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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.

Methods pertaining to experiments presented as supplementary figures.

Supplementary Tables S1-S4.

Supplementary Figures S1-S7.

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.

Glioblastoma (GBM) promotes immunosuppression through upregulation of PD-L1 and regulatory T cell (Treg) expansion, but the association of these suppressive factors has not been well elucidated. Here, we investigate a role of PD-L1 in expanding Tregs and the value of targeting the PD-1 receptor to inhibit Treg expansion. Quantitative RNA sequencing data from The Cancer Genome Atlas were evaluated for an association between CD274 and FOXP3 transcript expressions and impact of FOXP3 on clinical outcomes. Peripheral leukocytes from patients with newly diagnosed GBM were profiled for PD-L1+ myeloid expressions and Treg abundance. Healthy lymphocytes were assessed for impact of recombinant PD-L1 on expansion of the inducible Treg (iTreg) population. iTreg function was evaluated by the capacity to suppress effector T cell proliferation. Specificity of responses were confirmed by pharmacologic inhibition of the PD-1 receptor. Increased PD-L1 mRNA expression in GBM corresponded to increased FOXP3 mRNA (p = 0.028). FOXP3 elevation had a negative impact on overall survival (HR = 2.0; p < 0.001). Peripheral PD-L1 positivity was associated with an increased Treg fraction (p = 0.008). Lymphocyte activation with PD-L1co-stimulation resulted in greater iTreg expansion compared to activation alone (18.3% vs. 6.5%; p < 0.001) and improved preservation of the Treg phenotype. Suppressive capacity on naïve T cell proliferation was sustained. Nivolumab inhibited PD-L1-induced Treg expansion (p < 0.001). These results suggest that PD-L1 may expand and maintain immunosuppressive Tregs, which are associated with decreased survival in glioma patients. Blockade of the PD-L1/PD-1 axis may reduce Treg expansion and further improve T cell function beyond the direct impact on effector cells.

Given the continued poor clinical outcomes and refractory nature of glioblastoma multiforme to traditional interventions, immunotherapy is gaining traction due to its potential for specific tumor-targeting and long-term antitumor protective surveillance. Currently, development of glioma immunotherapy relies on overall survival as an endpoint in clinical trials. However, the identification of surrogate immunologic biomarkers can accelerate the development of successful immunotherapeutic strategies. Immunomonitoring techniques possess the potential to elucidate immunological mechanisms of antitumor responses, monitor disease progression, evaluate therapeutic effect, identify candidates for immunotherapy, and serve as prognostic markers of clinical outcome. Current immunomonitoring assays assess delayed-type hypersensitivity, T cell proliferation, cytotoxic T-lymphocyte function, cytokine secretion profiles, antibody titers, and lymphocyte phenotypes. Yet, no single immunomonitoring technique can reliably predict outcomes, relegating immunological markers to exploratory endpoints. In response, the most recent immunomonitoring assays are incorporating emerging technologies and novel analysis techniques to approach the goal of identifying a competent immunological biomarker which predicts therapy responsiveness and clinical outcome. This review addresses the current status of immunomonitoring in glioma vaccine clinical trials with emphasis on correlations with clinical response.

Glioblastoma is a grade IV astrocytoma that is widely accepted in clinical neurosurgery as being an extremely lethal diagnosis. Long-term survival rates remain dismal, and even when tumors undergo gross resection with confirmation of total removal on neuroimaging, they invariably recur with even greater virulence. Standard therapeutic modalities as well as more contemporary treatments have largely resulted in disappointing improvements. However, the therapeutic potential of vaccine immunotherapy for malignant glioma should not be underestimated. In contrast to many of the available treatments, vaccine immunotherapy is unique because it offers the means of delivering treatment that is highly specific to both the patient and the tumor. Peptide, heat-shock proteins, and dendritic cell vaccines collectively encapsulate the majority of research efforts involving vaccine-based treatment modalities. In this review, important recent findings for these vaccine types are discussed in the context of ongoing clinical trials. Broad challenges to immunotherapy are also considered.