Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade.

The success in lung cancer therapy with Programmed Death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between Epidermal Growth Factor Receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased cytotoxic T cells and increased markers of T cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape, and mechanistically link treatment response to PD-1 inhibition.

Immunosuppression is frequently associated with malignancy and is particularly severe in patients with malignant glioma. Anergy and counterproductive shifts toward T H 2 cytokine production are long-recognized T-cell defects in these patients whose etiology has remained elusive for >30 years. We show here that absolute counts of both CD4 + T cells and CD4 + CD25 + FOXP3 + CD45RO + T cells (T regs ) are greatly diminished in patients with malignant glioma, but T regs frequently represent an increased fraction of the remaining CD4 compartment. This increased T reg fraction, despite reduced counts, correlates with and is sufficient to elicit the characteristic manifestations of impaired patient T-cell responsiveness in vitro. Furthermore, T reg removal eradicates T-cell proliferative defects and reverses T H 2 cytokine shifts, allowing T cells from patients with malignant glioma to function in vitro at levels equivalent to those of normal, healthy controls. Such restored immune function may give license to physiologic antiglioma activity, as in vivo, T reg depletion proves permissive for spontaneous tumor rejection in a murine model of established intracranial glioma. These findings dramatically alter our understanding of depressed cellular immune function in patients with malignant glioma and advance a role for T regs in facilitating tumor immune evasion in the central nervous system. (Cancer Res 2006; 66(6): 3294-302)

T cell dysfunction contributes to tumor immune escape in patients with cancer and is particularly severe amidst glioblastoma (GBM). Among other defects, T cell lymphopenia is characteristic, yet often attributed to treatment. We reveal that even treatment-naïve subjects and mice with GBM can harbor AIDS-level CD4 counts, as well as contracted, T cell-deficient lymphoid organs. Missing naïve T cells are instead found sequestered in large numbers in the bone marrow. This phenomenon characterizes not only GBM but a variety of other cancers, although only when tumors are introduced into the intracranial compartment. T cell sequestration is accompanied by tumor-imposed loss of S1P1 from the T cell surface and is reversible upon precluding S1P1 internalization. In murine models of GBM, hindering S1P1 internalization and reversing sequestration licenses T cell-activating therapies that were previously ineffective. Sequestration of T cells in bone marrow is therefore a tumor-adaptive mode of T cell dysfunction, whose reversal may constitute a promising immunotherapeutic adjunct.

STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer (NSCLC), especially in tumors harboring KRAS mutations. Many oncogenes promote immune escape, undermining the effectiveness of immunotherapies, but it is unclear whether inactivation of tumor suppressor genes such as STK11/LKB1 exert similar effects. In this study, we investigated the consequences of STK11/LKB1 loss on the immune microenvironment in a mouse model of KRAS-driven NSCLC. Genetic ablation of STK11/LKB1 resulted in accumulation of neutrophils with T cell suppressive effects, along with a corresponding increase in the expression of T cell exhaustion markers and tumor-promoting cytokines. The number of tumor-infiltrating lymphocytes was also reduced in LKB1-deficient mouse and human tumors. Furthermore, STK11/LKB1 inactivating mutations were associated with reduced expression of PD-1 ligand PD-L1 in mouse and patient tumors as well as in tumor-derived cell lines. Consistent with these results, PD-1 targeting antibodies were ineffective against Lkb1-deficient tumors. In contrast, treating Lkb1-deficient mice with an IL-6 neutralizing antibody or a neutrophil-depleting antibody yielded therapeutic benefits associated with reduced neutrophil accumulation and proinflammatory cytokine expression. Our findings illustrate how tumor suppressor mutations can modulate the immune milieu of the tumor microenvironment, and they offer specific implications for addressing STK11/LKB1 mutated tumors with PD-1 targeting antibody therapies.

Author Manuscript Published OnlineFirst on February 7, 2018; DOI: 10.1158/1078-0432.CCR-17-1846 Statement of Translational Relevance 44Primary malignant brain neoplasms are responsible for over 15,000 deaths annually in the United

Gliomas, the most common malignant primary brain tumours, remain universally lethal. Yet, seminal discoveries in the past 5 years have clarified the anatomy, genetics and function of the immune system within the central nervous system (CNS) and altered the paradigm for successful immunotherapy. The impact of standard therapies on the response to immunotherapy is now better understood, as well. This new knowledge has implications for a broad range of tumours that develop within the CNS. Nevertheless, the requirements for successful therapy remain effective delivery and target specificity, while the dramatic heterogeneity of malignant gliomas at the genetic and immunological levels remains a profound challenge.

Glioblastoma (World Health Organization grade IV) is an aggressive adult brain tumor that is inevitably fatal despite surgery, radiation, and chemotherapy. Treatment failures are attributed to combinations of cellular heterogeneity, including a subpopulation of oftenresistant cancer stem cells, aberrant vasculature, and noteworthy immune suppression. Current preclinical models and treatment strategies do not incorporate or address all these features satisfactorily. Herein, we describe a murine glioblastoma stem cell (GSC) model that recapitulates tumor heterogeneity, invasiveness, vascularity, and immunosuppressive microenvironment in syngeneic immunocompetent mice and should prove useful for a range of therapeutic studies. Using this model, we tested a genetically engineered oncolytic herpes simplex virus that is armed with an immunomodulatory cytokine, interleukin 12 (G47Δ-mIL12). G47Δ-mIL12 infects and replicates similarly to its unarmed oncolytic herpes simplex virus counterpart in mouse 005 GSCs in vitro, whereas in vivo, it significantly enhances survival in syngeneic mice bearing intracerebral 005 tumors. Mechanistically, G47Δ-mIL12 targets not only GSCs but also increases IFN-γ release, inhibits angiogenesis, and reduces the number of regulatory T cells in the tumor. The increased efficacy is dependent upon T cells, but not natural killer cells. Taken together, our findings demonstrate that G47Δ-mIL12 provides a multifaceted approach to targeting GSCs, tumor microenvironment, and the immune system, with resultant therapeutic benefit in a stringent glioblastoma model.