In acute myeloid leukemia, B7-H1 (PD-L1) protection of blasts from cytotoxic T cells is induced by TLR ligands and interferon-gamma and can be reversed using MEK inhibitors
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- Berthon, C., Driss, V., Liu, J. et al. Cancer Immunol Immunother (2010) 59: 1839. doi:10.1007/s00262-010-0909-y
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B7-H1 (PD-L1) is a B7-related protein that inhibits T-cell responses. B7-H1 participates in the immunoescape of cancer cells and is also involved in the long-term persistence of leukemic cells in a mouse model of leukemia. B7-H1 can be constitutively expressed by cancer cells, but is also induced by various stimuli. Therefore, we examined the constitutive and inducible expression of B7-H1 and the consequences of this expression in human acute myeloid leukemia (AML). We analyzed B7-H1 expression in a cohort of 79 patients with AML. In addition, we studied blast cells after incubation with interferon-gamma or toll-like receptors (TLR) ligands. Finally, we evaluated functionality of cytotoxic T-cell activity against blast cells. Expression of B7-H1 upon diagnosis was high in 18% of patients. Expression of TLR2, 4 and 9 was detected in one-third of AML samples. Expression of TLR2 and TLR4 ligands or IFN-γ induced by B7-H1 was found to protect AML cells from CTL-mediated lysis. Spontaneous B7-H1 expression was also found to be enhanced upon relapse in some patients. MEK inhibitors, including UO126 and AZD6244, reduced B7-H1 expression and restored CTL-mediated lysis of blast cells. In AML, B7-H1 expression by blasts represents a possible immune escape mechanism. The inducibility of B7-H1 expression by IFN-γ or TLR ligands suggests that various stimuli, either produced during the immune response against leukemia cells or released by infectious microorganisms, could protect leukemic cells from T cells. The efficacy of MEK inhibitors against B7-H1-mediated inhibition of CTLs suggests a possible cancer immunotherapy strategy using targeted drugs.
KeywordsB7-H1 MEK TLR IFN-γ AML
Blast cells found in acute myeloid leukemia (AML) can grow in patients despite an autologous immune response. Numerous leukemia-associated antigens have been described, and cytotoxic T cells against these antigens have been detected . However, leukemia cells may escape autologous or allogenic immune responses through various mechanisms, such as indoleamine 2, 3 dioxygenase activity in blast cells, production of inhibitory cytokines, or active suppression of NK cells and dendritic cells [2, 3].
In AML, B7-H1 is a candidate molecule for inhibition of T cells. In a mouse model of AML, expression of B7-H1 allowed dormant tumor cells to escape from CTLs [4, 5, 6] [7, 8]. B7-H1 (also known as PD-L1 or CD274) is a B7 family member, and it is a ligand for PD-1 (programmed death-1, a member of the CD28 family) and B7.1 [9, 10, 11, 12]. B7-H1 is broadly distributed in various tissues and cell types and is often expressed following exposure to inflammatory cytokines, especially IFN-γ. B7-H1 can inhibit T-cell activation and CTL-mediated lysis. Various human carcinomas express B7-H1. We recently showed that malignant plasma cells from most multiple myeloma (MM) patients also express B7-H1, and expression is enhanced by IFN-γ- and toll-like receptor (TLR) stimulation via a MEK/ERK- and MyD88/TRAF6-dependent pathway . This induced expression can inhibit T-cell responses, indicating B7-H1 expression as a possible immune evasion mechanism in MM. Thus, the immune response against tumor cells via IFN-γ release or stimulation of TLR by pathogen-associated molecular patterns (PAMP) released by microorganisms may actually promote tumor growth by inducing B7-H1 expression, which then inhibits CTLs.
If blast cells in human AML are also able to spontaneously express B7-H1 spontaneously or upon stimulation, they would escape from the autologous immune response and from the graft-versus-leukemia effect after allogenic stem cell transplantation. To investigate whether B7-H1 may play such a role in human leukemia, we analyzed blast cells from a cohort of patients with AML. We studied spontaneous B7-H1 expression and expression after stimulation by TLR ligands or IFN-γ. Here, we show that B7-H1 expression in AML cells is mostly inducible via TLR stimulation and IFN-γ may appear upon relapse, thus protecting blast cells from CTLs. MEK inhibitors are able to inhibit B7-H1 expression and restore sensitivity of blast cells to CTL, thereby offering new opportunities to develop immunotherapy strategies via small, targeted molecules.
Materials and methods
Patients and cell lines
Total number of patients
Median age (range)
AML evolved from MDS
K562, U937, HL60, THP-1, KG1a, Jurkat, Raji, WEHI-3b (all purchased from ATCC) and DA1-3b cell lines were cultured in RPMI 1640 supplemented with 10% fetal calf serum, l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO2. Establishment of the DA1-3b BCR/ABL cell line has been described previously [8, 14].
Flow cytometry and Western blot analyses
Expression of B7 family molecules and TLR was evaluated by flow cytometry in blast cells gated with anti-CD45-PC5 (clone J33; Beckman Coulter, Miami, FL) monoclonal antibody (mAb). The following mAbs were used: anti-CD80-FITC (clone L307.4), anti-CD86-FITC (clone 2331), anti-B7-H1-PE (clone MIH1), anti-B7-DC-PE (clone MIH18), anti-B7-H4-PE (clone 7H3.1), anti-TLR2-FITC (clone TL2.1), anti-TLR 4-PE (HTA 125), anti-TLR9-PE (eBiosciences, San Diego, CA) and appropriate isotype controls with prior incubation with Fc-block reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). In some experiments, B7-H1 expression was also evaluated by Western blot using the anti-human B7-H1 antibody (clone 130002) or anti-mouse B7-H1 mAb (clone 179711) (R&D Systems, Minneapolis, MN).
Quantitative real-time PCR
Total RNA was extracted from cell lines and blast cells using conventional techniques. Levels of B7-H1 mRNA expression were measured using the forward primer (5′-AGG AGA TTA GAT CCT GAG GAA AAC C-3′), reverse primer (5′-GGA CTC ACT TGG TAA TTC TGG GA) and TaqMan probe (5′FAM-CTG GCA CAT CCT C–3′MGB), with the TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA). Real-time PCR was performed with an ABI PRISM 7700HT sequence detection system. A twofold serial dilution of cDNA from a lung carcinoma cell line (A549) was used to generate a standard curve. A negative control containing no RNA template was used in each run. 18S ribosomal RNA (PDAR 18S; Applied Biosystems) was amplified as an internal control. An equal quantity of cDNA prepared from 10 ng of RNA was loaded for PCR reaction. Results were measured via standard curves and expressed as ratios.
Cytokines, TLR ligands and signal transduction experiments
To explore signals that induced B7-H1 expression, the following cytokines and reagents were used: AG490 JAK2 inhibitor (25 μM), PD98059 MEK1 inhibitor (25 μM), SB203580 p38MAPK inhibitor (3 μM), LY294002 PI-3K inhibitor (25 μM), rapamycin mTOR inhibitor (10 nM), 1L6hydroxymethyl-chiro-inositol-2-R-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate AKT inhibitor (10 μM) (all from Calbiochem, San Diego, CA); SP600125 JNK inhibitor (25 μM; Biosource, Camarillo, CA), U0126 MEK1/2 inhibitor (20 μM; Cell Signaling Technology, Danvers, MA), AZD6244 MEK1/2 inhibitor (1 μM; kindly provided by Astra Zeneca), recombinant human IFN-γ (500 IU/ml; PeproTech, Rocky Hill, NJ), peptidoglycan TLR2 ligand (PGN, 2.5 μg/ml, from S. aureus), CpG oligonucleotide TLR9 ligand (type C, ODN M362, 5 μM) and its control CpG DNA, and TLR4 ligand Ultrapure lipopolysaccharide (500 ng/ml LPS, from E. coli strain O111:B4) (from InvivoGen/Cayla, Toulouse, France).
Generation of cytotoxic T cells
T cells from the peripheral blood of a healthy donor were isolated using a Pan T Cell Isolation Kit (Miltenyi Biotec) and cultured in RPMI 1640 (Life Technologies) supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 mg/ml streptomycin, 2 mM l-glutamine, 50 μM β-mercaptoethanol and 20 IU/ml interleukin 2 (PeproTech, Rocky Hill, USA). The culture medium was changed every 2 days, and irradiated AML cells (1/1 ratio) were added once a week . After 15 days, dead cells were removed and CD8a+ cells were purified using a CD8a+ T-cell Isolation Kit (Miltenyi Biotec). CTL activity was assessed with the Cytotox Non-Radioactive 96 kit (Promega, Madison, WI) using freshly thawed AML blasts as targets. To block B7-H1, target cells were pre-incubated with B7-H1 blocking antibodies (clone MIH1; eBiosciences) at 2 μg/ml 2 h before the CTL assay. The specificity of CTL-mediated lysis of AML cells was verified with HEK-293 cells as targets. MHC class I-restricted lysis was verified with an anti-HLA-ABC (clone W6/32; eBiosciences) or isotype control. The absence of NK cell-mediated cytotoxicity was verified with K562 cells as targets.
Statistical analyses were performed with the Sigma Stat 3.11 software (SPSS Sciences, Chicago, IL).
Expression of B7-H1 in leukemic cell lines
Expression of B7 family molecules in blast cells from AML
These results prompted us to study B7 family molecules in blasts from a large cohort of AML patients under basal conditions or after stimulation. On diagnosis, spontaneous expression of B7-H1 was detected in >30% of blast cells in 18% of patients. No correlations with age, FAB type, karyotype, leukocyte count or NPM, FLT3, MLL, CEBPA or WT1 mutations were found upon diagnosis. B7-DC, another PD-1 ligand (PD-1 is the B7-H1 receptor), was not detected. B7-H4, currently the only other known immunosuppressive B7 molecule [10, 12], was also absent (Fig. 1b). According to previous reports [16, 17, 18], B7.2 expression is high in most patients, and B7.1 expression resembles that of B7-H1.
As B7-H1 is usually inducible in normal cells, we investigated whether several stimuli known to play a role in the immune response could induce expression. TLR2, TLR4 and TLR9, which induce B7-H1 in MM , were expressed in 29, 27 and 36% of AML samples, respectively (Fig. 1c). No correlation between TLR expression and AML characteristics was observed. IFN-γ significantly enhanced B7-H1 expression (Fig. 1d). PGN and LPS, the TLR2 and TLR4 ligands, respectively, also induced B7-H1. However, ODN, the TLR9 ligand, had no effect on B7-H1 expression. Significant positive correlations were found between LPS-induced B7-H1 expression and TLR4 (r = 0.41, p = 0.01, Pearson’s correlation) and between PGN-induced expression and TLR2 (r = 0.51, p = 0.01), but not between ODN and TLR9 (r = 0.1, p = 0.5) (Fig. 1e). A significant correlation between PGN- and LPS-induced B7-H1 expression (r = 0.9, p = 0.001, Supplementary Figure S1a) and between IFNγ- and PGN- or LPS-induced B7-H1 expression (r = 0.8, p = 0.01, Supplementary Figure S1c,d) was also observed, indicating that in most samples B7-H1 expression was inducible through different stimuli. Correlation between TRL2 and TLR4 expression was significant, but less pronounced (r = 0.75, p = 0.01, Supplementary Figure S1b). These data suggest that blast cells that are refractory to stimulation with one B7-H1-inducing ligand are usually refractory to others. However, we cannot rule out that TLR ligands may induce B7-H1 in some patients via receptors other than TLRs.
Enhanced B7-H1 expression upon relapse
B7-H1-mediated resistance to CTLs is induced by TLR stimulation
The differential expression of B7-H1 between the time of diagnosis and relapse seen in some patients may also have a functional consequence. Blasts from a patient during AML relapse were more resistant to CTL-mediated killing than blasts isolated upon diagnosis, and this difference was reversible by incubation with an anti-B7-H1 antibody (Fig. 3b).
Blocking MEK but not PI3K/mTOR decreases B7-H1 expression
MEK inhibitors enhance CTL-mediated lysis of blast cells
AML blasts are known to be immunosuppressive, but the precise nature of the signals produced by these cells to inhibit T-cell function remains elusive [20, 21, 22]. B7 family molecules may be responsible for this immunosuppressive effect. B7.1 and B7.2, which both activate T cells via binding to CD28 and suppress T cells via CTLA-4, are expressed by leukemic cells [17, 18]. Furthermore, both ligands activate or inhibit T cells in AML, depending on their level of expression, which can be modified in vitro and in vivo by chemotherapy . Other more recently described members of the B7 family have not been investigated as thoroughly. B7-H2, which interacts with ICOS on activated T cells, is expressed on AML cells and induces CD4+ proliferation as well as IL4 and IL10 production . B7-H1 may suppress T-cell function through its receptor, PD-1, and possibly through direct interaction with B7.1 .
Marked expression of B7-H1 has been reported in several human and mouse tumors [13, 24, 25]. However, reports on AML are conflicting. Tamura et al. detected B7-H1 expression only in a minority of 69 de novo AML cases . Another study reported significant expression in 17 of 30 cases of leukemia of various types , although inhibition of T-cell proliferation mediated by B7-H1 was not observed. However, both reports demonstrated B7-H1 expression in several leukemic cell lines. Our data support the conclusion that B7-H1 expression upon AML at diagnosis is restricted; upon diagnosis, only 18% of patients expressed B7-H1 in most cells.
B7-H1 is an inducible molecule; IFN-γ, TNF-α and GM-CSF can enhance B7-H1 expression under physiological conditions [27, 28, 29]. Here, we confirmed previous reports showing that IFN-γ induces B7-H1 in leukemic cell lines [16, 26]. We previously demonstrated in the DA1-3b mouse leukemic model that B7-H1 dose dependently inhibits CTL . Here, we observed that IFN-γ induces and enhances B7-H1 expression in most AML samples. Thus, in addition to basal B7-H1 expression observed in some patients, we hypothesize that IFN-γ produced by T cells in the AML microenvironment may induce T-cell inhibition by inducing B7-H1 expression.
We also observed that TLR ligands induced B7-H1 expression in blast cells and increased resistance to CTLs. B7-H1 expression induced by PGN, a TLR2 ligand, correlated positively with B7-H1 expression induced by LPS, a TLR4 ligand, and by IFNγ. AML blast cells that are refractory to stimulation with one ligand are usually refractory to others. Activation of TLR2, TLR4 and TLR9 induces B7-H1 in MM, leading to resistance to CTLs . Activation of TLR4 on mouse tumor cells can also induce B7-H1 expression, leading to immunoescape. TLR4 activation in Langerhans cells has recently been shown to induce B7-H1 and favor tolerogenic properties in the oral cavity [30, 31]. Until now, TLR expression in hematological malignancies has been mostly reported in B-cell tumors [32, 33, 34, 35, 36, 37, 38]. Data regarding TLR expression and function in AML remain scarce. Numerous publications have reported the possibility that AML blast cells differentiate into leukemic dendritic cells (DC) using various combinations of agents, including LPS [1, 15]. These reports imply that AML blast cells may recognize LPS in vitro when combined with cytokines, such as GM-CSF and IL4. However, LPS can stimulate target cells through other receptors besides the TLRs, and LPS alone is not sufficient to mature blast cells into dendritic cells. In the THP-1 leukemic cell line, induction of apoptosis by LPS plus IFN-α has been reported, but primary AML samples seemed less sensitive to this combination . Altogether, these experiments provide little information about the role of TLRs in AML.
Recently, using gene expression profiling in a large cohort of cytogenetically normal AMLs, Marcucci et al. reported that TLR2, TLR4 and TLR8 expression was inversely correlated with the levels of microRNA-181 family, and this correlation was associated with a worse prognosis . Here, we observed that TLR2, TLR4 and TLR9 proteins were commonly expressed on AML blast cells. Stimulation of TLR2 and TLR4 induced B7-H1 expression and increased resistance of blast cells to CTLs. These data indicate that receptors of innate immunity could play a role in the development of AML.
A recent report studying gene expression profiles in mouse hematopoietic stem cells during aging showed that genes involved in inflammation and stress responses were up-regulated in aged mice [41, 42], and Tlr4 was among these genes. Thus, aging stem cells may be more sensitive to inflammatory response. An attractive hypothesis is that aging patients might be more prone to AML, in part because their stem cells are more sensitive to stimulation by TLR ligands, leading to immunoevasion of emerging leukemic clones; but this hypothesis remains untested.
In some patients, blasts studied upon relapse showed increased levels of B7-H1. This increase may have allowed blasts to resist CTL-mediated killing, perhaps during the complete remission period. However, given the low number of paired samples analyzed during diagnosis and relapse, further investigations using larger cohorts and on blast cells isolated from minimal residual disease are necessary to confirm this hypothesis.
We also confirmed that in leukemic cells, B7-H1 expression requires MEK, as previously shown in MM and dermal fibroblasts, and as recently confirmed in bladder carcinoma [13, 43, 44]. In addition to MAPK pathways, PI3K/AKT plays a critical role in B7-H1 expression in malignant gliomas, prostate and breast carcinoma, and dermal fibroblasts [19, 45]. We tested different inhibitors of PI3K and mTOR and observed the effect on B7-H1 expression. We found no inhibition in leukemic cell lines or in AML blast cells; rather, we found a slight enhancement. This finding suggests that regulation of B7-H1 expression varies widely between cell types; thus, drugs that target signal transduction pathways might have different immunological effects in different tumors. MEK inhibition not only inhibited B7-H1 expression, but also sensitized blast cells expressing B7-H1 after stimulation with LPS to CTL-mediated lysis. Current immunotherapy strategies primarily use monoclonal antibodies or cell therapy. Another approach would consist of using small, targeted molecules to block immunoescape mechanisms developed by cancer cells. MEK inhibitors could represent a means to this end. Several of these molecules are currently under clinical development, including AZD6244. These drugs have been developed to block tumor cell proliferation or induce cell death by blocking growth and survival signals, but our data show that they could also be used to kill tumor cells indirectly via suppression of B7-H1 expression and CTL-mediated killing. We can also imagine targeting B7-H1 via MEK after allogenic stem cell transplantation to facilitate a graft-versus-leukemia effect, but this strategy must be explored by further experiments.
In conclusion, we have identified B7-H1 as an immunoescape molecule in blast cells from patients with AML. Expression of B7-H1 increases when these cells are exposed to immune response, pathogens and sometimes upon relapse, and increased expression may be targeted via MEK inhibitors to facilitate CTL-mediated killing. These findings suggest that B7-H1 may be a possible target for immunotherapy via small molecules.
This work was supported by the Ligue Nationale Contre le Cancer (Comité du septentrion, and Equipe Labellisées program) and Institut National du Cancer «IMMUDORM» project. JL is the recipient of a grant from the Fondation pour la Recherche Médicale and from the Institut de Recherche sur le Cancer de Lille. VD received a grant from the Fondation de France. We thank AstraZeneca for providing AZD6244.
Conflict of interest
BQ received AZD6244 from AstraZeneca.
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