Blockade of the TIGIT-CD155/CD112 axis enhances functionality of NK-92 but not cytokine-induced memory-like NK cells toward CD155-expressing acute myeloid leukemia.

TIGIT is an alternative checkpoint receptor (CR) whose inhibition promotes Graft-versus-Leukemia effects of NK cells. Given the significant immune-permissiveness of NK cells circulating in acute myeloid leukemia (AML) patients, we asked whether adoptive transfer of activated NK cells would benefit from additional TIGIT-blockade. Hence, we characterized cytokine-induced memory-like (CIML)-NK cells and NK cell lines for the expression of inhibitory CRs. In addition, we analyzed the transcription of CR ligands in AML patients (CCLE and Beat AML 2.0 cohort) in silico and evaluated the efficacy of CR blockade using in vitro cytotoxicity assays, CD69, CD107a and IFN-γ expression. Alternative but not classical CRs were abundantly expressed on healthy donor NK cells and even further upregulated on CIML-NK cells. In line with our finding that CD155, one important TIGIT-ligand, is reliably expressed on AMLs, we show improved killing of CD155+-AML blasts by NK-92 but interestingly not CIML-NK cells in the presence of TIGIT-blockade. Additionally, our in silico data (n = 671) show that poor prognosis AML patients rather displayed a CD86low CD112/CD155high phenotype, whereas patients with a better outcome rather exhibited a CD86high CD112/CD155low phenotype. Collectively, our data evidence that the complex CR ligand expression profile on AML blasts may be one explanation for the intrinsic NK cell exhaustion observed in AML patients which might be overcome with adoptive NK-92 transfer in combination with TIGIT-blockade.

Gold Nanorods Induce Endoplasmic Reticulum Stress and Autocrine Inflammatory Activation in Human Neutrophils.

Gold nanorods (AuNRs) are promising agents for diverse biomedical applications such as drug and gene delivery, bioimaging, and cancer treatment. Upon in vivo application, AuNRs quickly interact with cells of the immune system. On the basis of their strong intrinsic phagocytic activity, polymorphonuclear neutrophils (PMNs) are specifically equipped for the uptake of particulate materials such as AuNRs. Therefore, understanding the interaction of AuNRs with PMNs is key for the development of safe and efficient therapeutic applications. In this study, we investigated the uptake, intracellular processing, and cell biological response induced by AuNRs in PMNs. We show that uptake of AuNRs mainly occurs via phagocytosis and macropinocytosis with rapid deposition of AuNRs in endosomes within 5 min. Within 60 min, AuNR uptake induced an unfolded protein response (UPR) along with induction of inositol-requiring enzyme 1 α (IREα) and features of endoplasmic reticulum (ER) stress. This early response was followed by a pro-inflammatory autocrine activation loop that involves LOX1 upregulation on the cell surface and increased secretion of IL8 and MMP9. Our study provides comprehensive mechanistic insight into the interaction of AuNRs with immune cells and suggests potential targets to limit the unwanted immunopathological activation of PMNs during application of AuNRs.

Coating of cochlear implant electrodes with bioactive DNA-loaded calcium phosphate nanoparticles for the local transfection of stimulatory proteins.

Calcium phosphate nanoparticles were loaded with nucleic acids to enhance the on-growth of tissue to a cochlear implant electrode. The nanoparticle deposition on a metallic electrode surface is possible by electrophoretic deposition (EPD) or layer-by-layer deposition (LbL). Impedance spectroscopy showed that the coating layer did not interrupt the electrical conductance at physiological frequencies and beyond (1-40,000 Hz). The transfection was demonstrated with the model cell lines HeLa and 3T3 as well as with primary explanted spiral ganglion neurons (rat) with the model protein enhanced green fluorescent protein (EGFP). The expression of the functional protein brain-derived neurotrophic factor (BDNF) was also shown. Thus, a coating of inner-ear cochlear implant electrodes with nanoparticles that carry nucleic acids will enhance the ongrowth of spiral ganglion cell axons for an improved transmission of electrical pulses.

Immunophenotyping of Circulating Myeloid-Derived Suppressor Cells (MDSC) in the Peripheral Blood of Cancer Patients.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of pathologically expanded myeloid cells with immunosuppressive activity. According to their phenotype, MDSC can be divided into three major subpopulations: early stage MDSC (e-MDSC), lacking myeloid lineage markers, monocytic MDSC (M-MDSC), and granulocytic MDSC (PMN-MDSC). Additionally, PMN-MDSC can be subdivided based on their activation and differentiation status, although it is not clear how this status contributes to immunosuppression and disease pathology. Here, we describe an immunophenotyping and gating strategy for the identification and isolation of MDSC subsets based on fluorescence-activated cell sorting. This method allows direct comparison of MDSC subsets in clinical settings.

Isolation of Human Circulating Myeloid-Derived Suppressor Cells and Analysis of Their Immunosuppressive Activity.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of myeloid cells with potent immunosuppressive activity and characterized by a pathological state of activation. The T cell suppression assay is the most common method to evaluate the suppressive capacity of MDSC. Identifying the suppressive potential of different MDSC subsets within individual donors is key for understanding the biology of MDSC and their clinical relevance. Here we describe assays to ascertain and quantify the suppression of autologous T cells by human MDSC. These include the dye dilution proliferation assay for flow cytometry and the detection of IFNγ production by T cells using flow cytometry and sandwich ELISA.