Purpose: Increased frequencies of myeloid-derived suppressor cells (MDSC) correlate with poor prognosis in patients with cancers. Tumor-derived prostaglandin-E2 (PGE2) plays an important role in inducing MDSCs. However, the detailed mechanisms of this induction remain unknown. To develop targeted therapies for MDSCs, we sought to investigate the molecular basis of PGE2-regulated accumulation of MDSCs and their functional consequence on natural killer (NK) cell activity. Experimental Design: The effects of PGE2 in inducing phenotypic, signaling, and functional alternations on monocytes were analyzed in vitro. Suppression of NK-cell activity by PGE2-treated monocytes was compared with that of freshly isolated CD14⁺HLA-DR^low/− monocytic MDSCs (moMDSC) from patients with melanoma. In addition, to explore the in vivo relevance of targeting PGE2 to reduce MDSC-mediated suppression of NK cells, we established a murine model, where tumor cells were disabled from cyclooxygenase-2 (COX-2) production. Results: Patient-derived moMDSCs inhibited NK-cell activity through the production of TGFβ. In vitro, binding of PGE2 to EP2 and EP4 receptors on monocytes activated the p38MAPK/ERK pathway and resulted in elevated secretion of TGFβ. Similar to moMDSCs, PGE2-treated monocytes potently suppressed NK-cell activity through production of TGFβ. Furthermore, silencing COX-2 in murine 4T1 tumor cells reduced the accumulation of CD11b⁺Gr1⁺ MDSCs in the spleen, resulting in concomitant improved in vivo clearance of NK-cell sensitive YAC-1 cells. Conclusions: Our results reveal an indispensable role of tumor-derived PGE2 in inducing MDSCs and suggest a favorable outcome of combining COX-2–targeted therapy and adoptive NK-cell transfer in patients with cancer. Clin Cancer Res; 20(15); 4096–106. ©2014 AACR.

The progression and metastatic capacity of solid tumors are strongly influenced by immune cells in the tumor microenvironment. In non–small cell lung cancer (NSCLC), accumulation of anti-inflammatory tumor-associated macrophages (TAM) is associated with worse clinical outcome and resistance to therapy. Here we investigated the immune landscape of NSCLC in the presence of protumoral TAMs expressing the macrophage receptor with collagenous structure (MARCO). MARCO-expressing TAM numbers correlated with increased occurrence of regulatory T cells and effector T cells and decreased natural killer (NK) cells in these tumors. Furthermore, transcriptomic data from the tumors uncovered a correlation between MARCO expression and the anti-inflammatory cytokine IL37. In vitro studies subsequently showed that lung cancer cells polarized macrophages to express MARCO and gain an immune-suppressive phenotype through the release of IL37. MARCO-expressing TAMs blocked cytotoxic T-cell and NK-cell activation, inhibiting their proliferation, cytokine production, and tumor killing capacity. Mechanistically, MARCO+ macrophages enhanced regulatory T (Treg) cell proliferation and IL10 production and diminished CD8 T-cell activities. Targeting MARCO or IL37 receptor (IL37R) by antibody or CRISPR knockout of IL37 in lung cancer cell lines repolarized TAMs, resulting in recovered cytolytic activity and antitumoral capacity of NK cells and T cells and downmodulated Treg cell activities. In summary, our data demonstrate a novel immune therapeutic approach targeting human TAMs immune suppression of NK- and T-cell antitumor activities. Significance: This study defines tumor-derived IL37 and the macrophage scavenger receptor MARCO as potential therapeutic targets to remodel the immune-suppressive microenvironment in patients with lung cancer.

The study of human macrophages and their ontogeny is an important unresolved issue. Here, we use a humanized mouse model expressing human cytokines to dissect the development of lung macrophages from human hematopoiesis in vivo. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) generated three macrophage populations, occupying separate anatomical niches in the lung. Intravascular cell labeling, cell transplantation, and fate-mapping studies established that classical CD14+ blood monocytes derived from HSPCs migrated into lung tissue and gave rise to human interstitial and alveolar macrophages. In contrast, non-classical CD16+ blood monocytes preferentially generated macrophages resident in the lung vasculature (pulmonary intravascular macrophages). Finally, single-cell RNA sequencing defined intermediate differentiation stages in human lung macrophage development from blood monocytes. This study identifies distinct developmental pathways from circulating monocytes to lung macrophages and reveals how cellular origin contributes to human macrophage identity, diversity, and localization in vivo.

Human cytomegalovirus (CMV)-induced adaptive natural killer (NK) cells display distinct phenotypic and functional characteristics, including properties of immune memory. We hypothesized that these cells may be more resistant to suppression mediated by immunoregulatory cell subsets, making them attractive for use in cancer therapy. Here we report that relative to conventional NK cells, adaptive NK cells express lower levels of the inhibitory receptor T-cell Ig and ITIM domain (TIGIT), which results in resistance to immune suppression mediated by myeloid-derived suppressor cells (MDSC), as derived from cytokine induction in normal blood or patients with myelodysplastic syndrome. In contrast, conventional NK cells were potently suppressed by MDSCs, an effect abrogated completely by TIGIT blockade. Mechanistically, TIGIT signaling in NK cells after MDSC coculture led to a decrease in the phosphorylation of ZAP70/Syk and ERK1/2. These effects were reversed by blocking TIGIT on NK cells or by inhibiting production of reactive oxygen species (ROS) by MDSCs, the latter of which upregulated the TIGIT ligand CD155 on MDSCs. Accordingly, the blunted cytotoxicity of NK cells cocultured with MDSCs against tumor cells could be reversed by blocking TIGIT or ROS production. Overall, our results show how adaptive NK cells arising in response to CMV infection can escape MDSC-mediated suppression, and defined TIGIT antagonists as a novel type of checkpoint inhibitor to enhance NK-cell–mediated responses against cancer and infection. Cancer Res; 76(19); 5696–706. ©2016 AACR.

Natural killer (NK) cells were discovered 40 years ago, by their ability to recognize and kill tumor cells without the requirement of prior antigen exposure. Since then NK cells have been seen as promising agents for cell-based cancer therapies. However, NK cells represent only a minor fraction of the human lymphocyte population. Their skewed phenotype and impaired functionality during cancer progression necessitates the development of clinical protocols to activate and expand to high numbers ex vivo to be able to infuse sufficient numbers of functional NK cells to the cancer patients. Initial NK cell-based clinical trials suggested that NK cell-infusion is safe and feasible with almost no NK cell-related toxicity, including graft-versus-host disease (GvHD). Complete remission and increased disease free survival is shown in a small number of patients with hematological malignances. Furthermore, successful adoptive NK cell based therapies from haploidentical donors have been demonstrated. Disappointingly, only limited anti-tumor effects have been demonstrated following NK cell infusion in patients with solid tumors. While NK cells have great potential in targeting tumor cells, the efficiency of NK cell functions in the tumor microenvironment is yet unclear. The failure of immune surveillance may in part be due to sustained immunological pressure on tumor cells resulting in the development of tumor escape variants that are invisible to the immune system. Alternatively, this could be due to the complex network of immune suppressive compartments in the tumor microenvironment, including myeloid-derived suppressor cells, tumor associated macrophages, and regulatory T cells. Although the negative effect of the tumor microenvironment on NK cells can be transiently reverted by ex vivo-expansion and long-term activation, the abovementioned NK cell/tumor microenvironment interactions upon reinfusion are not fully elucidated. Within this context, genetic modification of NK cells may provide new possibilities for developing effective cancer immunotherapies by improving NK cell responses and making them less susceptible to the tumor microenvironment. Within this review we will discuss clinical trials using NK cells with a specific reflection on novel potential strategies, such as genetic modification of NK cells and complementary therapies aimed at improving the clinical outcome of NK cell-based immune therapies.

Maturation of human natural killer (NK) cells as defined by accumulation of cell-surface expression of CD57 is associated with increased cytotoxic character and TNF and IFNγ production upon target-cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacologic inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK-cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFNγ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity. In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy. Cancer Res; 77(20); 5664–75. ©2017 AACR.