International Immunology, Vol. 12, No. 10, 1439-1447,
October 2000
© 2000 Japanese Society for Immunology
Characterization of a new human B7-related protein: B7RP-1 is the ligand to the co-stimulatory protein ICOS
Exploratory Research,
1 Pharmacology and
2 Process Development, Amgen Inc., Thousand Oaks, CA 91320, USA
Correspondence to: S. K. Yoshinaga
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Abstract |
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Optimal T cell activation requires the interactions of co-stimulatory molecules, such as those in the CD28 and B7 protein families. Recently, we described the co-stimulatory properties of the murine ligand to ICOS, which we designated as B7RP-1. Here, we report the co-stimulation of human T cells through the human B7RP-1 and ICOS interaction. This ligand–receptor pair interacts with a KD ~ 33 nM and an off-rate with a t
> 10 min. Interestingly, tumor necrosis factor (TNF)-
differentially regulates the expression of human B7RP-1 on B cells, monocytes and dendritic cells (DC). TNF- enhances B7RP-1 expression on B cells and monocytes, while it inhibits it on DC. The human B7RP-1–Fc protein or cells that express membrane-bound B7RP-1 co-stimulate T cell proliferation in vitro. Specific cytokines, such as IFN-
and IL-10, are induced by B7RP-1 co-stimulation. Although IL-2 levels are not significantly increased, B7RP-1 co-stimulation is dependent on IL-2. These experiments define the human ortholog to murine B7RP-1 and characterize its interaction with human ICOS.
Keywords: antigen-presenting cells, dendritic cells, cytokines, co-stimulation, TCR
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Introduction |
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The interaction of co-stimulatory proteins is required for an optimal response of T cells stimulated by MHC–peptide complexes expressed on antigen-presenting cells (APC). The most extensively characterized co-stimulatory pathway is mediated by CD28 and CTLA-4 on T cells (1). CD28 and CTLA-4 interact with the B7 proteins (CD80 and CD86) on either professional or non-professional APC (2,3). CD28 is considered to be a positive regulator and CTLA-4 a negative regulator of this signaling pathway. T cell activation in the absence of CD28 co-stimulation leads to anergy, a state of antigen-specific T cell non-responsiveness (4). These and other properties of CD28 regulation make co-stimulatory proteins attractive therapeutic targets. For example, it has been shown that activators of this pathway, such as inhibitory antibodies to the negative regulator CTLA-4 (5) or B7 ligands (6–8), enhance cytolytic T lymphocyte (CTL) responsiveness and anti-tumor immunity. Conversely, inhibitors of CD28 regulation, such as soluble CTLA-4–Ig protein or neutralizing antibodies to B7-1 or B7-2, repress disease progression in many spontaneous or experimentally induced animal models of autoimmunity (4).
Several in vivo experiments have indicated that aspects of T cell function are independent of CD28 regulation. Mice either lacking CD28, both B7-1 and B7-2, or expressing a soluble CTLA-4–Ig protein show reduced Th cell activity and class switching; however, many other T cell functions remain intact (9,10). Allografts transplanted into CD28 knockout mice or mice treated with CTLA-4–Ig demonstrate prolonged survival (11); however, the grafts are eventually rejected. Therefore, co-stimulation mediated through the CD28/B7 pathway cannot account for all of the T cell effector functions.
Additional T cell co-stimulatory proteins have been identified. For example, OX40, 4-1BB and B7-H1 have been shown to co-stimulate T cells (12,13). These co-stimulatory proteins have functions similar to, but not completely overlapping with, CD28. Recently, ICOS was described as an inducible CD28-related molecule with co-stimulatory functions on human T cells in vitro (14). Human ICOS is the ortholog of murine CRP-1 (CD28-related protein-1). We recently defined B7RP-1 (B7-related protein-1) as the ligand to murine ICOS, and demonstrated that B7RP-1 has both in vivo and in vitro co-stimulatory functions (15). Here we describe the cloning and characterization of human B7RP-1, and demonstrate its co-stimulatory functions through ICOS.
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Methods |
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Full-length cloning of human B7RP-1
A GenBank blast homology search (GCG, University of Wisconsin) using the murine B7RP-1 sequence (15) retrieved a clone (AB014553) containing a 4358 bp sequence with a 1679 bp open reading frame. PCR cloning primers were designed according to this sequence, and a DNA fragment of 1313 bp was obtained by 5' and 3' RACE using Human Lymph Node Marathon-Ready cDNA (Clontech, Palo Alto, CA). The DNA fragment was used to probe a cDNA library from normal human circulating peripheral blood (PB) lymphocytes. The DNA fragment (125 ng) was labeled with [32P]dCTP (Amersham, Piscataway, NJ) following the Redi-Prime 2 (Amersham) random prime labeling system protocol. The colony lift filters were then allowed to hybridize with the probe at 42°C in the following buffer overnight: 50% formamide, 5xSSPE, 2xDenhardt's solution, 0.5% SDS and 100 mg/ml single-stranded DNA. The sp. act. of the probe was 2.38x109 c.p.m./µg DNA, in ~2 ng labeled probe/ml hybridization buffer. The filters were then successively washed in 2xSSC, 0.1% SDS at room temperature for 15 min, followed by 1xSSC/0.1% SDS at 55°C for 15 min and 1xSSC/0.1% SDS at 60°C for 10 min. The filters were wrapped in plastic and exposed to autoradiography film overnight at –80°C. Three independent clone colonies were chosen, and the DNA was isolated and sequenced for each clone in triplicate.
The full-length coding region for human B7RP-1 was inserted in the DNA vector pDSR and stably transfected into CHO cells as previously described (15). Expression of human B7RP-1 was verified using FACS analysis with the human or mouse ICOS–Fc protein.
Construction of Fc proteins
A DNA fragment encoding the human B7RP-1 extracellular domain amino acids 1–246 (Fig. 1
) was fused in-frame upstream of DNA encoding the C-terminal 235 amino acids of human IgG1 in the pDSR vector. A DNA fragment encoding the human ICOS-1 extracellular domain amino acids 1–146 (14,15) was fused in-frame upstream of DNA encoding the C-terminal 235 amino acids of human IgG1 in the pDSR vector. The DNAs were stably transfected into CHO cells as previously described (15). A DNA fragment encoding the DR4 extracellular domain amino acids 1–239 (16) was fused in frame upstream of human IgG1 in a modified pCEP4 vector (Invitrogen, Carlsbad, CA). The resultant DR4–Fc/pCEP4 plasmid was transfected into 293-EBNA-1 cells using lipofectamine (Gibco, Gaithersberg, MD) and transfected cells were selected with 100 µg/ml hygromycin. Drug-resistant clones were pooled and cultured in serum-free media for 72 h. The non-fused human Fc fragment, hFc, contains the C-terminal 235 amino acids of human IgG1. hB7RP-1–Fc, hICOS–Fc, hFc and DR4–Fc proteins were purified by Protein G- or Protein A-affinity chromatography.
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Biacore 2000 analysis
Molecular interactions were analyzed by surface plasmon resonance utilizing a Biacore 2000 (Biacore, Uppsala, Sweden). For each experiment, the protein was immobilized onto the dextran layer of CM5 sensor chips via random amine coupling chemistry. The dextran surface was activated with NHS/EDC and then the proteins, in 10 mM sodium acetate buffer, pH 4.5, at 1 µg/ml, were injected. Proteins were exposed to the activated surface until ~50–100 response units (RU) were observed. The remaining active groups on the dextran were inactivated with ethanolamine. Activated and blocked dextran was generated as a reference surface. In order to determine kinetic values for the interactions described, CM5 chip surfaces were immobilized with counter-analytes at very low densities (<250 RU) to omit possible mass transport effects within the flow cell of the instrument. Furthermore, association and dissociation rates were determined globally, i.e. as dependent variables within the full sensogram data set. This type of data analysis is more robust than an independent analysis of the on and off rates of the interaction. Although not analyzed directly, the sensogram data that were obtained indicated a mono-dispersed sample at all analyte concentrations analyzed, based on the data's convergence with a theoretical 1:1 interaction. Proteins were diluted in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% Tween 20 to nanomolar concentrations, and were injected across the immobilized surfaces at 50 µl/min for 1 min. Double referencing was employed to obtain subtracted data, utilizing the signals from the reference flow cell and buffer alone injections (17). Subtracted data were analyzed using the BIAevaluation software Version 3.0 (Biacore).
Generation of monocyte-derived DC
Peripheral blood mononuclear cells (PBMC) were obtained from random, normal donors by leukophoresis or by venipuncture and collection into heparin-coated tubes. DC were grown from adherent PBMC as previously described, except that human recombinant granulocyte macrophage colony stimulating factor, IL-4 and tumor necrosis factor (TNF)- (Amgen Inc., Thousand Oaks, CA) were added at 100 ng/ml in EX VIVO serum-free media (Amgen Inc.) on day 0 (18). Loosely adherent and non-adherent cells were harvested on day 7.
PBMC preparation
PBMC from healthy donors were isolated by Ficoll-Paque (Sigma) centrifugation, and the cells were washed twice. B cells and monocytes were purified using mAb-labeled magnetic beads (Miltenyi Biotec, Auburn, CA). Cells were stimulated with either 25 µg/ml of lipopolysaccharide (LPS)/dextran or 200 ng/ml TNF- in RPMI 1640 medium containing 10% FCS, PSG, non-essential amino acids, sodium pyruvate, HEPES and 2-mercaptoethanol.
FACS analysis
The antigen-specific mAb anti-HLA-DR–FITC, anti-HLA-DR–phycoerythrin (PE), anti-CD19–FITC, anti-CD80–PE (BD PharMingen, San Diego, CA), anti-CD1a–FITC, anti-CD3–FITC, anti-CD11c–PE, anti-CD14–FITC, anti-CD14–PE and anti-CD86–PE (BD PharMingen), and matched isotype controls mIgG2a–FITC, mIgG1–FITC and mIgG1–PE (Becton Dickinson) were used at the concentrations recommended by the respective manufacturers. To detect ICOS expression on T cells, biotinylated human B7RP-1–Fc protein (B7RP-1–Fc–biotin) was used at 10 µg/ml. Specific binding was detected using streptavidin (SA)–PE (BD PharMingen) at 1 µg/ml. Human T cells (>98% CD3+) were isolated by negative selection of fresh or thawed, adherence depleted PBMC using mAb-labeled magnetic beads (Miltenyi Biotec), and were activated with phorbol myristate acetate (5 ng/ml) and ionomycin (250 ng/ml) overnight. To detect B7RP-1 expression, biotinylated ICOS–Fc (ICOS–Fc–biotin) was used at 10–20 µg/ml and specific binding was detected using SA–PE at 1 µg/ml. Identical concentrations of biotinylated hFc (hFc–biotin) and SA–PE were used as non-specific binding controls. The B cells, monocytes and DC were pre-incubated with 50 µg/ml polyclonal IgG (Chemicon, Temecula, CA) in 60 µl of 1% BSA/1xPBS (FACS buffer) on ice for 5 min. For competition assays demonstrating specificity of ICOS–Fc binding to purified monocytes (>99% pure), cells were also pre-incubated with various concentrations of unlabeled ICOS–Fc or the negative control Fc construct, DR4–Fc, for 10 min prior to incubation with 10 µg/ml ICOS–Fc–biotin. Cells (1x105 cells/reaction) were incubated in FACS buffer on ice with mAb and human Fc–biotin fusion proteins at the described concentrations for 30 min. The cells were washed once and SA–PE was added to the human Fc–biotin fusion protein stained cells for 30 min. The stained cells were washed twice and then fixed in 1% formaldehyde. Data were acquired on a FACStation calibrated with CaliBRITE beads and were analyzed using the CellQuest software (BD Pharmingen). The change in mean florescence intensity (
MFI) of ICOS–Fc–biotin binding as compared to Fc controls was calculated for TNF- or LPS treated and untreated cells. DC data were acquired using the following settings:
DC cultures were gated by FSC and SSC (>98% HLA-DR+/CD11c+ DC) for analysis of DC phenotypes. Student's t-test was used to determine the statistical difference between MFI for TNF- or LPS treated and untreated cells. P < 0.05 was considered significant.
In vitro T cell co-stimulation assays
Highly purified human T cells (>98% CD3+) were isolated by negative selection of fresh or thawed, adherence depleted PBMC using mAb-labeled magnetic beads (Miltenyi Biotec). T cells were cultured in triplicate wells in 96-well plates at 1x105 cells/well in 200 µl/well RPMI + 10% FCS. To evaluate B7RP-1–Fc co-stimulation, various concentrations of anti-CD3 (BD PharMingen) and 10 µg/ml anti-human IgG Fc (Sigma) in 100 µl 1xPBS were pre-coated onto U-bottom plates by an incubation at 4°C overnight. The unbound anti-CD3 and anti-human IgG Fc were removed and the cells were cultured in the presence or absence of various concentrations of B7RP-1–Fc, OPG–Fc control or anti-CD28 (BD PharMingen). For ICOS–Fc inhibition of B7RP-1–Fc co-stimulation, T cells were cultured in 0.33 µg/ml anti-CD3 and 10 µg/ml anti-human IgG Fc pre-coated wells with 0.5 µg/ml B7RP-1–Fc in the presence of serially diluted ICOS–Fc or OPG–Fc starting at 10 µg/ml. To evaluate the effects of combined anti-CD28 and B7RP-1-Fc co-stimulation, T cells were incubated in wells pre-coated with 0.5 µg/ml anti-CD3 and 10 µg/ml anti-human Fc in the presence of various concentrations of B7RP-1–Fc and 25 ng/ml anti-CD28 or various concentrations of anti-CD28 and 12.5 ng/ml B7RP-1–Fc. To evaluate the contribution of IL-2 for B7RP-1-induced growth, 3 µg/ml anti-IL-2 or isotype-matched control mAb (BD PharMingen) were added on day 0 to cultures stimulated with 0.5 µg/ml anti-CD3 and 0.5 µg/ml B7RP-1–Fc. To evaluate co-stimulation by CHO cells expressing B7RP-1, T cells were cultured in flat-bottom plates with various concentrations of soluble anti-CD3 in the presence or absence of various amounts of mitomycin C-treated CHO B7RP-1 cells or CHO vector cells. To test for T cell proliferation, cultures were pulsed with 1 µCi/well [3H]thymidine during the last 18 h of a 72 h culture. T cell proliferation was determined by [3H]thymidine incorporation. The results of one representative experiment from three random donors are expressed as mean c.p.m. incorporated ± SD. For analyses of cytokine production, cells were cultured for 48 and 72 h, and supernatants were collected for ELISA.
Cytokine production assays
The cytokine assays were conducted on the cell culture supernatants of cells under T cell proliferation conditions described above. Supernatants from T cell cultures stimulated for 48 and 72 h were analyzed for IL-2, IL-10 and IFN- by ELISA according to the manufacturer's specifications (BioSource International, Camarrillo, CA).
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Results |
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Cloning of the full-length human B7RP-1 gene
A homology search using the mouse B7RP-1 sequence (15) identified a putative human clone (AB014553) in a public database. Sequences from this clone were used to isolate a full-length version of the human B7RP-1 gene from a human PBL library. The alignment of the human and mouse B7RP-1 proteins is shown in Fig. 1
. The length of the human B7RP-1 protein is 302 amino acids. The polypeptide length and the relative position of the transmembrane domain are consistent with those of other B7 family members. The human B7RP-1 protein has 43% amino acid identity with the mouse protein. This degree of homology is significant, since the mouse and human CD80 (B7-1) proteins are only 41% identical. Notably conserved between the mouse and human proteins are the cysteine residues at amino acid positions 37, 113, 158, 215 and 216. These cysteine residues are presumably involved in disulfide bonds that form Ig homology loops.
Binding analysis by Biacore
The human B7RP-1 and ICOS protein interaction was analyzed by surface plasmon resonance utilizing a Biacore 2000 instrument. For comparison, a kinetic analysis was first performed with CTLA-4 and B7-2. B7-2–Fc was immobilized to the Biacore sensor chip surface and CTLA-4–Ig was injected as the analyte. The data were fit using the 1:1 Langmuir binding model to generate an association rate (ka) of 9.31x105 M–1 s–1 and a dissociation rate (kd) of 7.34x10–3 s–1, and therefore the equilibrium binding constant (KD) was predicted to be 7.77x10–9 M (Table 1). This is in general agreement with the previously described affinities of the CTLA-4 and B7 proteins (19). This system was then applied to the ICOS–Fc and B7RP-1–Fc constructs. In all cases, B7RP-1–Fc was immobilized to the chip surface and different concentrations of ICOS–Fc were injected as an analyte. The kinetic constants from a representative experiment are summarized in Table 1 and show that B7RP-1 binds to ICOS. The calculated equilibrium binding constant for each interaction is in the low nanomolar range, indicating a strong affinity that is consistent with other described T cell interactions. However, differences are observed in the association and dissociation rates. Interestingly, murine B7RP-1 interacts well with human ICOS; however, neither B7RP-1 nor ICOS interacts with proteins in the CD28 regulated pathway.
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FACS analysis of B7RP-1–Fc binding to ICOS
The B7RP-1–Fc fusion protein was used in FACS analysis to detect human PBMC that express its receptor, presumably ICOS. ICOS is expressed on few CD45RA+ naive T cells and a small population of CD45RO+ memory T cells (Fig. 2
). A large majority of T cells express ICOS after stimulation overnight with PMA and ionomycin. No binding of B7RP-1–Fc to B cells or monocytes was observed (data not shown). This expression is consistent with murine studies (15) and with ICOS antibody binding data (14).
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FACS analysis of B7RP-1 expression on APC
Biotinylated ICOS–Fc fusion protein was used to detect B7RP-1 expression on APC. Human PBMC were purified and DC were cultured as described in Methods. Specificity of ICOS–Fc–biotin binding to human monocytes was shown by a competition FACS analysis (Fig. 3
). Pre-incubating the cells with a 10-fold excess of unlabeled ICOS–Fc (Fig. 3A) inhibited the majority of the ICOS–Fc–biotin binding (83%), but pre-incubating with an excess of unlabeled control Fc construct (DR4–Fc, Fig. 3B) did not. B7RP-1 is expressed on PB B cells and monocytes, and on PB monocyte-derived DC (Table 2). TNF--treatment enhanced the ICOS–Fc fusion protein binding to B cells (1.7-fold increase in mean MFI, three donors) and monocytes (3.9-fold increase in mean MFI; P < 0.05, four donors). The increase in the magnitude of the mean MFI of ICOS–Fc binding to TNF- stimulated B cells, compared to unstimulated B cells, was not statistically significant (P > 0.05) due to large donor to donor variability; nevertheless, TNF- consistently enhanced ICOS–Fc binding to B cells with all donors. Statistically relevant results were obtained when B cells (2.2-fold increase in mean MFI; P < 0.05) and monocytes (3.2-fold increase in mean MFI; P < 0.05) were activated with LPS (20). Interestingly, TNF- treatment reduced ICOS–Fc binding to monocyte-derived DC (>50% reduction in mean MFI; P < 0.05). The effects of TNF- stimulation on B7-1 and B7RP-1 expression were also analyzed on CD1a+ and CD1a– DC (Fig. 4). These DC were CD14–, CD1a+/–, CD11chi, CD40+, MHC class II (HLA-DR)hi, CD86+ and CD80+/– as has been previously described (18). CD1a+ DC (10–50% of total DC generated) were typically 92% B7RP-1+ and CD1a– DC were typically 70% B7RP-1+. DC cultured with TNF- had a similar phenotype, except there were fewer CD1a+ DC (10–18% of total DC), with slightly enhanced B7-1 expression (1.3-fold increase in mean MFI, P < 0.02), and reduced B7RP-1 expression on both CD1a+ and CD1a– DC. Therefore, TNF- treatment increased the expression of B7RP-1 on B cells and monocytes, but decreased the expression of B7RP-1 on DC.
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T cell proliferation assays
To determine whether the human B7RP-1 protein has co-stimulatory activity on human T cells, we tested human B7RP-1-expressing CHO cells and B7RP-1–Fc fusion protein in T cell proliferation assays. B7RP-1–Fc demonstrated co-stimulatory activities that are dependent on anti-CD3 stimulation (Fig. 5A
). In addition, this activity can be specifically inhibited with soluble ICOS–Fc protein (Fig. 5B). The co-stimulatory effects by the B7RP-1–Fc fusion protein are dose dependent, and simultaneous co-stimulation of both the ICOS and CD28 pathways results in additive effects, suggesting similar, but non-overlapping functions (Fig. 5C and D). Similar co-stimulatory effects were obtained using CHO cells that express membrane-bound B7RP-1 (Fig. 6).
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), 0.5 µg/ml OPG–Fc (
) and 5 µg/ml anti-CD28 (
) at different concentrations of anti-CD3 primary stimulation. Data show that B7RP-1–Fc co-stimulated anti-CD3 primed T cells to similar levels as co-stimulation using anti-CD28 antibodies. Data shown are mean [3H]thymidine incorporated ± SD in triplicate wells from one representative experiment of several experiments generated with T cells isolated from three normal donors. (B) Dose-dependent inhibition of B7RP-1 Fc co-stimulation by ICOS–Fc. T cells were cultured with anti-CD3 coated at 0.3 and 0.5 µg/ml B7RP-1 Fc. Serially diluted concentrations of ICOS–Fc (•) or OPG–Fc (
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Cytokine production
The production of cytokines by human T cells under the above in vitro proliferation conditions was determined by ELISA using cell culture supernatants. We found IFN-
and IL-10 levels were significantly increased; however, unlike CD28 co-stimulation, IL-2 was only slightly increased at the 72 h timepoint (Fig. 7). Generally, this is consistent with previous results using ICOS antibodies; however, the magnitude of the induction of IL-10, relative to anti-CD28 antibody induction, was variable between donors (data not shown). In addition, a blockade using antibodies to IL-2 inhibited B7RP-1-induced co-stimulation (Fig. 8). This suggests that, although IL-2 levels were not substantially elevated by B7RP-1 co-stimulation, the IL-2 that was produced was required for B7RP-1-induced T cell proliferation.
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Discussion |
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We have described the cloning of human B7RP-1 and have demonstrated that it is an authentic ligand for the recently described T cell co-stimulatory protein, ICOS (14). The establishment of in vitro human T cell assays using the human B7RP-1 ligand is essential to identify inhibitors with potential clinical relevance and to determine if the natural human B7RP-1 ligand functions similarly to mouse B7RP-1 or an ICOS mAb. B7RP-1 co-stimulation of anti-CD3-stimulated human PB T cells results in dose-dependent proliferation and cytokine production. These results are consistent with our recent report on murine B7RP-1 (15), which positively regulates splenic T cell growth and cytokine production, and results by Hutloff et al. that showed positive co-stimulatory function by mAb-mediated (F44) ICOS cross-linking on CD3-stimulated human T cells (14). We found that B7RP-1 ligand stimulation of human T cells generates a pattern of cytokine production similar to that generated with the anti-ICOS mAb F44 or with mouse B7RP-1–Fc, although we found that the B7RP-1–Fc consistently induced less IL-10 than CD28-activating antibodies. Since the F44 mAb induced more IL-10 than CD28 antibodies (14), it may have greater effects on IL-10 than the natural ligand.
In order to clarify the role of B7RP-1 in immunoregulation, we sought to identify the factors that regulate B7RP-1 expression in human immune cells. Swallow et al. recently demonstrated that TNF- enhanced murine B7RP-1 mRNA expression in mouse fibroblasts (21). We show that APC maturation-inducing agents (TNF- and LPS) regulate B7RP-1 expression on APC. Unstimulated PB B cells and monocytes constitutively express B7RP-1, and its expression was further enhanced by TNF- or LPS activation.
Interestingly, TNF- enhanced B7-1 expression as expected (22), but reduced B7RP-1 expression, on both CD1a+ and CD1a– monocyte-derived DC. This differential regulation of B7RP-1 expression by TNF- on monocytes, B cells and DC may provide clues as to the different roles of the CD28 and ICOS pathways. Furthermore, we show that B7RP-1 is co-expressed with B7 molecules on human DC, and the simultaneous stimulation of both the CD28 and ICOS pathways has complementary effects on T cell proliferation in vitro. Taken together, our data indicate that B7RP-1 is a positive regulating ligand for ICOS-mediated T cell co-stimulation. The possibility remains that, like CTLA-4, ICOS is a negative regulator and B7RP-1 may function via an unknown, positive factor. As with the characterization of CTLA-4 (23,24), conclusive functional data can be obtained by ICOS gene deletion studies in mice. These studies are currently in progress.
It is interesting to consider how B7RP-1/ICOS co-stimulation relates to B7/CD28 co-stimulation. We suggest CD28 signaling may play a dominant role during T cell priming and ICOS signaling may emphasize effector functions. The temporal expression patterns of CD28 and ICOS are consistent with this hypothesis. CD28 is constitutively expressed, while ICOS expression requires induction on CD45RA+ naive T cells and most CD45RO+ memory T cells. The modulation of the expression patterns of B7RP-1 on DC, B cells and monocytes shown here is also consistent with the putative B7RP-1 functions on effector cells. Naive T cells respond optimally to mature DC that express high levels of B7 (25,26) and these cells appear to express low levels of B7RP-1. On the other hand, effector T cells are adequately stimulated by B cells or macrophages, which express relatively low levels of B7 and high levels of B7RP-1 (26,27).
The fact that the proteins in the CD28 and ICOS pathways do not interact, i.e. that they are separate receptor–ligand pairs, may suggest they have inherent differences in their functions. It is also possible that the CD28 and ICOS pathways have complementary functions. Our in vitro assays show additive effects when both pathways are stimulated. Furthermore, while the receptors or ligands of the two pathways are sometimes expressed on the same cells, they are differentially regulated by TNF-. Accumulating data indicate that B7 co-stimulation may be more important to naive T cell priming, while B7RP-1 co-stimulation may be primarily involved in maintaining or modulating primed and memory T cell effector functions. Further investigations are focused on the aspects of this pathway that may be amenable to therapeutic intervention.
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Acknowledgments |
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Special thanks to Dr Brenda Yoshinaga for assistance with the preparation of this manuscript. We also thank Dr Hailing Hsu for the DR-4–Fc protein and Amgen's DNA sequencing group for excellent technical support.
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Abbreviations |
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| APC antigen-presenting cell |
| B7RP-1 B7 related protein-1 |
| CRP-1 CD28-related protein-1 |
| CTL cytolytic T lymphocyte |
| DC dendritic cell |
| ICOS inducible co-stimulator |
| LPS lipopolysaccharide |
| PB peripheral blood |
| PBMC peripheral blood mononuclear cell |
| PE phycoerythrin |
| PMA phorbol myristate acetate |
| RU response unit |
| SA streptavidin |
| TNF tumor necrosis factor |
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Notes |
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Transmitting editor: T. Hünig
Received 3 February 2000, accepted 26 June 2000.
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