Structural and Functional Analysis of the Costimulatory Receptor Programmed Death-1

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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...

Section: B7-2 Exists As a Monomer On The Cell Surfacementioning

confidence: 82%

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

Bhatia

Edidin

Almo

et al. 2005

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“…In comparison with other TNFR͞TNF crystal structures, engagement of LIGHT or LT␣ by HVEM is predicted to impose a rigid 3-fold symmetrical structure spanning a length of Ϸ80-100 Å (34). The extracellular IgV domain of BTLA, which is Ϸ120 aa long, likely adopts a structure similar to CTLA-4, which spans Ϸ50 Å on its longest dimension (35)(36)(37)(38). This distance in addition to the number of linker residues (Ͻ10 aa) between the IgV domain and the transmembrane domain of BTLA is unlikely to provide enough distance to accommodate a complex where HVEM͞LIGHT͞LT␣ and BTLA interact on the same membrane.…”

Section: Discussionmentioning

confidence: 99%

A coreceptor interaction between the CD28 and TNF receptor family members B and T lymphocyte attenuator and herpesvirus entry mediator

Gonzalez¹,

Loyet²,

Calemine-Fenaux³

et al. 2005

226

231

The primary cosignaling receptors belong to either the Ig CD28-like or TNF receptor (TNFR) superfamilies (1-5). Currently, the CD28 family consists of five lymphoid-specific coreceptors [CD28, inducible T cell costimulator (ICOS), cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death-1 (PD-1), and B and T lymphocyte attenuator (BTLA)] (1-3). CD28 and ICOS are single Ig-variable (IgV) domain glycoproteins that promote T cell activation, whereas the structurally related CTLA-4, PD-1, and BTLA receptors function to attenuate T cell activation. To date, all of the ligands that have been described for the CD28-like family members belong to the B7 superfamily (1-3, 6-9). Six B7 family members have been described, all of which have conserved extracellular IgV and Ig-constant domains (3). In the TNFR-TNF superfamily, five receptor-ligand interactions have been described that act as positive regulators. These include OX40-OX40L, 4-1BB-4-1BBL, CD27-CD70, CD30-CD30L, and herpesvirus entry mediator (HVEM)-homologous to lymphotoxin, which shows inducible expression and competes with herpes simplex virus (HSV) glycoprotein D (gD) for HVEM, a receptor expressed by T lymphocytes (HVEM-LIGHT) (4).The most recently identified CD28 family member is the inhibitory coreceptor, BTLA (10-12). BTLA is expressed on developing B and T cells, all mature lymphocytes, splenic macrophages, and mature marrow-derived dendritic cells (10,11). BTLA contains a single IgV domain and two intracellular immunoreceptor tyrosine-based inhibitory motifs that are phosphorylated after BTLA coligation to antigen receptors, resulting in recruitment of protein tyrosine phosphatases SHP-1 and SHP-2 (13). Because of this, and that coligation of BTLA to the TCR inhibits T cell activation, BTLA is implicated as a negative regulator of T cell activation (10, 11). This finding is further supported by the observation that BTLA-deficient T cells show increased proliferation and that BTLA Ϫ͞Ϫ mice have increased Ab response and show increased incidence and severity to an autoimmune disorder (9, 10).Initially, BTLA was proposed to interact with a B7 family member called B7x (10, 14). This conclusion, however, was based on an indirect binding study testing the interaction of B7x-Ig fusion to spleen and lymph node cells from either WT or BTLA-deficient mice. Furthermore, we have been unable to detect any specific binding of BTLA-Fc to B7x-transfected cells (data not shown). Spurred by the inability to confirm the BTLA-B7x interaction, we screened a secreted protein library (15) by using surface plasmon resonance (SPR) and identified HVEM as a coreceptor for human BTLA.Here we show that BTLA and HVEM interact with high affinity and can form a trimeric complex with TNF ligands LIGHT or lymphotoxin ␣ (LT␣). Our binding studies suggest that BTLA interacts with the outer surface of the HVEM͞TNF complex, suggesting structural models for how HVEM might engage BTLA on the cell surface. Finally, we demonstrate that binding of HVEM to BTLA results in the inhibition of T cel...

“…The mouse GITRL extracellular domain (amino acids 46 -173), with an N-terminal 6ϫHis tag, was expressed in E. coli as insoluble inclusion body, refolded, and purified to homogeneity as described before (25). Alternatively, when grown at 25°C, a significant fraction of the protein remained soluble after bacterial cell lysis and was purified by Ni-NTA affinity chromatography and conventional gel-filtration chromatography.…”

Section: Methodsmentioning

confidence: 99%

Evolution of GITRL immune function: Murine GITRL exhibits unique structural and biochemical properties within the TNF superfamily

Chattopadhyay

Ramagopal²,

Brenowitz³

et al. 2008

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Glucocorticoid-induced TNF receptor ligand (GITRL), a recently identified member of the TNF superfamily, binds to its receptor, GITR, on both effector and regulatory T cells and generates positive costimulatory signals implicated in a wide range of T cell functions. In contrast to all previously characterized homotrimeric TNF family members, the mouse GITRL crystal structure reveals a previously unrecognized dimeric assembly that is stabilized via a unique ''domain-swapping'' interaction. Consistent with its crystal structure, mouse GITRL exists as a stable dimer in solution. Structureguided mutagenesis studies confirmed the determinants responsible for dimerization and support a previously unrecognized receptor-recognition surface for mouse GITRL that has not been observed for any other TNF family members. Taken together, the unique structural and biochemical behavior of mouse GITRL, along with the unusual domain organization of murine GITR, support a previously unrecognized mechanism for signaling within the TNF superfamily.crystal structure ͉ domain swap ͉ oligomerization ͉ T cell costimulation