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Blood, 1 April 2008, Vol. 111, No. 7, pp. 3635-3643. Prepublished online as a Blood First Edition Paper on January 25, 2008; DOI 10.1182/blood-2007-11-123141.
IMMUNOBIOLOGY B7-H1 is a ubiquitous antiapoptotic receptor on cancer cells1 Department of Dermatology and Oncology, Johns Hopkins University School of Medicine, Baltimore, MD
B7-H1 is an immunoglobulin-like immune suppressive molecule broadly detectable on the majority of human and rodent cancers, and its functions have been attributed to delivering an inhibitory signal to its counter-receptor programmed death-1 (PD-1) on T cells. Here we report that B7-H1 on cancer cells receives a signal from PD-1 to rapidly induce resistance against T cell–mediated killing because crippling signaling capacity of B7-H1 but not PD-1 ablates this resistance. Importantly, loss of B7-H1 signaling is accompanied by increased susceptibility to immune-mediated tumoricidal activity. In addition to resistance against T-cell destruction, B7-H1+ cancer cells also become refractory to apoptosis induced by Fas ligation or the protein kinase inhibitor Staurosporine. Our study reveals a new mechanism by which cancer cells use a receptor on immune cells as a ligand to induce resistance to therapy.
Cancer cells display altered surface molecular signatures that distinguish them quantitatively and qualitatively from their normal derivatives. These modifications in receptor and ligand expression commonly facilitate tumor growth and progression or to evasion of host defense mechanisms.1,2 For example, some tumor cells down-regulate their cell surface major histocompatibility complex (MHC), which is required for recognition by tumor antigen-specific T lymphocytes.3 As a result, these tumor cells become less recognizable by the immune system and more resistant to immune-mediated destruction. Another example is that during progression, cancer cells frequently overexpress proteases and modify glycosylation of cell surface proteins that are normally involved in tissue repair, remodeling, and homeostasis to facilitate invasion and metastasis.4,5 In general, these modifications in cell membrane ligands and receptors regulate interactions between tumor cells and nontransformed cells in the microenvironment in a fashion that enhances tumor growth, invasion, and immune resistance We previously identified an immunoglobulin (Ig)–like molecule termed B7-H1,6 which is either constitutively or inducibly expressed by the majority of human and rodent cancer cells.7,8 Ample evidence demonstrates that B7-H1 acts as a ligand for the receptor programmed death-1 (PD-1) to deliver an inhibitory signal to T cells, leading to inhibition of immune responses.9 The mechanisms underlying B7-H1/PD-1–mediated suppression include induction of apoptosis, anergy, unresponsiveness, and exhaustion of T cells.7,10–14 Interaction between B7-H1 and PD-1 is also shown to participate in the suppression of autoimmune diseases and transplantation rejection in animal models.15–18. A recent study suggests that B7-H1, in addition to PD-1, also binds B7-1 (CD80) on T cells to inhibit their activation.19 We and others have observed that B7-H1+ tumor cells are much more resistant to CD8+ cytolytic T cell (CTL)–mediated destruction in vitro than their B7-H1–negative parental cells, and this resistance is correlated with decreased efficacy of immunotherapy in mouse tumor models.20–22 Ablation of B7-H1 and PD-1 interaction by neutralizing antibodies could restore CTL-mediated lysis of tumor cells in vitro, suggesting that B7-H1/PD-1 interaction forms a barrier between tumor cells and CTL, and this phenomenon has been termed "molecular shield."20 These results have been interpreted as inhibition of CTL activity induced by unidirectional engagement of PD-1 on the T cell by B7-H1 on the tumor cells. However, there are alternate interpretations for this molecular shield phenomenon. Although interaction between B7-H1+ tumor cells and PD-1 on T cells has been shown to induce T-cell suppression, it is possible that the molecular shield is simply attributable to rapid loss of CTL cytolytic function. However, when B7-H1+ and B7-H1– tumor cells were mixed together with antigen-specific CD8+ CTL in short-term in vitro assays, preferential lysis of B7-H1– cells is observed.20 This experiment suggests that overall cytolytic function of CD8+ CTL upon exposure to B7-H1 in short-term assays is not impaired. Another possibility is that B7-H1 and PD-1 simply form a physical barrier to prevent interaction of T-cell receptor (TCR) and tumor antigen presented in the MHC class I. Finally, it is possible that B7-H1 could act as a receptor to transmit a signal from T cells to tumor cells, leading to resistance of lysis. To test these hypotheses, we specifically engineered B7-H1 and PD-1 molecules with normal binding capacity but impaired ability to transmit signals to tumor cells or T cells, respectively, to examine their effects on molecular shielding of tumor cells from T-cell killing. The results support a mechanism whereby PD-1 on T cells acts as a ligand for B7-H1, whereas B7-H1 acts as a receptor to transmit signals to the tumor cells, thereby enhancing its resistance to apoptosis induction by both immune effectors and proapoptotic drugs.
Mice and tumor lines
Female DBA/2, C57BL/6 (B6) mice were purchased from the National Cancer Institute (Frederick, MD). Age-matched mice, 6 to 10 weeks old, were used for all experiments. 2C transgenic mice (a gift from Dr Larry Pease, Mayo Clinic, Rochester, MN) and PD-1–deficient mice in B6 background (a gift from Dr Tasuko Honjo, Kyoto University, Kyoto, Japan) were described previously.16 The 2C x PD-1KO mice were obtained by backcrossing between 2C transgenic mice and PD-1KO mice. All mice were maintained in the animal facility at Johns Hopkins Hospital under approved protocol by the Institutional Animal Care and Use Committee. P815 mastocytoma cells were purchased from the American Type Culture Collection (Rockville, MD). A subclone, which does not express B7-H1, even in the presence of interferon- Monoclonal antibodies, fusion proteins, and flow cytometric analysis Purified monoclonal antibody (mAb) against mouse H-2Dd and CD95 (clone Jo2) was purchased from BD PharMingen (San Diego, CA) and H-2Ld from BioLegend (San Diego, CA). Rat mAb (clone TY25) against B7-DC, fluorescein isothiocyanate (FITC)– or phycoerythrin-conjugated goat antimouse antibodies and FITC-conjugated goat antihamster antibodies were purchased from eBioscience (San Diego, CA). An Armenian hamster mAb (clone 10B5) against mouse B7-H1,20 a hamster mAb (clone G4) against mouse PD-1,20 a rat mAb (clone 2A) against mouse CD137,23 mouse PD-1Ig fusion protein,20 and mouse B7-H1Ig fusion protein20 were all described previously. Fluorescence was detected by FACScalibur flow cytometry and analyzed with Cell Quest software (BD Biosciences, Mountain View, CA). Plasmids
To generate truncated chimeric murine Gene transfection and lentivirus-mediated transduction
To generate CTL growth and functional assay For the generation of alloreactive CTLs or 2C CTLs, lymph node cells (5 x 106/mL) from C57BL/6 mice were stimulated with irradiated spleen cells (2 x 106/mL) from DBA/2 mice in 24-well plates for 5 days. Cells were incubated with 51Cr-labeled target cells at the indicated effector/target (E/T) ratios for 4 hours as described previously.20 The spontaneous releases of 51Cr are less than 10%. In cold target competition assay, mock/P815 and B7-H1/P815 cells were premixed and incubated with activated 2C CTLs in 24-well plates for 4 hours. Cells were stained with phosphatidylethanolamine-conjugated anti–H-2Dd mAb and 10B5 mAb plus FITC-conjugated goat antihamster Ig antibodies or green fluorescence protein (GFP), and all cells were counted by flow cytometry. Wells containing only target cells were included as controls. To prepare PD-102C CTL clone, the lymph node cells from 2C x PD-1KO mice were maintained in the complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 25 mM HEPES, 100 units/mL penicillin G, 100 µg/mL streptomycin sulfate, 55 µM 2-ME, and 30 units/mL human IL-2 by stimulating with 50 Gy-irradiated spleen cells from DBA/2 mice every 10 to 14 days. Cell apoptosis assays PD-1 Ig fusion protein or control IgG were coated on 96 well-plates for 18 hours at 4°C. Full-length B7-H1/P815 cells were cultured with Staurosporine (STP; 1 µM) in the well-coated PD-1Ig or control IgG. Cells were harvested after 24 hours and stained with annexin-V (BD PharMingen) at 5 µL/test, propidium iodide (PI; Sigma-Aldrich, St Louis, MO) at 5 µg/mL for 1 hours and mAbs against H-2Dd. Apoptosis was calculated as the percentage of annexin V+ PI– cells gated in the H-2Dd+ fractions.
Expression of B7-H1 confers resistance of tumor cells to specific CTL-mediated lysis
Previous studies showed that expression of B7-H1 rendered P815 cells resistant to tumor-specific CD8+ CTL, and this resistance requires B7-H1 and PD-1 interaction.20 To determine whether this observation could be generalized, we first established an allogeneic CTL line by stimulation of B6-derived T cells with irradiated spleen cells from DBA/2 mice (H-2d) and subsequently examined the susceptibility of mock/P815 and B7-H1/P815 cells to lysis in a 4-hour 51Cr release assay. As predicted, whereas mock/P815 cells were susceptible to lysis by allogeneic CTL in a wide range of E/T ratios, B7-H1/P815 cells were much more resistant. However, this resistance could be completely eliminated by inclusion of murine B7-H1–neutralizing mAb. (Figure 1A). Next, we used Renca cells as targets for lysis by alloantigen-specific TCR transgenic 2C T cells. Renca is a murine renal cell carcinoma and does not constitutively express B7-H1 on cell surface. However, expression of B7-H1 could be induced by IFN-
PD-1 signaling is not required for molecular shielding of tumor cells from T-cell killing
Our previous study showed that interaction between B7-H1 and PD-1 is required for the formation of a molecular shield.20 It is widely accepted that signaling from B7-H1 to PD-1 on T cells delivers a negative signal to suppress T-cell responses.11 Therefore, one possible explanation for molecular shield is tumor-associated B7-H1 signaling through PD-1 into T cells, leading to transient loss of T-cell cytolytic activity. To test this possibility, we first prepared truncated PD-1, in which the intracellular domain of PD-1 was replaced by GFP gene (Figure 2A) to eliminate its intracellular signaling. This truncated PD-1 (
Intracellular domain of B7-H1 is required for molecular shielding
To determine the role of tumor-associated B7-H1 signaling in the formation of the molecular shield, we constructed a vector to express truncated B7-H1 (
Although our previous data suggest that intracellular domain of B7-H1 is important in determining the formation of molecular shield, it could not be excluded that the extracellular domain of B7-H1 may also contribute to the formation of molecular shield. To address this issue, we replaced the extracellular domain of B7-H1 with corresponding region from B7-DC, another counter-receptor for PD-124 (Figure 4A). The chimeric gene B7-DC/H1 was used for the transfection to establish stable B7-DC/H1/P815 line. P815 cells expressing wild-type B7-DC (B7-DC/P815) were also established as the control. The expression of B7-DC and H-2 Ld (data not shown) as well as ability of these cell lines to bind PD-1Ig was the same in both B7-DC/P815 and B7-DC/H1/P815 lines based on flow cytometry analysis (Figure 4B). As shown in Figure 4C, B7-DC/P815 cells could be lysed equally as well as mock/P815 cells by 2C T cells. This result indicates that B7-DC is not capable of forming a molecular shield even though B7-DC is capable of engaging PD-1 on T cells with affinity at least as high as B7-H1. In contrast, B7-DC/H1/P815 cells were resistant to 2C CTLs, and this resistance was completely blocked by anti–B7-DC mAb (clone TY25). Our findings thus demonstrate that intracellular but not extracellular domain of B7-H1 has unique character to form molecular shield against T-cell lysis.
Formation of molecular shield is correlated with tumor resistance to immunotherapy
We next evaluated whether reverse signaling by B7-H1 conferred resistance to immune killing of tumors in vivo. We showed previously that B7-H1/P815 tumors were resistant to the treatment by anti-CD137 mAb, whereas mock/P815 or wild-type P815 was sensitive in vivo.20 Anti-CD137 immunotherapy of tumors has been shown to be attributable to enhanced stimulation of endogenous tumor-specific CD8 T cells in vivo. Because
B7-H1 transmits an antiapoptotic signal to tumor cells To explore the mechanism of molecular shielding by B7-H1 signaling, we developed a T cell–free system to induce resistance of tumor cells to CTL lysis. In this system, PD-1Ig was immobilized on plastic plates and subsequently incubated with tumor cells to engage B7-H1. Twenty-four hours later, tumor cells were incubated with PD-102C T cells to test susceptibility of tumor cells to lysis. After 4 hours of incubation, the lysis of PD-1Ig–treated B7-H1/P815 cells by PD-102C T cells was significantly lower than control IgG-treated B7-H1/P815 cells in a wide range of E/T ratios (Figure 6A). This provides a simple system to explore the mechanism of B7-H1–induced molecular shielding in the absence of T cells in the induction phase.
Fas has been shown to participate in the death of some cancer cells. Mock/P815 and B7-H1/P815 cells express comparable levels of Fas on cell surface based on flow cytometric analysis using specific mAb against murine Fas. Incubation of these tumor lines in the presence of PD-1Ig does not affect expression of Fas (Figure 6B). Treatment of mock/P815 and B7-H1/P815 cells by anti-Fas mAb (and control Ig) also induced a comparable apoptosis (54.9% vs 57.2%). However, pretreatment by PD-1Ig significantly decreased the death of B7-H1/P815 cells (35.8%), whereas the death of mock/P815 cells remained the same (52.4%; Figure 6C). This represents 32% inhibition. We next tested whether B7-H1 confers resistance to drugs that induce apoptosis of tumor cells. STP (Streptomyces staurospores) is a relatively nonselective protein kinase inhibitor. STP is often used as a general method for inducing apoptosis of tumor cells.25 PD-1Ig pretreatment for 8 hours drastically decreased apoptosis of B7-H1/P815 cells (16.9%) compared with control Ig-treated cells (74.9%; Figure 6D). Taken together, our results support that B7-H1 is an antiapoptotic receptor against death of cancer cells.
Our study provides the evidence that B7-H1 can act as a receptor to transmit antiapoptotic signal to cancer cells, leading to resistance to cytolysis by CTL as well as Fas and drug-induced apoptosis. In addition, we demonstrate that elimination of the intracellular domain of B7-H1 can ablate cancer resistance to immune destruction, which is accompanied with regression of tumors in mouse models. In the context of existence of multiple ligands or counter-receptors for B7-H1 in hematopoietic cells, our findings suggest a new mechanism contributing to tumor escape from immune destruction in vivo.
Several previous studies by us and others indicate that B7-H1 can evade immune destruction by engaging inhibitory receptor(s) on T cells.20–22 These studies are particularly intriguing because up-regulation of B7-H1 is found in the majority of human cancers. However, previous studies focused on the function of B7-H1 as a ligand (ie, on interaction with the PD-1 receptor on T cells, inhibitory signal would be delivered to T cells, leading to apoptosis, suppression, anergy, and exhaustion).7,10–13 This immune inhibitory function occurs during the induction of T-cell activation in lymphoid organs and/or in the effector phase after migration of activated T cells to peripheral organs.15,22 However, the current study reveals a completely different mechanism for B7-H1 inhibition of antitumor immune responses by transmitting an antiapoptotic signal to tumor cells. However, in these in vitro CTL assays, PD-1 signaling does not seem to suppress cytolytic function of CTL. It is possible that PD-1–mediated dysfunction of CTL may require more than 4 to 6 hours of exposure, which, however, is sufficient for B7-H1 to deliver antiapoptotic function on tumor cells. Although it appears that the predominant mechanism in vitro appears to be B7-H1–mediated tumor resistance, both B7-H1–mediated tumor resistance and PD-1–mediated T-cell dysfunction could play the roles simultaneously in vivo. For example, immunotherapy of established tumors induced by The intracellular domain of B7-H1 has 30 amino acids and does not contain any obvious motifs for signaling to known antiapoptotic molecules. One possible explanation is that intracellular domain of B7-H1 binds adaptor molecules that deliver antiapoptotic signal. By preparation of truncated B7-H1, our data demonstrate that intracellular domain of B7-H1 on cancer cells is required for the formation of molecular shield. In contrast, expression of truncated PD-1 or wild-type PD-1 into PD-1–/– 2C transgenic T cells confers CTL resistance compared with nontransduced PD-1–/– 2C transgenic T cells (Figure 2D). An alternative interpretation is that transduced PD-1 may interfere with the TCR–MHC interaction. However, this is considered unlikely because PD-1–/– 2C transgenic T cells expressing truncated or wild-type PD-1 could lyse B7-H1– P815 equally well (Figure 2C). Taken together, our data indicate that PD-1 acts as a ligand in this system to stimulate B7-H1. This notion is further supported by the experiments showing that B7-DC, which is capable of binding to PD-1, on tumor cells does not induce resistance of tumor cells to CTL lysis, whereas replacement of its intracellular domain with that from B7-H1 does (Figure 4C). By comparison with B7-H1, murine B7-DC has a very short cytoplasmic tail (4 amino acids) and would not be expected to signal equivalently to B7-H1. Finally, incubation of B7-H1+ tumor cells in the presence of immobilized PD-1 is sufficient to induce resistance to apoptosis (Figure 6). It has been shown that binding to B7-H1 to PD-1 induces an inhibitory signal toward T cells. It is unclear why this effect is not evident in our coculture systems. It is likely that molecular shield occurs very quickly within a couple of hours after exposure, whereas induction of T-cell suppression through PD-1 as a receptor requires more time. This is supported by our previous study showing that induction of apoptosis of human tumor antigen-specific T cells by B7-H1+ cancer cells required 2 to 3 days.7 Although our data demonstrate a role of B7-H1 in the induction of antiapoptotic mechanism on cancer cells, underlying biochemical pathways remain to be defined. We examined several major antiapoptotic and apoptotic pathways using the Apoptosis Oligo GEArray (Superarray Bioscience Corporation, Frederick, MD) and did not find major difference between control Ig versus PD-1Ig treatment on B7-H1/P815 cells in the expression of apoptosis-related genes, including tumor necrosis factor (TNF) receptor family, Bcl-2 family, caspase family, integrin-associated protein family, TNF receptor–associated factor family, caspase recruitment domain family, death domain family, death effector domain family, CIDE family and antiapoptosis genes, etc (T.A., unpublished data, 2007). It has been shown that CTL-mediated lysis of target cells is mediated by at least 2 mechanisms: granule-mediated cytolysis and membrane receptor-mediated apoptosis. After contact of target cells, perforin could be released from CTL to form channels on target cells, which allows passage of important molecules such as granzyme B. Granzyme B will activate caspase 3 and/or cause mitochondrial disruption after cleavage of Bid,28 leading to apoptosis. In addition, cell surface apoptotic receptors such as Fas or TNF–related apoptosis-inducing ligand may also induce apoptosis of target cells after engaging their ligands or counter-receptor.29,30 B7-H1 appears to have a broad role in the resistance of apoptosis of tumor cells, including CTL-mediated death as well as Fas- and STP-mediated apoptosis. It is thus possible that B7-H1 represents an early signal in the cascade to induce the inhibition of both pathways. In the context of broad expression of B7-H1 on tumor cells, this mode of antiapoptosis may play significant role in the prevention of tumor cell death by immune therapy and chemotherapy. Our findings have important implications in design of cancer therapy. Because B7-H1 has more than one receptor, blockade of a single receptor such as anti–PD-1 mAb may not be sufficient to eliminate the molecular shield. When designing blocking mAb to B7-H1, it is desirable that this mAb should block all receptor binding sites that could trigger formation of molecular shield. Because the role of B7-H1–mediated molecular shield is mediated through prevention of apoptosis, these findings support a combined therapy using blockade of B7-H1 and apoptosis-inducing drugs. Finally, it is desirable to design small molecule inhibitors that could block the signal pathway mediated by the intracellular domain of B7-H1. Our findings thus provide an excellent opportunity to enhance cancer immunity and to prevent tumor-mediated escape of host immune responses.
Contribution: T.A., S.Y., G.Z., A.S.F., and S.J.F. performed the experiments. T.A. and L.C. wrote this paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Lieping Chen, Johns Hopkins Medical Institutions, 209 David H. Koch Cancer Research Building, 1550 Orleans St, Baltimore, MD 21231; e-mail: lchen42{at}jhmi.edu.
We thank Dr Drew M. Pardoll for his careful reading and comments and Jennifer Osborne for editing this manuscript. We would also like to extend a special thanks to those who provided us with mice, cell lines, and reagents, and we cite their gifts in the text. This study was supported in part by National Institutes of Health grants CA85721, CA97085, and CA113341.
Submitted November 14, 2007; accepted January 12, 2008.
Prepublished online as Blood First Edition Paper, January 25, 2008
DOI: 10.1182/blood-2007-11-123141
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Abstract
Introduction
(IFN-
B7-H1, full-length B7-DC, and PD-1, polymerase chain reaction (PCR) fragments of each gene were digested with XhoI and BglII, XhoI and EcoRI, or BamH1 and XhoI restriction sites, respectively (5'primer for truncated B7-H1; 5'-ccgctcgaggccaccatgaggatatttgctg -3', 3'primer for truncated B7-H1; 5'-gaagatcttcttgttttctcaagaaga-3', 5'primer for full-length B7-DC; 5'-ccgctcgaggccaccatgctgctcctgctgccga-3', 3'primer for full-length B7-DC; 5'-ggaattcctagatcctctttctct-3', 5'primer for full-length PD-1; 5'-cgcggatccgccaccatgtgggtccggcag-3' and 3'primer for full-length PD-1; 5'-ccgctcgagtcaaagaggccaagaac-3'). Subsequent ligations of these fragment were performed into XhoI/BamHI-digested pEGFP-N1 (Clontech, Mountain View, CA), XhoI/EcoRI-digested pcDNA3.1 (Invitrogen, Carlsbad, CA) or BamHI/XhoI-digested pHR' cytomegalovirus (CMV) vectors. Chimeric murine B7-DC/H1 and truncated chimeric murine 
