Wang, Shengdian

Abstract Contemporary approaches for vaccination and immunotherapy are often capable of eliciting strong T-cell responses against tumor antigens. However, such responses are not parallel to clinical tumor regression. The development of evasion mechanisms within tumor microenvironment may be responsible for poor therapeutic responses. We report here that constitutive or inducible expression of B7-H1, a B7 family molecule widely expressed by cancers, confers resistance to therapeutic anti-CD137 antibody in mice with established tumors. The resistance is accompanied with failure of antigen-specific CD8+ CTLs to destroy tumor cells without impairment of CTL function. Blockade of B7-H1 or PD-1 by specific monoclonal antibodies could reverse this resistance and profoundly enhance therapeutic efficacy. Our findings support that B7-H1/PD-1 forms a molecular shield to prevent destruction by CTLs and implicate new approaches for immunotherapy of human cancers.

Abstract This report describes a new human B7-like gene designatedB7-H2. Cell surface expression of B7-H2 protein is detected in monocyte-derived immature dendritic cells. Soluble B7-H2 and immunoglobulin (Ig) fusion protein, B7-H2Ig, binds activated but not resting T cells and the binding is abrogated by inducible costimulator Ig (ICOSIg), but not CTLA4Ig. In addition, ICOSIg stains Chinese hamster ovary cells transfected with B7-H2 gene. By suboptimal cross-linking of CD3, costimulation of T-cell proliferation by B7-H2Ig is dose-dependent and correlates with secretion of interleukin (IL)-2, whereas optimal CD3 ligation preferentially stimulates IL-10 production. The results indicate that B7-H2 is a putative ligand for the ICOS T-cell molecule.

This report describes a new human B7-like gene designatedB7-H2. Cell surface expression of B7-H2 protein is detected in monocyte-derived immature dendritic cells. Soluble B7-H2 and immunoglobulin (Ig) fusion protein, B7-H2Ig, binds activated but not resting T cells and the binding is abrogated by inducible costimulator Ig (ICOSIg), but not CTLA4Ig. In addition, ICOSIg stains Chinese hamster ovary cells transfected with B7-H2 gene. By suboptimal cross-linking of CD3, costimulation of T-cell proliferation by B7-H2Ig is dose-dependent and correlates with secretion of interleukin (IL)-2, whereas optimal CD3 ligation preferentially stimulates IL-10 production. The results indicate that B7-H2 is a putative ligand for the ICOS T-cell molecule.

Abstract B7-H3 is a B7 family molecule with T cell costimulatory function in vitro. The in vivo role of B7-H3 in the stimulation of tumor immunity is unclear. We report here that expression of B7-H3 by transfection of the mouse P815 tumor line enhances its immunogenicity, leading to the regression of tumors and amplification of a tumor-specific CD8+ CTL response in syngeneic mice. Tumor cells engineered to express B7-H3 elicit a rapid clonal expansion of P1A tumor Ag-specific CD8+ CTL in lymphoid organs in vivo and acquire the ability to directly stimulate T cell growth, division, and development of cytolytic activity in vitro. Our results thus establish a role for B7-H3 in the costimulation of T cell immune responses in vivo.

B7-H1 and B7-DC are ligands for PD-1, a receptor implicated in negative regulation of T and B cell functions. These ligands, however, also costimulate T cell responses. It remains elusive whether or not costimulation is mediated through PD-1. By comparative molecular modeling and site-directed mutagenesis, we found that nonconserved residues between these ligands on the A′GFCC′C′′ face mediate interaction with PD-1. This indicates significant structural heterogeneity of the interactions between PD-1 and its ligands. Importantly, ligand mutants with abolished PD-1 binding capacity could still costimulate proliferation and cytokine production of T cells from normal and PD-1–deficient mice. Our results reveal unique binding characteristics of B7-H1 and B7-DC and provide direct evidence for an independent costimulatory receptor other than PD-1.

Abstract Tissue structural cells are known in some situations to play a role in the presentation of antigen and in immunoregulation. We assessed the expression of B7 homologs, known to be involved in antigen presentation and lymphocyte costimulation, in human airway epithelial cells. Flow cytometry performed on the airway epithelial cell line BEAS-2B, as well as primary bronchial epithelial cells (PBEC), showed that B7-H2 was constitutively expressed on both BEAS-2B and PBEC, whereas B7-1 and B7-2 were undetectable on either epithelial cell type. B7-H2 expression was confirmed by Western blot using a specific antibody. Stimulation with various cytokines, including tumor necrosis factor-α, interferon-γ, and interleukin-4, slightly downregulated B7-H2 expression detected by flow cytometry, but did not significantly alter the apparent level of protein as assessed by Western blotting. Northern blotting detected mRNA for B7-H2 and B7-1, but not B7-2. B7-H2 was cloned from BEAS-2B cells and the sequence verified. Expression of B7-H2 mRNA was detected by real-time reverse transcriptase–polymerase chain reaction in PBEC from three independent donors. Immunohistochemical analysis of airway derived from autopsies revealed expression of B7-H2 in human airway epithelial cells. These results demonstrate that airway epithelial cells express the costimulatory molecule B7-H2, and suggest the possibility that B7-H2 may participate in antigen presentation by epithelial cells.

Interaction between inducible costimulator (ICOS) and its ligand is implicated in the induction of cell-mediated and humoral immune responses. However, the molecular details of this interaction are unknown. We report here a mutagenesis analysis of residues in ICOS that are critical for ligand binding. A three-dimensional model of the extracellular immunoglobulin-like domain of ICOS was used to map the residues conserved within the CD28 family. This analysis identified a surface patch containing the characteristic “PPP” sequence and is conserved in human and mouse ICOS. Mutations in this region of human ICOS reduce or abolish ligand binding. Our results suggest that the ligand binding site in ICOS maps to a region overlapping yet distinct from the CD80/CD86 binding sites in CD28 and cytotoxic T lymphocyte antigen (CTLA)-4. Thus, the analysis suggests that differences in ligand binding specificity between these related costimulatory molecules have evolved by utilization of overlapping regions with different patterns of conserved and nonconserved residues. Two site-specific mutants generated in the course of our studies bound ICOS ligand with higher avidity than wild-type ICOS. An S76E mutant protein of ICOS blocked T cell costimulatory function of ICOS ligand and inhibited T cell response to allogeneic antigens superior to wild-type ICOS. Our studies thus identified critical residues involving in ICOS receptor–ligand interaction and provide new modulators for immune responses.