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In the presence of antigen signal, engagement of CD28 by eitherspecific antibodies or its natural ligands B7-1 and B7-2 promotesantigen-specific T-cell proliferation, enhances production ofcytokines, and induces the differentiation of effector T cells1–3.Another effect of B7–CD28 co-stimulation is the induction of bcl-xL, which promotes T-cell survival4. Signaling through the homologous cytotoxic T-lymphocyte antigen 4 (CTLA4) counter-receptor of B7-1 and B7-2 on activated T cells, however, may de-liver a negative signal that inhibits T-cell proliferation, interleukin(IL)-2 production, IL-2 receptor expression and cell cycle progres-sion5,6. An additional CD28-like molecule, called ICOS (inducibleco-stimulator), has been described7. Although its natural ligandhas not yet been identified, a monoclonal antibody against ICOScan co-stimulate T-cell growth and induce IL-10 and IL-4 produc-tion. An increase in IL-10 production is usually associated with re-duced cellular immune response, increased immunoglobulinproduction and anergy of antigen-specific T cells8.

B7-1 and B7-2 belong to an emerging family of the im-munoglobulin superfamily, and have amino acid sequences thatare about 25% identical mainly in the immunoglobulin V- and C-like extracellular domains, whereas they have profound differ-ences in their cytoplasmic domains9,10. Despite this limitedhomology, B7-1 and B7-2 have similar tertiary structures and co-stimulatory functions11–13. Both B7-1 and B7-2 bind to CD28 withlow affinity and to CTLA4 with high affinity. The differences be-tween the binding of B7-1 and B7-2 to either CD28 or CTLA4 aresmall, although B7-1 binds more weakly to CD28 and has a fasteroff-rate from CTLA4 than B7-2 has14. B7-2 is constitutively ex-pressed on dendritic cells, and is upregulated rapidly, comparedwith B7-1 (refs. 2,15). These findings indicate that B7-2 partici-pates in initiating an immune response, whereas B7-1 may be in-volved in sustaining or regulating the activation process.

By searching for molecules that share homology with the im-munoglobulin V and C domains of B7-1 and B7-2 among thehuman cDNA expressed sequence tags in the National Center forBiotechnology Information database, we identified a previouslyunknown gene called B7-H1 (B7 homolog 1). Not only does B7-H1share an overall structure similarity with B7-1 and B7-2, but also

the ligation of B7-H1 co-stimulates the growth of human T cellsthrough a receptor different from CD28, CTLA4 or ICOS.However, co-stimulation mediated by B7-H1 leads to secretion ofIL-10.

Cloning and expression pattern of human B7-H1 geneA homology search of the human cDNA expressed sequence tagdatabase using published human B7-1 and B7-2 amino-acid se-quences produced one expressed sequence tag sequence(GeneBank accession number AA292201) encoding a homolog tothe human B7-1 and B7-2 molecules. We obtained the 5′- and 3′-end sequences by several independent series of RT–PCR with ahuman placenta cDNA library, using vector sequences as primers.We cloned and sequenced the full length of the gene. The extra-cellular domain of B7-H1 shares more homology with B7-1 (20%amino-acid identity) than with B7-2 (15% amino-acid identity)(Fig. 1b), whereas its cytoplasmic domain is very diverse, based onanalysis using McVector 6.5 software. The open reading frame ofthe B7-H1 gene encodes a putative type I transmembrane proteinof 290 amino acids, consisting of immunoglobulin V-like and C-like domains, a hydrophobic transmembrane domain and a cyto-plasmic tail of 30 amino acids (Fig. 1a). Four structural cysteines(Fig. 1b, stars), which are apparently involved in forming the disul-fide bonds of the immunoglobulin V and C domains9–13, are wellconserved in all B7 members (Fig. 1b).

Northern blot analysis showed that the expression of B7-H1mRNA was abundant in heart, skeletal muscle, placenta and lungtissues, but was weak in thymus, spleen, kidney and liver tissues.B7-H1 mRNA was not detectable in brain, colon and small intes-tine tissues and peripheral blood mononuclear cells (PBMCs) (Fig.1c). In most of the tissues containing B7-H1 mRNA, we found twotranscripts of approximately 4.1 and 7.2 kb (Fig.1c). We also con-structed a plasmid containing the extracellular domain of B7-H1fused in-frame with the Fc portion of the mouse immunoglobulinG2a. We purified the resulting product, the fusion protein B7-H1Ig, from the supernatants of CHO cells transfected with theplasmid and used this to immunize mice to prepare specific anti-serum against B7-H1. Fluorescence-activated cell sorting (FACS)

B7-H1, a third member of the B7 family, co-stimulates T-cellproliferation and interleukin-10 secretion

HAIDONG DONG, GEFENG ZHU, KOJI TAMADA & LIEPING CHEN

Department of Immunology, Mayo Graduate and Medical Schools, Mayo Clinic, 200 First Street SW,Rochester, Minnesota 55905, USA

Correspondence should be addressed to L.C.; email: chen.lieping@mayo.edu

The B7 family members B7-1 and B7-2 interact with CD28 and constitute an essential T-cell co-stim-ulatory pathway in the initiation of antigen-specific humoral and cell-mediated immune response.Here, we describe a third member of the B7 family, called B7-H1 that does not bind CD28, cytotoxicT-lymphocyte A4 or ICOS (inducible co-stimulator). Ligation of B7-H1 co-stimulated T-cell re-sponses to polyclonal stimuli and allogeneic antigens, and preferentially stimulated the productionof interleukin-10. Interleukin-2, although produced in small amounts, was required for the effect ofB7-H1 co-stimulation. Our studies thus define a previously unknown co-stimulatory molecule thatmay be involved in the negative regulation of cell-mediated immune responses.

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Fig. 1 Characterization and expression pattern of human B7-H1. a, Predicted amino-acid sequence of human B7-H1. Signal peptide, thepredicated signal peptide; Ig-V-like, the immunoglobulin V-like domain; Ig-C-like, the immunoglobulin C-like domain; TM (underlined), the transmem-brane region; *, the potential N-linked glycosylation site. b, Alignment ofthe B7 family members. Shaded and bolded, identical amino-acid residues;boxed, conserved residues; stars, cysteine residues that may be important inthe formation of disulfide bonds inside the immunoglobulin V or C do-mains. c, Distribution of B7-H1 mRNA in tissues, by northern blot analysis.Each lane contains approximately 1 µg poly A+ RNA. Left margin, mRNA sizemarkers; β-actin, a control to ensure equal loading of mRNA. d, Inducibleexpression of B7-H1 on T cells, B cells and macrophages. Freshly isolatedhuman PBMCs (Resting) and treated cell subsets (Activated) were stainedwith antibody against B7-H1 (Anti-B7-H1) or ‘pre-bleed’ sera (Pre-sera) incombination with monoclonal antibody against CD3, CD14 or CD19.Numbers (upper right corners), percentages of double-positive cells. Datarepresent one experiment of three.

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analysis using the antibody against B7-H1 showed that freshly iso-lated T and B cells expressed negligible amounts of B7-H1,whereas a fraction (about 16%) of CD14+ monocytes constitu-tively expressed B7-H1. However, B7-H1 can be upregulated bycell activation. Approximately 30% of phytohemagglutinin-treated CD3+ T cells and 90% of CD14+ monocytes (treated withgamma interferon (IFN-γ) and lipopolysaccharide) expressed B7-H1. Only 6% of CD19+ B cells expressed B7-H1 after lipopolysac-charide activation (Fig. 1d). We also obtained similar results usingRT–PCR analysis (data not shown).

Transfection of the plasmid pcDNA3-B7-H1 into 293 cells (B7-H1/293) led to the expression of B7-H1, as detected by antibodyagainst B7-H1 (Fig. 2a). This binding could be eliminated by theinclusion of B7-H1Ig (Fig. 2a, arrow), demonstrating the speci-ficity of the antibody. Neither CTLA4 immunoglobulin (CTLA4Ig)nor ICOS immunoglobulin (ICOSIg) bound to B7-H1/293 cells.Although both CTLA4Ig and ICOSIg bound to Raji cells, the bind-ing was not blocked by the inclusion of B7-H1Ig (Fig. 2a, arrows).Along with the observation that B7-H1Ig did not bind to Jurkatcells despite their constitutive expression of CD28 (Fig. 2b), our re-sults indicate that B7-H1 is not a ligand for CD28, CTLA4 or ICOS.

Co-stimulation of T-cell proliferation by B7-H1 ligationDespite having only about 25% amino-acid sequence homology,B7-1 and B7-2 have similar equilibrium receptor binding proper-ties and both co-stimulate T-cell growth. To determine whetherB7-H1 has the same functions, we purified T cells (about 95% pu-rity) from human PBMCs of healthy donors and stimulated themwith B7-H1Ig in the presence of suboptimal doses of monoclonalantibody against CD3. We determined T-cell proliferation in 3-daycultures by measuring the incorporation of 3H-thymidine. B7-H1Ig, when immobilized on culture plates, enhanced T-cell prolif-eration up to 1,000%, compared with proliferation with thecontrol immunoglobulin in the presence of 5–20 ng/ml of mono-clonal antibody against CD3 (Fig. 3a). In the absence of antibodyagainst CD3, B7-H1Ig at concentrations up to 5 µg/ml had no ef-fect on T-cell proliferation (Fig. 3a). Inclusion of B7-H1Ig in thecultures without immobilization mostly decreased its co-stimula-tory effect (data not shown). Consistent with this, the inclusion of

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Fig. 3 Co-stimulation of T-cell growth by B7-H1Ig. a, Purified T cells were cultured in thepresence of pre-coated antibody against CD3(horizontal axis (anti-CD3), concentrations) andimmobilized B7-H1Ig (�) or control im-munoglobulin (�). Data represent one experi-ment of five. cpm, counts per minute. b, Purified T cells were stimulated with irradi-ated allogeneic PBMCs in the presence of solu-ble B7-H1Ig (�) or control immunoglobulin (�) (horizontal axis, concentrations) for 5 d. cpm,counts per minute. Data represent one experi-ment of three.

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B7-H1Ig at concentrations of 0.6–5 µg/ml in allogeneic mixedlymphocyte reactions moderately increased (about 200%) the pro-liferation of T cells (Fig. 3b). We conclude that B7-H1 can promoteand co-stimulate proliferative responses of T cells to polyclonal T-cell stimuli and to allogeneic antigens.

B7-H1 co-stimulation preferentially induces IL-10 productionWe measured the amounts of IL-2, IL-4, IFN-γ and IL-10 producedby T cells after stimulation with B7-H1Ig, B7-1 immunoglobulin(B7-1Ig) and monoclonal antibody against CD28 in the presenceof antibody against CD3 (Fig. 4). As with B7-1Ig and antibodyagainst CD28, IL-10 production was increased considerably in theculture supernatants of T cells after stimulation with immobilizedB7-H1Ig and antibody against CD3 at 48 and 72 hours. We did notdetect IL-10 when we treated T cells with control immunoglobulinplus antibody against CD3. The amounts of IFN-γ were also in-creased. In contrast to B7-1Ig and antibody against CD28, B7-H1Iginduced small or negligible amounts of IL-2 and IL-4. We obtainedsimiliar results in six independent experiments. Our findings indi-cate that B7-H1 co-stimulation preferentially stimulates the pro-duction of IL-10.

Co-stimulatory effect of B7-H1 requires IL-2The production of IL-2, although in small amounts, peaked at 24hours after B7-H1 co-stimulation (Fig. 4), whereas IL-10 secretionstarted to increase after 48 and 72 hours. Increasing concentra-tions of B7-H1Ig led to a small increase (less than 1 ng/ml) of IL-2secretion (Fig. 4, inset). To determine the roles of the early-pro-duced IL-2, we tested the effects of monoclonal antibody againstIL-2 on T-cell proliferation and IL-10 production in B7-H1-medi-ated co-stimulation. As with B7-1-COS cells, the T-cell prolifera-tion induced by B7-H1-COS cells was blocked by the inclusion ofmonoclonal antibody against IL-2 (Fig. 5a). Furthermore, IL-10 se-cretion from T cells co-stimulated with B7-H1Ig was also inhibitedby monoclonal antibody against IL-2 (Fig. 5b). Therefore, the ef-fect of B7-H1 co-stimulation in T-cell growth and IL-10 secretion isan IL-2-dependent process.

DiscussionWe have cloned and characterized a previously unknown memberof the B7 co-stimulatory molecule family, called B7-H1. AlthoughB7-H1 shares only about 20% amino-acid identity with B7-1 andB7-2 in its extracellular domain, the secondary structures of thesemolecules are very similar. In addition to four conserved structuralcysteines, the tyrosine residue in B7-1 (at position 87) and in B7-2(at position 82) of the immunoglobulin V-like domain is also con-served in B7-H1 (at position 81) (Fig. 1b). The motif SQDXXXELYin the immunoglobulin C-like domain (positions 190–198 in B7-1and 189–197 in B7-2; Fig. 1b) is required for the binding of B7-1and B7-2 to their counter-receptors CD28 and CTLA4 (ref. 12).However, this motif was not found in B7-H1 (Fig. 1b). FACS analy-sis showed that CTLA4Ig did not bind to the 293 cells expressingB7-H1 (Fig. 2a) but bound to cells transfected with B7-1 (data notshown), indicating that B7-H1 is not a counter-receptor forCTLA4. Additional experiments showed that ICOSIg did not bindto the 293 cells expressing B7-H1 (Fig. 2a), indicating that B7-H1 isalso not a ligand for ICOS molecule. Definitive prove awaits thecloning and characterization of ICOS ligand or B7-H1 counter-receptor.

We demonstrated the co-stimulatory functions of B7-H1 by thestimulation of T-cell proliferation in the presence of suboptimal

Fig. 2 Binding of B7-H1 to CTLA4, ICOS and CD28. a, 293 cells trans-fected with B7-H1 (middle) or vector (left), as well as Raji cells (right), werestained with control immunoglobulin (open), fusion proteins (CTLA4Ig orICOSIg; filled) or antibody against B7-H1 (filled). To block binding, 10µg/ml B7-H1Ig was included during incubation with fusion protein or anti-body (bold lines and arrows). b, Jurkat T cells. Left, stained with controlmonoclonal antibody (open) or antibody against CD28 (Anti-CD28; filled).Right, stained with control immunoglobulin (open) or B7-H1Ig (filled), fol-lowed by fluorescein isothiocyanate-conjugated monoclonal antibodyagainst mouse immunoglobulin. Data represent one experiment of three.

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doses of antibody against CD3 (Fig. 3a) and in allogeneic mixedlymphocyte reactions (Fig. 3b). Cells transfected with either B7-1or B7-2 co-stimulate the production of large amounts of IFN-γ andIL-2 (ref. 16). In addition, transfection with B7-2 also increases theproduction of IL-4 and IL-10 (refs. 16,17). Here, we showed thatB7-H1Ig, although it stimulated the production of IL-10, inducedsmall amounts of IL-2 (Fig. 4). This pattern seems to be differentfrom that induced by transfection with B7-1 or B7-2, or by mono-clonal antibody against ICOS, in that large amounts of IL-10 andIL-4 are induced7. Therefore, B7-H1 co-stimulation seems to be rel-atively selective for inducing IL-10 production, and this does notfit into the classic T-helper cell type 1 or 2 cytokine pattern. IL-2production, although in small amounts, is essential in the B7-H1-mediated co-stimulatory effect on T cells, as blockade of IL-2 by aspecific monoclonal antibody completely inhibited T-cell prolifer-ation and IL-10 production responding to B7-H1-mediated co-stimulation (Fig. 5). IL-2 is an essential factor for the preventionand rescue of T-cell anergy18. Our preliminary studies indicate thatafter B7-H1 co-stimulation, T cells can still respond to stimulationwith antibody against CD3 (Dong et al., unpublished data).Therefore, the effect of IL-2 seems to maintain T-cell growth andprevent T-cell anergy to antigens in our experimental system.

Lymphocytes from patients with systemic lupus erythematosusundergo spontaneous cell death, which is associated with in-creased IL-10 and upregulated Fas ligand expression19. Blockade of

Fas ligand or IL-10 by a specific monoclonal antibody partially in-creases cell survival19. We found a large amount of B7-H1 mRNA inseveral normal organs such as lung and placenta (Fig. 1c) that areusually protected from unwanted inflammatory or immune reac-tions20. Furthermore, B7-H1 engagement stimulated increased IL-10 production (Fig. 4) and increased apoptosis of T cells (Dong etal. unpublished data). Together, our results indicate that B7-H1may be involved in organ-specific negative regulation of cellularimmune responses.

MethodsCloning of human B7-H1 cDNA and construction of immunoglobulin fu-sion proteins. The 5′ and 3′ ends of B7-H1 cDNA were amplified by PCR froma human placenta cDNA library synthesized by SMART PCR cDNA synthesiskit (Clontech, Palo Alto, California). The cDNA sequence of B7-H1 was verifiedby DNA sequencing. The primer pairs used for the PCR were derived from theplacenta library plasmid and from the expressed sequence tag cloneAA292201. Full-length B7-H1 cDNA was amplified by PCR from the samecDNA library by specific primers and cloned into the pcDNA3 vector(Invitrogen, Carlsbad, California). The amino-acid sequences of B7-H1, B7-1and B7-2 were analyzed using the ClustalW algorithm with the BLOSUM 30matrix (MacVector; Oxford Molecular Group, Beaverton, Oregon). B7-H1Igwas prepared by fusing the extracellular domain of human B7-H1 to theCH2–CH3 domain of mouse immunoglobulin G2a in the expression plasmid

Fig. 4 Cytokine secretion of resting T cells by B7-H1 co-stimulation. PurifiedT cells were stimulated by pre-coated antibody against CD3 at a concentra-tion of 1 µg/ml in the presence of 10 µg/ml B7-H1Ig (�), 10 µg/ml B7-1Ig(�) or control immunoglobulin (�). Monoclonal antibody against CD28 (5µg/ml; �) was used in soluble form. Supernatants were collected at 24, 48and 72 h and captured by a specific monoclonal antibody in a sandwichELISA. Inset, IL-2 production after 24 h of stimulation by pre-coated antibodyagainst CD3 in the presence of B7-H1Ig (�) or control immunoglobulin (�);horizontal axis, concentration. Data represent one experiment of three.

Fig. 5 B7-H1-co-stimulated T-cell proliferation and IL-10 production is IL-2-dependent. a, Purified T cells were stimulated by pre-coated suboptimal anti-body against CD3 (12.5 ng/ml) and irradiated B7-1 COS cells or B7-H1 COScells. Specific neutralizing monoclonal antibody against human IL-2 (anti-IL-2)was added at the beginning of culture. Mock COS, Parental vector-transfectedCOS cells treated with control monoclonal antibody, used as a control; Ab, an-tibody; cpm, counts per minute. b, Purified T cells were stimulated by immobi-lized antibody against CD3 (�, 100 ng/ml; �, 12 ng/ml) and B7-H1Ig. Specificneutralizing monoclonal antibody against human IL-2 was added at the begin-ning of culture. IL-10 production after 36 h of stimulation was determined inthe supernatant by sandwich ELISA. Ig, immunoglobulin; Ab, antibody. Datarepresent one experiment of three.

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pmIgV (G.Z., unpublished data), and was transfected into CHO cells. An iden-tical method was used for the preparation of the B7-1Ig, CTLA4Ig and ICOSIgfusion proteins. The protein in the culture supernatants was purified by aProtein G–Sepharose column (Pharmacia) and dialyzed in lipopolysaccharide-free PBS.

DNA transfection. Plasmids containing full-length B7-H1 (pcDNA3-B7-H1),B7-1 (pCDM8-B7.1) or parental vectors were transfected into 293 cells orCOS cells by the calcium phosphate or DEAE–dextran transfection systems(Promega). After 48 h of incubation, the expression levels of B7-H1 or B7-1 ontransfected cells were determined by FACS analysis with antibody against B7-H1 or monoclonal antibody against B7-1 (PharMingen, San Diego,California), respectively.

T-cell and cytokine assays. PBMCs of healthy donors were isolated byFicoll–Hypaque gradient centrifugation and were passed through a nylonwool column to obtain purified T cells (about 85% CD3+ cells), or were puri-fied further (more than 95% CD3+ cells) using the antibody against CD4/8MACS beads system (Miltenyl Biotec, Auburn, California). For co-stimulationassays, purified T cells (1 × 105 cells/well in triplicate) were cultured in 96-wellflat-bottomed plates that were pre-coated overnight with antibody againstCD3 (HIT3a; PharMingen, San Diego, California) in the presence of 5 µg/mlB7-H1Ig or control immunoglobulin (purified mouse immunoglobulin G2a ormurine 4-1BBIg fusion protein). COS cells transfected with B7-1 or B7-H1were used at a concentration of 1 × 104 cells/well to co-stimulate the growthof T cells in the presence of pre-coated antibody against CD3. To detect cy-tokines, supernatants were collected at 24, 48 and 72 h of culture, and theconcentrations of IL-2, IL-4, IFN-γ and IL-10 were determined by sandwichenzyme-linked immunosorbent assay (ELISA; PharMingen, San Diego,California) according to manufacturer’s instructions. B7-1Ig and antibodyagainst CD28 (CD28.2; PharMingen, San Diego, California) were included forcomparison and as a positive control, respectively. T-cell proliferation was de-termined by the addition of 1.0 µCi 3H-thymidine on day 2 for at least 18 h(ref. 21). The incorporation of 3H-thymidine was measured in a MicroBetaTriLux liquid scintillation counter (Wallac, Turku, Finland). For mixed lympho-cyte reactions assays, purified T cells (2 × 105 cells/well in triplicate) were co-cultured with allogeneic PBMCs (irradiated with 4,000 rad) at a concentrationof 2 × 105 cells/well in the presence of soluble B7-H1Ig or control im-munoglobulin. Then, 4 d later, the T-cell proliferation was determined by 3H-thymidine incorporation. Neutralizing monoclonal antibody against humanIL-2 (8 µg/ml, clone MQ1-17H12; PharMingen, San Diego, California) wasadded at the beginning of T-cell cultures. Polymyxin B (10 µg/ml) was also in-corporated in the assays of cell proliferation and cytokine secretion to com-pletely neutralize potential endotoxin contamination.

Nucleic acid analysis. Northern blot analysis was done using human multipletissue northern blots (Clontech, Palo Alto, California). The membrane was in-cubated for 30 min at 68 °C in ExpressHyb hybridization solution (Clontech,Palo Alto, California). A random-primed cDNA probe was synthesized usingthe entire human B7-H1 cDNA (870 bp), and was labeled using 32P-dCTP. Ahuman β-actin cDNA probe (2.0 kb) was used as a control. The blot was hy-bridized for 1 h at 68 °C, washed three times in 2× SSC containing 0.05%SDS, and was exposed at –70 °C to X-ray films.

Flow cytometry analysis. For the preparation of antiserum, mice were im-munized with purified B7-H1Ig mixed with complete Freund’s adjuvant(Sigma) and boosted three times with B7-H1Ig in incomplete Freund’s adju-vant. Serum was collected and the specificity was determined by ELISA andby FACS staining (1:1,000 dilution) of B7-H1 cDNA-transfected 293 cells orCOS cells. ‘Pre-bleed’ mouse serum (collected before immunization) wasused as the control. To prepare activated T and B cells, freshly isolated humanPBMCs (10 × 106 cells/ml) were stimulated with 5 µg/ml phytohemagglutinin(Sigma) or 10 µg/ml lipopolysaccharide (Sigma), respectively. For prepara-tion of activated monocytes, adherent PBMCs were cultured in 1,500 IU/mlrecombinant human IFN-γ (Biosource, Camarillo, California) and 100 ng/mllipopolysaccharide. All cultures were collected and analyzed at 48 h aftertreatment. For direct immunofluorescence staining22,23, T cells were incubatedfor 30 min at 4 °C with 1 µg monoclonal antibody conjugated with fluores-cein isothiocyanate or phycoerythrin, and were analyzed by FACScan flow cy-tometry with Cell Quest software (both from Becton Dickinson, Mountain

View, California) as described22. The monoclonal antibodies specific for CD3(UCHT1), CD4 (RPA-T4), CD8 (RPA-T8), CD14 (M5E2), CD19 (B43), CD28(CD28.2) and CD80 (BB1) were purchased from PharMingen (San Diego,California). For indirect immunofluorescence staining22,23, cells were first incu-bated at 4 °C with antibody against B7-H1(1:1,000 dilution), 5 µg ICOSIg orCTLA4Ig. After 30 min, the cells were washed and further incubated for 30min at 4 °C with goat against human or mouse immunoglobulin G F(ab′)2

conjugated with fluorescein isothiocyanate (Biosource, Camarillo, California)or phycoerythrin (Southern Biotechnology Associates, Birmingham,Alabama). Human or mouse immunoglobulin G1 protein (Sigma) or mouse4-1BBIg (the mouse 4-1BB extracellular domain fused with the Fc of humanimmunoglobulin G1 or mouse immunoglobulin G2a) was used as a controlimmunoglobulin. In some experiments, Fc receptors were blocked by humanor mouse immunoglobulin before incubation with monoclonal antibodiesconjugated with fluorescein isothiocyanate or phycoerythrin.

AcknowledgmentsWe thank J. Lau and K. Jensen for editing the manuscript. This work wassupported in part by the Mayo Foundation and National Institutes of Healthgrant CA79915. G.Z. is supported by National Institutes of Health training grantCA09127. The accession number for the human B7-H1 sequence in the GenBankis AF177937.

RECEIVED 17 AUGUST; ACCEPTED 20 OCTOBER 1999

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