Distinct effects of human glioblastoma immunoregulatory molecules programmed cell death ligand-1 (PDL-1) and indoleamine 2,3-dioxygenase (IDO) on tumour-specific T cell functions

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Abstract

Immunotherapy is a promising new treatment for patients suffering from glioma, in particular glioblastoma multiforme (GBM). However, tumour cells use different mechanisms to escape the immune responses induced by the treatment. As many other tumours, gliomas express or secrete several immunosuppressive molecules that regulate immune cell functions. In this study, we first analysed FasL, HLA-G, IDO, PDL-1 and TGF-β1, -β2 and -β3 expression by transcriptomic microarray analysis in a series of 20 GBM samples and found respectively 15%, 60%, 85%, 30%, 70%, 80% and 35% of positive specimens. mRNA expression was then confirmed in 10 GBM primary cell lines and 2 immortalised cell lines U251 and U87MG. Furthermore, the protein expression of PDL-1, IDO activity and TGF-β2 secretion were found on most of the untreated GBM primary cell lines. Remarkably, treatment with IFN-γ increased the PDL-1 cell surface expression and the IDO activity, but reduced the TGF-β2 secretion of GBM cell lines. We finally analysed the immunosuppressive effects of IDO, PDL-1 and TGF-β1-3 by measuring IFN-γ production and cell cytotoxicity activity of tumour antigen-specific T cells. PDL-1 partially affected the IFN-γ production of antigen-specific T cells in response to GBM primary cell lines, and IDO inhibited lymphocyte proliferation induced by lectins. None of these molecules directly affected the T cell cytotoxicity function. Due to the functional role of PDL-1 and IDO molecules expressed by GBM cells, one could expect that blocking these molecules in the immunotherapy strategies would reinforce the efficiency of these treatments of GBM patients.

Introduction

Glioblastoma multiforme, the most common primary brain tumour in adults, is an attractive target for novel therapies regarding its poor prognosis with standard treatment including total surgical resection, radiotherapy and adjuvant chemotherapy (Stupp et al., 2005). Indeed, despite intensive investigations for novel drugs, less than a third of patients survive more than two years (Stupp et al., 2009). Recurrence of GBM occurs most often within few months due to the re-growth of remaining tumour cells that are resistant to both radiotherapeutic and chemotherapeutic agents.

Cellular immunotherapy in gliomas, one of the promising new therapies, has shown convincing results in terms of induction of anti-tumour immune responses and prolonged survival compared with control groups receiving conventional chemotherapy and radiotherapy (Yamanaka, 2008). In particular, patients treated with dendritic cell vaccinations have demonstrated systemic antigen-specific cytotoxicity and intra-tumour infiltration of cytotoxic T cells (Yamanaka et al., 2003, Kikuchi et al., 2004, Yu et al., 2004, Yamanaka et al., 2005). Liau et al. recently reported that T cell infiltration within the tumour was inversely correlated with the secretion of Transforming Growth Factor-beta 2 (TGF-β2) by the glioma cells (Liau et al., 2005), highlighting the impact of the immunosuppressive properties of the tumour on the clinical efficiency of the treatment.

Local and systemic immune suppression has been involved in glioma progression (Weller and Fontana, 1995, Walker et al., 2002). TGF-β2, originally defined as the glioblastoma cell-derived T cell suppressor factor, is a molecule that suppresses T cell growth and inhibits T cell activation and differentiation into effector cells (Gorelik and Flavell, 2002). Several other intracellular, membrane and soluble molecules such as FasL, HLA-G, PDL-1 (for Programmed cell Death Ligand-1, CD274), IDO (for Indoleamine2,3-DiOxygenase) and TGF-β1-3 have been described as taking part in this phenomenon. The Fas ligand (FasL, CD178), which is a member of the TNF family, induces apoptosis in Fas (CD95)-expressing cells (Nagata and Golstein, 1995). Although FasL is expressed by immune cells as a cytotoxic molecule, it is also found on the surface of different types of cancer cells (Walker et al., 1997). FasL is expressed in human malignant glioma cells (Gratas et al., 1997). In addition, FasL expressed on glioma cell lines is active and induces the death of sensitive target cells such as Fas-transfected P815 cells (Saas et al., 1997), JURKAT T cell line (Weller et al., 1997) and T cell lines derived from infiltrating T lymphocytes from astrocytoma (Walker et al., 1997). HLA-G is a non-classical MHC class I molecule mainly expressed on placenta by the trophoblastic cells (Kovats et al., 1990). Although HLA-G mRNA expression has also been found in tumour cells such as melanoma and leukaemia cells, its protein expression is still controversial because of the heterogeneity of the results observed in the different studies (Bainbridge et al., 2001, Chang and Ferrone, 2003). HLA-G can interact directly or indirectly (via the HLA-E molecule) with inhibitory receptors expressed on cytotoxic lymphocytes, which induces the abrogation of effector cell activation (Carosella et al., 2008). Wiendl et al. (2002) describe the expression of HLA-G in brain tumour tissues and glioma cell lines, and demonstrate that cytotoxicity, proliferation and T cell priming of alloreactive PBMC are inhibited by HLA-G. PDL-1 belongs to the B7 family, and is expressed on antigen presenting cells (Greenwald et al., 2005). Engagement of its receptor PD-1 inhibits T cell functions such as proliferation, cytokine production and cytotoxicity, and promotes apoptosis (Freeman et al., 2000, Dong et al., 2002). PDL-1 has been observed on glioma and astrocytoma cell lines, and reduces IFN-γ production of allogeneic T cells (Wintterle et al., 2003, Wilmotte et al., 2005). Finally, IDO is an intracellular enzyme that degrades tryptophan along the kynurenine pathway. IDO activity is essential in mouse placenta for preventing the rejection of allogeneic fetuses (Munn et al., 1998). Its activity suppresses T cell activation and proliferation (Munn et al., 1999). IDO expression has also been described in cancer of different origins (Munn and Mellor, 2007, Katz et al., 2008), including gliomas (Uyttenhove et al., 2003) and malignant glioma cell lines (Grant and Kapoor, 2003, Miyazaki et al., 2009), in which both IDO expression and activity have recently been reported. To our knowledge, the effect of glioma cell IDO activity on T cell functions has not been investigated.

While the expression of FasL, HLA-G, PDL-1, IDO and TGF-β1-3 molecules has been reported in glioma cells, their role in modulating GBM-specific T cell effector functions is not fully understood. This is particularly relevant with regard to cellular immunotherapy, whereby tumour-specific T cells would be generated to specifically kill GBM cells. In this study, we first describe the expression pattern of FasL, HLA-G, PDL-1, IDO and TGF-β1-3 in a series of 20 GBM specimens using data derived from our previous transcriptomic analysis (de Tayrac et al., 2009). The mRNA expression of these molecules is then analysed in 10 GBM primary cell lines, as well as the immortalised cell lines U251 and U87MG. We then focus on the immunosuppressive effects of PDL-1, IDO and TGF-β1-3 on GBM-specific CD8+ T cell effector functions, i.e. IFN-γ secretion and cell cytotoxicity in response to GBM primary cell lines. We show that none of these molecules have a direct effect on cell cytotoxicity functions, but we provide evidence that PDL-1 affects GBM-specific CD8+ T cell IFN-γ secretion in response to the tumour, and that GBM expresses functional IDO that inhibits the lectin-induced proliferation of lymphocytes.

Section snippets

Reagents and antibodies

All reagents not specified below were purchased from Sigma-Aldrich (St Quentin Fallavier, France). Antibodies against CD3, CD8, HLA-ABC and PDL-1 were obtained from BD Biosciences (Le Pont de Claix, France); Melan-A/HLA-A2 tetramers from Beckman Coulter (Marseille, France); anti-EGFRvIII antibody from Skybio (Bedfordshire, United Kingdom); anti-IDO antibody from Millipore (St Quentin Fallavier, France); anti-IL13Rα2 antibody from Diaclone (Besançon, France); anti-gp100 and anti-vimentin

FasL, HLA-G, IDO, PDL-1 and TGF-β1-3 mRNA expression in a series of GBM samples by expression microarray analysis

We have recently reported a DNA target transcriptomic profiling on 20 GBM samples (de Tayrac et al., 2009). Using the complete microarray data set, FasL, HLA-G, IDO, PDL-1 and TGF-β1-3 transcripts expression was compared between GBM and non-tumour brain samples. A significant increase (expression fold change greater than two) is observed in 15%, 60%, 85%, 30%, 70%, 80%, and 35% of GBM samples for FasL, HLA-G, IDO, PDL-1, TGF-β1, TGF-β2 and TGF-β3, respectively (Fig. 1).

FasL, HLA-G, IDO, PDL-1 and TGF-β1-3 mRNA expression in GBM cell lines

For in vitro assays, we

Discussion

In this study, we analyse the expression of five immunoregulatory molecules FasL, HLA-G, PDL-1, IDO and TGF-β1-3 in human GBM. Using GBM primary cell lines, we confirm the mRNA and protein expression of PDL-1, IDO and TGF-β2 molecules in most of the GBM cell lines. Furthermore, functional studies highlight the role of PDL-1, TGF-β1-3 and IDO in the inhibition of IFN-γ production by tumour-specific T cells and lectin-induced lymphocyte proliferation, respectively. None of these molecules are

Acknowledgments

We thank persons and organizations which have contributed to this work: Stéphane Moiteaux for his excellent technical assistance; L. Amiot, V. Catros and A. Clavreul for their kind gifts of the JEG-3 trophoblastic cell line, U251 and U87MG cell lines, respectively; the Biogenouest® transcriptomic platform from Rennes for the microarray analysis; Alain Feutrel and Pascale Bellaud from the Biogenouest® histopathology platform (IFR 140, Université de Rennes 1) for the immunohistochemistry analysis

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      Thus, overexpression of PDL1 in patients with tumors suggests poor prognosis. Similarly, the expression level of PD-L1 in gliomas is also closely related to the grades of gliomas [9,10]. Except expressed on glioma cells, PD-L1 also expressed in the around of tumors, for example the infiltrative lymphocytes or adjacent neurons [11,12], it implies the poor prognosis [13].

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