Purpose Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and radiation is one of the main treatment modalities. However, cure rates remain low despite best available therapies. Immunotherapy is a promising modality that could work synergistically with radiation, which has been shown to increase antigen presentation and promote a proinflammatory tumor microenvironment. Programmed-death-1 (PD-1) is a surface receptor expressed on activated and exhausted T cells, which mediate T cell inhibition upon binding with its ligand PD-L1, expressed on many tumor types including human GBMs. We tested the combination of anti-PD-1 immunotherapy with stereotactic radiosurgery in a mouse orthotopic GBM model. Methods and Materials We performed intracranial implantation of mouse glioma cell line GL261 transfected with luciferase into C57BL/6 mice. Mice were stratified into 4 treatment groups: (1) control; (2) radiation only; (3) anti-PD-1 antibody only; and (4) radiation plus anti-PD-1 antibody. Overall survival was quantified. The mice were killed on day 21 after implantation to assess immunologic parameters in the brain/tumor, cervical lymph nodes, and spleen. Results Improved survival was demonstrated with combination anti-PD-1 therapy plus radiation compared with either modality alone: median survival was 25 days in the control arm, 27 days in the anti-PD-1 antibody arm, 28 days in the radiation arm, and 53 days in the radiation plus anti-PD-1 therapy arm (P<.05 by log-rank Mantle-Cox). Long-term survival was seen only in the combined treatment arm, with a fraction (15%–40%) of animals alive at day 180+ after treatment. Immunologic data on day 21 after implantation showed increased tumor infiltration by cytotoxic T cells (CD8+/interferon-γ+/tumor necrosis factor-α+) and decreased regulatory T cells (CD4+/FOXP3) in the combined treatment group compared with the single modality arms. Conclusions The combination of PD-1 blockade and localized radiation therapy results in long-term survival in mice with orthotopic brain tumors. These studies provide strong preclinical evidence to support combination trials in patients with GBM.

STAT3 activation has been observed in several autoimmune diseases, suggesting that STAT3-mediated pathways promote pathologic immune responses. We provide in vivo evidence that the fundamental role of STAT3 signaling in autoimmunity relates to its absolute requirement for generating TH17 T cell responses. We show that STAT3 is a master regulator of this pathogenic T cell subtype, acting at multiple levels in vivo, including TH17 T cell differentiation and cytokine production, as well as induction of RORγt and the IL-23R. Neither naturally occurring TH17 cells nor TH17-dependent autoimmunity occurs when STAT3 is ablated in CD4 cells. Furthermore, ablation of STAT3 signaling in CD4 cells results in increased TH1 responses, indicating that STAT3 signaling skews TH responses away from the TH1 pathway and toward the TH17 pathway. Thus, STAT3 is a candidate target for TH17-dependent autoimmune disease immunotherapy that could selectively inhibit pathogenic immune pathways.

Lymphocyte activation gene-3 (LAG-3) is a cell-surface molecule with diverse biologic effects on T cell function. We recently showed that LAG-3 signaling is important in CD4 + regulatory T cell suppression of autoimmune responses. Here, we demonstrate that LAG-3 maintains tolerance to self and tumor antigens via direct effects on CD8 + T cells using 2 murine systems. Naive CD8 + T cells express low levels of LAG-3, and expression increases upon antigen stimulation. Our data show increased levels of LAG-3 protein on antigen-specific CD8 + T cells within antigen-expressing organs or tumors. In vivo antibody blockade of LAG-3 or genetic ablation of the Lag-3 gene resulted in increased accumulation and effector function of antigen-specific CD8 + T cells within organs and tumors that express their cognate antigen. Most notably, combining LAG-3 blockade with specific antitumor vaccination resulted in a significant increase in activated CD8 + T cells in the tumor and disruption of the tumor parenchyma. A major component of this effect was CD4 independent and required LAG-3 expression by CD8 + T cells. Taken together, these data demonstrate a direct role for LAG-3 on CD8 + T cells and suggest that LAG-3 blockade may be a potential cancer treatment.

SUMMARY Interactions between tumor and immune cells either enhance or inhibit cancer progression. We show here that Stat3 signaling within the tumor microenvironment induces a pro-carcinogenic cytokine, IL-23, while inhibiting a central anti-carcinogenic cytokine, IL-12, thereby shifting the balance of tumor immunity towards carcinogenesis. Stat3 induces expression of IL-23, which is mainly produced by tumor-associated macrophages, via direct transcriptional activation of the IL-23/p19 gene. Furthermore, Stat3 inhibits NF-κB/c-Rel-dependent IL-12/p35 gene expression in tumor-associated dendritic cells. Tumor-associated regulatory T cells (Treg) express IL-23 receptor (IL-23R) which activates Stat3 in this cell type, leading to upregulation of the Treg-specific transcription factor, Foxp3, and the immunosuppressive cytokine, IL-10. These results demonstrate that Stat3 promotes IL-23-mediated pro-carcinogenic immune responses while inhibiting IL-12-dependent anti-tumor immunity. SIGNIFICANCE Recent studies suggest that two related cytokines, IL- 23 and IL- 12, play opposite roles in carcinogenesis. However, the underlying mechanisms regulating the balance between these cytokines in the tumor microenvironment have not been elucidated. Mechanisms by which IL-23 promotes tumor immune evasion also remain to be explored. Our results reveal that Stat3 signaling in the tumor microenvironment regulates the IL-12/IL-23 balance and further, that IL- 23 enhances the immunosuppressive activity of regulatory T cells within the tumor microenvironment, in part via IL-23 receptor dependent Stat3 activation. Because Stat3 is a point of convergence for signaling pathways commonly activated in cancer, our data reveal a mechanism by which oncogenic pathways regulate the immune microenvironment to promote tumor development.