Advances in the understanding of the tumor microenvironment have led to devel-opment of immunotherapeutic strategies, such as chimeric antigen receptor T cells (CAR-Ts). However, despite success in blood malignancies, CAR-T therapies in solid tumors have been hampered by their restricted infiltration. Here, we used our understanding of early cytotoxic lymphocyte infiltration of human lymphocytes in solid tumors in vivo to investigate the receptors in normal, adjacent, and tumor tissues of primary non-small-cell lung cancer specimens. We found that CX3CL1-CX3CR1 reduction restricts cytotoxic cells from the solid-tumor bed, contributing to tumor escape. Based on this, we designed a CAR-T construct using the well-established natural killer group 2, member D (NKG2D) CAR-T expression together with overexpression of CX3CR1 to promote their infiltration. These CAR-Ts infiltrate tumors at higher rates than control-activated T cells or IL-15-overexpressing NKG2D CAR-Ts. This construct also had similar functionality in a liver-cancer model, demonstrating potential efficacy in other solid malignancies.

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.

One of the primary drivers of Phosphorus (P) limitation in aquatic systems is P adsorption to sediments. Sediments adsorb more P in freshwater compared to other natural solutions, but the mechanism driving this difference is poorly understood. To provide insights into the mechanism, we conducted batch experiments of P adsorption to calcite in freshwater and seawater, and used computer software to develop complexation models. Our simulations revealed three main reasons that, combining together, may explain the greater P adsorption to calcite in freshwater vs. seawater. First, aqueous speciation of P makes a difference. The ion pair CaPO4- is much more abundant in freshwater; although seawater has more Ca2+ ions, MgHPO40 and NaHPO40 are more thermodynamically favored. Second, the adsorbing species of P make a difference. The ion pair CaPO4 (the preferred adsorbate in freshwater) is able to access adsorption sites that are not available to HPO42- (the preferred adsorbate in seawater), thereby raising the maximum concentration of P that can adsorb to the calcite surface in freshwater. Third, water chemistry affects the competition among ions for surface sites. Other ions (including P) compete more effectively against CO32- when immersed in freshwater vs. seawater, even when the concentration of HCO3/CO32- is higher in freshwater vs. seawater. In addition, we found that under oligotrophic conditions, P adsorption is driven by the higher energy adsorption sites, and by the lower energy sites in eutrophic conditions. This study is the first to model P adsorption mechanisms to calcite in freshwater and seawater.

We have reported previously that CD33hi myeloid-derived suppressor cells (MDSCs) play a direct role in the pathogenesis of myelodysplastic syndromes (MDSs) and that their sustained activation contributes to hematopoietic and immune impairment, including modulation of PD1/PDL1. MDSCs can also limit the clinical activity of immune checkpoint inhibition in solid malignancies. We hypothesized that depletion of MDSCs may ameliorate resistance to checkpoint inhibitors and, hence, targeted them with AMV564 combined with anti-PD1 in MDS bone marrow (BM) mononuclear cells (MNCs) enhanced activation of cytotoxic T cells. AMV564 was active in vivo in a leukemia xenograft model when co-administered with healthy donor peripheral blood MNCs (PBMCs). Our findings provide a strong rationale for clinical investigation of AMV564 as a single agent or in combination with an anti-PD1 antibody and in particular for treatment of cancers resistant to checkpoint inhibitors.

Dendritic cells (DCs) are professional antigen presenting cells with a great capacity for cross-presentation of exogenous antigens from which robust anti-tumor immune responses ensue. However, this function is not always available and requires DCs to first be primed to induce their maturation. In particular, in the field of DC vaccine design, currently available methodologies have been limited in eliciting a sustained anti-tumor immune response. Mechanistically, part of the maturation response is influenced by the presence of stimulatory receptors relying on ITAM-containing activating adaptor molecules like DAP12, that modulates their function. We hypothesize that activating DAP12 in DC could force their maturation and enhance their potential anti-tumor activity for therapeutic intervention. For this purpose, we developed constitutively active DAP12 mutants that can promote activation of monocyte-derived DC. Here we demonstrate its ability to induce the maturation and activation of monocyte-derived DCs which enhances migration, and T cell stimulation in vitro using primary human cells. Moreover, constitutively active DAP12 stimulates a strong immune response in a murine melanoma model leading to a reduction of tumor burden. This provides proof-of-concept for investigating the pre-activation of antigen presenting cells to enhance the effectiveness of anti-tumor immunotherapies.

Single-cell transcriptomes are established by transcription factors (TFs), which determine a cell's gene-expression complement. Post-transcriptional regulation of single-cell transcriptomes, and the RNA binding proteins (RBPs) responsible, are more technically challenging to determine, and combinatorial TF-RBP coordination of single-cell transcriptomes remains unexplored. We used fluorescent reporters to visualize alternative splicing in single Caenorhabditis elegans neurons, identifying complex splicing patterns in the neuronal kinase sad-1. Most neurons express both isoforms, but the ALM mechanosensory neuron expresses only the exon-included isoform, while its developmental sister cell the BDU neuron expresses only the exon-skipped isoform. A cascade of three cell-specific TFs and two RBPs are combinatorially required for sad-1 exon inclusion. Mechanistically, TFs combinatorially ensure expression of RBPs, which interact with sad-1 pre-mRNA. Thus a combinatorial TF-RBP code controls single-neuron sad-1 splicing. Additionally, we find ‘phenotypic convergence,’ previously observed for TFs, also applies to RBPs: different RBP combinations generate similar splicing outcomes in different neurons.