Acute myeloid leukaemia triggering via CD40 induces ... - Nature

Leukemia (2000) 14, 123–128  2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu

Acute myeloid leukaemia triggering via CD40 induces leukocyte chemoattraction and cytotoxicity against allogenic or autologous leukemic targets RT Costello1,2, F Mallet2, H Chambost3, D Sainty1, C Arnoulet1, J-A Gastaut1 and D Olive2,4 1

De´partement d’He´matologie; 2Unite´ d’Immunologie des Tumeurs, Institut Paoli-Calmettes; 3He´matologie Pe´diatrique, Hoˆpital de la Timone, Universite´ de la Me´diterraneé; and 4Unite´ INSERM U119, Marseille, France

The CD40 antigen is a member of the tumor necrosis factor receptor superfamily which interacts with its ligand and regulates the immune response via a dialogue between T-lymphocytes and antigen-presenting or tumor cells. Tumor triggering via CD40 exerts direct effects on cancer cells, which have mainly been investigated in terminally differentiated hematological malignancies such as low-grade lymphoma. We focused our attention on minimally differentiated acute myeloid leukemia (AML-M0), an aggressive hematological malignancy in which severe prognosis suggests the requirement for innovative therapeutic strategies. Here we demonstrate, for the first time to our knowledge, a CD40-triggered IL-8, RANTES and IL12 secretion by leukemic cells. Supernatants from CD40-stimulated leukemia cells had chemoattractant effects on T-lymphocytes, natural killer cells and monocytes. Moreover, these supernatants, when complemented with low-dose IL-2, induced significant lymphokine-activated and natural killer cytotoxicity, leading to leukemia lysis both in allogenic HLA-matched and autologous settings. Stimulation of leukemia cells via CD40 could participate significantly to the anti-leukemia immune response by contributing to the development of an inflammatory response and to in situ cytotoxicity. Leukemia (2000) 14, 123–128. Keywords: acute myeloid leukemia; CD40; chemokines; IL-12; immunotherapy

Introduction The CD40/CD40L system regulates the immune response via interactions between T-lymphocytes and antigen-presenting cells.1–3 Recently, tumor cell triggering via CD40 has been proposed to be an efficient mean to restore deficient alloantigen APC functions, more particularly in low-grade malignancies, via surface up-regulation of adhesion/costimulatory molecules expression.4,5 Nonetheless, due to the variable effects of CD40 stimulation on tumor biology and its pleiotropic putative targets,6 the precise role of CD40 in AML immunotherapy has still to be defined. Minimally differentiated AML-M0 have very poor complete remission (CR) rates and CR duration, suggesting this subtype is a good candidate for non-conventional approaches.7–11 We have previously demonstrated that CD40 stimulation of AML-M0 induces an up-regulation of adhesion/costimulatory molecules and improves their immune recognition,12 although less efficiently than in low-grade lymphoid malignancies4 or differentiated AMLs.13 Here we further extend our observations on these poorly differentiated AMLs by focusing our attention on CD40-triggered cytokine/chemokine secretion by leukemic cells. We first studied the CD40-induced secretion of two chemokines with potent chemoattractant propertides, ie IL-8 and RANTES. We also assessed the CD40-driven IL-12 Correspondence: D Olive, Immunologie des Tumeurs, Institut PaoliCalmettes, Universite´ de la Me´diterraneé, 232 bd Sainte Marguerite, 13009 Marseille, France; Fax: 33 491223610 Received 11 August 1999; accepted 15 September 1999

secretion, since this cytokine plays a central role in the generation of cytotoxic T-lymphocytes. Then, we tested the putative effects of CD40-driven blast cytokine secretion by evaluating the effects of CD40-stimulated leukemia supernatants on immune effector chemotaxis and cytotoxicity against blast cells.

Materials and methods

Patient samples Peripheral blood (PB) samples were part of diagnostic and clinical follow-up procedures, and were obtained after informed consent before specific anti-leukemia therapy. Diagnosis was established by cytological criteria based on the French– American–British (FAB) classification14 and specific recommendations.8,15,16 Revision of slides was independently performed by two morphologists (DS and CA).

Cell separation and blast purification Leukemic cells from peripheral blood and peripheral blood mononucleated cells (PBMCs) from normal donors were isolated on Ficoll–Hypaque gradients and viably frozen in liquid nitrogen until use. The median percentage of blast cells after Ficoll–Hypaque gradients was 90% (range 80–99). Highly purified leukemic cells were obtained by negative selection using magnetic beads (Beckman Coulter, Marseille, France) coated with anti-CD3, anti CD19, anti-CD56 and, depending on the phenotype at diagnosis, anti-CD14 mAbs (Beckman Coulter). The purity of the preparation (⬎98%) was assessed by flow cytometry analysis and Giemsa stain of cytocentrifuge preparations. For cytotoxicity assays, T-lymphocytes were isolated from healthy blood donors or in an AML patient in CR phase, and purified by sheep erythrocyte rosetting;17 this preparation contains both T cell and a subfraction (⬍5%) of natural killer (NK) cells.

Flow cytometry The following antibodies were used in flow cytometric studies: anti-CD3, anti-CD14 and anti-CD56 (Beckman Coulter). For each mAb, an isotype-matched immunoglobulin was used as control in all experiments. Cells were processed following standard procedures and analysis was performed on a FacScan flow cytometer (Becton Dickinson, Immunocytometry Systems, San Jose, CA, USA) with a gate corresponding to viable leukemic cells.

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Cell lines and co-incubation conditions

Statistical analysis

The L cell stable transfectants for human CD40L and CD32 expression were kindly provided by Dr J Banchereau (Schering-Plough, Lyon, France). Leukemic blasts (5 × 106 per well) were incubated with transfected cells at a 10/1 ratio. The ␥-IFN (kind gift of Roussel-Uclaf, Paris, France) was used at 20 IU/ml. The IL-2 (Roussel-Uclaf) was used at 20 IU/ml as ‘cytotoxicity costimulus’ added to AML supernatants (SNs).

Statistical analysis was performed using the SPSS software.19 The Kolmogorov–Smirnov test was used to determine if the data fitted a normal distribution. As this test rejected the assumption of normality for all variables, comparisons were made using non-parametric tests. For comparison of two samples, we used the Wilcoxon matched-pairs signed-ranks test. Results

Cytokine production and assay To assess blast secretion, highly purified leukemic cells were pre-incubated for 96 h with medium, ␥-IFN, CD32 or CD40Ltransfected cells as previously described. Cytokines were tested using immunoenzymatic assays with sensitivity of 5 pg/ml for IL-8 (R&D Systems Europe, Abingdon, UK), 5 pg/ml for IL-12 (Diaclone, Besançon, France) and 5 pg/ml for the Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) chemokine (Quantikine; R&D Systems Europe).

Chemotaxis assay Cell migration was assessed using Transwell chambers with 3 ␮m pores (Corning Costar, Cambridge, MA, USA) as previously described.18 Briefly, 106 cells/well were placed in the upper compartment in RPMI supplemented with 0.5% FCS in a final 100 ␮l volume. Then, 600 ␮l of the culture supernatant (SN) to be tested was placed in the lower compartment, at 1/5 dilution. After incubation at 37°C for 4 h, cells migrated to the lower chambers were counted and phenotype analyzed by flow cytometry. All experiments were done in duplicate and the results were compared to the migration of the same cells induced with culture medium alone in the lower chambers. Additional control of spontaneous motility after a 18 h incubation was also performed (data not shown).

Cytotoxicity assays T-lymphocytes from peripheral blood were incubated with 1/5 dilution of AML cell SNs for 18 h (cytotoxicity against DAUDI or K562 cell lines and against allogenic human leukocyte antigen (HLA)-matched AML blasts) or 5 days (cytotoxicity against autologous AML blasts), with or without exogenous IL-2 at 20 IU/ml. The targets cells were labelled with 100 ␮Ci of 51Cr (NEN, Boston, MA, USA) for 2 h at 37°C, and then incubated (2 × 103/well) with the effector cells at various ratios. All experiments were done in triplicate, using 96-well V-bottom plates and RPMI with 10% FCS as incubation medium. Plates were centrifuged at 500 g for 3 min and incubated for 6 h. Then, 100 ␮l of SN was collected and radioactivity release was performed with a ␥ counter (Topcount; Packard, Rungis, France). Specific lysis was determined for each triplicate as: Specific lysis =

冉

冊

Experimental 51Cr release − spontaneous 51Cr release × 100 Maximum 51Cr release − spontaneous 51Cr release

Maximal release was determined by the addition of 2% detergent NP40 (Sigma, St Louis, MO, USA) to target cells. Leukemia

CD40 stimulation induces chemokine and IL-12 secretion by leukemia cells For the whole study, we selected seven AML-M0 for the expression of CD40 (data not shown). Since we have previously shown that ␥-IFN contributes to the modulation of the immunologic properties of AML blasts,12,13 we compared blast cytokine secretion regulation via CD40 stimulation to ␥-IFN. Incubation with CD32 positive fibroblasts or medium alone were the respective controls of CD40L and ␥-IFN stimulations. We chose to focus on IL-8 and RANTES since they have potent chemoattractant properties and are prototypic chemokines representative of the two main subfamily, ie respectively CXC and CC.20 The dosage of IL-12 was also chosen as a prototypic cytokine responsible for cytotoxicity generation21,22 and prevention of anergy,23 after we had concluded that IL-2 was absent (data not shown). In order to eliminate the possibility of cell contamination, virtually pure (⬎98%) blast cell preparations were used in our study. Moreover, we only used AML samples from peripheral blood and not from bone marrow aspirates which can have more important contamination by stromal cells. Finally, as control, we failed to detect the presence of the tested cytokines (IL-8, RANTES and IL-12) in SNs from our transfected fibroblasts (data not shown). As seen in Figure 1a, leukemic cell incubation with CD40Ltransfected fibroblasts induced significantly higher (P ⬍ 0.05) IL-8 secretion (2723 pg/ml ± 259) than medium alone (820 pg/ml ± 847), ␥-IFN (475 pg/ml ± 489) or CD32expressing fibroblasts (522 pg/ml ± 381). The secretion of RANTES (Figure 1b) was also higher (P ⬍ 0.05) following leukemic cell stimulation via CD40 (1500 pg/ml ± 742) than after incubation with medium alone (835 pg/ml ± 366), ␥-IFN (485 pg/ml ± 212) or CD32-expressing fibroblasts (456 pg/ ml ± 202). As seen in Figure 1c, leukemic cell incubation with CD40L-transfected fibroblasts induced significantly higher (P ⬍ 0.05) IL-12 secretion (792 pg/ml ± 192) than medium alone (165 pg/ml ± 121), ␥-IFN (188 pg/ml ± 156) or CD32expressing fibroblasts (194 pg/ml ± 185).

Supernatants from CD40-stimulated AML cells have chemoattractant properties The evidence for CD40-triggered chemokine secretion by blast cells prompted us to test the effects of AML SNs on the migration of normal PBMC through cell-permeable membranes. As seen in Figure 2a, SNs from CD40-stimulated leukemic cells induced greater (P ⬍ 0.05) chemoattraction (6.3% ± 1.6 migrating cells) than SNs from unstimulated leukemic cell (3.1% ± 1.6) and ␥-IFN (3.5% ± 1.2) or CD32expressing fibroblast (2.8% ± 1.4) stimulated leukemic cells. We then analyzed by flow cytometry the subpopulations of


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Figure 1 Chemokine secretion by purified leukemic cells. The secretion by purified AML blasts (5 × 105/ml) of IL-8 (a), RANTES (b) and IL-12 (c) was dosed after a 4-day incubation with medium, ␥IFN, CD32 L cells or CD40L transfected cells. The data are expressed as the mean concentration (in pg/ml) ± standard deviation from seven representative experiments. We failed to detect any secretion of the tested cytokines in SNs from CD32 or CD40L-transfected cells (data not shown).

leukocytes chemoattracted by SNs from CD40-stimulated leukemic cells. The monocytes, corresponding to CD14-positive cells (Figure 2b, upper panel) represented 14% ± 2 of migrated cells. The CD56-positive cells (Figure 2b, middle panel) represented 15% ± 3 of migrated cells. The whole T-lymphocyte population (Figure 2b, lower panel), as evaluated by CD3 expression, represented 65% ± 4 of migrated cells. These subpopulations were further subdivided in true natural killer (NK) cells which are CD3-negative (Figure 2c, upper left quadrant, 9.5% ± 2) or CD56-positive T-lymphocytes (Figure 2c, upper right quadrant, 4.5% ± 0.5). The respective percentage of cell T-lymphocytes, NK cells and monocytes in the migrated cells did not significantly differ from the original PBMC subpopulation (data not shown).

Figure 2 Leukocyte chemoattraction and phenotypic analysis of migrated cells. To evaluate the chemoattractant properties of SNs from AMLs (a), a 1/5 dilution of these SNs was added to the lower wells of Transwell cell culture chambers, and PBMCs responding cells (1 × 106/ml) were placed in the upper wells. After incubation at 37°C for 4 h, the cells that migrated into the bottom wells were counted and the percentage of migrating cells was calculated as follows; % = (number of cells in the lower chamber/initial input) × 100. Results are expressed after subtraction of the background corresponding to ‘spontaneous’ migration of the PBMCs through the membrane. Data represent mean from seven experiments performed in duplicates. We then analyzed by flow cytometry the phenotype of the cells chemoattracted by SNs from CD40-stimulated AML cells. Panel b corresponds to the histogram analysis of CD14, CD56 and CD3 markers (filled area), with the corresponding isotype control (unfilled area). Panel c represents a double staining with CD56 marker (phycoerythrin, PE) and CD3 (fluorescein iosthiocyanate, FITC). These data are representative of four experiments performed with PBMCs from three different normal donors.

Supernatants from CD40-stimulated AML cells induce leukocyte cytotoxicity against leukemia cells As seen in Figure 3, we tested the effects of SNs obtained after leukemic cell stimulation by CD40L-transfected fibroblasts, in comparison with control SNs obtained from blast cultured with CD32-expressing fibroblasts, regarding cytotoxicity induction by responding lymphocytes. These SNs were tested either alone or complemented with low concentration IL-2 (20 IU/ml) in order to sensitize the test. At this concentration, IL-2 did not induce detectable cytotoxicity (data not shown). T-lymphocyte preparations (containing a majority of T cell and a small fraction of NK cells (⬍5%)) were incubated with 1/5 dilution of leukemia SNs and tested at different effector/cell ratios. In order to evaluate the induction of lymphokine-activated killer (LAK) activity, we first tested the SN-induced (18-h incubation) cytoxicity of lymphocytes against the DAUDI cell line. As seen in Figure 3a, SNs from CD40L-stimulated blasts either alone (12% ± 2 cell lysis at 30/1 ratio) or complemented with low-dose IL-2 (25% ± 3 cell lysis at 30/1 ratio) signifiLeukemia


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Figure 3 Cytotoxicity of T-lymphocytes incubated with AML supernatants. Responding T-lymphocytes were obtained from normal donors (panels a and b, cytotoxicity against DAUDI and K562 cells), from HLA-matched intra-familial siblings (panel c, cytotoxicity against HLAmatched blasts) or from the patient himself after completion of complete remission (panel d, cytotoxicity against autologous AML blasts). Tlymphocytes (including a small fraction of NK cells (⬍5%)) were incubated for 18 h (cytotoxicity against DAUDI, K562 and HLA-matched AML blasts) or 5 days (cytotoxicity against autologous AML blasts) with SNs of AML cells incubated with control CD32 fibroblasts of CD40L-transfected fibroblasts for 96 h. These SNs (1/5 dilution) were tested either alone or complemented with low concentration IL-2 (20 IU/ml) in order to sensitize the test; at these concentrations, IL-2 alone did not induce detectable cytotoxicity (data not shown). T-lymphocytes were tested at different effector/target (E/T) ratio (30/1 to 2/1) for their LAK cytotoxicity against the DAUDI cell line (a), for NK cytotoxicity against the K562 cell line (b), for cytotoxicity against allogenic but HLA-matched AML blasts (c) and for cytotoxicity against autologous AML blasts (d). SNs from three different AMLs were tested in triplicates. The figure corresponds to one representative experiment. Squares correspond to CD40L-stimulated blasts SN alone (䊐) or complemented with low-dose IL-2 (䊏). Circles correspond to CD32 fibroblast-stimulated leukemic cells SNs either alone (䊊) or complemented with low-dose IL-2 (䊉).

cantly induced greater LAK activity (P ⬍ 0.05) than SNs from CD32-expressing fibroblast-stimulated leukemic cells either without (0% ± 2 cell lysis at 30/1 ratio) or with IL-2 (1% ± 3 cell lysis at 30/1 ratio). In order to evaluate the induction of NK activity, we tested the SN-induced (18-h incubation) lymphocyte cytoxicity against the K562 cell line. As seen in Figure 3b, SNs from CD40L-stimulated blasts either alone (12% ± 2 cell lysis at 30/1 ratio) or complemented with low-dose IL-2 (28% ± 4 cell lysis at 30/1 ratio) significantly induced greater NK activity (P ⬍ 0.05) than SNs from CD32 fibroblast-stimulated leukemic cells either without (0% ± 1 cell cytotoxicity at 30/1 ratio) or with IL-2 (6% ± 1 cell cytotoxicity at 30/1 ratio). We then tested the SN-induced (18-h incubation) cytotoxicity against leukemia cells in an allogenic but HLA-matched setting using PBMCs from an intra-familial HLA-compatible sibling. As seen in Figure 3c, SNs from CD40L-stimulated blasts alone failed to induce significant cytotoxicity (2% ± 1 cell lysis at 30/1 ratio) in contrast with the potent effect observed after low-dose IL-2 complementation (28% ± 4 cell lysis at 30/1 ratio). CD32-expressing fibroblast-stimulated AML SNs did not induce significant cytotoxicity (2% ± 1 lysis at 30/1 ratio), while complementation with IL-2 induced a measurable cytotoxicity (7% ± 3 lysis at 30/1 ratio) which was significantly (P ⬍ 0.01) lower than the cytotoxicity induced by CD40-triggered AML SNs. We finally tested the SN-induced (5-day incubation) cytotoxicity against leukemia cells in an autologous setting using Leukemia

lymphocytes from a patient in remission. As seen in Figure 3d, SNs from CD40L-stimulated blast alone failed to induce significant cytotoxicity (0% ± 1 cell lysis at 30/1 ratio) in contrast with the effect observed after low-dose IL-2 complementation (12% ± 3 cell lysis at 30/1 ratio). CD32-expressing fibroblaststimulated AML SNs did not induce significant cytotoxicity (0% ± 2 at 30/1 ratio), even with IL-2 complementation (0% ± 2 at 30/1 ratio). Discussion Although most human tumors are antigenic, the normal immune system is often unable to efficiently inhibit their outgrowth.24–26 In accordance with the ‘danger model’, a local inflammation,27 which involves in situ recruitment and activation of leukocytes, is considered to be required to induce effective antitumor immunity.28 The failure of tumor to initiate this inflammatory response can probably be considered as a mechanism of immune escape. We wanted to test, in the setting of a poor-prognosis leukemic proliferation, if tumor triggering via CD40 could contribute, at least in vitro, to attract and activate immune effector cells. Our study demonstrates, for the first time to our knowledge, that CD40 triggering of minimally differentiated leukemia cells induces a chemoattractant activity in blast SNs. That corresponds to the completion of the first step of the inflammatory response. To gain more insight into the mechanisms of this


chemotaxis, we dosed the secretion of two prototypic chemokines, ie IL-8 and RANTES.29,30 The IL-8 is particularly interesting in AMLs since (1) it is spontaneously released by myeloid blasts showing monocytic differentiation;31,32 (2) endothelial IL-8 exerts apoptotic activity on some leukemic cell line,33,34 although this notion is debated;35 (3) in a mouse model, local lymphocyte infiltration but not systemic activation of Tlymphocytes is associated with the IL-8-driven antitumor response.36 RANTES is also a chemokine of interest in the antitumor response since it attracts various leukocyte subtypes (Tlymphocytes, monocytes, dendritic cells or NK cells) and contributes to T-lymphocyte proliferation.37 Since we observed a very significant secretion of IL-8 and RANTES following CD40 stimulation of leukemia cells, both chemokines may participate in T-lymphocyte, monocytes and NK cell recruitment, although we cannot exclude the involvement of other chemokines. Tumor leukocyte infiltration is considered to reflect the cellular immune response against cancer and is associated with favorable prognosis in some malignancies.38–40 Nonetheless immune effectors can also contribute to tumor growth24 more particularly in hematological malignancies via cytokine release.41 This putative protumor effect of immune effector cells could be increased in the absence of efficiently activated cytotoxic T-lymphocytes, a phenomenon which can be induced by the tumor itself via TGF-␤ or IL-10 secretion, for example.26 In our study we demonstrate that SNs from CD40triggered minimally differentiated blasts induce the generation of NK and LAK cytotoxic activity by responding lymphocytes. This cytotoxicity is further enhanced by addition of low-dose IL-2, leading to AML blast cell lysis both in allogenic HLAmatched and autologous settings. We failed to detect IL-2 in SNs from CD40-triggered AMLs (data not shown), while a significant IL-12 secretion was detected. IL-12 is a cytokine that induces cytotoxicity, proliferation and cytokine secretion by NK cells and T-lymphocytes,21,22 directs T cell differentiation towards a Th1 phenotype,42,43 prevents the induction of anergy23 and participate in dendritic cell physiology.44 We thus speculate that CD40-triggered IL-12 secretion, in addition to its participation in the cytotoxicity we observed, may also have other putatively interesting effects regarding the induction of an anti-tumor immune response. In addition, IL-12 secretion could synergize with CD40-triggered up-regulation of adhesion/costimulation molecule observed in AML-M012 to induce anti-tumor immunity mediated by T-lymphocytes or NK cells.22,43,45–47 Nonetheless, our in vitro data suggest the requirement for additional T cell stimulation or exogenous IL2 supplementation in addition to blast stimulation via CD40 in order to elicit an efficient anti-tumor response. Altogether, our data suggest that immature leukemia cell triggering via CD40, putatively in association with IL-2 infusion, could induce in close proximity to the tumor some sort of inflammatory milieu of therapeutic interest by providing the ‘danger signal’ necessary to initiate an anti-tumor response via the increased secretion by leukemic cells of IL8, RANTES, IL-12 and other pro-inflammatory cytokines (TNF␣, GM-CSF and ␥-IFN, data not shown). The generation of an inflammatory response could contribute to a new mechanism of in vivo immune-stimulation of CD40 triggering48–50 in addition to other mechanisms such as, more particularly, direct inhibition of tumor growth.51 This hypothesis will have to be further tested using an in vivo leukemia model in mice.

Acknowledgements

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This research was supported by Groupement Entreprise Français Lutte Cancer, Association pour la Recherche contre le Cancer, Ligues contre le Cancer des Bouches-du-Rhoˆne et du Var, Fe´de´ration Nationale des Centres de Lutte Contre le Cancer, Fondation Contre la Leuce´mie and Ligue Nationale contre le Cancer. We would specially thank MA Gougerot-Pocidalo for expert advice. We also thank C Mawas, Y Collette, B Gaugler and C Cerdan for helpful comments. We thank R Bouabdallah, J Camerlo, D Coso, AM Stoppa and N Vey for providing the samples.

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