Tom Horan

T-cell co-stimulation through B7RP-1 and ICOS.

T-cell activation requires co-stimulation through receptors such as CD28 (refs 1,2,3) and antigen-specific signalling through the T-cell antigen receptor. Here we describe a new murine co-stimulatory receptor–ligand pair. The receptor, which is related to CD28 and is the homologue of the human protein ICOS, is expressed on activated T cells and resting memory T cells. The ligand, which has homology to B7 molecules and is called B7-related protein-1 (B7RP-1), is expressed on B cells and macrophages. ICOS and B7RP-1 do not interact with proteins in the CD28–B7 pathway, and B7RP-1 co-stimulates T cells in vitro independently of CD28. Transgenic mice expressing a B7RP-1–Fc fusion protein show lymphoid hyperplasia in the spleen, lymph nodes and Peyers patches. Presensitized mice treated with B7RP-1–Fc during antigen challenge show enhanced hypersensitivity. Therefore, B7RP-1 exhibits co-stimulatory activities in vitro and in vivo. ICOS and B7RP-1 define a new and distinct receptor–ligand pair that is structurally related to CD28–B7 and is involved in the adaptive immune response.

ICOS is essential for effective T-helper-cell responses

The outcome of T-cell responses after T-cell encounter with specific antigens is modulated by co-stimulatory signals, which are required for both lymphocyte activation and development of adaptive immunity. ICOS, an inducible co-stimulator with homology to CD28, is expressed on activated, but not resting Txa0cells, and shows T-cell co-stimulatory function in vitro. ICOS binds specifically to its counter-receptor B7RP-1 (refs 5,6,7), but not to B7-1 or B7-2. Here we provide in vivo genetic evidence that ICOS delivers a co-stimulatory signal that is essential both for efficient interaction between T and B cells and for normal antibody responses to T-cell-dependent antigens. To determine the physiological function of ICOS, we generated and characterized gene-targeted ICOS-deficient mice. In vivo, a lack of ICOS results in severely deficient T-cell-dependent B-cell responses. Germinal centre formation is impaired and immunoglobulin class switching, including production of allergy-mediating IgE, is defective. ICOS-deficient T cells primed in in vivo and restimulated in vitro with specific antigen produce only low levels of interleukin-4, but remain fully competent to produce interferon-γ.

The B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses.

We investigated the in vivo function of the B7 family member B7-H3 (also known as B7RP-2) by gene targeting. B7-H3 inhibited T cell proliferation mediated by antibody to T cell receptor or allogeneic antigen-presenting cells. B7-H3-deficient mice developed more severe airway inflammation than did wild-type mice in conditions in which T helper cells differentiated toward type 1 (TH1) rather than type 2 (TH2). B7-H3 expression was consistently enhanced by interferon-γ but suppressed by interleukin 4 in dendritic cells. B7-H3-deficient mice developed experimental autoimmune encephalomyelitis several days earlier than their wild-type littermates, and accumulated higher concentrations of autoantibodies to DNA. Thus, B7-H3 is a negative regulator that preferentially affects TH1 responses.

Essential Role of NF-κB-Inducing Kinase in T Cell Activation Through the TCR/CD3 Pathway

NF-κB-inducing kinase (NIK) is involved in lymphoid organogenesis in mice through lymphotoxin-β receptor signaling. To clarify the roles of NIK in T cell activation through TCR/CD3 and costimulation pathways, we have studied the function of T cells from aly mice, a strain with mutant NIK. NIK mutant T cells showed impaired proliferation and IL-2 production in response to anti-CD3 stimulation, and these effects were caused by impaired NF-κB activity in both mature and immature T cells; the impaired NF-κB activity in mature T cells was also associated with the failure of maintenance of activated NF-κB. In contrast, responses to costimulatory signals were largely retained in aly mice, suggesting that NIK is not uniquely coupled to the costimulatory pathways. When NIK mutant T cells were stimulated in the presence of a protein kinase C (PKC) inhibitor, proliferative responses were abrogated more severely than in control mice, suggesting that both NIK and PKC control T cell activation in a cooperative manner. We also demonstrated that NIK and PKC are involved in distinct NF-κB activation pathways downstream of TCR/CD3. These results suggest critical roles for NIK in setting the threshold for T cell activation, and partly account for the immunodeficiency in aly mice.

Dickkopf-1 regulates bone formation in young growing rodents and upon traumatic injury.

The physiological role of Dickkopf‐1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1‐Ab) that blocked Dkk1 binding to both low density lipoprotein receptor‐related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1‐Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury.

Stimulatory Effects of B7-Related Protein-1 on Cellular and Humoral Immune Responses in Mice

Inducible costimulator (ICOS) and B7-related protein-1 (B7RP-1) constitute a receptor-ligand pair involved in T cell costimulation. In this study, the stimulatory effects of B7RP-1 on cellular and humoral immune responses were investigated giving mice a construct with the extracellular domain of murine B7RP-1 fused with human IgG1 Fc (B7RP-1-Fc). B7RP-1-Fc stimulated contact hypersensitivity (CH) given near either the time of sensitization or challenge with oxazolone. When given near challenge time, B7RP-1-Fc stimulated CH more than a construct containing the extracellular domain of murine B7.2 and Fc (B7.2-Fc). B7RP-1-Fc increased the number of cells in lymph nodes draining the skin sensitized with oxazolone, especially activated T cells. B7RP-1-Fc also increased the ability of the cells in these lymph nodes to induce CH when transfused into naive mice. B7RP-1-Fc stimulated the production of anti-keyhole limpet hemocyanin (KLH) Ab, increasing anti-KLH IgG, IgG2a, and IgE, whereas B7.2-Fc did not affect this production. B7RP-1-Fc also increased the number of cells in lymph nodes draining the skin immunized with KLH and their production of IFN-γ, IL-4, and IL-10 in response to KLH. Finally, B7RP-1-Fc increased the presence of eosinophils in the bronchoalveolar lavage and lungs of mice sensitized and challenged with OVA so to mount an asthmatic reaction. B7RP-1-Fc stimulates both cellular and humoral immune responses in vivo by increasing number and function of T and B cells reacting to Ag exposure.

A role for inducible costimulator protein in the CD28- independent mechanism of resistance to Toxoplasma gondii.

Long-term resistance to Toxoplasma gondii is dependent on the development of parasite-specific T cells that produce IFN-γ. CD28 is a costimulatory molecule important for optimal activation of T cells, but CD28−/− mice are resistant to T. gondii, demonstrating that CD28-independent mechanisms regulate T cell responses during toxoplasmosis. The identification of the B7-related protein 1/inducible costimulator protein (ICOS) pathway and its ability to regulate the production of IFN-γ suggested that this pathway may be involved in the CD28-independent activation of T cells required for resistance to T. gondii. In support of this hypothesis, infection of wild-type or CD28−/− mice with T. gondii resulted in the increased expression of ICOS by activated CD4+ and CD8+ T cells. In addition, both costimulatory pathways contributed to the in vitro production of IFN-γ by parasite-specific T cells and when both pathways were blocked, there was an additive effect that resulted in almost complete inhibition of IFN-γ production. Although in vivo blockade of the ICOS costimulatory pathway did not result in the early mortality of wild-type mice infected with T. gondii, it did lead to increased susceptibility of CD28−/− mice to T. gondi associated with reduced serum levels of IFN-γ, increased parasite burden, and increased mortality compared with the control group. Together, these results identify a critical role for ICOS in the protective Th1-type response required for resistance to T. gondii and suggest that ICOS and CD28 are parallel costimulatory pathways, either of which is sufficient to mediate resistance to this intracellular pathogen.

Paradoxical role of programmed death-1 ligand 2 in Th2 immune responses in vitro and in a mouse asthma model in vivo

Programmed death‐1 ligandu20042 (PD‐L2) is a ligand for programmed death‐1 (PD‐1), a receptor that plays an inhibitory role in Tu2004cell activation. Since previous studies have shown up‐regulation of PD‐L2 expression by Th2 cytokines, and asthma is driven by a Th2 response, we hypothesized that PD‐L2 might be involved in regulation of the immune response in this disease. We have found that lungs from asthmatic mice had sustained up‐regulation of PD‐1 and PD‐L2, with PD‐L2 primarily on dendritic cells. Although addition of PD‐L2‐Fc in vitro led to decreased Tu2004cell proliferation and cytokine production, administration of PD‐L2‐Fc in vivo in a mouse asthma model resulted in elevated serum IgE levels, increased eosinophilic and lymphocytic infiltration into bronchoalveolar lavage fluid, higher number of cells in the draining lymph nodes, and production of IL‐5 and IL‐13 from these cells. Although PD‐1 was expressed on regulatory Tu2004cells, PD‐L2‐Fc did not affect regulatory Tu2004cell activity in vitro. This study provides in vivo evidence of an exacerbated inflammatory response following PD‐L2‐Fc administration and indicates a potential role for this molecule in Th2‐mediated diseases such as asthma.

Potent activity of soluble B7RP-1-Fc in therapy of murine tumors in syngeneic hosts.

We have characterized a receptor:ligand pair, ICOS:B7RP‐1, that is structurally and functionally related to CD28:B7.1/2. We reported previously that B7RP‐1 costimulates T cell proliferation and immune responses (Yoshinaga et al., Nature 1999;402:827–32; Guo et al., J Immunol 2001;166:5578–84; Yoshinaga et al., Int Immunol 2000;12:1439–47). We report that B7RP‐1‐Fc causes rejection or growth inhibition of Meth A, SA‐1 and EMT6 tumors in syngeneic mice. Established Meth A tumors were rejected effectively with a single dose of B7RP‐1‐Fc, however, the treatment was less effective on larger tumors. Mice that rejected Meth A tumors previously by Day 30, also rejected a subsequent Meth A challenge on Day 60, without additional B7RP‐1‐Fc treatment, indicating a long‐lived memory response. Tumor cells believed to be less immunogenic, such as P815 and EL‐4 cells, were less responsive to this treatment. The EL‐4 responsiveness to the B7RP‐1‐Fc treatment was enhanced, however, by pre‐treatment of the mice with cyclophosphamide. As expected, T cells appeared to be targeted by B7RP‐1‐Fc treatment. Thus, the administration of soluble B7RP‐1‐Fc may have therapeutic value in generating or enhancing anti‐tumor activity in a clinical setting.

CD28 and ICOS play complementary non-overlapping roles in the development of Th2 immunity in vivo

Previous work has shown ICOS can function independently of CD28, but whether either molecule can compensate for the other in vivo is not known. Since ICOS is a potent inducer of Th2 cytokines and linked to allergy and elevated serum IgE in humans, we hypothesized that augmenting ICOS costimulation in murine allergic airway disease may overcome CD28 deficiency. While ICOS was expressed on T cells from CD28(-/-) mice, Th2-mediated airway inflammation was not induced in CD28(-/-) mice by increased ICOS costimulation. Further, we determined if augmenting CD28 costimulation could compensate for ICOS deficiency. ICOS(-/-) mice had a defect in airway eosinophilia that was not overcome by augmenting CD28 costimulation. CD28 costimulation also did not fully compensate for ICOS for antibody responses, germinal center formation or the development of follicular B helper T cells. CD28 and ICOS play complementary non-overlapping roles in the development of Th2 immunity in vivo.