The costimulatory molecules B7-1 and B7-2 regulate T lymphocyte activation by delivering activating signals through CD28 and inhibitory signals through cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The importance of CTLA-4-mediated inhibition was demonstrated by the uncontrolled T cell activation and lymphoproliferative disease that develops in CTLA-4-deficient (-/-) mice. To examine the role of B7 signaling in the activation of CTLA-4-deficient T cells, we bred CTLA-4(-/-) mice with mice lacking B7-1, B7-2, or both B7 molecules. The CTLA-4/B7-1(-/-) and the CTLA-4/B7-2(-/-) mice develop lymphoproliferation and enhanced T cell activation. Mice lacking CTLA-4, B7-1, and B7-2 have a normal life-span, and do not have lymphocytic infiltrates in any organs, or increased T cell activation. Therefore, the two B7 molecules have overlapping functions, since either B7-1 or B7-2 alone can cause the CTLA-4(-/-) phenotype. Elimination of both B7-1 and B7-2 from the CTLA-4- deficient mouse abrogates the lymphocyte activation and disease, and does not reveal evidence for additional stimulatory CD28 ligands. The CTLA-4(-/-) phenotype can be reproduced with anti-CD28 antibody in mice lacking CTLA-4, B7-1, and B7-2, but wild-type mice are unaffected by the same treatment. This suggests that the inhibitory function of CTLA-4 can overcome strong CD28-mediated signaling in vivo.

Ligation of cytotoxic T lymphocyte antigen 4 (CTLA4) appears to inhibit T cell responses. Four mechanisms have been proposed to explain the inhibitory activity of CTLA4: competition for B7-1 and B7-2 binding by CD28; sequestration of signaling molecules away from CD28 via endocytosis; delivery of a signal that antagonizes a CD28 signal; and delivery of a signal that antagonizes a T cell receptor (TCR) signal. As three of these potential mechanisms involve functional antagonism of CD28, an experimental model was designed to determine whether CTLA4 could inhibit T cell function in the absence of CD28. TCR transgenic/recombinase activating gene 2-deficient/CD28-wild-type or CD28-deficient mice were generated and immunized with an antigen-expressing tumor. Primed T cells from both types of mice produced cytokines and proliferated in response to stimulator cells lacking B7 expression. However, whereas the response of CD28+/+ T cells was augmented by costimulation with B7-1, the response of the CD28-/- T cells was strongly inhibited. This inhibition was reversed by monoclonal antibody against B7-1 or CTLA4. Thus, CTLA4 can potently inhibit T cell activation in the absence of CD28, indicating that antagonism of a TCR-mediated signal is sufficient to explain the inhibitory effect of CTLA4.

B7-1 transgene expression on the pancreatic islets in nonobese diabetic (NOD) mice leads to accelerated diabetes, with >50% of animals developing diabetes before 12 wk of age. The expression of B7-1 directly on the pancreatic beta cells, which do not normally express costimulator molecules, converts the cells into effective antigen-presenting cells leading to an intensified autoimmune attack. The pancreatic islet infiltrate in diabetic mice consists of CD8 T cells, CD4 T cells, and B cells, similar to diabetic nontransgenic NOD mice. To elucidate the relative importance of each of the subsets of cells, the NOD-rat insulin promoter (RIP)-B7-1 animals were crossed with NOD.beta2microglobulin -/- mice which lack major histocompatibility complex class I molecules and are deficient in peripheral CD8 T cells, NOD.CD4 -/- mice which lack T cells expressing CD4, and NOD.muMT -/- mice which lack B220-positive B cells. These experiments showed that both CD4 and CD8 T cells were necessary for the accelerated onset of diabetes, but that B cells, which are needed for diabetes to occur in normal NOD mice, are not required. It is possible that B lymphocytes play an important role in the provision of costimulation in NOD mice which is unnecessary in the NOD-RIP-B7-1 transgenic mice.

The association of B7-1/CD28 between antigen presenting cells (APCs) and T-cells provides a second signal to proliferate and activate T-cell immunity at the induction phase. Many reports indicate that tumor cells transfected with B7-1 induced augmented antitumor immunity at the induction phase by mimicking APC function; however, the function of B7-1 on antitumor immunity at the effector phase is unknown. Here, we report direct evidence of enhanced T-cell antitumor immunity at the effector phase by the B7-1 molecule. Our experiments in vivo and in vitro indicated that reactivity of antigen-specific monoclonal and polyclonal T-cell effectors against a Lass5 epitope presented by RMA-S cells is increased when the cells expressed B7-1. Use of either anti-B7-1 or anti-CD28 antibodies to block the B7-1/CD28 association reduced reactivity of the T effectors against B7-1 positive RMA-S cells. Transfection of Lass5 cDNA into or pulse of Lass5 peptide onto B7-1 positive RMA-S cells overcomes the requirement of the B7-1/CD28 signal for T effector response. To our knowledge, the data offers, for the first time, strong evidence that supports the requirement of B7-1/CD28 secondary signal at the effector phase of antitumor T-cell immunity being dependent on the density of an antigenic peptide.

Recent research on podocytes has proposed B7-1 as an important player in podocyte biology and as a potential new therapeutic target. B7-1 was upregulated in injured podocytes and described as a biomarker to identify patients who may benefit from abatacept, a B7-1 blocker. However, after this initial enthusiasm, several reports have not confirmed the efficiency of abatacept at inducing proteinuria remission in patients. In order to resolve these discrepancies, we explored the role of B7-1 in the injured podocyte. Both primary cultured and immortalized podocytes were exposed to lipopolysaccharides, but this failed to induce B7-1 expression at the mRNA and protein levels. Importantly, TLR-4 engagement confirmed lipopolysaccharide efficacy. We then evaluated B7-1 expression in several mouse models of podocyte injury including treatment with lipopolysaccharide or Adriamycin, a lupus prone model (NZB/W F1) and subtotal nephrectomy. Using 3 commercially available anti-B7-1 antibodies and appropriate controls, we could not find B7-1 expression in podocytes, whereas some infiltrating cells were positive. Thus, our findings do not support a role for B7-1 in podocyte biology. Hence, further studies are mandatory before treating proteinuric patients with B7-1 blockers.

Invariant natural killer T (iNKT) cell development in the thymus depends on T cell receptor recognition of CD1d ligand on CD4/CD8 double-positive thymocytes. We previously reported that B7-CD28 co-stimulation is required for thymic iNKT cell development, but the cellular and molecular mechanisms underlying this co-stimulatory requirement are not understood. Here we report that CD28 expression on CD1d-expressing antigen-presenting T cells is required for thymic iNKT cell development. Mechanistically, antigen-presenting T cells provide co-stimulation through an unconventional mechanism, acquiring B7 molecules via CD28-dependent trogocytosis from B7-expressing thymic epithelial cells, dendritic cells, and B cells and providing critical B7 co-stimulation to developing iNKT cells. Thus, the present study demonstrates a mechanism of B7 co-stimulation in thymic T cell development by antigen-presenting T cells.

Programmed cell death ligand 1 (PD-L1) status has been reported to be different between metastatic and primary lesions in some cases. Therefore, the interaction between carcinoma and immune cells could influence their expression in the tumor microenvironment. PD-L1 is known to bind not only to Programmed cell death 1 (PD-1) but also to B7-1 (CD80). In this study, we examined the interaction between lung carcinoma cell lines and peripheral blood mononuclear cells (PBMCs) in vitro. We then examined the significance of B7-1 expression non-small cell lung cancer (NSCLC) microenvironment.

B7-1 proteins are routinely expressed on the surface of antigen presenting cells (APC) and within the innate immune system. They function to establish a biologically optimal and dynamic balance between immune activation and inhibition or self-tolerance. Interactions between B7-1 and its receptors, which include CD28, CTLA4 and PD-L1, contribute to both stimulatory as well as inhibitory or homeostatic regulation. In the current study, we investigated whether the tumor-promoting actions of transforming growth factor beta (TGF-β) disrupted this equilibrium in pancreatic cancer to promote malignant progression and an enhanced means to evade immune detection. The data show that B7-1 is (i) upregulated following treatment of pancreatic carcinoma cells with TGF-β; (ii) induced by TGF-β via both Smad2/3-dependent and independent pathways; (iii) required for pancreatic tumor cell in vitro migration/invasion; and (iv) necessary for TGF-β regulated epithelial-mesenchymal transition (EMT) through induction of Snail family members. Results from the proposed studies provide valuable insights into mechanisms whereby TGF-β regulates both the innate immune response and intrinsic properties of pancreatic tumor growth.

Atherosclerosis is based on a chronic inflammatory process including the innate and adaptive immune response. Costimulatory molecules and their receptors provide decisive signals for antigen-specific cell activation. The contribution of B7-related pathways to atherosclerosis has hardly been explored.

The structurally related T cell surface molecules CD28 and CTLA-4 interact with cell surface ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC) and modulate T cell antigen recognition. Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (Kd values approximately 12 and approximately 200 nM, respectively) that are high when compared with other molecular interactions that contribute to T cell-APC recognition. In the present study, we use surface plasmon resonance to measure the affinity and kinetics of CD80 binding to CD28 and CTLA-4. At 37 degrees C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 microM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (k(off)); sCD80 dissociated from CD28 and CTLA-4 with k(off) values of > or = 1.6 and > or = 0.43 s-1, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate-dynamic T cell-APC contacts and to facilitate scanning of APC for antigen.

To assess the role of T cell antigen (Ag) presentation in multiple sclerosis (MS), proteolipid protein (PLP) peptide reactive CD4+ T cell clones (TCCs) from MS patients and normal subjects were studied. TCCs derived from chronic progressive (CP) MS patients were able to proliferate and secret cytokines in response to PLP peptide stimulation in the absence of professional antigen presenting cells (APCs), suggesting that these T cells can simultaneously present and respond to Ags. However, they did not respond to total PLP protein, suggesting that PLP-peptide TCCs were unable to process and present the whole PLP molecule. The ability of the different TCCs to act as APCs in response to Ag stimulation did not correlate with expression of HLA-class II molecules. However, the degree of expression of B7-1 and B7-2 co-stimulatory molecules showed a significant correlation with APC capacity. Furthermore, a combination of anti-B7-1 and anti-B7-2 mAbs effectively inhibited proliferative responses as well as secretion of IL-10, IFN gamma and TGF beta induced by antigen presenting T cells. By contrast, IL-4 secretion was not affected. Finally, IL-12 significantly enhanced the efficiency of T cell Ag presentation by a pathway independent of Ag processing, suggesting that IL-12 might act as an additional co-stimulatory signal for T cell activation during T-T cell interactions. Together, these observations suggest that Ag presentation by T cells might amplify and perpetuate an autoimmune response previously initiated by professional APCs. These properties may account for progression of MS into a CP phase.

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor-mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon gamma, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

Lupus nephritis (LN) is the most common complication that causes mortality in patients with systemic lupus erythematosus. The B7-1/B7-2 and CD28/cytotoxic T-lymphocyte associated protein 4 co-stimulatory pathway serves a key role in autoimmune disease and organ transplantation. The aim of the present study was to generate and characterize a monoclonal antibody (mAb; clone 4E5) against human B7-1 and to investigate its potential use for the treatment of LN. The results demonstrated that the 4E5 mAb was successfully generated and able to recognize both human and mouse B7-1. After injection of this mAb into a mouse model with chronic graft-vs.-host disease (cGVHD)-induced lupus-like disease, the expression of CD21, CD23, CD80 and CD86 on B220+ B-cells in the spleen, and the concentrations of serum autoantibodies and urine protein, were decreased. Direct immunofluorescence analysis of the kidneys revealed that immunofluorescence of immune complex deposits was weaker in the 4E5-treated mice and electron microscopy analyses of renal tissues indicated that pathological injury of the kidneys of 4E5-treated mice was decreased compared with that in the model control mice. The results of the present study demonstrated that inhibition of the B7-1/CD28 co-stimulatory signaling pathway with the 4E5 mAb may represent a promising strategy to decelerate the progression of LN that is induced by cGVHD with potential for use in the treatment of other autoimmune diseases.

CD28 and CTLA-4 are cell surface cosignaling molecules essential for the control of T cell activation upon the engagement of their ligands B7-1 and B7-2 from antigen-presenting cells. By employing a receptor array assay, we have demonstrated that B7-H2, best known as the ligand of inducible costimulator, was a ligand for CD28 and CTLA-4 in human, whereas these interactions were not conserved in mouse. B7-H2 and B7-1 or B7-2 interacted with CD28 through distinctive domains. B7-H2-CD28 interaction was essential for the costimulation of human T cells' primary responses to allogeneic antigens and memory recall responses. Similar to B7-1 and B7-2, B7-H2 costimulation via CD28 induced survival factor Bcl-xL, downregulated cell cycle inhibitor p27(kip1), and triggered signaling cascade of ERK and AKT kinase-dependent pathways. Our findings warrant re-evaluation of CD28 and CTLA-4's functions previously attributed exclusively to B7-1 and B7-2 and have important implications in therapeutic interventions against human diseases.

Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids) and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

The inflammatory response is indispensable for protective immunity, yet microbial pathogens often trigger an excessive response, 'cytokine storm', harmful to the host. Full T-cell activation requires interaction of costimulatory receptors B7-1(CD80) and B7-2(CD86) expressed on antigen-presenting cells with CD28 expressed on the T cells. We created short peptide mimetics of the homodimer interfaces of the B7 and CD28 receptors and examined their ability to attenuate B7/CD28 coligand engagement and signaling through CD28 for inflammatory cytokine induction in human immune cells, and to protect from lethal toxic shock in vivo.

It has been proposed that some bystander T cell activation may in fact be due to T cell antigen receptor (TCR) cross-reactivity that is too low to be detected by the effector cytotoxic T lymphocyte (CTL). However, this hypothesis is not supported by direct evidence since no TCR ligand is known to induce T cell proliferation and differentiation without being recognized by the effector CTL. Here we report that transgenic T cells expressing a T cell receptor to influenza virus A/NT/68 nucleoprotein (NP) 366-374:Db complexes clonally expand and become effector CTLs in response to homologous peptides from either A/PR8/34 (H1N1), A/AA/60 (H2N2), or A/NT/68 (H3N2). However, the effector T cells induced by each of the three peptides kill target cells pulsed with NP peptides from the H3N2 and H2N2 viruses, but not from the H1N1 virus. Thus, NP366-374 from influenza virus H1N1 is the first TCR ligand that can induce T cell proliferation and differentiation without being recognized by CTLs. Since induction of T cell proliferation was mediated by antigen-presenting cells that express costimulatory molecules such as B7, we investigated if cytolysis of H1N1 NP peptide-pulsed targets can be restored by expressing B7-1 on the target cells. Our results revealed that this is the case. These data demonstrated that costimulatory molecule B7 modulates antigen specificity of CTLs, and provides a missing link that explains some of the bystander T cell activation.

Cytotoxic CD8 +T lymphocytes (CTLs) are key players of adaptive anti-tumor immunity based on their ability to specifically recognize and destroy tumor cells. Many cancer immunotherapies rely on unleashing CTL function. However, tumors can evade killing through strategies which are not yet fully elucidated. To provide deeper insight into tumor evasion mechanisms in an antigen-dependent manner, we established a human co-culture system composed of tumor and primary immune cells. Using this system, we systematically investigated intrinsic regulators of tumor resistance by conducting a complementary CRISPR screen approach. By harnessing CRISPR activation (CRISPRa) and CRISPR knockout (KO) technology in parallel, we investigated gene gain-of-function as well as loss-of-function across genes with annotated function in a colon carcinoma cell line. CRISPRa and CRISPR KO screens uncovered 187 and 704 hits, respectively, with 60 gene hits overlapping between both. These data confirmed the role of interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α) and autophagy pathways and uncovered novel genes implicated in tumor resistance to killing. Notably, we discovered that ILKAP encoding the integrin-linked kinase-associated serine/threonine phosphatase 2 C, a gene previously unknown to play a role in antigen specific CTL-mediated killing, mediate tumor resistance independently from regulating antigen presentation, IFN-γ or TNF-α responsiveness. Moreover, our work describes the contrasting role of soluble and membrane-bound ICAM-1 in regulating tumor cell killing. The deficiency of membrane-bound ICAM-1 (mICAM-1) or the overexpression of soluble ICAM-1 (sICAM-1) induced resistance to CTL killing, whereas PD-L1 overexpression had no impact. These results highlight the essential role of ICAM-1 at the immunological synapse between tumor and CTL and the antagonist function of sICAM-1.

Protein nanoparticle scaffolds are increasingly used in next-generation vaccine designs, and several have established records of clinical safety and efficacy. Yet the rules for how immune responses specific to nanoparticle scaffolds affect the immunogenicity of displayed antigens have not been established. Here we define relationships between anti-scaffold and antigen-specific antibody responses elicited by protein nanoparticle immunogens. We report that dampening anti-scaffold responses by physical masking does not enhance antigen-specific antibody responses. In a series of immunogens that all use the same nanoparticle scaffold but display four different antigens, only HIV-1 envelope glycoprotein (Env) is subdominant to the scaffold. However, we also demonstrate that scaffold-specific antibody responses can competitively inhibit antigen-specific responses when the scaffold is provided in excess. Overall, our results suggest that anti-scaffold antibody responses are unlikely to suppress antigen-specific antibody responses for protein nanoparticle immunogens in which the antigen is immunodominant over the scaffold.

The inhibitory T-cell surface-expressed receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which belongs to the class of cell surface proteins phosphorylated by extrinsic tyrosine kinases that also includes antigen receptors, binds the related ligands, B7-1 and B7-2, expressed on antigen-presenting cells. Conformational changes are commonly invoked to explain ligand-induced "triggering" of this class of receptors. Crystal structures of ligand-bound CTLA-4 have been reported, but not the apo form, precluding analysis of the structural changes accompanying ligand binding. The 1.8-Å resolution structure of an apo human CTLA-4 homodimer emphasizes the shared evolutionary history of the CTLA-4/CD28 subgroup of the immunoglobulin superfamily and the antigen receptors. The ligand-bound and unbound forms of both CTLA-4 and B7-1 are remarkably similar, in marked contrast to B7-2, whose binding to CTLA-4 has elements of induced fit. Isothermal titration calorimetry reveals that ligand binding by CTLA-4 is enthalpically driven and accompanied by unfavorable entropic changes. The similarity of the thermodynamic parameters determined for the interactions of CTLA-4 with B7-1 and B7-2 suggests that the binding is not highly specific, but the conformational changes observed for B7-2 binding suggest some level of selectivity. The new structure establishes that rigid-body ligand interactions are capable of triggering CTLA-4 phosphorylation by extrinsic kinase(s).