Regulation of T-cell activity is dependent on antigen-independent co-stimulatory signals provided by the disulphide-linked homodimeric T-cell surface receptors, CD28 and CTLA-4 (ref. 1). Engagement of CD28 with B7-1 and B7-2 ligands on antigen-presenting cells (APCs) provides a stimulatory signal for T-cell activation, whereas subsequent engagement of CTLA-4 with these same ligands results in attenuation of the response. Given their central function in immune modulation, CTLA-4- and CD28-associated signalling pathways are primary therapeutic targets for preventing autoimmune disease, graft versus host disease, graft rejection and promoting tumour immunity. However, little is known about the cell-surface organization of these receptor/ligand complexes and the structural basis for signal transduction. Here we report the 3.2-A resolution structure of the complex between the disulphide-linked homodimer of human CTLA-4 and the receptor-binding domain of human B7-2. The unusual dimerization properties of both CTLA-4 and B7-2 place their respective ligand-binding sites distal to the dimer interface in each molecule and promote the formation of an alternating arrangement of bivalent CTLA-4 and B7-2 dimers that extends throughout the crystal. Direct observation of this CTLA-4/B7-2 network provides a model for the periodic organization of these molecules within the immunological synapse and suggests a distinct mechanism for signalling by dimeric cell-surface receptors.

Gelatinase A (MMP-2), a matrix metalloproteinase (MMP) involved in tumor invasion and angiogenesis, is secreted as an inactive zymogen (proMMP-2) and activated by proteolytic cleavage. Here we report that polymorphonuclear neutrophil (PMN)-derived elastase, cathepsin G, and proteinase-3 activate proMMP-2 through a mechanism that requires membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Immunoprecipitation of human PMN-conditioned medium with a mixture of antibodies to elastase, cathepsin G, and proteinase-3 abolished proMMP-2 activation, whereas individual antibodies were ineffective. Incubation of HT1080 cells with either purified PMN elastase or cathepsin G or proteinase-3 resulted in dose-and time-dependent proMMP-2 activation. Addition of PMN-conditioned medium to MT1-MMP expressing cells resulted in increased proMMP-2 activation and in vitro invasion of extracellular matrix (ECM), but had no effect with cells that express no MT1-MMP. MMP-2 activation by PMN-conditioned medium or purified elastase was blocked by the elastase inhibitor alpha(1)-antitrypsin but not by Batimastat, an MMP inhibitor, showing that elastase activation of MMP-2 is not mediated by MMP activities. The PMN-conditioned medium-induced increase in cell invasion was blocked by Batimastat as well as by alpha(1)-antitrypsin, showing that PMN serine proteinases trigger a proteinase cascade that entails proMMP-2 activation: this gelatinase is the downstream effector of the proinvasive activity of PMN proteinases. These findings indicate a novel role for PMN-mediated inflammation in a variety of tissue remodeling processes including tumor invasion and angiogenesis.

PD-1, a member of the CD28/CTLA-4/ICOS costimulatory receptor family, delivers negative signals that have profound effects on T and B cell immunity. The 2.0 A crystal structure of the extracellular domain of murine PD-1 reveals an Ig V-type topology with overall similarity to the CTLA-4 monomer; however, there are notable differences in regions relevant to function. Our structural and biophysical data show that PD-1 is monomeric both in solution as well as on cell surface, in contrast to CTLA-4 and other family members that are all disulfide-linked homodimers. Furthermore, our structure-based mutagenesis studies identify the ligand binding surface of PD-1, which displays significant differences compared to those present in the other members of the family.

B7-1 and B7-2 are homologous costimulatory ligands expressed on the surfaces of antigen-presenting cells. Their interactions with CD28͞CTLA-4 receptors expressed on T cell surfaces are crucial for the proper regulation of T cell activity. B7-1 and B7-2 display distinct roles in immune regulation, although they are usually considered to have redundant functions. Here, we report the crystal structure of the receptor-binding (Ig V-type) domain of human B7-2 at 2.7-Å resolution. Structures of unliganded and liganded B7-1 and B7-2 suggest a physical-chemical basis for the observed functional similarities and differences between these two costimulatory ligands. Of particular note, whereas the majority of the residues mediating B7-1 dimerization are hydrophobic, the B7-2 dimer observed in the B7-2͞CTLA-4 complex displays a very hydrophilic dimer interface. These differences provide a mechanism for preventing the formation of B7-1͞B7-2 heterodimers. The divergence at the putative dimer interface is also consistent with the lower tendency of B7-2 to dimerize, as shown by the monomeric state of unliganded B7-2 both in solution and crystalline form, and may result in detailed differences in signaling mechanisms associated with B7-1 and B7-2.T he initiation and regulation of the cell-mediated immune response depend on two distinct signals. The first signal results from the specific interaction between the T cell receptor and its antigenic peptide bound to an MHC molecule. Whether this signal leads to T cell activation or anergy is determined by the concurrent delivery of a second signal provided by costimulatory receptor-ligand interactions (1). CD28 and CTLA-4, which share Ϸ30% identity, are the most well characterized costimulatory receptors present on the surface of T cells (2, 3). Upon binding its B7-1 and B7-2 ligands, CD28 delivers a costimulatory signal to the responding T cell, enhancing T cell proliferation and cytokine secretion and preventing the induction of T cell anergy (4, 5). In contrast, the engagement of CTLA-4 by these same ligands results in down-regulation of the response that is essential for maintaining T cell homeostasis and self-tolerance (6-8).B7-1 and B7-2, which share Ϸ25% sequence identity, are type I transmembrane glycoproteins expressed on the surfaces of antigen-presenting cells (APCs; refs. 9 and 10). Both B7 ligands bind CTLA-4 with 100-to 1,000-fold higher affinity than they do CD28, and B7-1 has Ϸ10-fold higher affinity for both receptors than does B7-2 (11, 12). Whereas B7-1 and B7-2 mainly have overlapping functions in T cell costimulation, comparative studies have suggested distinct immunoregulatory roles for these two proteins. For instance, although B7-2 is constitutively expressed on dendritic cells and can be rapidly up-regulated upon immune activation, B7-1 is only poorly expressed on resting dendritic cells, and its up-regulation occurs significantly later than that of B7-2. Furthermore, B7-2 expression is predominantly restricted to hematopoietic cells, whereas B7-1 is also expr...

Recent studies have highlighted the structural requirements for T cell costimulation and have revealed unusual modes of dimerization for the cytolytic T lymphocyte-associated antigen 4 (CTLA-4) costimulatory receptor and its B7 ligands. These distinctive quaternary structures potentially endow both receptor and ligand with bivalent binding properties, which suggests a number of mechanistic features relevant to signaling. These include the potential to form a highly ordered, alternating network of CTLA-4 and B7 homodimers that may represent the organization of these molecules and their associated signaling partners within the immunological synapse. Primary sequence and structural considerations suggest that some aspects of the organizational and mechanistic features associated with the CTLA-4-B7 complexes may extend to other members of the costimulatory receptor-ligand family. An examination of the signaling mechanisms within the costimulatory receptor-ligand family provides an excellent framework to consider the general principles that are relevant to cell surface receptor-mediated signaling events.

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