti-CD3 mAb, T cell proliferation by CFSE tracking indicated that cross-linking of CD167 upon TCR signaling on resting T cells enhanced proliferation. Double-color stained and analyzed by flow cytometry indicated that cross-linking of CD167 upon TCR signaling on resting T cells up-regulated activation markers of T cell such as CD25 and CD69. Calcium mobilization assay showed that cross-linking of CD167 upon TCR signaling on resting T cells elevated intracellular Ca2+ levels. Western blot assay showed that cross-linking of CD167 upon TCR signaling on resting T cells induced intracellular tyrosine phoshorylation. These results suggest that CD167 provides a costimulatory signal for T cell activation through the TCR.Activation of resting T cells by specific antigen under appropriate conditions leads to functional differentiation into subsets of effector cells. This differentiation is accompanied by changes in homing receptor expression resulting in memory subsets displaying distinct homing properties. In this study, we observed that cross-linking of CD167 upon TCR signaling on resting T cells abrogates IL-4 secretion and leads to enhanced secretion of IFN-γby T cells, indicating that cross-linking of CD167 upon TCR signaling on resting T cells polarized T cell differentiation toward Th1/Tc1 cells.During T cell activation, the membrane lipid rafts which are biochemically characterized as detergent insoluble glycosphingolipid-enriched microdomains may serve as platforms for recruiting and concentrating signaling components and thus enable the close interaction of essential molecules. On the other hand, during antigen presentation, the interface between the T cell and APC membranes forms the hot spot for T-cell activation, a highly organized supramolecular structure known as the immunological synapse (IS), where signaling, adhesion and cytoskeleton molecules are concentrated within lipid raft microdomains following TCR co-aggregation. Namely, rafts may be required to organize a complete immunological synapse or for signal transduction associated with T-cell costimulatory molecules. In this study, Confocal microscopy and Western blot assay showed that CD167 is recruited to T cell lipid rafts within the immunological synapse after cross-linking through CD3 and CD28.In conclusion, we have identified a previously unknown function for CD167 in T cell activation and have demonstrated that CD167 acts as a novel costimulatory receptor and recruits to the lipid rafts in immunological synapse.