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 patientderived GBM explant cell cultures was assessed for cytokine expression and utilized to stimulate na€ ve 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 þ 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. Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001; ÃÃÃÃ , P < 0.0001.Lamano et al. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis):

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 ( = 0.028). FOXP3 elevation had a negative impact on overall survival (HR = 2.0; < 0.001). Peripheral PD-L1 positivity was associated with an increased Treg fraction ( = 0.008). Lymphocyte activation with PD-L1 co-stimulation resulted in greater iTreg expansion compared to activation alone (18.3% vs. 6.5%; < 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 ( < 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.

SummaryObjectiveReproductive dysfunction is a comorbidity that commonly occurs with temporal lobe epilepsy (TLE). Characterization of this comorbidity in various models of TLE in mice will greatly facilitate mechanistic investigations of the relationship between reproductive disorders and seizures initiated in the hippocampus. Here we investigate the impact on female reproductive estrous cyclicity in the intrahippocampal kainic acid mouse model of TLE and demonstrate the utility of using this model for future mechanistic studies.MethodsKainic acid (KA) or saline vehicle was stereotaxically injected in the right dorsal hippocampus of adult female C57BL/6J mice. Development of epilepsy was assessed by video monitoring for behavioral seizures. Reproductive function was assessed by daily estrous cycle monitoring and ovarian morphology. Estrous cycles were monitored for up to 2 months after injection. Ovarian morphology was examined by histological staining and assessment of follicular and luteal development.ResultsWe observed spontaneous behavioral seizures in 82% of kainic‐acid‐treated mice. Irregular estrous cycles developed within 2 months after kainic acid injection. Sixty‐seven percent of KA‐treated mice showed disrupted estrous cycles, typically characterized by increased estrous cycle length, increased time spent in diestrus (nonfertile stage), and decreased time spent in estrus by 42 days post‐KA injection. The estrous cycle disruption, however, was not accompanied by major changes in ovarian morphology or follicular development. KA‐treated mice also displayed increased weight gain compared to control mice.SignificanceThese data indicate that comorbid female irregular estrous cyclicity arises in the intrahippocampal kainic acid mouse model of TLE. This is the first demonstration of disrupted reproductive endocrine function in a mouse model of TLE initially produced by an insult specifically targeted to the hippocampus. This model should thus be useful for basic studies investigating the neural mechanisms driving comorbid reproductive dysfunction in epilepsy in women.