Dataset Information

Glioblastoma-Derived IL6 Induces Immunosuppressive Peripheral Myeloid Cell PD-L1 and Promotes Tumor Growth.

ABSTRACT:

Purpose

Upregulation of programmed death-ligand 1 (PD-L1) on circulating and tumor-infiltrating myeloid cells is a critical component of GBM-mediated immunosuppression that has been associated with diminished response to vaccine immunotherapy and poor survival. Although GBM-derived soluble factors have been implicated in myeloid PD-L1 expression, the identity of such factors has remained unknown. This study aimed to identify factors responsible for myeloid PD-L1 upregulation as potential targets for immune modulation.

Experimental design

Conditioned media from patient-derived GBM explant cell cultures was assessed for cytokine expression and utilized to stimulate naïve myeloid cells. Myeloid PD-L1 induction was quantified by flow cytometry. Candidate cytokines correlated with PD-L1 induction were evaluated in tumor sections and plasma for relationships with survival and myeloid PD-L1 expression. The role of identified cytokines on immunosuppression and survival was investigated in vivo utilizing immunocompetent C57BL/6 mice bearing syngeneic GL261 and CT-2A tumors.

Results

GBM-derived IL6 was identified as a cytokine that is necessary and sufficient for myeloid PD-L1 induction in GBM through a STAT3-dependent mechanism. Inhibition of IL6 signaling in orthotopic murine glioma models was associated with reduced myeloid PD-L1 expression, diminished tumor growth, and increased survival. The therapeutic benefit of anti-IL6 therapy proved to be CD8⁺ T-cell dependent, and the antitumor activity was additive with that provided by programmed death-1 (PD-1)-targeted immunotherapy.

Conclusions

Our findings suggest that disruption of IL6 signaling in GBM reduces local and systemic myeloid-driven immunosuppression and enhances immune-mediated antitumor responses against GBM.

SUBMITTER: Lamano JB

PROVIDER: S-EPMC6571046 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

Glioblastoma-Derived IL6 Induces Immunosuppressive Peripheral Myeloid Cell PD-L1 and Promotes Tumor Growth.

Lamano Jonathan B JB Lamano Jason Balquidera JB Li Yuping D YD DiDomenico Joseph D JD Choy Winward W Veliceasa Dorina D Oyon Daniel E DE Fakurnejad Shayan S Ampie Leonel L Kesavabhotla Kartik K Kaur Rajwant R Kaur Gurvinder G Biyashev Dauren D Unruh Dusten J DJ Horbinski Craig M CM James C David CD Parsa Andrew T AT Bloch Orin O

Clinical cancer research : an official journal of the American Association for Cancer Research 20190301 12

<h4>Purpose</h4>Upregulation of programmed death-ligand 1 (PD-L1) on circulating and tumor-infiltrating myeloid cells is a critical component of GBM-mediated immunosuppression that has been associated with diminished response to vaccine immunotherapy and poor survival. Although GBM-derived soluble factors have been implicated in myeloid PD-L1 expression, the identity of such factors has remained unknown. This study aimed to identify factors responsible for myeloid PD-L1 upregulation as potential ...[more]

PMID: 30824583

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Project description:BackgroundAdaptive immune resistance in the tumor microenvironment appears to attenuate the immunotherapeutic targeting of glioblastoma (GBM). In this study, we identified a tumor-infiltrating myeloid cell (TIM) population that expands in response to dendritic cell (DC) vaccine treatment. The aim of this study was to understand how this programmed death ligand 1 (PD-L1)-expressing population restricts activation and tumor-cytolytic function of vaccine-induced tumor-infiltrating lymphocytes (TILs).MethodsTo test this hypothesis in our in vivo preclinical model, we treated mice bearing intracranial gliomas with DC vaccination ± murine anti-PD-1 monoclonal antibody (mAb) blockade or a colony stimulating factor 1 receptor inhibitor (CSF-1Ri) (PLX3397) and measured overall survival. We then harvested and characterized the PD-L1+ TIM population and its role in TIL activation and tumor cytolysis in vitro.ResultsOur data indicated that the majority of PD-L1 expression in the GBM environment is contributed by TIMs rather than by tumor cells themselves. While PD-1 blockade partially reversed the TIL dysfunction, targeting TIMs directly with CSF-1Ri altered TIM expression of key chemotactic factors associated with promoting increased TIL infiltration after vaccination. Neither PD-1 mAb nor CSF-1Ri had a demonstrable therapeutic benefit alone, but when combined with DC vaccination, a significant survival benefit was observed. When the tripartite regimen was given (DC vaccine, PD-1 mAb, PLX3397), long-term survival was noted together with an increase in the number of TILs and TIL activation.ConclusionTogether, these studies elucidate the role that TIMs play in mediating adaptive immune resistance in the GBM microenvironment and provide evidence that they can be manipulated pharmacologically with agents that are clinically available. Development of immune resistance in response to active vaccination in GBM can be reversed with dual administration of CSF-1Ri and PD-1 mAb.

Project description:BackgroundGlioblastoma (GBM) characterized by immune escape is the most malignant primary brain tumors, which has strong immunosuppressive effect. Programmed death ligand-1 (PD-L1) is a recognized immunosuppressive member on the surface of tumor cells, and plays a crucial role in immune evasion of tumors. Actually, little is known about the regulation of PD-L1 expression in GBM. Insulin-like growth factor binding protein 3 (IGFBP3) is upregulated in GBM and is related to poor patient prognosis. However, it remains unclear whether IGFBP3 plays a role in the regulation of PD-L1 expression in GBM.MethodsThe role of IGFBP3 in the glioma immune microenvironment was investigated using the CIBERSORT algorithm. The correlation between IGFBP3 and PD-L1 expression was analyzed using TCGA and CGGA databases. QRT-PCR, immunoblotting and RNA-seq were used to examine the regulatory effect of IGFBP3 on PD-L1 expression. Co-culture assay, cell counting kit (CCK-8), qRT-PCR, ELISA and flow cytometry were performed to explore the function of IGFBP3 in inducing immunosuppression. The biological role of IGFBP3 was verified using immunohistochemical, immunofluorescence and mice orthotopic tumor model.ResultsIn this study, we analyzed immune cells infiltration in gliomas and found that IGFBP3 may be associated with an immunosuppressive microenvironment. Then, by analyzing TCGA and CGGA databases, our results showed that IGFBP3 and PD-L1 expression were positively correlated in GBM patients, but not in LGG patients. In vitro experiments conducted on different GBM cell lines revealed that the overexpression of IGFBP3 led to an increase in PD-L1 expression, which was reversible upon knockdown IGFBP3. Mechanistically, IGFBP3 activated the JAK2/STAT3 signaling pathway, leading to an increase in PD-L1 expression. Additionally, co-culture experiments results showed IGFBP3 overexpression induced upregulation of PD-L1 expression promoted apoptosis in Jurkat cells, and this effect was blocked by IGFBP3 antibody and PDL-1 inhibitors. Importantly, in vivo experiments targeting IGFBP3 suppressed tumor growth and significantly prolonged the survival of mice.ConclusionsThis research demonstrated IGFBP3 is a novel regulator for PD-L1 expression in GBM, and identified a new mechanism by which IGFBP3 regulates immune evasion through PD-L1, suggesting that IGFBP3 may be a potential novel target for GBM therapy.

Project description:Sample description: Anti-PD-L1 co-immunoprecipitation samples from cultured wild-type, PD-L1-VavCre or PD-L1-LysMCre bone marrow cells. Samples are: KO-VavCre-1 (775112) v.s. WT-1 (775113); KO-LysMCre-2 (775114) v.s. WT-2 (775115). Among these samples, KO-VavCre-1 and KO-LysMCre-2 are the respective blank controls (from knockout mice). Three 10-cm plates of bone marrow cells from each genotype (wild-type or PD-L1-VavCre, and wild-type or PD-L1-LysMCre) of 2-month-old male littermate mice at a density of 2.8 * 107 cells/plate were cultured in the presence of 40 ng/mL of mouse M-CSF for 3 days. Cells were washed with PBS and resuspend in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM PMSF, 1x protease inhibitor cocktail) for 4 hours at 4C. The lysate was centrifuged at 12,000 g for 10 min. The supernatant was mixed with 2 micro G of anti-PD-L1 antibody and incubated for overnight at 4C. Protein A/G agarose (Santa Cruz) was added and incubated for 2 hours at 4C. Agarose was washed with lysis buffer for four times. The samples were separated in SDS-PAGE gel (10-mm length per lane). Coomassie blue stained gel slices were analyzed by mass spectrometry. Samples were digested overnight with trypsin (Pierce) following reduction and alkylation with DTT and iodoacetamide (Sigma-Aldrich). The samples then underwent solid-phase extraction cleanup with an Oasis HLB plate (Waters) and were subsequently dried and reconstituted into 10 microL of 2% ACN, 0.1% TFA. 2 microL of these samples were injected onto a QExactive HF mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Samples were injected onto a 75 micro M i.d., 15-cm long EasySpray column (Thermo) and eluted with a gradient from 0-28% buffer B over 90 min with a flow rate of 250 nL/min. Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 2.2 kV and an ion transfer tube temperature of 275 C. MS scans were acquired at 120,000 resolution in the Orbitrap and up to 20 MS/MS spectra were obtained for each full spectrum acquired using higher-energy collisional dissociation (HCD) for ions with charges 2-8. Dynamic exclusion was set for 20 second after an ion was selected for fragmentation.

Dataset Information

Glioblastoma-Derived IL6 Induces Immunosuppressive Peripheral Myeloid Cell PD-L1 and Promotes Tumor Growth.

Purpose

Experimental design

Results

Conclusions

Publications

Glioblastoma-Derived IL6 Induces Immunosuppressive Peripheral Myeloid Cell PD-L1 and Promotes Tumor Growth.

Similar Datasets

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