Characterization of mouse and human B7-H3 genes

T cell activation and immune function are regulated by costimulatory molecules of the B7 superfamily. Human B7-H3 is a recent addition to this family and has been shown to mediate T cell proliferation and IFN-gamma production. In this work we describe the identification of the mouse B7-H3 homolog, which is ubiquitously expressed in a variety of tissues. Activated CD4 and CD8 T cells express a putative receptor that can be recognized by soluble mouse B7-H3-Ig molecules. While the mouse B7-H3 gene was found to contain a single copy, we discovered a novel isoform of human B7-H3 (named as B7-H3b hereafter) with four Ig-like domains that results from gene duplication and differential splicing. B7-H3b is the major isoform expressed in several tissues. This structural information suggests a genetic variation of the B7-H3 gene in mammalian species.     

Blockade of T cell costimulation by CTLA4-Ig inhibits lung inflammation in murine hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is characterized by an influx of activated T cells in the lungs. The CD28/B7 system provides costimulatory signals essential for complete T cell activation and differentiation. We have previously demonstrated that alveolar macrophages from patients with HP have an up-regulated expression of B7 molecules. In the present study, we investigated the effect of i. p. administration of CTLA4-Ig, a CD28/B7 antagonist, on the lung inflammation of mice inoculated with Saccharoplyspora rectivirgula (SR), a major causative agent of HP. Five groups of C57BL/6 mice were intranasally instilled with SR or saline for 3 consecutive days per wk during 3 wk. CTLA4-Ig was administered starting either after 1 wk of SR challenge or 6 h before the first antigenic exposure and continued during the whole period of sensitization. A control-IgG was given similarly during the 3 wk of SR exposure. The groups included: 1, saline; 2, SR; 3, SR + control-Ig; 4, SR + CTLA4-Ig for the last 2 wk; and 5, SR + CTLA4-Ig for 3 wk. CTLA4-Ig treatment markedly decreased lung inflammation as shown by significantly fewer inflammatory cells in the bronchoalveolar lavage and in lung tissue and reduced SR-specific serum and bronchoalveolar lavage Ig levels. Production of IL-4, IL-10, and IFN-gamma by IL-2-stimulated pulmonary T cells was also decreased by CTLA4-Ig. Administration of CTLA4-Ig did not affect the SR-induced up-regulation of B7-2 expression. These results show that blockade of CD28/B7 interactions by CTLA4-Ig inhibits SR-induced lung inflammation and immune response to SR Ag in mice and may provide a novel approach in the treatment of HP.     

Induction of tumor regression by administration of B7-Ig fusion proteins: mediation by type 2 CD8+ T cells and dependence on IL-4 production

CD28 signals contribute to either type 1 or type 2 T cell differentiation. Here, we show that administration of B7.2-Ig fusion proteins to tumor-bearing mice induces tumor regression by promoting the differentiation of antitumor type 2 CD8(+) effector T cells along with IL-4 production. B7.2-Ig-mediated regression was not induced in IL-4(-/-) and STAT6(-/-) mice. However, it was elicited in IFN-gamma(-/-) and STAT4(-/-) mice. By contrast, IL-12-induced tumor regression occurred in IL-4(-/-) and STAT6(-/-) mice, but not in IFN-gamma(-/-) and STAT4(-/-) mice. Moreover, B7.2-Ig treatment was effective in a tumor model not responsive to IL-12. B7.2-Ig administration elicited elevated levels of IL-4 production. Tumor regression was predominantly mediated by CD8(+) T cells, although the induction of these effector cells required CD4(+) T cells. Tumor regression induced by CD8(+) T cells alone was inhibited by neutralizing the IL-4 produced during B7.2-Ig treatment. Thus, these results indicate that stimulation in vivo of CD28 with B7.2-Ig in tumor-bearing mice results in enhanced induction of antitumor type 2 CD8(+) T cells (Tc2) leading to Tc2-mediated tumor regression.     

Cutting edge: the related molecules CD28 and inducible costimulator deliver both unique and complementary signals required for optimal T cell activation

Optimal T cell activation requires engagement of CD28 with its counterligands B7-1 and B7-2. Inducible costimulator (ICOS) is the third member of the CD28/CTLA4 family that binds a B7-like protein, B7RP-1. Administration of ICOS-Ig attenuates T cell expansion following superantigen (SAg) administration, but fails to regulate either peripheral deletion or anergy induction. ICOS-Ig, but not CTLA4-Ig, uniquely regulates SAg-induced TNF-alpha production, whereas IL-2 secretion is modulated by CTLA4-Ig, but not ICOS-Ig. In contrast, both ICOS and CD28 are required for complete attenuation of IL-4 production. Our data suggest that ICOS and CD28 regulate T cell expansion and that ligation of either CD28 or ICOS can either uniquely regulate cytokine production (IL-2/TNF-alpha) or synergize for optimal cytokine production (IL-4) after SAg administration.     

Porcine CTLA4-Ig lacks a MYPPPY motif, binds inefficiently to human B7 and specifically suppresses human CD4+ T cell responses costimulated by pig but not human B7

The CTLA4 receptor (CD152) on activated T lymphocytes binds B7 molecules (CD80 and CD86) on APC and delivers a signal that inhibits T cell proliferation. Several regions involved in binding to B7 are known, but the relative importance of these is not clear. We have cloned porcine CTLA4 (pCTLA4). Although highly homologous to human CTLA4 (hCTLA4), the predicted protein sequence contains a leucine for methionine substitution at position 97 in the MYPPPY sequence. A fusion protein constructed from the extracellular regions of pCTLA4 and the constant regions of human IgG1 (pCTLA4-Ig) bound porcine CD86 with equivalent affinity to that of hCTLA4-Ig. However, pCTLA4-Ig bound poorly to human CD80 and CD86 expressed on transfectants and EBV-transformed human B cells. In functional assays with MHC class II-expressing porcine endothelial cells and human B cells, pCTLA4-Ig blocked human CD4+ T cell responses to pig but not human cells, whereas control hCTLA4-Ig inhibited responses to both. Comparison between mouse, human, and porcine CTLA4-Ig suggests that the selective binding of pCTLA4-Ig to porcine CD86 molecules is due to the L for M substitution at position 97. Our results indicate that pCTLA4-Ig may be a useful reagent to define the precise nature of the interaction between B7 and CTLA4. By failing to inhibit the delivery of costimulatory signals provided by human B7, it may also prove to be a relatively specific inhibitor of the direct human T cell response to immunogenic pig tissue.     

BTNL2, a butyrophilin-like molecule that functions to inhibit T cell activation

B7 family members regulate T cell activation and tolerance. Although butyrophilin proteins share sequence homology with the B7 molecules, it is unclear whether they have any function in immune responses. In the present study, we characterize an MHC class II gene-linked butyrophilin family member, butyrophilin-like 2 (BTNL2), the mutation of which has been recently associated with the inflammatory autoimmune diseases sarcoidosis and myositis. Mouse BTNL2 is a type I transmembrane protein with two pairs of Ig-like domains separated by a heptad peptide sequence. BTNL2 mRNA is highly expressed in lymphoid tissues as well as in intestine. To characterize the function of BTNL2, we produced a BTNL2-Ig fusion protein. It recognized a putative receptor whose expression on B and T cells was significantly enhanced after activation. BTNL2-Ig inhibited T cell proliferation and TCR activation of NFAT, NF-kappaB, and AP-1 signaling pathways. BTNL2 is thus the first member of the butyrophilin family that regulates T cell activation, which has implications in immune diseases and immunotherapy.     

Cutting edge: silencing suppressor of cytokine signaling 3 expression in dendritic cells turns CD28-Ig from immune adjuvant to suppressant

CTLA-4-Ig and CD28-Ig are both agonist ligands of B7 coreceptor molecules on mouse dendritic cells (DCs), yet they bias the downstream response in opposite directions, and CTLA-4-Ig promotes tolerance, whereas CD28-Ig favors the onset of immunity. Although B7 engagement by either ligand leads to a mixed cytokine response, a dominant IL-6 production in response to CD28-Ig prevents the IFN-gamma-driven induction of immunosuppressive tryptophan catabolism mediated by IDO. In the present study, we show that silencing the expression of suppressor of cytokine signaling 3 (SOCS3) in DCs by RNA interference renders CD28-Ig capable of activating IDO, likely as a result of unrestrained IFN-gamma signaling and IFN-gamma-like actions of IL-6. Thus, in the absence of SOCS3, CD28-Ig becomes immunosuppressive and mimics the action of CTLA-4-Ig on tryptophan catabolism.     

Overexpression of activation-induced cytidine deaminase in B cells is associated with production of highly pathogenic autoantibodies

Defective receptor editing or defective B cell checkpoints have been associated with increased frequency of multireactive autoantibodies in autoimmune disease. However, Ig somatic hypermutation and/or class switch recombination may be mechanisms enabling the development of pathogenic multireactive autoantibodies. In this study, we report that, in the BXD2 mouse model of autoimmune disease, elevated expression of activation-induced cytidine deaminase (AID) in recirculating follicular CD86(+) subsets of B cells and increased germinal center B cell activity are associated with the production of pathogenic multireactive autoantibodies. CD4 T cells from BXD2 mice that expressed increased levels of CD28 and an increased proliferative response to anti-CD3 and anti-CD28 stimulation are required for this process. Inhibition of the CD28-CD86 interaction in BXD2 mice with AdCTLA4-Ig resulted in normalization of AID in the B cells and suppression of IgG autoantibodies. This treatment also prevented the development of germinal center autoantibody-producing B cells, suggesting that an optimal microenvironment enabling AID function is important for the formation of pathogenic autoantibodies. Taken together, our data indicate that AID expression in B cells is a promising therapeutic target for the treatment of autoimmune diseases and that suppression of this gene may be a molecular target of CTLA4-Ig therapy.     

The CD28/B7 interaction is not required for resistance to Toxoplasma gondii in the brain but contributes to the development of immunopathology.

Infection of C57BL/6 mice with Toxoplasma gondii leads to chronic encephalitis characterized by infiltration into the brain of T cells that produce IFN-gamma and mediate resistance to the parasite. Our studies revealed that expression of B7.1 and B7.2 was up-regulated in brains of mice with toxoplasmic encephalitis (TE). Because CD28/B7-mediated costimulation is important for T cell activation, we assessed the contribution of this interaction to the production of IFN-gamma by T cells from brains and spleens of mice with TE. Stimulation of splenocytes with Toxoplasma Ag or anti-CD3 mAb resulted in production of IFN-gamma, which was inhibited by 90% in the presence of CTLA4-Ig, an antagonist of B7 stimulation. However, production of IFN-gamma by T cells from the brains of these mice was only slightly reduced (20%) by the addition of CTLA4-Ig. To address the role of the CD28/B7 interaction during TE, we compared the development of disease in C57BL/6 wild-type (wt) and CD28-/- mice. Although the parasite burden was similar in wt and CD28-/- mice, CD28-/- mice developed less severe encephalitis and survived longer than wt mice. Ex vivo recall responses revealed that mononuclear cells isolated from the brains of chronically infected CD28-/- mice produced less IFN-gamma than wt cells, and this correlated with reduced numbers of intracerebral CD4+ T cells in CD28-/- mice compared with wt mice. Taken together, our data show that resistance to T. gondii in the brain is independent of CD28 and suggest a role for CD28 in development of immune-mediated pathology during TE.     

CTLA4-Ig interacts with cultured synovial macrophages from rheumatoid arthritis patients and downregulates cytokine production

Introduction  

Co-stimulatory signal B7(CD80/CD86):CD28 is needed in order to activate T cells in immune response. Cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig) binding to the B7 molecules on antigen-presenting cells downregulates this activation and represents a recent biological treatment in rheumatoid arthritis (RA). Objectives of the study were to investigate the presence of the B7.2 (CD86) molecule and its masking by CTLA4-Ig on cultures of both RA synovial macrophages (RA SM), and of macrophages differentiated from THP-1 cells (M). In addition, the anti-inflammatory effects of CTLA4-Ig on co-cultures of RA SM and M with activated T cells were tested.     

Construction, and in vitro and in vivo analyses of tetravalent immunoadhesins

Previous observations demonstrated that various immunosuppressive agents and their combination therapies can increase allograft survival rates. However, these treatments may have serious side effects and cannot substantially improve or prolong graft survival in acute graft-versus-host disease (GVHD). To improve the therapeutic potency of divalent immunoadhesins, we have constructed and produced several tetravalent forms of immunoadhesins comprising each of cytotoxic T-lymphocyte-associated antigen-4 (CTLA4), CD2, and lymphocyte activation gene-3 (LAG3). Flow cytometric and T cell proliferation analyses displayed that tetravalent immunoadhesins have a higher binding affinity and more potent efficacy than divalent immunoadhesins. Although all tetravalent immunoadhesins possess better efficacies, tetravalent forms of CTLA4-Ig and LAG3-Ig revealed higher inhibitory effects on T cell proliferation than tetravalent forms of TNFR2-Ig and CD2-Ig. In vitro mixed lymphocytes reaction (MLR) showed that combined treatment with tetravalent CTLA4-Ig and tetravalent LAG3-Ig was highly effective for inhibiting T cell proliferation in both human and murine allogeneic stimulation. In addition, both single tetravalent-form and combination treatments can prevent the lethality of murine acute GVHD. The results of this study demonstrated that co-blockade of the major histocompatibility complex class (MHC)II:T cell receptor (TCR) and CD28:B7 pathways by using tetravalent human LAG3-Ig and CTLA4-Ig synergistically prevented murine acute GVHD.     

A role for CD28 in lymphopenia-induced proliferation of CD4 T cells

The peripheral mechanisms that regulate the size and the repertoire of the T cell compartment during recovery from a lymphopenic state are incompletely understood. In particular, the role of costimulatory signals, such as those provided by CD28, which have a critical importance for the immune response toward foreign Ags in nonlymphopenic animals, has been unclear in lymphopenia-induced proliferation (LIP). In this study, we show that accumulation of highly divided CD4 T cells characterized by great potential to make IFN-gamma is significantly delayed in the absence of B7:CD28 costimulation during LIP. Furthermore, CD28-sufficient CD4 T cells show great competitive advantage over CD28-deficient CD4 T cells when transferred together into the same lymphopenic hosts. Administration of CTLA-4-Ig removed this competitive advantage. Interestingly, CTLA-4-Ig treatment resulted in modest inhibition of LIP by CD28-deficient responders, suggesting that some of its effects may be independent of mere B7 blockade.     

Cutting Edge: KIR3DS1, a gene implicated in resistance to progression to AIDS, encodes a DAP12-associated receptor expressed on NK cells that triggers NK cell activation

The killer cell Ig-like receptor (KIR) gene, KIR3DS1, has been implicated in slowing disease progression in HIV infection; however, little is known about its expression, function, or ligand specificity. Using retrovirally transduced NKL cells and peripheral blood NK cells from KIR3DS1-positive donors we assessed expression of this gene by flow cytometry and its function by in vitro assays measuring KIR3DS1-induced cell-mediated cytotoxicity and cytokine production. In the present study, we demonstrate that KIR3DS1 is expressed on peripheral blood NK cells and triggers both cytotoxicity and IFN-gamma production. Using cotransfection and coimmunoprecipitation, we found that KIR3DS1 associates with the ITAM-bearing adaptor, DAP12. Soluble KIR3DS1-Ig fusion proteins did not bind to EBV-transformed B lymphoid cell lines transfected with HLA-Bw4 80I or 80T allotypes, suggesting that if KIR3DS1 does recognize HLA-Bw4 ligands, this may be peptide dependent.     

Triggering of murine NK cells by CD40 and CD86 (B7-2)

NK cell-mediated cytotoxicity is regulated by both triggering and inhibitory signals. The interaction between MHC class I molecules expressed on target cells and specific MHC class I-binding receptors expressed by NK cells generally leads to inhibition of lysis. We have shown recently that CD80 (B7-1) in mice and CD40 in humans trigger NK cell-mediated cytotoxicity in vitro. In the present study, we show that murine CD40 and CD86 (B7-2) trigger murine NK cell-mediated cytotoxicity in vitro when expressed on tumor cells. Preincubation of the transfected cell lines with anti-CD40 F(ab')2 fragments or cytolytic T lymphocyte-associated Ag-4-Ig (CTLA-4-Ig) before the cytotoxic assay abolished the triggering effect. Furthermore, radiolabeled CD40- and B7-2-expressing cells were rapidly eliminated in vivo in an NK cell-dependent manner. NK cells from CD40 ligand (CD40L)-/- or CD28-/- mice were triggered by tumor cells transfected with CD40 and B7-2, respectively, and these transfectants were rapidly eliminated in vivo when inoculated into CD40L-/- and CD28-/- mice. This suggests that the CD40 and B7-2 molecules can interact with receptors on NK cells other than CD40L and CD28, respectively, and that these may account for some of the reactivities observed in the present study. Collectively, these data demonstrate that 1) costimulatory molecules, other than B7-1, can modulate NK cell responses in vitro, 2) they can also affect NK cell-dependent responses in vivo, and 3) parts of these reactions are independent of CD28 and CD40L.     

CD84 functions as a homophilic adhesion molecule and enhances IFN-gamma secretion: adhesion is mediated by Ig-like domain 1

CD84 is a member of the CD2 subset of the Ig superfamily of cell surface molecules. Its cytoplasmic tail binds to Src homology 2 domain-containing protein 1A (signaling lymphocytic activation molecule-associated protein), a protein encoded by the X-linked lymphoproliferative disease gene. It is preferentially expressed on B lymphocytes, monocytes, and platelets. We show that it is also expressed on thymocytes and T cells. CD84 was positive on CD4-CD8- thymocytes, and its expression decreased with cell maturation. It is expressed on mature T cells preferentially on CD45RO+. To identify the CD84 ligand, we generated a soluble Ig fusion protein containing the human CD84 extracellular domains (CD84-Ig). Because receptor-ligand interactions occur between several members of this subfamily, we assayed CD84-Ig binding with all members of the CD2 family. CD84-Ig bound to CD84-transfected cells, whereas no binding was detected with cells expressing other CD2 subfamily receptors, showing that CD84 binds to itself. Anti-CD84 mAbs recognizing epitopes wholly within domain 1 of CD84 blocked the binding of the CD84-Ig fusion protein to CD84-transfected cells and platelets. Data from CD84 domain human/mouse chimeras further revealed that only the first extracellular domain of the molecule is involved in the ligand receptor recognition. The CD84-CD84 interaction was independent of its cytoplasmic tail. Finally, concurrent ligation of human CD84 with mAbs or CD84-Ig and CD3 enhanced IFN-gamma secretion in human lymphocytes. Thus, CD84 is its own ligand and acts as a costimulatory molecule.     

Suppressive effects of CTLA4-Ig on nasal allergic reactions in presensitized murine model

Ag-specific T cell activation requires the engagement of T cell receptor (TCR) with antigen in the context of MHC, and the engagement of appropriate costimulatory molecules. It is well established that B7/CD28-CTLA4 costimulatory pathway plays an important role in the induction of T helper (Th) cells in T-cell dependent immune reactions. In this study, we evaluated the effects of blocking the costimulatory pathway by systemic administration of CTLA4-Ig during repeated nasal antigen challenges in systemically presensitized mouse. The antigen-induced early phase nasal symptoms, nasal hyperresponsiveness to histamine and nasal eosinophilia were significantly suppressed by CTLA4-Ig treatment. Elevation of serum level of antigen-specific IgE, but not IgG1 or IgG2a was inhibited by the treatment. In relation to cytokine levels in the tissue extracts of the nasal mucosa, an up-regulation of IL-4 was significantly inhibited, however, the levels of IL-5 and IFN-gamma were not affected by the treatment. These results suggest that B7/CD28-CTLA4 costimulatory pathway plays an important role in on-going Th2-related allergic reactions in the nose.     

Murine B7-H3 is a negative regulator of T cells

T cell activation is regulated by the innate immune system through positive and negative costimulatory molecules. B7-H3 is a novel B7-like molecule with a putative receptor on activated T cells. Human B7-H3 was first described as a positive costimulator, most potently inducing IFN-gamma production and cellular immunity. In this study we examined the expression and function of mouse B7-H3. B7-H3 is mostly expressed on professional APCs; its expression on dendritic cells appears to be up-regulated by LPS. In contrast to human B7-H3, we found that mouse B7-H3 protein inhibited T cell activation and effector cytokine production. An antagonistic mAb to B7-H3 enhanced T cell proliferation in vitro and led to exacerbated experimental autoimmune encephalomyelitis in vivo. Therefore, mouse B7-H3 serves as a negative regulator of T cell activation and function.     

Role of CD28-B7 interactions in generation and maintenance of CD8 T cell memory.

Although the role of CD28-B7 interaction in the activation of naive T cells is well established, its importance in the generation and maintenance of T cell memory is not well understood. In this study, we examined the requirement for CD28-B7 interactions in primary T cell activation and immune memory. Ag-specific CD8 T cell responses were compared between wild-type (+/+) and CD28-deficient (CD28(-/-)) mice following an acute infection with lymphocytic choriomeningitis virus (LCMV). During the primary response, there was a substantial activation and expansion of LCMV-specific CD8 T cells in both +/+ and CD28(-/-) mice. However, the magnitude of the primary CD8 T cell response to both dominant and subdominant LCMV CTL epitopes was approximately 2- to 3-fold lower in CD28(-/-) mice compared with +/+ mice; the lack of CD28-mediated costimulation did not lead to preferential suppression of CD8 T cell responses to the weaker subdominant epitopes. As seen in CD28(-/-) mice, blockade of B7-mediated costimulation by CTLA4-Ig treatment of +/+ mice also resulted in a 2-fold reduction in the anti-LCMV CD8 T cell responses. Loss of CD28/B7 interactions did not significantly affect the generation and maintenance of CD8 T cell memory; the magnitude of CD8 T cell memory was approximately 2-fold lower in CD28(-/-) mice as compared with +/+ mice. Further, in CD28(-/-) mice, LCMV-specific memory CD8 T cells showed normal homeostatic proliferation in vivo and also conferred protective immunity. Therefore, CD28 signaling is not necessary for the proliferative renewal and maintenance of memory CD8 T cells.