We have determined the high resolution crystal structure of the I domain from the ␣-subunit of the integrin ␣2␤1, a cell surface adhesion receptor for collagen and the human pathogen echovirus-1. The domain, as expected, adopts the dinucleotide-binding fold, and contains a metal ion-dependent adhesion site motif with bound Mg 2؉ at the top of the ␤-sheet. Comparison with the crystal structures of the leukocyte integrin I domains reveals a new helix (the C-helix) protruding from the metal ion-dependent adhesion site face of the domain which creates a groove centered on the magnesium ion. Modeling of a collagen triple helix into the groove suggests that a glutamic acid side chain from collagen can coordinate the metal ion, and that the Chelix insert is a major determinant of binding specificity. The binding site for echovirus-1 maps to a distinct surface of the ␣2-I domain (one edge of the ␤-sheet), consistent with data showing that virus and collagen binding occur by different mechanisms. Comparison with the homologous von Willebrand factor A3 domain, which also binds collagen, suggests that the two domains bind collagen in different ways.The integrins are a family of plasma membrane proteins that transduce bidirectional signals between the cytoplasm and the extracellular matrix or other cells (1). The integrin ␣2␤1 is expressed on a variety of cell types, serving as the collagen receptor on platelets and fibroblasts, and as both a collagen and laminin receptor on endothelial and epithelial cells (2, 3). It also acts as the receptor for the human pathogen echovirus-1 (4). In common with six other integrin ␣-chains (␣1, ␣D, ␣E, ␣L, ␣M, and ␣X) the ␣2 chain contains a 200-amino acid inserted domain, the I domain, that is homologous to the von Willebrand factor A domains (5). Recombinant ␣2-I domain recapitulates many of the ligand binding properties of the parent integrin (6 -8). It exhibits specific binding to various fibrillar collagens, and two groups have shown that, like collagen binding to the complete receptor (9, 10), binding to the I domain is cation-dependent, being supported by magnesium or manganese but not by calcium (7, 11). The triple-helical structure of collagen is required for recognition by ␣2␤1, but specific collagen sequences have not been identified (for review, see Ref. 12).The first crystal structure of an integrin I domain, from ␣M␤2, showed that it adopts the dinucleotide-binding fold, with a central parallel ␤-sheet surrounded on both sides by ␣-helices (13). In this class of fold, a functional surface of the domain always lies at the C-terminal end of the ␤-sheet (14). In the I domain, a novel cation coordination sphere is located there, and in the ␣M-I domain crystal structure with bound Mg 2ϩ , a glutamate side chain from a neighboring I domain in the crystal lattice completes the octahedral coordination sphere of the metal. This led to the suggestion that the glutamate behaves as a ligand mimetic, as most integrin ligands possess a critical aspartate residue (or glutamate) as a key fe...

Summary Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

Ly6G is a glycosylphosphatidylinositol (GPI)-anchored protein of unknown function that is commonly targeted to induce experimental neutrophil depletion in mice.In the present study, we found that doses of anti-Ly6G Abs too low to produce sustained neutropenia remained capable of inhibiting experimental arthritis, leaving joint tissues free of infiltrating neutrophils. Thioglycollate-stimulated peritonitis was also attenuated. No alteration in neutrophil apoptosis was observed, implicating impaired recruitment. Indeed, Ly6G ligation abrogated neutrophil migration toward LTB 4 and other chemoattractants in a transwell system. Exploring the basis for this blockade, we identified colocalization of Ly6G and ␤2-integrins by confocal microscopy and confirmed close association by both coimmunoprecipitation and fluorescence lifetime imaging microscopy. Anti-Ly6G Ab impaired surface expression of ␤2-integrins in LTB 4 -stimulated neutrophils and mimicked CD11a blockade in inhibiting both ICAM-1 binding and firm adhesion to activated endothelium under flow conditions. Correspondingly, migration of ␤2-integrindeficient neutrophils was no longer inhibited by anti-Ly6G. These results demonstrate that experimental targeting of Ly6G has functional effects on the neutrophil population and identify a previously unappreciated role for Ly6G as a modulator of neutrophil migration to sites of inflammation via a ␤2-integrindependent mechanism. (Blood. 2012; 120(7):1489-1498) IntroductionNeutrophils are one of the first cell types to reach sites of infection or acute inflammation. Recruitment involves an orchestrated sequence of events in which circulating neutrophils respond to chemotactic signals to become firmly adherent to activated endothelium, followed by transendothelial migration via either a paracellular or transcellular route. 1,2 Once at a site of inflammation, neutrophils contribute to ongoing leukocyte recruitment and tissue injury by releasing lipid mediators, proteases, reactive oxygen species (ROS), and other factors. [3][4][5] Whereas they are critical to immune defense, neutrophils can, also play a pathogenic role in chronic inflammatory diseases and therefore their recruitment is subject to numerous levels of control, not all of which are understood completely. 1 Delineating these regulatory pathways will provide insights into the mechanisms of tissue injury in inflammatory disorders and novel targets for drug development.Much of the experimental evidence implicating neutrophils in disease has been obtained through murine studies in which this lineage was selectively depleted using Abs that bind the neutrophil surface antigen Ly6G, such as RB6-8C5 (more typically termed anti-Gr-1). 6,7 However, the function of Ly6G remains unknown. Ly6G is a small protein of approximately 25 kDa that is tethered to the cell surface via a GPI linker. 7 In bone marrow (BM), peripheral blood, and wound exudates, the expression of Ly6G is limited to cells with granulocyte morphology. 7,8 Structurally, Ly6G belongs to the Ly6/urokinase pl...