Siqi Chen

Host miR155 Promotes Tumor Growth through a Myeloid-Derived Suppressor Cell–Dependent Mechanism

miR155 is a regulator of immune cell development and function that is generally thought to be immunostimulatory. However, we report here that genetic ablation of miR155 renders mice resistant to chemical carcinogenesis and the growth of several transplanted tumors, suggesting that miR155 functions in immunosuppression and tumor promotion. Host miR155 deficiency promoted overall antitumor immunity despite the finding of defective responses of miR155-deficient dendritic cells and antitumor T cells. Further analysis of immune cell compartments revealed that miR155 regulated the accumulation of functional myeloid-derived suppressive cells (MDSC) in the tumor microenvironment. Specifically, miR155 mediated MDSC suppressor activity through at least two mechanisms, including SOCS1 repression and a reduced ability to license the generation of CD4(+)Foxp3(+) regulatory T cells. Importantly, we demonstrated that miR155 expression was required for MDSC to facilitate tumor growth. Thus, our results revealed a contextual function for miR155 in antitumor immunity, with a role in MDSC support that appears to dominate in tumor-bearing hosts. Overall, the balance of these cellular effects appears to be a root determinant of whether miR155 promotes or inhibits tumor growth.

Graft-versus-Host Disease Is Enhanced by Selective CD73 Blockade in Mice

CD73 functions as an ecto-5′-nucleotidase to produce extracellular adenosine that has anti-inflammatory and immunosuppressive activity. We here demonstrate that CD73 helps control graft-versus-host disease (GVHD) in mouse models. Survival of wild-type (WT) recipients of either allogeneic donor naïve CD73 knock-out (KO) or WT T cells was similar suggesting that donor naïve T cell CD73 did not contribute to GVHD. By contrast, donor CD73 KO CD4+CD25+ regulatory T cells (Treg) had significantly impaired ability to mitigate GVHD mortality compared to WT Treg, suggesting that CD73 on Treg is critical for GVHD protection. However, compared to donor CD73, recipient CD73 is more effective in limiting GVHD. Pharmacological blockade of A2A receptor exacerbated GVHD in WT recipients, but not in CD73 KO recipients, suggesting that A2 receptor signaling is primarily implicated in CD73-mediated GVHD protection. Moreover, pharmacological blockade of CD73 enzymatic activity induced stronger alloreactive T cell activity, worsened GVHD and enhanced the graft-versus-leukemia (GVL) effect. These findings suggest that both donor and recipient CD73 protects against GVHD but also limits GVL effects. Thus, either enhancing or blocking CD73 activity has great potential clinical application in allogeneic bone marrow transplants.

Targeting Ornithine Decarboxylase by α-Difluoromethylornithine Inhibits Tumor Growth by Impairing Myeloid-Derived Suppressor Cells.

α-Difluoromethylornithine (DFMO) is currently used in chemopreventive regimens primarily for its conventional direct anticarcinogenesic activity. However, little is known about the effect of ornithine decarboxylase (ODC) inhibition by DFMO on antitumor immune responses. We showed in this study that pharmacologic blockade of ODC by DFMO inhibited tumor growth in intact immunocompetent mice, but abrogated in the immunodeficient Rag1−/− mice, suggesting that antitumor effect of DFMO is dependent on the induction of adaptive antitumor T cell immune responses. Depletion of CD8+ T cells impeded the tumor-inhibiting advantage of DFMO. Moreover, DFMO treatment enhanced antitumor CD8+ T cell infiltration and IFN-γ production and augmented the efficacy of adoptive T cell therapy. Importantly, DFMO impaired Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs) suppressive activity through at least two mechanisms, including reducing arginase expression and activity and inhibiting the CD39/CD73-mediated pathway. MDSCs were one primary cellular target of DFMO as indicated by both adoptive transfer and MDSC-depletion analyses. Our findings establish a new role of ODC inhibition by DFMO as a viable and effective immunological adjunct in effective cancer treatment, thereby adding to the growing list of chemoimmunotherapeutic applications of these agents.

Exogenous IL-33 Restores Dendritic Cell Activation and Maturation in Established Cancer

The role of IL-33, particularly in tumor growth and tumor immunity, remains ill-defined. We show that exogenous IL-33 can induce robust antitumor effect through a CD8+ T cell–dependent mechanism. Systemic administration of rIL-33 alone was sufficient to inhibit growth of established tumors in transplant and de novo melanoma tumorigenesis models. Notably, in addition to a direct action on CD8+ T cell expansion and IFN-γ production, rIL-33 therapy activated myeloid dendritic cells (mDCs) in tumor-bearing mice, restored antitumor T cell activity, and increased Ag cross-presentation within the tumor microenvironment. Furthermore, combination therapy consisting of rIL-33 and agonistic anti-CD40 Abs demonstrated synergistic antitumor activity. Specifically, MyD88, an essential component of the IL-33 signaling pathway, was required for the IL-33–mediated increase in mDC number and upregulation in expression of costimulatory molecules. Importantly, we identified that the IL-33 receptor ST2, MyD88, and STAT1 cooperate to induce costimulatory molecule expression on mDCs in response to rIL-33. Thus, our study revealed a novel IL-33–ST2–MyD88–STAT1 axis that restores mDC activation and maturation in established cancer and, thereby, the magnitude of antitumor immune responses, suggesting a potential use of rIL-33 as a new immunotherapy option to treat established cancer.

Cutting Edge: MicroRNA-223 Regulates Myeloid Dendritic Cell–Driven Th17 Responses in Experimental Autoimmune Encephalomyelitis

Myeloid cells play a crucial role in the induction and sustained inflammation in neuroinflammatory disorders, such as multiple sclerosis. miR-223, a myeloid cell–specific microRNA, is one of the most upregulated microRNAs in multiple sclerosis patients. We demonstrate that miR-223–knockout mice display significantly reduced active and adoptive-transfer experimental autoimmune encephalomyelitis that is characterized by reduced numbers of myeloid dendritic cells (mDCs) and Th17 cells in the CNS. Knockout mDCs have increased PD-L1 and decreased IL-1β, IL-6, and IL-23 expression, as well as a reduced capacity to drive Th17, but not Th1, cell differentiation. Thus, miR-223 controls mDC-induced activation of pathologic Th17 responses during autoimmune inflammation.

Exogenous IL-33 overcomes T cell tolerance in murine acute myeloid leukemia

Emerging studies suggest that dominant peripheral tolerance is a major mechanism of immune escape in disseminated leukemia. Using an established murine acute myeloid leukemia (AML) model, we here show that systemic administration of recombinant IL-33 dramatically inhibits the leukemia growth and prolongs the survival of leukemia-bearing mice in a CD8+ T cell dependent manner. Exogenous IL-33 treatment enhanced anti-leukemia activity by increasing the expansion and IFN-γ production of leukemia-reactive CD8+ T cells. Moreover, IL-33 promoted dendritic cell (DC) maturation and activation in favor of its cross presentation ability to evoke a vigorous anti-leukemia immune response. Finally, we found that the combination of PD-1 blockade with IL-33 further prolonged the survival, with half of the mice achieving complete regression. Our data establish a role of exogenous IL-33 in reversing T cell tolerance, and suggest its potential clinical implication into leukemia immunotherapy.

WEE1 inhibition by MK1775 as a single-agent therapy inhibits ovarian cancer viability

Wee1-like protein kinase (WEE1) physiologically serves a key function in maintaining the integrity of the cell genome through mediating the activation of cyclin-dependent kinase (CDK)1 and CDK2. Increased expression of WEE1 has been associated with the poor prognosis of patients with ovarian cancer. The present study aimed at examining the in vitro and in vivo antitumor activity of MK1775, a potent pharmacological inhibitor of WEE1, as a single agent against ovarian cancer cells. The cytotoxicity of MK1775 was examined in a panel of tumor cells using MTT in vitro. Subsequently, a cell apoptosis assay was performed in ovarian cancer SKOV3 and ID8 cells to characterize the function of MK1775 in tumor cell apoptosis, under either wild-type tumor protein 53 (p53) or null p53 status. In addition, cell cycle analysis and a western blot analysis were performed to validate the effect of MK1775 on cell cycle progression and to elucidate the underlying molecular mechanism of cell death. Finally, the in vivo antitumor efficacy of MK1775 as a single agent at a clinical well-tolerated dose was determined. A dose-dependent inhibitory effect of MK1775 on tumor cell viability was determined in distinct cell lines, including B16F10, LLC1, BPS1, EG7, ID8 and SKOV3. Results from the cell cycle analysis and western blotting indicated that MK1775 abrogated the G2/M checkpoint through inhibiting the phosphorylation of CDK1 and inducing the apoptosis of ovarian cancer cells that lacked mutations in p53 and breast cancer 1 (BRCA1). Additionally, a significant antitumor effect of MK1775 was observed in C57BL/6 mice bearing syngeneic ID8 ovarian tumors. The results of the present study supported the use of MK1775 as a monotherapy agent in ovarian cancer. MK1775 was effective at inducing mitotic catastrophe, independent of p53 and BRCA1 mutations. Therefore, WEE1 inhibition by MK1775 requires additional investigation to identify novel combination approaches in ovarian cancer therapy with the current DNA damaging agents, including irradiation treatment and cell cycle checkpoint inhibitors.

Comprehensive T-cell immunophenotyping and next-generation sequencing of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinomas

The success of programmed cell death 1 (PD‐1) inhibition in achieving a clinical response in a subset of head and neck squamous cell carcinoma (HNSCC) patients emphasizes the need to better understand the immunobiology of HNSCC. Immunophenotyping was performed for 30 HCSCC patients [16 human papillomavirus (HPV)‐positive; 14 HPV‐negative] on matched tissue from the primary tumour site, locally metastatic cervical lymph nodes (LNs), uninvolved local cervical LNs, and peripheral blood. CD4+ and CD8+ T‐cell lymphocytes obtained from tissue were analysed for expression levels of the inhibitory receptors PD‐1, TIM‐3 and CTLA‐4. Next‐generation sequencing of the T‐cell receptor (TCR) β chain was performed on patients (n = 9) to determine receptor repertoire diversity and for clonality analysis. HPV‐negative HNSCC patients, particularly those with stage IV disease, had significantly higher proportions of CD8+ T cells expressing CTLA‐4 in tumour tissue (P = 0.0013) and in peripheral blood (P = 0.0344) than HPV‐positive patients, as well as higher expression levels of TIM‐3+PD‐1+ CD8+ T cells (P = 0.0072) than controls. For all patients, PD‐1 expression on CD8+ T cells – particularly in HPV‐negative HNSCC cases – strongly correlated (r = 0.63, P = 0.013) with tumour size at the primary site. The top CD8+ TCR clones from tumour tissue significantly overlapped with circulating peripheral blood TCR clones (r = 0.946), and HPV‐positive patients had frequently expanded TCR clones that were more hydrophobic – and potentially more immunogenic – than those from HPV‐negative patients. Collectively, our findings demonstrate, for the first time, that high‐stage HPV‐negative HNSCC patients with primary tumours at different sites in the head and neck have elevated peripheral CTLA‐4+CD8+ T‐cell levels, that tumour‐familiar CD8+ T cells are detectable in peripheral blood from HNSCC patients, and that TCRs from HPV‐positive HNSCC patients potentially recognize distinctly immunogenic cognate antigens. However, our findings are preliminary, and need to be further confirmed in a larger patient cohort; also, how these factors affect patient response to immunotherapy needs to be determined. Copyright

[Corrigendum] WEE1 inhibition by MK1775 as a single‑agent therapy inhibits ovarian cancer viability

[This corrects the article DOI: 10.3892/ol.2017.6584.].

Abstract NTOC-117: CD73 BLOCKADE OVERCOMES RESISTANCE TO AGONISTIC ANTI-CD137 ANTIBODY THERAPY IN OVARIAN CANCER

Agonist antibodies (Ab) directed against costimulatory molecule CD137 (4-1BB) on the surface of antigen-primed T-lymphocytes are currently in various stages of pre-clinical and clinical development in advanced ovarian cancers, though therapeutic benefit has been limited as single agents. Here, we identified a novel mechanism of tumor-reactive CD8+ T cell unresponsiveness in ovarian cancer driven by the upregulation of CD73 (ecto-5′-nucleotidase), which prevents CD8+ T cell activity from engagement of the CD137 costimulatory molecule. Accordingly, CD73-deificient mice treated with anti-CD137 Ab survived significantly longer wild-type mice against an ovarian tumor challenge. To explore the therapeutic relevance of these observations, we combined anti-CD137 and anti-CD73 Ab to treat mice with established ovarian cancers. This combination therapy induced tumor regression associated with enhanced antitumor CD8+ T cell responses and reduced CD4+Foxp3+ regulatory T cells number and function. Mechanistically, CD137 engagement-triggered antitumor T cell activity was diminished in response to microenvironmental transforming growth factor-β (TGF-β) that was essential for CD73 induction. Furthermore, extracellular adenosine generation by CD73 abrogated T cell activity through A2BR activation in the face of agonistic anti-CD137 treatment. Our study demonstrates that CD73 blockade has critical implications for effective clinical targeting of CD137 and possibly other costimulatory molecules. Note: This abstract was not presented at the conference. Citation Format: Siqi Chen, Jie Fan, Donye Dominguez, Qin Lei, Alan Long, Deyu Fang, Bin Zhang. CD73 BLOCKADE OVERCOMES RESISTANCE TO AGONISTIC ANTI-CD137 ANTIBODY THERAPY IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-117.