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`IMMUNOBIOLOGY
`
`B7-H1 costimulation preferentially enhances CD28-independent
`T-helper cell function
`Hideto Tamura, Haidong Dong, Gefeng Zhu, Gabriel L. Sica, Dallas B. Flies, Koji Tamada, and Lieping Chen
`
`B7-H1 is a recently described B7-like mol-
`ecule that costimulates T-cell growth and
`cytokine secretion without binding to
`CD28, cytotoxic T-lymphocyte antigen-4
`(CTLA-4), and inducible costimulator
`(ICOS).
`In this report, a mouse homo-
`logue of human B7-H1 is identified, and
`its immunologic functions are studied in
`vitro and in vivo. Mouse B7-H1 shares
`69% amino acid homology to the human
`counterpart. Similar to human B7-H1,
`mouse B7-H1 can be induced to express
`Introduction
`
`on macrophages, T cells, and B cells
`and to enhance T-cell proliferation and
`secretion of interleukin-10 (IL-10), inter-
`feron-g, and granulocyte-macrophage
`colony-stimulating factor but not IL-2
`and IL-4. Furthermore, B7-H1 preferen-
`tially costimulates CD41 T cells indepen-
`dently of CD28 and enhances mixed lym-
`phocyte responses to allogeneic antigens.
`In contrast to B7-1, expression of B7-H1
`on murine P815 tumor cells by transfec-
`tion fails to increase allogeneic and syn-
`
`responses in
`geneic cytolytic T-cell
`vitro and in vivo. Administration of
`B7-H1Ig fusion protein, however, en-
`hances keyhole limpet hemocyanin–
`specific T-cell proliferation and 2,4,6-
`trinitrophenyl–specific immunoglobulin
`G2a antibody production. The study thus
`identifies a unique costimulatory path-
`way that preferentially affects T-helper
`cell functions. (Blood. 2001;97:1809-1816)
`
`© 2001 by The American Society of Hematology
`
`The antigen-specific induction of proliferation and differentia-
`tion of lymphocytes requires a first signal delivered through
`T-cell or B-cell receptor together with a second, costimulatory
`signal. Stimulation of T cells by antigen in the absence of
`costimulation can result in unproductive T-cell responses or
`T-cell anergy. Engagement of CD28 by its natural ligand B7-1 or
`B7-2 promotes T-cell activation, proliferation, and the differen-
`tiation of effector functions for both CD41 and CD81 T cells.1-3
`CD28 triggering augments both Th1 and Th2 cytokine secretion
`including interleukin-2 (IL-2), interferon-g (IFN-g), granulocyte-
`macrophage colony-stimulating factor (GM-CSF), tumor necro-
`sis factor-a (TNF-a), IL-4, IL-5, and IL-6 2,4 and up-regulates
`bcl-xL expression, which promotes T-cell survival.5 Expression
`of B7-1 or B7-2 by tumor cells can enhance CD81 cytotoxic T
`lymphocyte (CTL) generation and differentiation in vitro and in
`vivo.6-10 CTLA-4, the second receptor for B7-1 and B7-2, is
`believed to inhibit T-cell activation and IL-2 production by
`delivering an inhibitory signal.11 Recently, several new B7-like
`molecules have been described to costimulate T-cell growth and
`cytokine production by binding to receptors other than CD28
`and CTLA-4. Human and mouse B7h/B7RP-1 (GL50/LICOS/B7-
`H2) interact with an inducible receptor, ICOS, on T cells to
`costimulate T-cell growth in vitro.12-18 More importantly, mice
`treated with B7RP-1 fusion protein had enhanced experimental
`contact hypersensitivity, and overexpression of soluble B7RP-1
`in transgenic mice showed hyperplasia on several secondary
`lymphoid organs.13 Taken together, these data suggest that the
`interaction between B7h/B7RP-1 and ICOS regulate cell-
`mediated responses in vivo.
`
`We have recently identified a new member of human B7
`family designated B7-H1.19 B7-H1 gene encodes a putative type
`I transmembrane protein of 290 amino acids and consists of an
`immunoglobulin (Ig)V–like domain and an IgC-like domain in
`its extracellular portion. Although homology between B7-H1,
`B7h/B7RP-1, B7-1, and B7-2 is limited, the putative secondary
`and tertiary structures of these molecules are very similar (Chen
`et al, unpublished data, 2001). B7-H1 messenger RNA (mRNA)
`is found in various tissues,
`including lung, heart, skeletal
`muscle, and placenta, as well as several
`lymphoid organs,
`including spleen, thymus, and liver. B7-H1 can be detected on
`macrophages but not on T and B cells. Activation by various
`stimuli, however, can up-regulate the expression of B7-H1 on
`these cells. Stimulation of purified T cells by immobilized
`B7-H1Ig fusion protein in the presence of anti-CD3 antibodies
`induces a profound proliferative response and secretion of
`cytokines. Examination of cytokines on costimulation by B7-H1
`ligation reveals a unique pattern: B7-H1 selectively costimu-
`lates the production of IL-10 and IFN-g but not IL-2 and IL-4.
`Furthermore, soluble B7-H1Ig enhances mixed lymphocyte
`responses to allogeneic antigens.19 These results suggest that
`B7-H1 may participate in the regulation of cell-mediated and
`humoral
`immune responses. However,
`the potential role of
`B7-H1 in immune regulation in vivo is not known because of
`limitations of in vitro human T-cell culture system.
`By cloning mouse B7-H1, immunologic functions of B7-H1 are
`evaluated both in vitro and in vivo. We demonstrate that, in contrast
`to a more broad immunomodulatory functions of B7-1 and B7-2,
`B7-H1 appears to selectively enhance antigen-specific helper T-cell
`
`From the Department of Immunology, Mayo Graduate and Medical Schools,
`Mayo Clinic, Rochester, MN.
`
`Reprints: Lieping Chen, Department of Immunology, Mayo Clinic, 200 First St
`SW, Rochester, MN 55905; e-mail: chen.lieping@mayo.edu.
`
`Submitted July 26, 2000; accepted November 17, 2000.
`
`is supported by NIH
`Supported in part by the Mayo Foundation. G.Z.
`postdoctoral fellow training grant CA09127, and K.T. is a US Army Breast
`Cancer Research Program postdoctoral fellow.
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
`
`© 2001 by The American Society of Hematology
`
`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
`
`1809
`
`Petitioner
`Ex. 1110, p. 1809
`
`

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`by guest
`
`
`
`www.bloodjournal.orgFrom
`
`on June 3, 2015.
`
`For personal use only.
`
`1810
`
`TAMURA et al
`
`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
`
`responses but has minimal effect on the generation and differentia-
`tion of cytolytic T cells.
`
`Materials and methods
`
`Mice and cell lines
`
`Female C57BL/6 (B6), DBA/2, and BALB/c mice were purchased from the
`National Cancer Institute (Frederick, MD). CD282/2 mice with a B6
`background were kindly provided by Dr Moses Rodrigues (Mayo Clinic,
`Rochester, MN). P815 mastocytoma, L1210 lymphoma, EL4 mouse T-cell
`lymphoma, and 293 human kidney epithelial cells were purchased from the
`American Type Culture Collection (Rockville, MD). Cell
`lines were
`maintained in a complete medium of RPMI 1640 (Life Technologies,
`Rockville, MD) supplemented with 10% fetal bovine serum (FBS; Hy-
`Clone, Logan, UT), 25 mM HEPES, 100 U/mL penicillin G, and 100
`mg/mL streptomycin sulfate.
`
`Cloning and identification of mouse B7-H1
`complementary DNA
`
`A search of the NCBI database revealed that 2 mouse expressed sequence
`tags (ESTs) had high homology (GenBank accession no. AA823166 and
`AA896104) to the published human B7-H1 sequence.19 The missing 59 end
`and 39 end of mouse B7-H1 (mB7-H1) sequence was obtained by a rapid
`amplification of complementary DNA (cDNA) ends, using the SMART
`PCR cDNA synthesis kit (Clontech, Palo Alto, CA) from a mouse T-cell
`library. The full-length cDNA of mB7-H1 was cloned into pcDNA3 vector
`(mB7-H1.pcDNA3) (Invitrogen, Carlsbad, CA). The homology to human
`B7-H1, mouse B7-1, B7-2, and B7h/B7RP-1 was analyzed, using the
`ClustalW program of MacVector 6.5.
`
`RNA analysis
`
`Tissue distribution of mB7-H1 was carried out, using mouse RNA dot blot
`(Clontech) according to the manufacture’s instructions. The random-primed
`cDNA probe containing the full-length of mB7-H1 cDNA (895 base pairs)
`was labeled, using 32P-dCTP. The hybridization was performed for 16 hours
`at 65°C. After a 4-time wash with 23 SSC-sodium dodecyl sulfate, the
`membrane was exposed at 280°C to x-ray films.
`
`Antibodies and fusion proteins
`
`Rabbit antibodies against mB7-H1 protein were prepared by the Cocalico
`Biologicals (Reamstown, PA) by immunization with keyhole limpet
`hemocyanin (KLH)–conjugated hydrophilic peptides, spanning mB7-H1
`amino acid sequence 95-119 (GNAALQITDVKLQDAGVYCCIISYG).
`The antibodies were purified from rabbit serum by using peptide-
`conjugated affinity columns. Both enzyme-linked immunosorbent assay
`(ELISA) and flow-activated cell sorter (FACS) staining of mB7-H1
`transfected COS cells demonstrated that the antibodies bound specifically
`to mB7-H1. Purified monoclonal antibody (mAb) against CD3, CD4
`(GK1.5), and CD28; fluorescein isothiocyanate (FITC)–conjugated mAb
`against CD3, CD4, CD8, and CD40L; phycoerythrin (PE)–conjugated
`CD3; B220; and Mac-1 were purchased from Pharmingen (San Diego, CA).
`FITC- and PE-conjugated goat antibody against rabbit IgG was purchased
`from Southern Biotechnology Associates (Birmingham, AL). Purified
`rabbit IgG and hamster IgG (Rockland, Gilbertsville, PA) and rat IgG
`(Sigma, St Louis, MO) were used as controls. To prepare the mB7-H1Ig
`fusion protein, cDNA encoding mouse B7-H1 extracellular domain was
`generated by reverse transcriptase–polymerase chain reaction (RT-PCR),
`using the sense primer 59-CAGGAATTCACCATGAGGATATTTGCTG-39
`and the antisense primer 59-CATCAGATCTATGTGAGTCCTGTTCT-
`GTG-39 from mouse T-cell mRNA. After digestion with EcoRI and BglII,
`the PCR products were fused to CH2-CH3 domain of mouse IgG2a in the
`expression plasmid pmIgV.19 The resulting plasmid, pmB7-H1Ig, was
`transfected into Chinese hamster ovary (CHO) cells and cultured by using
`serum-free CHO media (Life Technologies). The mB7-H1Ig in the superna-
`
`tants was purified by a protein G–Sepharose column (Pierce, Rockford, IL)
`and dialyzed in lipopolysaccharide (LPS)–free phosphate-buffered saline
`(PBS). The endotoxin concentration was less than 1 pg/mg of purified
`protein according to limulus amebocyte lysate assays (Cape Cod, Woods
`Hole, MA). The mB7-1 immunoglobulin fusion protein (mB7-1Ig) was
`prepared by the same methods.
`
`Flow cytometric analysis
`
`The method for FACS analysis of surface molecules by antibodies has been
`previously described.6,20 For indirect immunofluorescence, the cells were
`incubated with the antibodies at 4°C for 30 minutes in the presence of
`blocking mAb to CD16/32 (Pharmingen). The cells were washed and
`further incubated with FITC- or PE-conjugated antirabbit IgG. For activa-
`tion of T cells, nylon-wool (NW)–purified T cells (. 75% CD31 cells) at
`2 3 106/mL were cultured with Con A (3 mg/mL), anti-CD3 (5 mg/mL), or a
`combination of anti-CD3 and anti-CD28 (5 mg/mL). For preparation of
`activated B cells, splenocytes were cultured with 10 mg/mL LPS for 24 to
`48 hours (Sigma). Monocytes were obtained from the peritoneal cavity of
`mice, which had been injected with thioglycollate 7 days before, and these
`cells were cultured with IFN-g at 10 U/mL and LPS at 100 ng/mL. All
`cultures were collected and analyzed at 48 hours. To detect CD40L
`expression, CD41 T cells were purified by magnetic sorting (see the
`followings) and further incubated with FITC-conjugated mAb to CD40L.
`Fluorescence was analyzed by a FACS Calibur flow cytometry and
`analyzed with Cell Quest software (Becton Dickinson, Mountain View, CA).
`
`T-cell costimulation and cytokine assay
`
`NW column-purified T cells were further positively selected by magnetic
`sorting, using FITC-conjugated mAb against CD4 or CD8 and anti-FITC
`microbeads according to the manufacture’s instructions (Miltenyi Biotec,
`Auburn, CA). The purity of isolated CD41 and CD81 T cells was more than
`95% by FACS analysis, using mAb to CD4 and CD8, respectively. Purified
`T cells at 2 3 106/mL from mouse spleens were cultured in 96-well plates
`that were precoated with anti-CD3 in the presence of 10 mg/mL mB7-H1Ig
`or control mouse IgG2a. mAb against CD28 (2.5 mg/mL) was used in
`soluble form as a positive control. Proliferation of T cells was determined
`by incorporation of 1 mCi/well of 3H-TdR during the last 15 hours of the
`3-day culture. 3H-TdR incorporation was counted by a MicroBeta TriLux
`liquid scintillation counter (Wallac, Turku, Finland). To detect cytokines,
`supernatants were collected at 18 to 72 hours of cultures, and the
`concentrations of IFN-g, IL-2, IL-10, IL-4, and GM-CSF were measured by
`sandwich ELISA following the manufacture’s instructions (Pharmingen).
`Assay for proliferative responses of CD41 T cells in mixed lymphocyte
`responses to allogeneic antigens has been described.19
`
`Generation of mB7-H1–expressing P815 cells and
`induction of cytotoxic lymphocyte
`
`P815 cells were transfected with mB7-H1.pcDNA3 plasmid by Fugene
`(Roche, Mannheim, Germany) according to manufacture’s instructions.
`The transfected cells were selected in complete medium containing 1
`mg/mL G418 (Life Technologies) and were subsequently cloned by limiting
`dilution. mB7-H11 P815 cells were identified by FACS analysis, using
`anti–B7-H1 antibody. A representative clone, mB7-H11 P815, was selected
`for further study. P815 clones transfected with pcDNA vector (mock.P815)
`and mB7-1.pcDNA (mB7-11 P815) were also generated as described
`previously.6
`To induce alloantigen-specific CTL activity in vitro, NW-purified T
`cells (2.5 3 106/mL) from B6 splenocytes were stimulated with irradiated
`(10 000 rad) mock.P815, mB7-11 P815, or mB7-H11 P815 cells (2.5 3 105/
`mL). After the 5-day stimulation, CTL activities against P815 (H-2d) and
`EL4 (H-2b) were measured by a standard 51Cr release assay.6,20 Alterna-
`tively, NW-purified T cells from BALB/c splenocytes at 3 3 106/mL were
`stimulated with irradiated B6 splenocytes at 1 3 106/mL in the presence of
`immobilized B7-H1Ig for 5 days, and CTL activity against allogeneic (EL4)
`or syngeneic target cells (P815) was measured by 51Cr release assay.
`To induce tumor-specific CTL activity in vivo, DBA/2 mice were
`
`Petitioner
`Ex. 1110, p. 1810
`
`

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`by guest
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`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
`
`B7-H1 AND T-HELPER CELL FUNCTIONS
`
`1811
`
`inoculated subcutaneously with 1 3 106 lived cells from mock.P815,
`mB7-11 P815, or mB7-H11 P815. The draining lymph nodes were
`removed 7 to 10 days after tumor injection, and the suspended lymph
`node cells (3 3 106/mL) were restimulated with P815 cells (3 3
`105/mL) for 5 days. The CTL activity was measured in a standard 51Cr
`release assay.6,20
`
`In vivo induction and assay of 2,4,6-trinitrophenyl–
`specific antibody
`
`2,4,6-trinitrophenyl (TNP)–KLH (Biosearch Technologies, Novato, CA) at
`100 mg/mouse in PBS was injected intraperitoneally into B6 mice on day 0.
`On days 1 and 4, the mice were injected intraperitoneally with 100 mg of
`control mIg, mB7-1Ig, or mB7-H1Ig. Sera were collected on days 7 and 14.
`To examine TNP-specific antibodies in sera, 0.3 mg/mL TNP-BSA
`(Biosearch Technologies) was bound to 96-well ELISA plates overnight at
`4°C. Nonspecific binding was blocked with 10% FBS in PBS for 90
`minutes at room temperature. After extensive washing, samples, diluted by
`1/200 to 1/2000 with PBS, were added and incubated for 2 hours. The plates
`were then washed, and biotinylated rat antimouse IgM, IgG1, IgG2a, or
`IgG3 (Pharmingen) was added to the wells. The plates were further
`incubated for 1 hour at room temperature. Bound biotinylated rat antibodies
`were detected by incubation with horseradish peroxidase-conjugated strepta-
`vidin (Caltag Laboratories, Burlingame, CA) for 1 hour at room tempera-
`ture. Color reactions were developed by 3,39,5,59-tetramethyl-benzidine
`substrate (Sigma) and the optical density (OD) reading at 450 nm
`was evaluated.
`
`T-cell proliferation to KLH
`
`B6 mice were immunized with 100 mg TNP-KLH in incomplete Freund’s
`adjuvant (IFA) subcutaneously or in PBS intraperitoneally on day 0 and
`were subsequently treated with fusion protein or control immunoglobulin
`intraperitoneally on days 1 and 4. To detect T-cell responses to KLH,
`draining lymph nodes and spleens were removed from immunized mice on
`days 7 and 14, respectively. Suspended cells were cultured with KLH at
`1.56 to 100 mg/mL as indicated. Proliferation of T cells to KLH was
`determined by addition of 1 mCi/well 3H-TdR during the last 15 hours of the
`3-day culture. 3H-TdR incorporation was counted by a Microbeta Trilux
`liquid scintillation counter (Wallac).
`
`Results
`
`Characterization of mouse B7-H1 gene
`
`The mB7-H1 gene encodes a putative type I transmembrane
`protein of 290 amino acids and has 69% overall amino acid
`homology to human B7-H1 (Figure 1A). Similar to other members
`of B7 family, mB7-H1 consists of an IgV-like domain, an IgC-like
`domain, a hydrophobic transmembrane domain, and a cytoplasmic
`tail. B7-H1 shares 20% homology to mB7-1, 14% to mB7-2, and
`19% to mB7h/B7RP-1, based on analysis using McVector 6.5
`software (Figure 1B). Four structural cysteines are well conserved,
`the same as other B7 members.
`RNA analysis revealed that mB7-H1 mRNA was abundant in
`heart, spleen, lung, skeletal muscle, and liver, less abundant but
`positive in kidney, liver, thymus, and thyroid (Figure 2A). There-
`fore, the expression pattern is similar to human B7-H1 mRNA.19
`Negligible expression of the B7-H1 mRNA was observed in
`pancreas and testis.
`FACS analysis using the antibody against mB7-H1 showed that
`freshly isolated as well as Con A-stimulated CD31 T cells express
`negligible amounts of B7-H1. However, stimulation by anti-CD3
`or a combination of anti-CD3/CD28 mAb moderately increased the
`expression of B7-H1 (Figure 2B). A small fraction of B2201 B cells
`and Mac-11 macrophages expressed a low level of B7-H1 (Figure
`
`2C, upper panel). Activation of B cells with LPS and macrophages
`with LPS plus IFN-g significantly increased the expression of
`B7-H1 on the cell surface (Figure 2C, lower panel). We conclude
`that B7-H1 is an inducible cell surface molecule.
`
`B7-H1 costimulation is CD28 independent and
`preferential for CD41 T cells
`
`To investigate the costimulatory effect of B7-H1, we stimulated
`NW-purified T cells with B7-H1Ig and suboptimal doses of
`anti-CD3 mAb. B7-H1 can enhance T-cell growth up to 5-fold
`compared to that of the control immunoglobulin (Figure 3A). The
`costimulatory effect of the B7-H1 is dose dependent and is
`dependent on anti-CD3 mAb because, in the absence of anti-CD3
`mAb, B7-H1Ig up to 10 mg/mL did not stimulate the proliferation
`of T cells (Figure 3B, left panel). When purified T cells were
`cultured with 293 cells transfected with mB7-H1.pcDNA3 or
`control vector in the presence of suboptimal doses of anti-CD3
`
`Figure 1. Putative amino acid sequence of mouse B7-H1 and alignment with
`human B7-H1. (A) Predicted amino acid sequence of mB7-H1 and alignment with
`human B7-H1. Signal peptide, IgV-like domain, IgC-like domain, transmembrane
`(TM), and cytoplasmic tail are indicated. (B) Alignment of mB7-H1 with other
`members of the B7 family. Conserved cysteine residues, which may be important to
`form the disulfide bounds of IgV and IgC domains, are marked by star signs. A few
`gaps (2) were introduced for the optimal alignment. Identical amino acid residues are
`shaded and conserved residues are boxed. The nucleic acid and amino acid
`sequences of mouse B7-H1 are available from GenBank under accession no.
`AAG31810.
`
`Petitioner
`Ex. 1110, p. 1811
`
`

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`From
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`1812
`
`TAMURA et al
`
`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
`
`Therefore, B7-H1 can costimulate T-cell growth in a CD28-
`independent fashion.
`We next examined the preference of B7-H1 costimulation on
`CD41 and CD81 T cells. Purified CD41 and CD81 T cells were
`stimulated with the same concentration of mB7-H1Ig and anti-
`CD3. Proliferation of CD41 T cells was up to 10-fold, and the
`proliferation of CD81 T cells was only enhanced 2- to 3-fold
`(Figure 3C). Thus, costimulatory effect of B7-H1 is more favorable
`for CD41 T cells.
`
`Effects of B7-H1 costimulation on cytokine secretion
`
`We have previously shown that human B7-H1 preferentially
`stimulated secretion of IL-10 and IFN-g but not IL-2 and IL-4.19 To
`
`Figure 2. Expression pattern of mB7-H1. (A) Tissue distribution of mB7-H1 mRNA
`in mouse tissue dot blot (Clontech). The random-primed cDNA probe containing the
`full length of mB7-H1 cDNA was prepared and labeled using 32P-dCTP. The tissues
`corresponding to the dot are indicated in the right panel. (B) Expression of mB7-H1 on
`resting and activated T cells. NW-purified T cells were analyzed without (control) or
`with stimulation of Con A, anti-CD3, or anti-CD3 plus anti-CD28 for 48 hours. The
`cells were stained by FITC-conjugated mAb against CD3 and rabbit anti–B7-H1
`antibodies (solid line) or control
`immunoglobulin (dotted line) followed by PE-
`conjugated goat antirabbit IgG. Positive cells for CD3 were gated and analyzed for
`the expression of mB7-H1 by FACS. (C) Expression of mB7-H1 on B cells and
`macrophages. Freshly isolated mouse splenocytes were analyzed without (upper
`panel) or with stimulation (Activated;
`lower left panel) by LPS for 48 hours.
`Macrophages obtained from the peritoneal cavity of B6 mice were stimulated with
`LPS and IFN-g (lower right panel). The cells were doubly stained by FITC-conjugated
`mAb against B220 or Mac-1 and rabbit anti–B7-H1 antibodies (solid line) or control
`immunoglobulin (dotted line) followed by PE-conjugated goat antirabbit IgG. Positive
`cells for B220 or Mac-1 were gated and analyzed for the expression of mB7-H1
`by FACS. The percentages of B7-H11 cells in the gated fraction are indicated in
`each histogram.
`
`mAb, B7-H1-transfected 293 cells also enhanced T-cell prolifera-
`tion substantially compared with that of the control vector-
`transfected 293 cells (data not shown). We conclude that, similar to
`human B7-H1, mB7-H1 costimulates T-cell proliferation.
`The role of CD28 in B7-H1 costimulation was evaluated by
`comparing the effects of mB7-H1Ig costimulation on T cells
`isolated from CD282/2 mice and from normal mice. As shown in
`Figure 3B, although there was no costimulatory effect of anti-
`CD28 mAb on CD282/2 T cells, mB7-H1Ig induced the prolifera-
`tion of both CD282/2 and CD281/1 T cells to a similar degree.
`
`Figure 3. Costimulatory effect of B7-H1 on T-cell growth. (A) The costimulatory
`effect of the B7-H1 is dose dependent. NW-purified T cells were cultured with the
`indicated doses of immobilized mB7-H1Ig (f) or control immunoglobulin (E) in the
`presence of a suboptimal dose (200 ng/mL) of precoated anti-CD3 mAb. The
`proliferation of T cells was determined by 3H-thymidine incorporation assay. (B)
`B7-H1 costimulation is CD28 independent. NW-purified T cells obtained from spleens
`of wild-type (wt, left panel) and CD282/2 (right panel) mice were cultured with control
`immunoglobulin (E), anti-CD28 mAb ((cid:130)), or mB7-H1Ig (f) at 10mg/mL in the
`presence of suboptimal doses (0.125 and 0.25 mg/mL) of immobilized anti-CD3. The
`proliferation of T cells was determined by 3H-TdR incorporation assay. (C) B7-H1
`preferentially costimulates CD41 T-cell growth. Affinity-purified CD41 or CD81 T cells
`were cultured in the presence of a suboptimal dose of anti-CD3 and immobilized
`B7-H1Ig. Control Ig, M; B7-H1Ig, f. The proliferation of T cells was determined by
`3H-TdR incorporation assay. Data represent one experiment of 3 and 3H-TdR
`incorporation in counts per minute (cpm) 6 SD.
`
`Petitioner
`Ex. 1110, p. 1812
`
`

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`From
`
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`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
`
`B7-H1 AND T-HELPER CELL FUNCTIONS
`
`1813
`
`contrast to anti-CD28 mAb, which induces high levels of IL-2 and
`IL-4, mB7-H1Ig induces low or negligible levels of IL-2 and IL-4
`up to 3 days (Figure 4A). We conclude that mouse B7-H1 and
`human B7-H1 have similar selectivity for cytokine production.
`Because IL-2 is undetectable in the presence of B7-H1 costimu-
`lation, it is thus possible that B7-H1 ligation inhibits IL-2 secretion.
`To test this possibility, we examined the effect of B7-H1 costimula-
`tion in IL-2 secretion from T cells stimulated by anti-CD3/28 mAb.
`Figure 4B shows that inclusion of immobilized mB7-H1Ig up to 10
`mg/mL in the culture results in a small but not significant decrease
`in IL-2 production during 18 to 48 hours. Similarly, mB7-H1Ig
`did not inhibit IL-2 production from the culture, in which T cells
`were stimulated with anti-CD3 alone (Figure 4B). Our results
`thus indicate that mB7-H1 ligation does not inhibit the production
`of IL-2.
`
`Effect of B7-H1 costimulation on antigen-specific
`T-cell responses
`
`To examine the effect of B7-H1 costimulation on the generation of
`CTL, we transfected mouse P815 tumor cells with mB7-
`H1.pcDNA3 plasmid. Clones that stably express mB7-H1 on their
`surface were selected for further experiments. A representative
`clone, B7-H11 P815, expresses high levels of mB7-H1 as indicated
`by FACS staining, using the anti–mB7-H1 antibodies (Figure 5A).
`The binding of the antibody is specific because mock.P815 cells
`(Figure 5A, left panel) and mB7-11 P815 cells (data not shown) are
`negative for the staining. Furthermore, inclusion of the peptide of
`B7-H1, which was used for immunization, completely blocks the
`binding of anti–B7-H1 antibodies to mB7-H11 P815 (Figure 5A,
`right panel).
`We showed previously that inclusion of B7-H1Ig fusion protein
`enhances mixed lymphocyte responses to allogeneic antigen in
`human T-cell culture.19 Similar to this observation, stimulation of
`purified T cells from B6 mice with allogeneic mB7-H11 P815 cells
`induces proliferative responses superior to that stimulated with
`mock.P815 cells. As a control, stimulation with mB7-11 P815 cells
`also induces high-level proliferative responses (Figure 5B). The
`proliferative responses enhanced by both mB7-H11 P815 as well
`as mB7-11 P815 cells can be completely inhibited by anti-CD4
`mAb (data not shown). However, mB7-H11 P815 cells as well as
`mock.P815 are poor stimulators of P815-specific CTL activity,
`whereas mB7-11 P815 elicited a strong P815-specific CTL activity
`as assayed in standard 51Cr release assay (Figure 5C). The CTL
`induced by mB7-1 was allogeneic antigen specific because it did
`not lyse H-2b EL4 cells. Similar results were also obtained in a
`different system in which purified T cells from BALB/c mice were
`stimulated with irradiated B6 spleen cells as allogeneic antigen-
`presenting cells. As shown in Figure 5D, proliferative responses are
`enhanced by inclusion of B7-H1Ig fusion protein, and the response
`can be completely blocked by an anti-CD4 antibody. CTL activity
`against allogeneic antigens in this system is not affected (Figure
`5E). Therefore, B7-H1 costimulation does not facilitate the genera-
`tion of allogeneic CTL in vitro.
`We also examined the ability of mB7-H11 P815 cells to
`stimulate P815-specific CTL in vivo. DBA/2 mice were injected
`subcutaneously with lived cells from mock.P815, mB7-11 P815, or
`mB7-H11 P815 lines. T cells from tumor-draining lymph nodes
`were removed 7 to 10 days later and cocultivated with wild-type
`(wt).P815 cells for 5 days. In mice injected with mB7-H11 P815, a
`small, but not significant increase in CTL activity against P815
`cells was detected compared to CTL from the mock.P815-injected
`mice. In contrast, mB7-11 P815 elicited a strong CTL activity in
`
`Figure 4. Cytokine secretion of mB7-H1-stimulated T cells. (A) NW-purified T
`cells were cultured with immobilized control immunoglobulin (E) or mB7-H1Ig (f,
`10mg/mL) in the presence of precoated anti-CD3 (2mg/mL). The mAb to CD28 ((cid:130), 2.5
`mg/mL) was used in soluble form. Supernatants were collected at days 1, 2, and 3 on
`the culture, and indicated cytokine concentrations in the supernatants were deter-
`mined by sandwich ELISA (Pharmingen). Data represent one experiment of 3. Error
`bars represent the SD of the mean of triplicate T-cell culture wells. (B) Effect of B7-H1
`costimulation on IL-2 production. The levels of
`IL-2 were determined in the
`supernatants, in which purified T cells were cultured in the presence of 2.5 mg/mL
`anti-CD28, 10 mg/mL mB7-H1Ig, or anti-CD28 plus mB7-H1Ig (r) in the precoated
`plate with immobilized anti-CD3 (1 mg/mL). Supernatants were collected at 18, 24,
`36, and 48 hours, and IL-2 concentrations in the supernatants were determined by
`sandwich ELISA. Data present one experiment of 3. Error bars represent the SD of
`the mean of triplicate T-cell culture wells.
`
`examine whether mB7-H1 functions similarly, we measured the
`levels of these cytokines as well as GM-CSF from T cells after
`stimulation with mB7-H1Ig or anti-CD28 mAb in the presence of
`anti-CD3 mAb. Figure 4A shows that mB7-H1, similar to anti-
`CD28 mAb, can induce high levels of IL-10 production in the
`72-hour culture supernatants. IL-10 was not detectable at the same
`time point when T cells were treated with control immunoglobulin
`and anti-CD3. mB7-H1 also promotes the secretion of IFN-g and
`GM-CSF, which are also induced by anti-CD28 mAb. In sharp
`
`Petitioner
`Ex. 1110, p. 1813
`
`

`
`From
`
`by guest
`www.bloodjournal.org
`
`
`
`
`For personal use only.on June 3, 2015.
`
`1814
`
`TAMURA et al
`
`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
`
`vivo. CTL activity is P815 specific since syngeneic but antigeni-
`cally irrelevant L1210 cells were not
`lysed (Figure 5F). We
`conclude that expression of B7-H1 in P815 cells does not enhance
`the induction of CTL activity against P815 tumor antigens.
`
`B7-H1 costimulation amplifies antigen-specific T-helper cell
`responses and T-cell–dependent humoral responses in vivo
`
`To investigate the effect of B7-H1 costimulation on T-helper cell
`function, we immunized B6 mice with TNP-conjugated KLH and
`subsequently injected the mice with mB7-H1Ig at day 1 and day 4.
`The proliferation of T cells obtained from both lymph nodes and
`spleens of the immunized mice (control mice) was examined by
`exposure to KLH in vitro. As shown in Figure 6, T cells from both
`spleens and lymph nodes of TNP-KLH–immunized mice prolifer-
`ated to KLH in a dose-dependent fashion. Administration of
`mB7-H1Ig to TNP-KLH–immunized mice amplified the prolifera-
`tive responses up to 2- to 3-fold. Our result indicates that B7-H1
`costimulation can enhance T-helper cell responses in vivo.
`We next examined the effect of B7-H1 costimulation in the
`generation of antigen-specific antibodies to TNP. This is a well-
`established system in which antibody production against TNP is
`dependent on helper T-cell response to KLH.21,22 The production of
`antibodies against TNP in the sera from TNP-KLH–immunized
`mice was measured after treatment with control immunoglobulin,
`
`Figure 6. T-helper cell response to KLH. B6 mice were injected with 100 mg
`TNP-KLH in IFA subcutaneously (left panel) or in PBS intraperitoneally (right panel)
`immunoglobulin (E) or
`on day 0, and subsequently treated with 100 mg control
`B7-H1Ig (f) on days 1 and 4. The draining lymph nodes were prepared at day 7 (left
`panel), and the splenocytes were prepared on day 14 (right panel). The suspension
`cells were stimulated with KLH at the indicated concentrations. The proliferative
`responses of T cells to KLH were determined by the addition of 1 mCi/well
`3H-thymidine during the last 15 hours of the 3-day culture.
`
`mB7-1Ig, or mB7-H1Ig. In preliminary experiments, we have
`found a significant increase of total IgG levels in mice treated with
`mB7-H1Ig compared to those treated with control mIg (data not
`shown). The subclasses of IgG, including IgG1, IgG2a, IgG2b, and
`IgG3 were further examined. As shown in Figure 7, TNP-specific
`IgG2a antibody was increased significantly in mice treated with
`mB7-H1Ig, and there were no significant changes on other
`immunoglobulin subtypes. This effect
`is different from mice
`treated with mB7-1Ig since other IgG components, including IgG1
`and IgG2b, were also increased significantly (Figure 7). Therefore,
`B7-H1 costimulation enhances T-helper cell proliferation and
`T-helper–dependent humoral immune responses.
`CD40–CD40L interaction is critical for T-helper cell–B-cell
`
`Figure 5. Effect of B7-H1 costimulation in T-cell responses to allogeneic and tumor
`antigens. (A) The expression of mB7-H1 by transfected P815 tumor cells. P815 tumor
`cells were transfected with pcDNA3 vector (Mock; left panel) or B7-H1.pcDNA3 (B7-H11
`P815; right panel). Cells were stained by rabbit anti–B7-H1 antibodies (bold line) or control
`rabbit immunoglobulin (dashed line) followed by FITC-conjugated goat antirabbit IgG. In
`addition, B7-H11 P815 cells were stained by anti–B7-H1 antibodies in the presence of the
`peptide of B7-H1, which had been used for immunization to make the antibodies (solid
`line). (B) The effect of mB7-H1 expression by P815 on T-cell proliferative responses to
`allogeneic antigen. NW-purified T cells obtained from B6 m