Different cell surface oligomeric states of B7-1 and B7-2: Implications for signaling

Abstract

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...

Crystal structure of the complex between programmed death-1 (PD-1) and its ligand PD-L2

“…Both CTLA-4 and CD28 exist as disulfide-linked homodimers. In addition, B7-1 exhibits a considerable propensity to form noncovalent oligomers in solution and on the plasma membrane (2,18). The higher order oligomeric states of these receptors and ligands afford the opportunity to assemble multicomponent signaling complexes with specific stoichiometries and geometries.…”

“…In vivo both CD28 and CTLA4 exist as covalent homodimers because of an interchain disulfide formed by a conserved cysteine residue in the linker region connecting the IgV domain to the transmembrane segment. The in vivo oligomeric states of the ligands are less characterized, but recent studies demonstrated that B7-1 has high propensity to oligomerize on the cell surface (2). The oligomeric and polyvalent features of these receptors and ligands may contribute to the formation and localization of multicomponent signaling assemblies at the immunological synapse.…”

The interchain disulfide linkage is not a prerequisite but enhances CD28 costimulatory function

“…Mouse and human CD28 cDNAs were cloned in-frame into ECFP-N1 and EYFP-N1 (Clontech) expression vectors that had been mutated (A206K) to prevent self-dimerization [11]. The C123S mutants of human and mouse CD28 were generated by site-directed mutagenesis.…”

“…(Figure 2B). Our FRET data were validated by using tandem B7-1-CFP-YFP expressing cells as positive control [11], and cells expressing non-interacting proteins as negative control (Supplementary Figure B).…”

“…Sample preparation and photobleaching FRET (pbFRET) studies were performed as previously described [11]. Fixed cells were examined with a Leica TCS SP II AOBS laser-scanning confocal microscope at the Analytical Imaging Facility at AECOM.…”