Margaretha Lambers

COX-2 inhibition improves immunotherapy and is associated with decreased numbers of myeloid-derived suppressor cells in mesothelioma. Celecoxib influences MDSC function

BackgroundMyeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells that accumulates in tumour-bearing hosts. These cells are induced by tumour-derived factors (e.g. prostaglandins) and have a critical role in immune suppression. MDSC suppress T and NK cell function via increased expression of arginase I and production of reactive oxygen species (ROS) and nitric oxide (NO). Immune suppression by MDSC was found to be one of the main factors for immunotherapy insufficiency. Here we investigate if the in vivo immunoregulatory function of MDSC can be reversed by inhibiting prostaglandin synthesis by specific COX-2 inhibition focussing on ROS production by MDSC subtypes. In addition, we determined if dietary celecoxib treatment leads to refinement of immunotherapeutic strategies.MethodsMDSC numbers and function were analysed during tumour progression in a murine model for mesothelioma. Mice were inoculated with mesothelioma tumour cells and treated with cyclooxygenase-2 (COX-2) inhibitor celecoxib, either as single agent or in combination with dendritic cell-based immunotherapy.ResultsWe found that large numbers of infiltrating MDSC co-localise with COX-2 expression in those areas where tumour growth takes place. Celecoxib reduced prostaglandin E2 levels in vitro and in vivo. Treatment of tumour-bearing mice with dietary celecoxib prevented the local and systemic expansion of all MDSC subtypes. The function of MDSC was impaired as was noticed by reduced levels of ROS and NO and reversal of T cell tolerance; resulting in refinement of immunotherapy.ConclusionsWe conclude that celecoxib is a powerful tool to improve dendritic cell-based immunotherapy and is associated with a reduction in the numbers and suppressive function of MDSC. These data suggest that immunotherapy approaches benefit from simultaneously blocking cyclooxygenase-2 activity.

Consolidative dendritic cell-based immunotherapy elicits cytotoxicity against malignant mesothelioma

RATIONALE We previously demonstrated that dendritic cell-based immunotherapy induced protective antitumor immunity with a prolonged survival rate in mice. However, the clinical relevance is still in question. To examine this, we designed a clinical trial using chemotherapy followed by antigen-pulsed dendritic cell vaccination in mesothelioma patients. OBJECTIVES The aim of this study was to assess the safety and immunological response induced by the administration of tumor lysate-pulsed dendritic cells in patients with mesothelioma. METHODS Ten patients with malignant pleural mesothelioma received three vaccinations of clinical-grade autologous dendritic cells intradermally and intravenously at 2-week intervals after chemotherapy. Each vaccine was composed of 50 x 10(6) mature dendritic cells pulsed with autologous tumor lysate and keyhole limpet hemocyanin (KLH) as surrogate marker. Delayed-type hypersensitivity activity to tumor antigens and KLH was assessed, both in vivo and in vitro. Peripheral blood mononuclear cells during the treatment were analyzed for immunological responses. MEASUREMENTS AND MAIN RESULTS Administration of dendritic cells pulsed with autologous tumor lysate in patients with mesothelioma was safe with moderate fever as the only side effect. There were no grade 3 or 4 toxicities associated with the vaccines or any evidence of autoimmunity. Local accumulations of infiltrating T cells were found at the site of vaccination. The vaccinations induced distinct immunological responses to KLH, both in vitro and in vivo. Importantly, after three vaccinations, cytotoxic activity against autologous tumor cells was detected in a subgroup of patients. CONCLUSIONS This study demonstrated that autologous tumor lysate-pulsed dendritic cell-based therapy is feasible, well-tolerated, and capable of inducing immunological response to tumor cells in mesothelioma patients. Clinical trial registered with www.clinicaltrials.gov (NCT00280982).

Zoledronic acid impairs myeloid differentiation to tumour-associated macrophages in mesothelioma

Background:Suppressive immune cells present in tumour microenvironments are known to augment tumour growth and hamper efficacy of antitumour therapies. The amino-bisphosphonate Zoledronic acid (ZA) is considered as an antitumour agent, as recent studies showed that ZA prolongs disease-free survival in cancer patients. The exact mechanism is a topic of debate; it has been suggested that ZA targets tumour-associated macrophages (TAMs).Methods:We investigate the role of ZA on the myeloid differentiation to TAMs in murine mesothelioma in vivo and in vitro. Mice were intraperitoneally inoculated with a lethal dose of mesothelioma tumour cells and treated with ZA to determine the effects on myeloid differentiation and survival.Results:We show that ZA impaired myeloid differentiation. Inhibition of myeloid differentiation led to a reduction in TAMs, but the number of immature myeloid cells with myeloid-derived suppressor cell (MDSC) characteristics was increased. In addition, ZA affects the phenotype of macrophages leading to reduced level of TAM-associated cytokines in the tumour microenvironment. No improvement of survival was observed.Conclusion:We conclude that ZA leads to a reduction in macrophages and impairs polarisation towards an M2 phenotype, but this was associated with an increase in the number of immature myeloid cells, which might diminish the effects of ZA on survival.

Arginase-1 mRNA expression correlates with myeloid-derived suppressor cell levels in peripheral blood of NSCLC patients

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with immunosuppressive activity that are increased in cancer patients. Until now, the characterization of MDSC in humans was very challenging. The aim of this study was to determine the characterization and optimal assessment of MDSC and to investigate their presence and function in blood of advanced-stage NSCLC patients. We determined MDSC and lymphocyte populations in peripheral blood mononuclear cells (PBMC) samples of 185 treatment-naïve NSCLC patients and 20 healthy controls (HC). NSCLC patients had an increased population of PMN-MDSC compared to HC (p < 0.0001). Frequencies of CD4(+) and CD8(+) T-cells were significantly decreased in NSCLC patients (p < 0.0001 and p = 0.05). We found that PMN-MDSC were able to suppress T-cell proliferation in vitro. qRT-PCR showed that arginase-1 (Arg-1) mRNA is mainly expressed by MDSC and that the level of Arg-1 in PBMC correlates with the frequency of MDSC in PBMC (Spearmans rho: 0.797). There were significant differences in MDSC and lymphocyte populations between NSCLC patients and HC. We found that MDSC frequencies are stable up to six hours at room temperature after blood was drawn and that cryopreservation leads to a strong decrease of MDSC in PBMC. We show that Arg-1 mRNA expression is a valuable method to determine the levels of MDSC in peripheral blood of cancer patients. This method is therefore a useful alternative for the complex flowcytometric analysis in large multicenter patient studies.

Low-Dose Cyclophosphamide Synergizes with Dendritic Cell-Based Immunotherapy in Antitumor Activity

Clinical immunotherapy trials like dendritic cell-based vaccinations are hampered by the tumors offensive repertoire that suppresses the incoming effector cells. Regulatory T cells are instrumental in suppressing the function of cytotoxic T cells. We studied the effect of low-dose cyclophosphamide on the suppressive function of regulatory T cells and investigated if the success rate of dendritic cell immunotherapy could be improved. For this, mesothelioma tumor-bearing mice were treated with dendritic cell-based immunotherapy alone or in combination with low-dose of cyclophosphamide. Proportions of regulatory T cells and the cytotoxic T cell functions at different stages of disease were analyzed. We found that low-dose cyclophosphamide induced beneficial immunomodulatory effects by preventing the induction of Tregs, and as a consequence, cytotoxic T cell function was no longer affected. Addition of cyclophosphamide improved immunotherapy leading to an increased median and overall survival. Future studies are needed to address the usefulness of this combination treatment for mesothelioma patients.

The DNA-binding factor Ctcf critically controls gene expression in macrophages

Macrophages play an important role in immunity and homeostasis. Upon pathogen recognition via specific receptors, they rapidly induce inflammatory responses. This process is tightly controlled at the transcriptional level. The DNA binding zinc-finger protein CCCTC-binding factor (Ctcf) is a crucial regulator of long-range chromatin interactions and coordinates specific communication between transcription factors and gene expression processes. In this study, the Ctcf gene was specifically deleted in myeloid cells by making use of the transgenic Cre-LoxP system. Conditional deletion of the Ctcf gene in myeloid cells induced a mild phenotype in vivo. Ctcf-deficient mice exhibited significantly reduced expression of major histocompatibility complex (MHC) class II in the liver. Ctcf-deficient macrophages demonstrated a normal surface phenotype and phagocytosis capacity. Upon Toll-like receptor (TLR) stimulation, they produced normal levels of the pro-inflammatory cytokines IL-12 and IL-6, but manifested a strongly impaired capacity to produce tumor-necrosis factor (TNF) and IL-10, as well as to express the IL-10 family members IL-19, IL-20 and IL-24. Taken together, our data demonstrate a role of Ctcf that involves fine-tuning of macrophage function.

Anti-inflammatory actions of phosphatidylinositol

Chronic inflammatory T‐cell‐mediated diseases such as inflammatory bowel disease (IBD) are often treated with immunosuppressants including corticosteroids. In addition to the intended T‐cell suppression, these farmacons give rise to many side effects. Recently, immunosuppressive phospholipids have been proposed as less‐toxic alternatives. We aimed to investigate the immunoregulatory capacities of the naturally occurring phospholipid phosphatidylinositol (PI). Systemic PI treatment dramatically reduced disease severity and intestinal inflammation in murine 2,4,6‐trinitrobenzene sulfonic acid (TNBS) colitis. Moreover, PI treatment inhibited the inflammatory T‐cell response in these mice, as T cells derived from colon‐draining LN of PI‐treated mice secreted less IL‐17 and IFN‐γ upon polyclonal restimulation when compared to those of saline‐treated mice. Further characterization of the suppressive capacity of PI revealed that the phospholipid suppressed Th cell differentiation in vitro irrespective of their cytokine profile by inhibiting proliferation and IL‐2 release. In particular, PI diminished IL‐2 mRNA expression and inhibited ERK1‐, ERK‐2‐, p38‐ and JNK‐phosphorylation. Crucially, PI did not ablate Treg differentiation or the antigen‐presenting capacity of DCs in vitro. These data validate PI as a pluripotent inhibitor that can be applied mucosally as well as systemically. Its compelling functions render PI a promising novel physiological immune suppressant.

Abstract 5394: Tumor-induced immunological changes in peripheral blood of untreated stage IV non-squamous NSCLC patients

Background Lung cancer is the most common cause of cancer mortality world wide. The five-year survival rate of stage III and IV non-small cell lung cancer (NSCLC) is poor with 16%. There is growing evidence that the immune system plays a paradoxical role in the development and progression of lung cancer. It influences tumor incidence, tumor growth, response to therapy, and the prognosis of the disease. Mouse models show that myeloid-derived suppressor cells (MDSCs) play a role in these processes. MDSCs are a heterogeneous population of immature myeloid cells and myeloid progenitor cells, which are present in the tumor micro-environment and the peripheral circulation of cancer patients. These cells are able to suppress features of immune responses, like T-cell proliferation and cytokine production. Aim In the present study we want to determine the role of pivotal immunological populations such as CD4+ T-cells, CD8+ T-cells, and MDSCs. Methods Fresh blood of 60 stage IV non-squamous-NSCLC patients and 15 healthy controls (HC) was studied. Mononuclear cells were purified from peripheral blood by density gradient centrifugation and analysed by flowcytometry. MDSCs were characterized as CD11b, CD15 CD33 positive and low expression of HLA-DR and CD16 cells. Suppressive activity of MDSCs was measured by CD3α-chain expression assays, and reactive-oxygen species (ROS) production using flowcytometry. Also T-cell proliferation assays were performed, in which sorted MDSCs were co-cultured with activated CD8-T-cells to investigate whether the MDSCs could inhibit the T-cell proliferation. Results All patients had a significantly increased MDSC population compared to healthy controls (p = 0.0009). Peripheral blood fractions possessed strong immunosuppressive functions, as shown by a high ROS production by MDSC, a decreased CD3α-chain expression in T cells compared to HC. In addition, the CD8+ T-cells showed no proliferation when co-cultured with MDSCs, indicating that MDSCs could block CD8+ -T cell proliferation. The populations of CD4+ T-cells and CD8+ T-cells were significantly decreased in lung cancer patients compared to healthy controls (p = 0.01 and p = 0.0024, respectively). Conclusion Circulating MDSCs are significantly increased, while CD4+ T-cells and CD8+ T-cells are significantly decreased in stage IV non-squamous-NSCLC patients compared to controls. MDSCs suppress the immune system by ROS production, down regulating the CD3α-chain expression and thereby inhibiting T-cell proliferation. This suggests that MDSCs may play an important role in disease progression and overall survival. Therefore, further investigation is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5394. doi:1538-7445.AM2012-5394