The B7 family consists of structurally related, cell-surface protein ligands, which bind to receptors on lymphocytes that regulate immune responses. Activation of T and B lymphocytes is initiated by engagement of cell-surface, antigen-specific T-cell receptors or B-cell receptors, but additional signals delivered simultaneously by B7 ligands determine the ultimate immune response. These 'costimulatory' or 'coinhibitory' signals are delivered by B7 ligands through the CD28 family of receptors on lymphocytes. Interaction of B7-family members with costimulatory receptors augments immune responses, and interaction with coinhibitory receptors attenuates immune responses. There are currently seven known members of the family: B7.1 (CD80), B7.2 (CD86), inducible costimulator ligand (ICOS-L), programmed death-1 ligand (PD-L1), programmed death-2 ligand (PD-L2), B7-H3, and B7-H4. Members of the family have been characterized predominantly in humans and mice, but some members are also found in birds. They share 20-40% amino-acid identity and are structurally related, with the extracellular domain containing tandem domains related to variable and constant immunoglobulin domains. B7 ligands are expressed in lymphoid and non-lymphoid tissues. The importance of the family in regulating immune responses is shown by the development of immunodeficiency and autoimmune diseases in mice with mutations in B7-family genes. Manipulation of the signals delivered by B7 ligands has shown potential in the treatment of autoimmunity, inflammatory diseases and cancer.

The seven known members of the B7 family - B7.1, B7.2, ICOS-L, PD-L1, PD-L.2, B7-H3, and B7-H4 - are all transmembrane or glycosylphosphatidylinositol (GPI)-linked proteins characterized by extracellular IgV and IgC domains related to the variable and constant domains of immunoglobulins (Table 1; see 123 for detailed reviews on the family). The IgV and IgC domains of B7-family members are each encoded by single exons, with additional exons encoding leader sequences, transmembrane and cytoplasmic domains. B7-H3 is unique in that the major human form contains two extracellular tandem IgV-IgC domains (Table 1) 4- Differentially spliced forms have been identified for B7.1, B7.2, ICOS-L, B7-H3, and PD-L2 456789, although the functional significance of these splice forms is unclear. The cytoplasmic domains are short, ranging in length from 19 to 62 amino-acid residues (see Additional data file 1), and can be encoded by multiple exons. A function for the intracellu-lar domains has not been established, although they all contain serine and threonine residues that could potentially serve as phosphorylation sites in signaling pathways. Recent evidence suggests that B7-family members can signal into the cell on which they are expressed (see below). One member, B7-H4, is GPI-linked to the cell surface 1011, whereas all others have transmembrane domains.

The genes for B7.1 and B7.2 are closely linked, as are the genes for PD-L1 and PD-L2, whereas the genes for ICOS-L, B7-H3 and B7-H4 are unlinked (Table 2). Orthologs of B7.1 and B7.2 have been identified in diverse mammalian species, but genes for other family members remain to be defined in most species beyond primates and rodents (Table 3). Interestingly, genes annotated as B7.1, B7.2, ICOS-L and B7-H4 orthologs have been described in the chicken, Gallus gallus, indicating that the family is conserved in birds (Figure 1; see also Table 3). A BLAST search of the NCBI Fugu rubripes (pufferfish) sequence database suggests that there may also be B7-like proteins in bony fish (data not shown), and CD28-like and CTLA-4 like molecules have been identified in trout (GenBank accession numbers AAW78853 and AAW78854). The CD28-receptor family is also conserved across mammalian species, and chicken orthologs have been described 12, suggesting that these receptor-ligand interactions are critical in the regulation of immune responses in mammals, birds and probably also fish.

Members of the B7 family (Table 1) are predicted to form homodimers at the cell surface, as has been shown for B7.1, although recent data indicate that B7.2 is likely to be a monomer 13. B7.1 and B7.2 share only about 25% amino-acid identity, yet each interacts with both CD28 and CTLA-4 receptors. Crystal structures have been solved for human B7.1 14 and human B7.2 13, both alone and complexed with the CTLA-4 receptor 1516. The structures of human B7.1 and B7.2 extracellular domains reveal typical IgV-like and IgC-like domains. Immunoglobulin-superfamily domains are typically about 100 amino acids in length and form a structure characterized by two anti-parallel β sheets, with each sheet composed of three to five anti-parallel strands 171819. The distinction between IgV and IgC domains is based on their structures, with IgV domains containing nine anti-parallel strands and IgC domains containing seven anti-parallel strands. Immunoglobulin-superfamily domains are found in many proteins and often serve as protein-protein interaction domains, as is frequently seen for cell-surface proteins in the immune system. For B7.1 and B7.2, the amino-terminal IgV domain is the receptor-binding domain. Interestingly, the amino-acid residues within B7.1 and B7.2 that bind to CTLA-4 are not highly conserved. A comparison of the two structures reveals, however, that the front faces of B7.1 and B7.2 each have shallow cavities that allow the highly conserved MYPPPY motif (in the single letter amino-acid code) in the CD28 and CTLA-4 receptors to fit and bind 13 (see Figure 2).

B7.1 and B7.2 have non-equivalent roles in immune modulation, in part because of their different interactions with CD28 and CTLA-4. The B7.1-CTLA-4 interaction is of higher affinity than the B7.2-CTLA-4 interaction, whereas CD28 is predicted to bind B7.2 more effectively than B7.1 20. Co-crystals of B7.1 with CTLA-4 reveal that B7.1 homodimers have CTLA-4-binding sites that are distal from the B7 dimer interface, allowing formation of a cross-linked B7-CTLA-4 lattice 15 (see Figure 3). In contrast, the data support a monovalent interaction between B7.2 and CD28 20. These models predict that the B7.1-CTLA-4 and B7.2-CD28 interactions will be of the greatest functional significance (see 2122 for further discussion). A crystal structure of the CD28 extracellular domain has recently been reported and shows similarities with that of CTLA-4 23. Crystal structures for other B7-family members have not yet been reported. Binding affinities of ICOS-L to its receptor have been reported to be similar to those of CD28-B7.1 interactions 24. PD-L1 and PD-L2 both have IgV-IgC extracellular domains, and both bind the same PD-1 receptor, although PD-L2 has been reported to bind PD-1 with an affinity 2-6 times higher than the affinity with which PD-L1 binds PD-1 25. The crystal structure of the extracellular domain of PD-1 has recently been reported 26.

The B7 family has a critical role in controlling immune responses, as was initially shown for B7.1 and B7.2 and extended by the recent identification and characterization of new B7-family members. Inflammatory stimuli modulate expression of B7 ligands, resulting in the delivery of activating and attenuating signals that determine the nature and extent of subsequent immune responses. Crystal structures, affinity measurements and biological assays have demonstrated how B7.1 and B7.2 deliver specific signals to immune cells resulting in precise regulation of immune function. This detailed level of knowledge is still to be obtained for additional members of the family. The receptors for B7-H3 and B7-H4 remain to be identified, and the available data suggest that there may be additional receptors for PD-L1 and PD-L2. Further clarification is needed before we understand how signals delivered by B7-family ligands are integrated spatially and temporally with those derived from antigen-specific receptors. Therapeutic manipulation of B7 signaling has shown clinical promise 4546, underscoring the key role that B7-family ligands play in regulating immune responses.

A sequence alignment for the proteins that were used to make the phylogenetic tree in Figure 1 is available in Additional data file 1. This alignment was made using Pileup and the Blosum62 scoring matrix.