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.

Rationale Multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), are inflammatory disorders of the central nervous system (CNS). The function of platelets in inflammatory and autoimmune pathologies is thus far poorly defined. Objective Here we addressed the role of platelets in mediating CNS inflammation in EAE. Results We found that platelets were present in human MS lesions as well as in the CNS of mice subjected to EAE but not in the CNS from control non-diseased mice. Platelet depletion at the effector-inflammatory phase of EAE in mice resulted in significantly ameliorated disease development and progression. EAE suppression upon platelet depletion was associated with reduced recruitment of leukocytes to the inflamed CNS, as assessed by intravital microscopy, and with a blunted inflammatory response. The platelet-specific receptor glycoprotein Ib alpha (GPIbα) promotes both platelet adhesion as well as inflammatory actions of platelets, and, targeting of GPIbα attenuated EAE in mice. Moreover, targeting another platelet adhesion receptor, glycoprotein IIb/IIIa (GPIIb/IIIa) also reduced EAE severity in mice. Conclusions Thus, platelets contribute to the pathogenesis of EAE by promoting CNS inflammation. Targeting platelets may therefore represent an important new therapeutic approach for MS treatment.

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The costimulatory ligands B7-1 and B7-2 are expressed on the surface of antigen-presenting cells and interact with the costimulatory receptors CD28 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expressed on T cells. Although B7-1 and B7-2 are homologous ligands having common receptors, they exhibit distinct biochemical features and roles in immune regulation. Several biochemical and structural studies have indicated differences in the oligomeric state of B7-1 and B7-2. However, the organization of B7 ligands on the cell surface has not been examined. By using photobleaching-based FRET (pbFRET), we demonstrate that B7-1 and B7-2 adopt different oligomeric states on the cell surface. Our study shows that B7-2 exists as a monomer on the cell surface whereas B7-1 exists predominantly as dimers on the cell surface. A series of mutations in B7-1 result in the expression of a predominantly monomeric species on the cell surface and validate the dimer interface proposed by prior crystallographic analysis. The difference in the oligomeric states of B7-1 and B7-2 provides insight into the geometric organization of the costimulatory receptorligand complexes in the immunological synapse and suggests constraints on signal transduction mechanisms involved in T cell activation.oligomerization ͉ photobleaching FRET ͉ T cell costimulation T he engagement of B7-1 and B7-2 with CD28 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) represents the best characterized T cell costimulatory interactions. CTLA-4 and CD28 share a similar molecular architecture, existing as disulfide linked homodimers of Ig variable (IgV) domains (1, 2). CTLA-4 and CD28 bind the B7 molecules with different affinities (3) and direct different T cell functional outcomes. Interaction of CD28 with B7 ligands delivers a positive signal to T cells that promotes proliferation and cytokine secretion and prevents the induction of T cell tolerance (4, 5). In contrast, the interaction of CTLA-4 with B7 ligands attenuates T cell activation and induces T cell anergy (6-8). Furthermore, recent studies have shown that CD28 and CTLA-4 binding to B7 ligands delivers different signals to dendritic cells. Binding of CD28 to B7 ligands leads to up-regulation of IL-6 production by dendritic cells, resulting in immunostimulatory activity (9). On the other hand, binding of CTLA-4 to B7 ligands up-regulates IFN-␥, which, in turn, up-regulates the expression of the enzyme indolamine, 2,3-dioxygenase in dendritic cells, resulting in tryptophan catabolism and suppression of T cell proliferation (10, 11).B7-1 and B7-2 are both type 1 transmembrane proteins with a membrane distal IgV and a membrane proximal Ig constant (IgC) domain and share Ϸ25% sequence identity. Although interacting with the same receptors, B7-1 and B7-2 show several distinct features (12, 13). B7-1 binds CTLA-4 and CD28 with equilibrium dissociation constants (K d ) of 0.2 M and 4 M, respectively, whereas B7-2 exhibits Ϸ5-to 10-fold lower affinities, with K d values of 2.6 M and 20 M for CTLA-4 a...