Cancer Immunol Immunother (2013) 62:1797–1809 DOI 10.1007/s00262-013-1484-9
Original Article
CD137 stimulation and p38 MAPK inhibition improve reactivity in an in vitro model of glioblastoma immunotherapy Caspar Kühnöl · Monique Herbarth · Jürgen Föll · Martin S. Staege · Christof Kramm
Received: 13 February 2013 / Accepted: 3 October 2013 / Published online: 16 October 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract Dendritic cell vaccination has become an interesting option for cancer immunotherapy. Tumorlysate-pulsed dendritic cells (DC) can prime naïve T cells and induce the regression of established tumors including gliomas as shown in various animal models. Despite hopeful results even in clinical studies, the outcome for many patients is still unsatisfying. In the present study, we tested the combination of tumor-lysate-pulsed dendritic cells (TPDC) with a monoclonal antibody against CD137, a monoclonal antibody against CD25 (daclizumab) and a specific p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580) for improving immunostimulation in an in vitro model of immunotherapy for human gliomas. We observed a higher secretion of interferon gamma by TPDC-primed peripheral blood mononuclear cells (PBMC) that were incubated with an antibody against CD137 or the p38 MAPK inhibitor. In addition, we observed higher specific lysis of tumor cells after incubation of PBMC with the p38 MAPK inhibitor or the anti-CD137 antibody.
Electronic supplementary material The online version of this article (doi:10.1007/s00262-013-1484-9) contains supplementary material, which is available to authorized users. C. Kühnöl · M. Herbarth · M. S. Staege (*) · C. Kramm Department of Pediatrics, Martin-Luther-University HalleWittenberg, Ernst‑Grube‑Str. 40, 06097 Halle, Germany e-mail:
[email protected]‑halle.de J. Föll Department of Pediatrics and Juvenile Medicine, University Hospital Regensburg, 93053 Regensburg, Germany C. Kramm Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, University Medical Center Göttingen, 37075 Göttingen, Germany
In contrast, incubation of TPDC-primed PBMC with the anti-CD25 antibody did enhance neither interferon gamma secretion nor cellular cytotoxicity. Cell depletion experiments demonstrated that the immune reaction induced by TPDC is strongly dependent on CD4-positive and CD8positive cells. Incubation of DC during maturation and antigen loading with the anti-CD137 antibody did not enhance cytotoxicity and interferon gamma secretion in comparison with application of the anti-CD137 antibody during priming. In conclusion, our data suggest that p38 MAPK inhibition and anti-CD137 antibodies can enhance the immune response against glioblastoma cells. Keywords Glioblastoma · Dendritic cells · CD137 · P38MAPK inhibitors · CD25
Introduction Dendritic cell vaccination (DCV) represents an interesting option for treatment for high-grade gliomas and has already been used in various clinical trials [1]. Although promising results with prolonged event-free and overall survival after DCV have been reported for certain patient subgroups with glioblastoma multiforme (GBM), the general prognosis for patients with GBM still remains very poor [2]. Further improvements in DCV strategies are obviously needed. Thus, we investigated in the present study whether the combination of DCV with immunomodulatory signals can improve the overall immune response against glioma cells. To this end, we combined DCV with an anti-CD137 antibody, a p38 mitogen-activated protein kinase (MAPK) inhibitor and an anti-CD25 antibody (Fig. 1). The co-stimulatory receptor CD137 (a type I transmembrane protein) is a member of the tumor necrosis factor
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Fig. 1 Overview of the used immunomodulatory elements. Tumorlysate-pulsed dendritic cells were used for priming of PBMC with the aim of stimulating cytotoxic T cells (CTL) that can kill tumor cells. The simultaneous activation of helper T cells can increase the activity of these cells, whereas regulatory T cells might inhibit the activity of effector cells. Antibodies directed at CD137 can increase survival of effector T cells. Antibodies against CD25 can inhibit CD25-positive regulatory cells. The p38 MAPK inhibitor can inhibit regulatory T cells. In addition, this agent might positively affect the TH1 polarization activity of dendritic cells and inhibit apoptosis of CTL
receptor superfamily (TNFRSF), a group of molecules that play key roles in the control of survival of immune cells during immune reactions [3]. CD137, also known as ILA (induced by lymphocyte activation), was first detected on activated human and murine T cells [4]. In addition to activated T cells, also dendritic cells, NK cells and mast cells express CD137 [5–8]. The natural ligand of CD137 is CD137L (4-1BBL), a type II transmembrane protein [9, 10]. CD137L was detected on activated antigen-presenting cells (APC), e.g., on dendritic cells [10–12]. Stimulation of CD137 with its natural ligand or a monoclonal antibody (mAB) leads to an activation of nuclear factor kappa B (NFκB) and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pathways and finally to the regulation of gene transcription [13–15]. Stimulation of CD137 on T cells leads to cell proliferation and cytokine production [16–18]. Another effect of stimulation of CD137 on activated T cells is the protection from activation-induced cell death (AICD) [19]. The p38 MAPK was first discovered in mice [20], followed by the identification of the human p38 MAPK [21]. Several p38 MAPK isoforms were described (reviewed in [22]); the one that is characterized best is isoform p38α, encoded by the MAPK14 gene. For CD4-positive cells, it is known that full activation of p38 MAPK needs ligation of the T-cell receptor (TCR) and co-stimulatory molecules such as CD137, CD28, or inducible T-cell co-stimulator (ICOS) (reviewed in [23]). p38 MAPK is necessary for production of interferon gamma and differentiation of CD4positive cells into effector TH1 cells [24]. In CD8-positive cells, p38 MAPK activation can induce apoptosis [25]. It
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was shown that p38 MAPK is necessary for maturation of dendritic cells [26–29]. Furthermore, p38 MAPK has a negative effect on expression of MHC class II proteins [30]. Production of interleukin (IL) 10 after incubation of dendritic cells with Toll-like receptor (TLR) agonists is also dependent on p38 MAPK. Inhibition of p38 MAPK increased IL12 production by dendritic cells and amplified the therapeutic effects of a TLR-ligand-activated DC immunotherapy against tumors by suppression of regulatory T cells (Treg) [31]. It was shown that p38 MAPK inhibition can also increase the ratio of IL12–IL10 in human DC. Inhibition of p38 MAPK reduces expression of IL10 by human DC. Increased as well as decreased expression of IL12 has been described and might depend on the activation status of the human DC [32, 33]. CD25 (alpha chain) belongs together with CD122 (beta chain) and CD132 (gamma chain) to the trimeric highaffinity IL2 receptor [34]. It is expressed on activated T cells, on forkhead box P3 (FOXP3)-positive CD4+CD25+ Treg, and at lower levels on pre-B cells, thymocytes and NK cells [35]. CD25 is up-regulated after ligation of the TCR on CD8-positive T cells and on activated CD4-positive T cells. Binding of IL2–CD25 leads to proliferation of CD4-positive and CD8-positive cells (reviewed in [36]). Neutralization of IL2 leads to autoimmune diseases with evidences of a dysfunction or deficiency of Treg [35, 37]. Other experiments showed a long-term immunity against glioma in mice after blocking CD25 by daclizumab, a mAB against human CD25 [38].
Materials and methods Cell culture The HLA-A0201-positive human glioma cell lines U87MG [39] and T98G [40] and the human fibrosarcoma cell line HT1080 [41] were grown at 37 °C in a humidified atmosphere with 5 % CO2 in Dulbecco’s modified Eagle medium (DMEM, PAA, Coelbe, Germany) with 10 % fetal calf serum (FCS, Biochrom, Berlin, Germany), 100 U/mL penicillin, and 100 μg/mL streptomycin (PAA) in cell culture flasks. Twice a week, cells were washed with phosphatebuffered saline (PBS) and detached with trypsin/EDTA solution (0.05 % trypsin, 0.02 % EDTA in PBS, PAA). After centrifugation at 350×g for 7 min, cells were resuspended in DMEM at a ratio of 1:10 in new cell culture flasks. Generation and maturation of dendritic cells Human dendritic cells were generated from monocytes from peripheral blood mononuclear cells (PBMC) of voluntary healthy HLA-A2-positive donors. PBMC and
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CD14-positive cells were isolated as previously described [42]. Isolated monocytes were then cultured with IL4, granulocyte–macrophage colony-stimulating factor (GM-CSF), IL1β, IL6, tumor necrosis factor alpha (TNFα), and prostaglandin E2 (PGE2) for 7 days in a humidified atmosphere with 5 % CO2 at 37 °C. On day 6 of culture, tumor lysate from human glioma cell line U87MG (50–100 μg/mL) was added for 2 days. Tumor-lysate-loaded dendritic cells (TPDC) were harvested, washed with PBS, and suspended in DMEM with 1 % human AB serum (Lonza, Basel, Switzerland), 100 U/mL penicillin, and 100 μg/mL streptomycin. Purity of DC was assessed by flow cytometry. A representative result is shown in Supplementary Figure 1. Stimulation of PBMC HLA-A2-positive PBMC (1 × 106/mL) from voluntary healthy donors were stimulated for 5 or 7 days in 6-well plates with TPDC (2.5 × 105/mL) of the same donors in 5 mL DMEM with 1 % human AB serum, 100 U/mL penicillin, and 100 μg/mL streptomycin). Depletion of singlecell populations was performed as described before [42]. When monoclonal antibodies (mAB) against CD137 (clone 26G6, a kind gift from R. Mittler, Atlanta, USA) were used, 6-well plates were coated with mAB solution (10 μg/mL in PBS) overnight at 4 °C before starting the co-incubation of PBMC and TPDC. This antibody increases the proliferation of T cells that have been stimulated with suboptimal concentrations of αCD3 (data not shown), suggesting that it has an intrinsic stimulating activity. The p38 MAPK inhibitor SB203580 (Biaffin, Kassel, Germany) was dissolved in dimethyl sulfoxide (DMSO) and was used at a concentration of 1 μM. SB203580 was added either at the beginning of the co-incubation of TPDC with PBMC or together with the tumor lysate during pulsing of dendritic cells. As control we added dimethyl sulfoxide (DMSO, Sigma, Taufkirchen, Germany). Antibodies against CD25 (Daclizumab, Roche, Mannheim, Germany) were used at 5 μg/mL and added at the beginning of the co-incubation of PBMC and TPDC. For analyses cells were harvested and centrifuged at 300×g. Supernatants were stored at −80 °C for further usage; cells were washed with PBS and analyzed.
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30 min at 4 °C. FACS analysis was performed using a FACSscan or FACSCalibur (Becton–Dickinson). Representative plots are presented in Supplementary Figures 1–3. ELISPOT analysis Interferon gamma ELISPOT was used to quantify tumorcell-specific interferon-gamma-releasing cells. On days 5 and 7 of the PBMC/TPDC co-culture experiments, cells were harvested, washed, and resuspended in cell culture medium (DMEM with 1 % human AB serum, 100 U/mL penicillin, and 100 μg/mL streptomycin) at a concentration of 4 × 105 cells/mL. Tumor cells from cell lines U87MG, T98G, and HT1080 were harvested, washed with PBS, and resuspended in cell culture medium (1 × 105 cells/ mL). ELISPOT analyses were performed with an interferon gamma ELISPOT Set (Becton–Dickinson) according to the manufacturer’s instructions. Spots were counted with software KS ELISPOT (Zeiss, Jena, Germany). Cytotoxicity assay Cytotoxicity exerted by PBMC after priming with TPDC was assessed by lactate dehydrogenase (LDH) release essentially as described [44]. On days 5 and 7 of PBMC/TPDC co-culture, cells were harvested, washed, and resuspended in cell culture medium (DMEM with 1 % human AB serum, 100 U/mL penicillin, and 100 μg/mL streptomycin) at a concentration of 5 × 106 cells/mL. Tumor cells from cell lines U87MG, T98G, and HT1080 were harvested, washed with PBS, and resuspended in cell culture medium (2 × 105 cells/ mL). Tumor cells and primed PBMC were co-incubated in triplicates in 96-well plates for 16 h. Thereafter, LDH release from the cells was analyzed by using a LDH assay kit (Roche) according to the manufacturer’s instructions. Cytometric bead array Cell culture supernatants were harvested on days 5 and 7, centrifuged at 300×g, and stored for further usage at −80 °C. Cytokines were measured with a cytometric bead array (CBA) using the Human TH1/TH2 Cytokine Kit (Becton–Dickinson) according to the manufacturer’s instructions.
Fluorescence‑activated cell scanning (FACS) analysis FACS analysis was performed essentially as described before [43]. About 0.5–1 × 106 cells were stained with fluorochrome-labeled antibodies (αCD3, αCD4, αCD8, αCD25, αCD56, αCD137L, αFOXP3, and appropriate isotype controls, Becton–Dickinson, Heidelberg, Germany). After incubation for 30 min in the dark at 4 °C, cells were washed twice with PBS and resuspended in 0.5 mL PBS. For detection of FOXP3, cells were permeabilized and stained for
Results Effects of anti‑CD137 stimulation on PBMC priming with tumor‑lysate‑pulsed DC Priming of PBMC with DC that had been loaded with tumor lysate from human glioma cell line U87MG led to a significant increase in U87MG-reactive cells as detected by IFNγ
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Fig. 2 Characterization of the response elicited by tumor-lysatepulsed DC. a HLA-A2-positive PBMC were cultured with U87MG tumor-lysate-pulsed DC (DC/TL), immature DC (imDC), or without DC (w/o DC). After 5 days of incubation in a humidified atmosphere, cells were re-stimulated with tumor cells from lines U87MG, T98G, and HT1080 and interferon gamma ELISPOT analysis was performed. b Before culture of PBMC (HLA-A2-positive) with TPDC, depletion of CD4-positive, CD8-positive, or CD56-positive cells was performed. Depleted and non-depleted PBMC were cultured for 5 and 7 days in a humidified atmosphere. During interferon gamma ELISPOT analyses, PBMC were re-stimulated with U87MG tumor cells. The differences between non-depleted and CD4-depleted cells as well as between non-depleted and CD8-depleted cells are significant (p