Expression profiling of immune inhibitory Siglecs and their ligands in patients with glioma.

Gliomas appear to be highly immunosuppressive tumors, with a strong myeloid component. This includes MDSCs, which are a heterogeneous, immature myeloid cell population expressing myeloid markers Siglec-3 (CD33) and CD11b and lacking markers of mature myeloid cells including MHC II. Siglec-3 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family and has been suggested to promote MDSC expansion and suppression. Siglecs form a recently defined family of receptors with potential immunoregulatory functions but only limited insight in their expression on immune regulatory cell subsets, prompting us to investigate Siglec expression on MDSCs. We determined the expression of different Siglec family members on monocytic-MDSCs (M-MDSCs) and polymorphnuclear-MDSCs (PMN-MDSCs) from blood of glioma patients and healthy donors, as well as from patient-derived tumor material. Furthermore, we investigated the presence of sialic acid ligands for these Siglecs on MDSCs and in the glioma tumor microenvironment. Both MDSC subsets express Siglec-3, -5, -7 and -9, with higher levels of Siglec-3, -7 and -9 on M-MDSCs and higher Siglec-5 levels on PMN-MDSCs. Similar Siglec expression profiles were found on MDSCs from healthy donors. Furthermore, the presence of Siglec-5 and -9 was also confirmed on PMN-MDSCs from glioma tissue. Interestingly, freshly isolated glioma cells predominantly expressed sialic acid ligands for Siglec-7 and -9, which was confirmed in situ. In conclusion, our data show a distinct Siglec expression profile for M- and PMN-MDSCs and propose possible sialic acid-Siglec interactions between glioma cells and MDSCs in the tumor microenvironment.

T-Cell Lymphopenia in Patients with Advanced Thyroid Carcinoma Is Associated with Poor Prognosis.

BACKGROUND: Aggressive forms of thyroid carcinoma (TC) show an abundant infiltration of immune cells, and this correlates with prognosis. However, little is known about circulating immune cell levels in advanced TC. OBJECTIVE: Investigate T-cell and myeloid-derived suppressor cell (MDSC) levels in peripheral blood of patients with advanced TC and correlate them with survival. METHODS: T cells and MDSCs were quantified by flow cytometry in peripheral blood from nine patients with advanced TC and nine healthy volunteers. RESULTS: No significant differences in MDSC or regulatory T-cell levels were detected between patients with TC and healthy controls. CD3, CD4, and CD8 T-cell levels were significantly lower in patients with TC. CD3 and CD4 T-cell levels further decreased in patients with survival of less than 1 month. CONCLUSION: These data suggest that T-cell lymphopenia in patients with TC indicates an aggressive tumor behavior and might influence therapeutic choices in the future. Restoring T-cell levels may become a potential therapeutic option within the multitarget approaches.

Enterovirus Exposure Uniquely Discriminates Type 1 Diabetes Patients with a Homozygous from a Heterozygous Melanoma Differentiation-Associated Protein 5/Interferon Induced with Helicase C Domain 1 A946T Genotype.

In children at risk for type 1 diabetes, innate immune activity is detected before seroconversion. Enterovirus infections have been linked to diabetes development, and a polymorphism (A946T) in the innate immune sensor recognizing enterovirus RNA, interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5, predisposes to disease. We hypothesized that the strength of innate antienteroviral responses is affected in autoimmune type 1 diabetes patients and linked to the A946T polymorphism. We compared induction of interferon-stimulated genes (ISGs) in peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) in healthy individuals and diabetes patients upon stimulation with enterovirus, enterovirus-antibody complexes, or ligands mimicking infection in relation to the A946T polymorphism. Overall, PBMCs of diabetes patients and healthy donors showed comparable ISG induction upon stimulation. No differences were observed in DCs. Interestingly, the data imply that the magnitude of responses to enterovirus and enterovirus-antibody complexes in PBMCs is critically influenced by the A946T polymorphism and elevated in heterozygotes compared to TT homozygous individuals in autoimmune diabetes patients, but not healthy controls. These data imply an intrinsic difference in the responses to enterovirus and enterovirus-antibody complexes in diabetes patients carrying a TT risk genotype compared to heterozygotes that may influence control of enterovirus clearance.

Anti-GD2 mAb and Vorinostat synergize in the treatment of neuroblastoma.

Neuroblastoma (NBL) is a childhood malignancy of the sympathetic nervous system. For high-risk NBL patients, the mortality rate is still over 50%, despite intensive multimodal treatment. Anti-GD2 monoclonal antibody (mAB) in combination with systemic cytokine immunotherapy has shown clinical efficacy in high-risk NBL patients. Targeted therapy using histone deacetylase inhibitors (HDACi) is currently being explored in cancer treatment and already shows promising results. Using our recently developed transplantable TH-MYCN NBL model, we here report that the HDAC inhibitor Vorinostat synergizes with anti-GD2 mAb therapy in reducing NBL tumor growth. Further mechanistic studies uncovered multiple mechanisms for the observed synergy, including Vorinostat-induced specific NBL cell death and upregulation of the tumor antigen GD2 on the cell surface of surviving NBL cells. Moreover, Vorinostat created a permissive tumor microenvironment (TME) for tumor-directed mAb therapy by increasing macrophage effector cells expressing high levels of Fc-receptors (FcR) and decreasing the number and function of myeloid-derived suppressor cells (MDSC). Collectively, these data imply further testing of other epigenetic modulators with immunotherapy and provide a strong basis for clinical testing of anti-GD2 plus Vorinostat combination therapy in NBL patients.

Elevated levels of polymorphonuclear myeloid-derived suppressor cells in patients with glioblastoma highly express S100A8/9 and arginase and suppress T cell function.

BACKGROUND: Gliomas are primary brain tumors that are associated with a poor prognosis. The introduction of new treatment modalities (including immunotherapy) for these neoplasms in the last 3 decades has resulted in only limited improvement in survival. Gliomas are known to create an immunosuppressive microenvironment that hampers the efficacy of (immuno)therapy. One component of this immunosuppressive environment is the myeloid-derived suppressor cell (MDSC). METHODS: We set out to analyze the presence and activation state of MDSCs in blood (n = 41) and tumor (n = 20) of glioma patients by measuring S100A8/9 and arginase using flow cytometry and qPCR. Inhibition of T cell proliferation and cytokine production after stimulation with anti-CD3/anti-CD28 coated beads was used to measure in vitro MDSC suppression capacity. RESULTS: We report a trend toward a tumor grade-dependent increase of both monocytic (M-) and polymorphonuclear (PMN-) MDSC subpopulations in the blood of patients with glioma. M-MDSCs of glioma patients have increased levels of intracellular S100A8/9 compared with M-MDSCs in healthy controls (HCs). Glioma patients also have increased S100A8/9 serum levels, which correlates with increased arginase activity in serum. PMN-MDSCs in both blood and tumor tissue demonstrated high expression of arginase. Furthermore, we assessed blood-derived PMN-MDSC function and showed that these cells have potent T cell suppressive function in vitro. CONCLUSIONS: These data indicate a tumor grade-dependent increase of MDSCs in the blood of patients with a glioma. These MDSCs exhibit an increased activation state compared with MDSCs in HCs, independent of tumor grade.

Increase in both CD14-positive and CD15-positive myeloid-derived suppressor cell subpopulations in the blood of patients with glioma but predominance of CD15-positive myeloid-derived suppressor cells in glioma tissue.

Myeloid-derived suppressor cells (MDSCs), defined as CD33-positive major histocompatibility complex class II-negative cells, are increased in a variety of human tumors and are associated with immunosuppression. Myeloid-derived suppressor cells can be further subdivided into CD14-positive monocytic MDSC and CD15-positive granulocytic MDSC (polymorphonuclear MDSC) subpopulations. Here we analyzed MDSC subsets in the blood and tumor tissue of patients with glioma, including the most malignant variant, glioblastoma multiforme (GBM). CD33-positive major histocompatibility complex class II-negative MDSCs in blood from 21 patients with glioma and 12 healthy individuals were phenotyped and quantified by flow cytometry. Myeloid populations of the monocytic MDSC and polymorphonuclear MDSC phenotypes were both significantly increased in the blood of patients with GBM versus healthy controls. The myeloid activation markers CD80 and PD-L1 could not be detected on either of these MDSC subsets; CD124, CD86, and CD40 were detected at similar levels on MDSCs in patients with glioma and healthy donors. By contrast, in tumor cell suspensions, the MDSC population consisted almost exclusively of CD15-positive cells. Immunohistochemistry confirmed infiltration of CD15-positive major histocompatibility complex class II-negative cells in glioma tissue samples. These data support a role for cells with an MDSC phenotype in the blood and tumor microenvironment of patients with GBM.

Enterovirus-infected ß-cells induce distinct response patterns in BDCA1+ and BDCA3+ human dendritic cells.

Enteroviruses often cause mild disease, yet are also linked to development of autoimmune diabetes. Dendritic cells (DCs) shape both innate and adaptive immune responses, including anti-viral responses. How different human DC subsets shape anti-viral responses, whether they have complementary or overlapping functions and how this relates to autoimmune responses is largely unknown. We used enterovirus-infected ß-cells and freshly isolated human myeloid DC (mDC) subsets as a model for autoimmune type 1 diabetes. Our data show that both the BDCA1+ and BDCA3+ mDC subsets engulf mock- as well as virus-infected ß-cells, albeit BDCA1+ mDCs are more efficient. Uptake of enterovirus-infected, but not mock-infected cells, activated both DC subsets as indicated by the induction of co-stimulatory molecules and secretion of type I and type III interferons. Both subsets produced similar amounts of interferon-α, yet the BDCA3+ DC were superior in IFN-λ production. The BDCA1+ mDCs more strongly upregulated PD-L1, and were superior in IL-12 and IL-10 production as compared to the BDCA3+ DC. Despite lack of IL-12 production by the BDCA3+ DC, both BDCA1+ and BDCA3+ DCs activated T cells in allogeneic mixed lymphocyte reaction towards a Th1-type reactivity while suppressing Th2-associated cytokines.

Connexin43 confers Temozolomide resistance in human glioma cells by modulating the mitochondrial apoptosis pathway.

Glioblastoma multiforme (GBM) is the most aggressive astrocytoma, and therapeutic options are generally limited to surgical resection, radiotherapy, and Temozolomide (TMZ) chemotherapy. TMZ is a DNA alkylating agent that causes DNA damage and induces cell death. Unfortunately, glioma cells often develop resistance to TMZ treatment, with DNA de-methylation of the MGMT promoter identified as the primary reason. However, the contributions from proteins that normally protect cells against cytotoxic stress in TMZ-induced apoptosis have not been extensively explored. Here, we showed that increasing the level of the gap junction protein, Cx43, in human LN18 and LN229 glioma cells enhances resistance to TMZ treatment while knockdown of Cx43 in these same cells sensitizes them to TMZ treatment. By expressing a channel-dead or a C-terminal truncation mutant of Cx43, we show that Cx43-mediated TMZ resistance involves both channel dependent and independent functions. Expression of Cx43 in LN229 cells decreases TMZ-induced apoptosis, as determined by Annexin V staining. Cx43-mediated chemoresistance appears to be acting via a mitochondrial apoptosis pathway as manifested by the reduction in Bax/Bcl-2 ratio and the release of cytochrome C. Our findings highlight additional mechanisms and proteins that contribute to TMZ resistance, and raise the possibility of increasing TMZ efficiency by targeting Cx43 protein. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.