Phosphatidylinositol-3-kinase gamma (PI3Kγ) is a leukocyte-specific lipid kinase with signaling function downstream of G protein-coupled receptors to regulate cell trafficking, but its role in T cells remains unclear. To investigate the requirement of PI3Kγ kinase activity in T-cell function, we studied T cells from PI3Kγ kinase-dead knock-in (PI3KγKD/KD) mice expressing the kinase-inactive PI3Kγ protein. We show that CD4+ and CD8+ T cells from PI3KγKD/KD mice exhibit impaired TCR/CD28-mediated activation that could not be rescued by exogenous IL-2. The defects in proliferation and cytokine production were also evident in naïve and memory T cells. Analysis of signaling events in activated PI3KγKD/KD T cells revealed a reduction in phosphorylation of protein kinase B (AKT) and ERK1/2, a decrease in lipid raft formation, and a delay in cell cycle progression. Furthermore, PI3KγKD/KD CD4+ T cells displayed compromised differentiation toward Th1, Th2, Th17, and induced Treg cells. PI3KγKD/KD mice also exhibited an impaired response to immunization and a reduced delayed-type hypersensitivity to Ag challenge. These findings indicate that PI3Kγ kinase activity is required for optimal T-cell activation and differentiation, as well as for mounting an efficient T cell-mediated immune response. The results suggest that PI3Kγ kinase inhibitors could be beneficial in reducing the undesirable immune response in autoimmune diseases.
CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47’s ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention.
Phosphoinositide 3‐kinase gamma (PI3Kγ) catalyzes the production of phosphatidylinositol‐3,4,5‐triphosphate by phosphorylating phosphatidylinositol (PI), phosphatidylinositol‐4‐ phosphate (PIP) and phosphatidylinositol‐4,5‐bisphosphate (PIP2). Catalytic subunit of PI3Kγ is activated by βγ subunits of heterotrimeric G proteins in response to chemokines, growth factors and hormones, which in turn coordinates cell migration, cell growth, cell cycle entry, and cell survival. PI3Kγ has been implicated in T cell function, but direct effect of its kinase function has not been evaluated. Using mice expressing a catalytically inactive mutant of PI3Kγ(PI3KγKD/KD) we were able to demonstrate that defect in kinase activity leads to T cell developmental defect in the thymus and results in decreased peripheral T cell numbers. PI3KγKD/KD peripheral CD4 and CD8 T cells demonstrated impaired TCR/CD28‐induced proliferation and IL‐2 production, reduced chemokine driven chemotaxis and impaired T cell differentiation. Thus, our study demonstrated that kinase activity of PI3Kγ amend status of T cell response by contributing to thymic development, T cell activation and differentiation as well as lymphocyte chemotaxis.
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