Dr Bruno Quesnel, Service des Maladies du Sang, CHU Lille, 1 Place de Verdun, 59037 Lille, France. E-mail: email@example.com.
Expression of B7 molecules provides co-stimulatory signals to T lymphocytes, which prevent the induction of anergy. It has been previously reported that B7.1 gene transfer in a murine leukaemia model induced a potent antileukaemic immunity and that relative expression of B7.1 and B7.2 in human acute myeloid leukaemia (AML) had prognostic significance. As ex vivo engineering of leukaemic cells for immunotherapy protocols would require prior irradiation of these cells before reinjection to the patient, we investigated in murine and leukaemic cell lines and in 20 ex vivo primary cultured acute myeloid leukaemic cells the effect of γ-irradiation on the expression of B7 molecules. We observed that γ-irradiation enhanced B7.1 molecule expression in murine leukaemic cell lines and in B7.2 molecules in human HL60 and K562 cell lines. γ-Irradiation induced B7.1 molecule expression in 90% AML samples but only 21% showed B7.2 molecule expression enhancement. B7.1 expression was increased both at the protein and RNA level in human AML cells but only at the protein level in the DA1-3b murine cell line. Oxidative stress increased B7.1 expression in the murine DA1-3b cell line but human cell lines and AML samples remained unaffected both by heat shock and oxidative stress, suggesting different pathways of B7.1 induction between mouse and human cells. Our data show that B7.1 expression can be induced by ex vivo irradiation of AML cells, indicating that these cells can express co-stimulatory molecules without gene transfer.
B7.1 and B7.2 molecules are cell-surface proteins involved in the delivery of a co-stimulatory signal to T lymphocytes ( Greenfield et al, 1998 ). Antigen presentation by the MHC class I complex in the absence of a B7 co-stimulatory signal leads to T-cell anergy. The level of expression of B7.1 also seems to determine the type of antigen recognition by the immune system ( Dorfman et al, 1997 ). Weak expression of B7.1 can reverse T-cell anergy against lymphoma cells, whereas its strong expression can induce an efficient cytotoxic T-cell (CTL) response. B7.1 and B7.2 proteins are poorly expressed in myeloid leukaemic cells and their expression has been correlated with the prognosis of acute myeloid leukaemia (AML) ( Hirano et al, 1996 ; Maeda et al, 1998 ).
We therefore analysed the effect of ex vivo irradiation on B7 molecules expression in primary cultured AML cells and leukaemic cell lines.
Materials and methods
AML blast cells and cell lines Fresh cells were obtained from peripheral blood of 20 patients with AML classified according to French–American–British (FAB) criteria (four M0, four M1, three M2, three M4, two M5, two AML post-myelodysplastic syndromes and two AML post-CML), and with > 90% circulating blast cells. AML cells were maintained in liquid culture in Iscove’s medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% FCS, 100 IU penicillin, 100 µg/ml streptomycin, 2 m m l-glutamine, 20 ng/ml human recombinant stem cell factor (SCF) (Amgen, Thousand Oaks, CA, USA), 8 ng/ml interleukin 3 (IL-3) (Novartis, Basel, Switzerland) and 20 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF) (Schering Plough, Levallois Perret, France) at 37°C with 5% CO2. K562, HL60 human cell line, WEHI-3B murine myelomonocytic leukaemic cell line obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and DA1–3b murine leukaemic cell line were maintained in RPMI-1640 supplemented with 10% FCS (Life Technologies), 100 IU penicillin, 100 µg/ml streptomycin and 2 m m l-glutamine. Cells were tested for mycoplasma contamination using a mycoplasma polymerase chain reaction enzyme-linked immunosorbent assay (PCR ELISA) kit (Boehringer Mannheim, Germany).
Irradiation and stress induction Primary cultured leukaemic cells were cultured for 24 h after isolation, then cells were γ-irradiated in a 137caesium irradiator at the minimal lethal dose of 25 Gy. Additionally, DA1-3b and WEHI-3B cell lines and four AML samples were also irradiated at 10 Gy. Cells were exposed to heat shock and oxidative stress as previously described ( Morel et al, 1998 ). Briefly, cells were exposed at 42°C for 2 h and then returned to 37°C for 18 h for flow cytometry analysis. For oxidative stress, H2O2 at 1 m m final concentration was added to cultured medium and cells were further incubated for 18 h before flow cytometric analysis.
Flow cytometry AML blast cells were washed twice with phosphate-buffered saline (PBS) and stained with mouse anti-human CD80–FITC, mouse anti-human CD86–FITC, mouse anti-human CD33–PE monoclonal antibodies (Pharmingen, San Diego, CA, USA) and control isotypic antibodies. WEHI-3B and DA1-3b cell lines were first incubated with CD16/CD32 Fc blocking reagent (Pharmingen) followed by staining with the hamster anti-mouse CD80–FITC monoclonal or isotypic control antibodies. Cells were washed twice again with PBS before being analysed using a Coulter XL 3C flow cytometer (Beckman-Coulter, Miami, USA). A total of 10 000 gated events were collected and analysed with Expo II software (Beckman-Coulter).
Northern blot analysis Total RNA was isolated from living AML and DA1-3b cells 48 h after irradiation, using lysis with Trizol reagent (Life technologies) followed by ethanol precipitation. Total RNA (25 µg) was loaded onto a RNA formaldehyde denaturing gel and run overnight followed by an overnight transfer to Hybond C membrane. Membranes were hybridized with murine B7.1, human B7.1 and control β-actin cDNA probes labelled with [α-32P]-dCTP (Amersham). The intensity of specific hybridization signals was evaluated using a phosphorimager (Molecular Dynamics).
B7 molecule expression in irradiated leukaemic cell lines and AML samples
In DA1-3b and WEHI-3B murine myeloid leukaemic cells lines, γ-irradiation at 25 Gy induced strong induction of B7.1 molecule expression, in agreement with previously published results in other murine tumour cell lines ( Fig 1) ( Morel et al, 1998 ; Seo et al, 1999 ). However, Northern blot analysis of the DA1-3b cell line showed that B7.1 RNA level was not modified by 10 or 25 Gy irradiation ( Fig 2). B7.2 molecules were not detected before or after irradiation. However, irradiation of the human K562 and HL60 cell lines did not modify B7.1 but increased B7.2 molecule expression in both cell lines ( Fig 1).
In 20 primary cultured AML samples, γ-irradiation at 25 Gy induced B7.1 molecule overexpression in 18 (90%) samples, a slight decrease in expression in one and no modification of expression in the remaining sample, and the difference between irradiated and non-irradiated samples was significant (P = 0·001, paired t-test; ( Fig 3). B7.2 molecule expression slightly increased in 8/14 (57%) samples but decreased in 6/14, and the difference between irradiated and non-irradiated samples was not significant (P = 0·967, paired t-test; Fig 3). Northern blot analysis in one AML sample showed a threefold increase of B7.1 mRNA expression after 25 Gy γ-irradiation ( Fig 4).
Analysis of B7.1 molecule expression at 0, 10 and 25 Gy irradiation in four AML samples and the DA1-3b and WEHI-3B cell lines showed only minor correlation between dose and expression, indicating that even sublethal doses of irradiation could induce B7.1 molecule expression ( Fig 5).
Time-course analysis of B7.1 molecule expression in three AML samples showed that ex vivo culture itself increased expression in non-irradiated AML cells, however B7.1 expression levels induced by cell culture always remained much lower than those induced by γ-irradiation ( Fig 6).
To investigate a possible link between the effect of irradiation and other types of cellular stress, we analysed B7.1 molecule expression in murine DA1-3b and human K562 and HL60 cell lines and in two primary cultured AML samples (one M1, one M2) after heat shock and oxidative stress. B7.1 molecule expression was not modified by heat shock and oxidative stress in any sample and oxidative stress slightly increased B7.1 expression only in the DA1-3b cell line ( Fig 7). Oxidative stress could not be analysed in the HL60 cell line, as addition of 1 m m H2O2 to the culture medium induced massive cell death.
The purpose of this study was to investigate whether co-stimulatory molecules are expressed in myeloid leukaemic cells after γ-irradiation. We observed that B7.1 co-stimulatory molecule expression could be induced in murine leukaemic cell lines by γ-irradiation, as previously reported in other murine cell lines ( Morel et al, 1998 ; Seo et al, 1999 ). These findings suggest that immunostimulatory experiments in murine tumour models, including leukaemia, could be affected by irradiation, leading to cautious interpretation of the results. However, human leukaemic cells were somewhat different. B7.1 molecule expression remained stable in human HL60 and K562 cell lines, but B7.2 molecules were overexpressed after γ-irradiation. B7.1 molecule expression increased after γ-irradiation in most primary cultured AML samples, but B7.2 molecule expression was not significantly modified. The mechanisms leading γ-irradiation to enhance B7.1 expression are unclear. B7.1 mRNA increased after irradiation in one AML sample but remained stable in the DA1-3b cell line. Gamma- and alpha-interferons, which induce a signal that interferes with heat shock protein, are strong inducers of B7.1 molecule expression in leukaemic cell lines and monocytes ( Chang et al, 1991 ; Stephanou & Latchman, 1999; Vokes et al, 1989 ). It has been also reported that oxidative stress induced B7.1 expression in murine tumour cell lines ( Morel et al, 1998 ). We observed an effect of oxidative stress on B7.1 expression only in the murine DA1-3b cell line, but human cell lines and AML samples remained unaffected and heat shock was completely ineffective in all experiments. This might suggest that the B7.1 expression pathway differs between mouse and human cells.
Many reports have demonstrated the induction of an efficient antileukaemic immunity in mice after B7.1 gene transfer, and induction of B7.1 in human B-lymphoma cells by CD154 has demonstrated an efficient ex vivo T-cell stimulation ( Matulonis et al, 1995 , 1996; Dunussi-Joannopoulos et al, 1996 , 1997a, 1998; Gajewski, 1996; Dilloo et al, 1997a ; Hirano et al, 1997a , 1997b; Hirst et al, 1997 ; Mutis et al, 1998 ; Zajac et al, 1998 ). Maeda et al (1998) reported that B7.2 expression in leukaemic cells had prognostic value for disease-free and overall survival in AML, and suggested that the relative expression of B7.1 and B7.2 might affect the Th1/Th2 balance. Our experiments showed that in vitro irradiation of cultured AML cells modified the B7.1/B7.2 relative expression. It may be also hypothesized that irradiation of leukaemic cells at 10–25 Gy would affect the immune response against leukaemic cells in the recipient. Such doses of irradiation were chosen in our experiments because they are the minimal doses required to prevent regrowth of the cells in the patient in gene therapy protocols using ex vivo gene transfer and in vivo reinfusion of tumour cells. Lower doses of irradiation would therefore not be compatible with clinical experiments using in vivo reinfusion of leukaemic cells. Other groups have reported very conflicting data about the effect of irradiation of tumour cells on the immune response in animal models. For instance, Dunussi-Joannopoulos et al (1997b) reported an efficient antileukaemic response after gene transfer of B7.1 in murine leukaemic cells, which was not affected by high-dose irradiation (at 32 Gy). In most animal models, irradiation of tumour cells enhanced immunogenicity even in untransfected cells. Doses of irradiation chosen in those experiments, even with leukaemic cells, were always high (sometimes > 100 Gy) to prevent spontaneous regrowth of the tumour in the recipient host. Thus, the doses of irradiation used in our experiments, which were lower, could have left the immunogenicity of human myeloid leukaemic cells unaltered. Further experiments are, however, needed to investigate to what extent ex vivo irradiation of AML cells can affect the immune recognition.
AML samples were also affected by culture conditions. To maintain maximum cell viability, we cultured AML cells in the presence of IL-3, SCF and GM-CSF, which also induced a stronger baseline B7.1 expression level than previously reported in fresh AML cells ( Hirano et al, 1996 ). Although B7.1 gene transfer has been reported as a good candidate procedure for the gene therapy protocol in myeloid leukaemias, our data indicate that at least cell culture and irradiation of leukaemic cells per se already induce B7.1 expression. It might suggest that the choice of B7.1 gene for a gene therapy protocol in AML would be not optimal, as substantial levels of expression of this gene could be obtained without gene transfer. However, we cannot rule out that massive transgene expression which can presently be obtained with recently described modified viral vectors targeting specific cell types could lead to a therapeutic effect because the level of B7.1 molecule expression has been reported to influence the type of immune response in lymphoma ( Dilloo et al, 1997b ; Dorfman et al, 1997 ; Gonzalez et al, 1999 ).
This study was supported by the Ligue Contre le Cancer (Comité du Nord and Comité du Pas de Calais), the Association de Recherche sur le Cancer, the Association Recherche Transfusion, the Fondation contre la Leucémie, and the GEFLUC.