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NK cell migration and activation are crucial elements of tumor immune surveillance. In mammary carcinomas, the number and function of NK cells is diminished, despite being positively associated with clinical outcome. MicroRNA-155 (miR-155) has been shown to be an important regulator of NK cell activation through its interaction with SHIP-1 downstream of inhibitory NK receptor signaling, but has not been explored in regard to NK cell migration. Here, we explored the migratory potential and function of NK cells in subcutaneous AT3 in mice lacking miR-155. Without tumor, these bic/miR-155-/mice possess similar numbers of NK cells that exhibit comparable surface levels of cytotoxic receptors as NK cells from wildtype (WT) mice. Isolated miR-155-/-NK cells also exhibit equivalent cytotoxicity towards tumor targets in vitro compared to isolated WT control NK cells, despite overexpression of known miR-155 gene targets. NK cells isolated from miR-155-/mice exhibit impaired Factin polymerization and migratory capacity in Boyden-chamber assays in response chemokine (C-C motif) ligand 2 (CCL2). This migratory capacity could be normalized in the presence of SHIP-1 inhibitors. Of note, miR-155-/mice challenged with mammary carcinomas exhibited heightened tumor burden which correlated with a lower number of tumor-infiltrating NK1.1 + cells. Our results support a novel, physiological role for SHIP-1 in the control of NK cell tumor trafficking, and implicate miR-155 in the regulation of NK cell chemotaxis, in the context of mammary carcinoma. This may implicate dysfunctional NK cells in the lack of tumor clearance in mice.

Myelodysplastic Syndromes (MDSs) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, including NLRP3 inflammasome driven pyroptotic cell death. We recently reported that the danger-associated molecular pattern (DAMP) oxidized mitochondrial DNA (ox-mtDNA) is diagnostically increased in MDS plasma although the functional consequences remain poorly defined. We hypothesized that ox-mtDNA is released into the cytosol, upon NLRP3 inflammasome pyroptotic lysis, where it propagates and further enhances the inflammatory cell death feed-forward loop onto healthy tissues. This activation can be mediated via ox-mtDNA engagement of Toll-like receptor 9 (TLR9), an endosomal DNA sensing pattern recognition receptor known to prime and activate the inflammasome propagating the IFN-induced inflammatory response in neighboring healthy hematopoietic stem and progenitor cells (HSPCs), which presents a potentially targetable axis for the reduction in inflammasome activation in MDS. We found that extracellular ox-mtDNA activates the TLR9-MyD88-inflammasome pathway, demonstrated by increased lysosome formation, IRF7 translocation, and interferon-stimulated gene (ISG) production. Extracellular ox-mtDNA also induces TLR9 redistribution in MDS HSPCs to the cell surface. The effects on NLRP3 inflammasome activation were validated by blocking TLR9 activation via chemical inhibition and CRISPR knockout, demonstrating that TLR9 was necessary for ox-mtDNA-mediated inflammasome activation. Conversely, lentiviral overexpression of TLR9 sensitized cells to ox-mtDNA. Lastly, inhibiting TLR9 restored hematopoietic colony formation in MDS BM. We conclude that MDS HSPCs are primed for inflammasome activation via ox-mtDNA released by pyroptotic cells. Blocking the TLR9/ox-mtDNA axis may prove to be a novel therapeutic strategy for MDS.

We have previously reported that the overexpression of S100A9 drives the development of Myelodysplastic Syndrome (MDS) through expansion of Myeloid Derived Suppressor Cells (MDSC) and promotion of pyroptosis. Despite the identified role of S100A9's effects on MDSC, and hematopoietic stem and progenitor cells (HSPC), as well as their establishment of an immunosuppressive microenvironment, the effects of S100A9 on adaptive immunity in MDS progression are less clear. Here, we report for the first time the unidentified role of S100A9 on T cell function in MDS that may lead to impaired immunosurveillance in the disease. Danger Associated Molecular Pattern (DAMP) S100A9 is a known ligand for the Pattern Recognition Receptor (PRR) Receptor for Advanced Glycation Endproducts (RAGE). We investigated RAGE surface expression by flow cytometry on MDS bone marrow resident T cells vs those derived from healthy donor bone marrow. We found significantly (p=0.04) higher RAGE surface expression on T cells from MDS bone marrow, and this expression was restricted to the CD4 lineage. To ascertain the effects of S100A9 on RAGE+CD4+ T cell function, we performed flow cytometry in a time course experiment post-T cell activation. Without S100A9 treatment, cell surface RAGE expression was low in activated T cells from healthy donors, but extended treatment with recombinant human S100A9 resulted in increased RAGE expression, suggesting a positive feedback loop for this DAMP. Unlike activated T cells, T cells not exposed to activating conditions did not display upregulated RAGE expression after S100A9 treatment. This indicates that this may be a post-activation switch, acting as a checkpoint for the T cell in the context of excessive damage signaling by DAMP S100A9. In order to further characterize the functional consequences of RAGE engagement, we performed transcription factor staining paired with a cytometric bead array for secreted cytokines in activated T cells treated with S100A9. Tumor Necrosis Factor Alpha (TNFa), IL-10 and IL-6 were induced by S100A9, indicating perhaps some degree of polarization induced by this DAMP. Commercially available RAGE V-domain inhibitor FPS-ZM1 blunted this cytokine signaling, indicating a significant portion of this cytokine production is indeed mediated through RAGE. In addition, we performed lipophilic dye dilution assays to track the effects S100A9 has on T cell proliferation following activation. S100A9 significantly decreased proliferative response under normal stimulatory conditions. Similar inhibition was seen in T cells derived from PBMC, MDS, or healthy donor bone marrow resident T cells, suggesting that the consequences of RAGE engagement are not disease specific. To rule out apoptosis as a potential cause for this halt in proliferation, we stained the cells with Annexin V and Propidium Iodide. To further elucidate how S100A9 might be affecting T cell proliferation, we analyzed cell cycle profiles following activation and S100A9 treatment. T cells treated with S100A9 showed a repressed cell cycle prior to G2, compared to T cells activated without any S100A9 treatment, suggesting a possible G1/S arrest. The evidence obtained in our study suggests any role of T cell dysfunction mediated by RAGE in MDS may be directly linked to the increased levels of S100A9 in the bone marrow microenvironment. Our work represents a novel mechanism of T cell dysfunction that may lead to a lack of responsiveness in the context of a disease known to overexpress the RAGE ligand S100A9. Capitalizing on this novel checkpoint can potentially be used both as novel biomarker and as a therapeutic target in the future to restore T cell immunosurveillance to a functional state in MDS. Disclosures List: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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