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Inhibitor of apoptosis protein (IAP) antagonists are in clinical trials for a variety of cancers, and mouse models show synergism between IAP antagonists and anti-PD-1 immunotherapy. Although IAP antagonists affect the intrinsic signaling of tumor cells, their most pronounced effects are on immune cells and the generation of antitumor immunity. Here, we examined the effects of IAP antagonism on T-cell development using mouse fetal thymic organ culture and observed a selective loss of iNKT cells, an effector cell type of potential importance for cancer immunotherapy. Thymic iNKT-cell development probably failed due to increased strength of TCR signal leading to negative selection, given that mature iNKT cells treated with IAP antagonists were not depleted, but had enhanced cytokine production in both mouse and human ex vivo cultures. Consistent with this, mature mouse primary iNKT cells and iNKT hybridomas increased production of effector cytokines in the presence of IAP antagonists. In vivo administration of IAP antagonists and α-GalCer resulted in increased IFNγ and IL-2 production from iNKT cells and decreased tumor burden in a mouse model of melanoma lung metastasis. Human iNKT cells also proliferated and increased IFNγ production dramatically in the presence of IAP antagonists, demonstrating the utility of these compounds in adoptive therapy of iNKT cells. Cancer Immunol Res; 6(1); 25-35. ©2017 AACR ., (©2017 American Association for Cancer Research.)

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 the inactivation of tumor suppressor genes, such as STK11/LKB1, exerts 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 IL6-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., (©2016 American Association for Cancer Research.)

Purpose: The graft-versus-leukemia (GVL) reaction is an important example of immune-mediated tumor destruction. A coordinated humoral and cellular response accomplishes leukemia cell killing, but the specific targets remain largely uncharacterized. To learn more about the antigens that elicit antibodies during GVL reactions, we analyzed patients with advanced myelodysplasia (MDS) and acute myelogenous leukemia (AML) who received an autologous, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell vaccine early after allogeneic hematopoietic stem cell transplantation (HSCT)., Experimental Design: A combination of tumor-derived cDNA expression library screening, protein microarrays, and antigen-specific ELISAs were used to characterize sera obtained longitudinally from 15 patients with AML/MDS who were vaccinated early after allogeneic HSCT., Results: A broad, therapy-induced antibody response was uncovered, which primarily targeted intracellular proteins that function in growth, transcription/translation, metabolism, and homeostasis. Unexpectedly, antibodies were also elicited against eight secreted angiogenic cytokines that play critical roles in leukemogenesis. Antibodies to the angiogenic cytokines were evident early after therapy, and in some patients manifested a diversification in reactivity over time. Patients that developed antibodies to multiple angiogenic cytokines showed prolonged remission and survival., Conclusions: These results reveal a potent humoral response during GVL reactions induced with vaccination early after allogeneic HSCT and raise the possibility that antibodies, in conjunction with natural killer cells and T lymphocytes, may contribute to immune-mediated control of myeloid leukemias., (©2014 American Association for Cancer Research.)

Unlabelled: 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 EGF receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, CTL antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased CTLs 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., Significance: We show that autochthonous EGFR-driven lung tumors inhibit antitumor immunity by activating the PD-1/PD-L1 pathway to suppress T-cell function and increase levels of proinflammatory cytokines. These findings indicate that EGFR functions as an oncogene through non-cell-autonomous mechanisms and raise the possibility that other oncogenes may drive immune escape., (©2013 AACR.)

In an attempt to explore novel treatment modalities in acute myeloid leukemia (AML), we studied the role of costimulatory and cytokine gene immunotherapy in murine AML. We have previously shown that leukemic mice can be cured with CD80 transfected leukemic cells (B7. 1-AML vaccine) administered early in the course of the disease and that the failure B7.1-AML vaccines administered late cannot be attributed to immunosuppression induced by tumor growth. CD8+ T cells, which are necessary for tumor rejection, are activated rather than suppressed during the first half of the leukemic course in nonvaccinated mice. In this report, we question whether CD86 (B7.2) or the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), or tumor necrosis factor-alpha (TNF-alpha) can improve the vaccination potential of AML cells. The choice of cytokines was based on their combined and alone as well ability to direct the differentiation of CD34+ cells into potent antigen-presenting dendritic cells in vitro. Our studies show that (1) mice vaccinated with a leukemogenic number of AML cells engineered to express B7.2 (B7.2-AML) or to secrete GM-CSF, IL-4, or TNF-alpha (GM-, IL-4-, TNF-alpha-AML) do not develop leukemia; (2) GM-AML cells are tumorigenic in sublethally irradiated SJL/J mice but not in Swiss nu/nu mice, indicating that killing of tumor cells is not T-cell-dependent; (3) vaccines with irradiated GM-AML, but not B7.2-, IL-4-, or TNF-alpha-AML cells, can elicit leukemia-specific protective and therapeutic immunity; and (4) in head-to-head comparison experiments, vaccination with irradiated GM-AML is more potent than B7.1-AML, curing 80% and providing 20% prolonged survival of the leukemic mice at week 2, as opposed to cures only up to 1 week with B7.1-AML vaccines. These preclinical data emphasize that GM-CSF gene immunotherapy deserves clinical evaluation in AML.

Objective: The promise of immunotherapies developed against brain tumors in animal models has not been realized in human clinical trials. This may be because of the routine use of rodent tumors artificially induced by chemicals or viruses that do not accurately portray the intrinsic qualities of spontaneously arising human tumors and that often fail to incorporate the role of immunosuppressants, such as transforming growth factor-beta, that are secreted by human gliomas. From an astrocytoma that arose spontaneously in inbred VM/Dk mice, we have characterized a highly tumorigenic spontaneous murine astrocytoma cell line (SMA-560) that retains features of glial differentiation and naturally produces high levels of biologically active transforming growth factor-beta. We have used this model to determine whether cytokine production by tumor cells will inhibit intracerebral astrocytoma growth., Methods: Packaging cell lines producing replication-incompetent retroviral vectors were used to transfect the SMA-560 cell line in vitro with the genes encoding the murine cytokines interleukin (IL)-2, IL-3, IL-4, IL-6, tumor necrosis factor-alpha, gamma-interferon, or granulocyte-macrophage colony-stimulating factor or the costimulatory molecule B7.1 (CD80)., Results: Mice challenged intracerebrally with 5000 untransfected SMA-560 cells all succumbed to tumor within 30 days, with a median survival of 25 days. In contrast, mice challenged with SMA-560 cells producing IL-2, IL-4, or tumor necrosis factor-alpha each had a more than 400% increase in median survival (P < 0.0001). In these groups, 78.3% (18 of 23 mice), 66.7% (10 of 15 mice), and 60% (6 of 10 mice) of the mice, respectively, remained alive without evidence of tumor for longer than 100 days after the initial tumor challenge. All other cytokines tested and the expression of B7.1 failed to result in an increase in median survival., Conclusion: Using a spontaneous astrocytoma model in an inbred mouse strain, we have shown that cytokine production by glial tumors can abrogate their tumorigenicity in vivo despite production of transforming growth factor-beta. These results predict that approaches directed at cytokine production within intracerebral astrocytomas may be efficacious in human trials and that the "immunological privilege" of the brain may not be absolute under such conditions.

Vaccination with cytokine-producing tumor cells generates potent immune responses against tumors outside the central nervous system (CNS). The CNS, however, is a barrier to allograft and xenograft rejection, and established tumors within the CNS have failed to respond to other forms of systemic immunotherapy. To determine what barriers the "immunologically privileged" CNS would pose to cytokine-assisted tumor vaccines and what cytokines would be most efficacious against tumors within the CNS, we irradiated B16 murine melanoma cells producing murine interleukin 2 (IL-2), IL-3, IL-4, IL-6, gamma-interferon, or granulocyte-macrophage colony stimulating factor (GM-CSF) and used these cells as subcutaneous vaccines against tumors within the brain. Under conditions where untransfected B16 cells had no effect, cells producing IL-3, IL-6, or GM-CSF increased the survival of mice challenged with viable B16 cells in the brain. Vaccination with B16 cells producing IL-4 or gamma-interferon had no effect, and vaccination with B16 cells producing IL-2 decreased survival time. GM-CSF-producing vaccines were also able to increase survival in mice with pre-established tumors. The response elicited by GM-CSF-producing vaccines was found to be specific to tumor type and to be abrogated by depletion of CD8+ cells. Unlike the immunity generated against subcutaneous tumors by GM-CSF, however, the effector responses generated against tumors in the CNS were not dependent on CD4+ cells. These data suggest that cytokine-producing tumor cells are very potent stimulators of immunity against tumors within the CNS, but effector responses in the CNS may be different from those obtained against subcutaneous tumors.