Mechanisms Driving Neutrophil-Induced T-cell Immunoparalysis in Ovarian Cancer.

T-cell activation and expansion in the tumor microenvironment (TME) are critical for antitumor immunity. Neutrophils in the TME acquire a complement-dependent T-cell suppressor phenotype that is characterized by inhibition of T-cell proliferation and activation through mechanisms distinct from those of myeloid-derived suppressor cells. In this study, we used ascites fluid supernatants (ASC) from patients with ovarian cancer as an authentic component of the TME to evaluate the effects of ASC on neutrophil function and mechanisms for neutrophil-driven immune suppression. ASC prolonged neutrophil life span, decreased neutrophil density, and induced nuclear hypersegmentation. Mass cytometry analysis showed that ASC induced 15 distinct neutrophil clusters. ASC stimulated complement deposition and signaling in neutrophils, resulting in surface mobilization of granule constituents, including NADPH oxidase. NADPH oxidase activation and phosphatidylserine signaling were required for neutrophil suppressor function, although we did not observe a direct role of extracellular reactive oxygen species in inhibiting T-cell proliferation. Postoperative surgical drainage fluid also induced a complement-dependent neutrophil suppressor phenotype, pointing to this effect as a general response to injury. Like circulating lymphocytes, ASC-activated neutrophils caused complement-dependent suppression of tumor-associated lymphocytes. ASC-activated neutrophils adhered to T cells and caused trogocytosis of T-cell membranes. These injury and signaling cues resulted in T-cell immunoparalysis characterized by impaired NFAT translocation, IL2 production, glucose uptake, mitochondrial function, and mTOR activation. Our results demonstrate that complement-dependent priming of neutrophil effector functions in the TME induces a T-cell nonresponsiveness distinct from established checkpoint pathways and identify targets for immunotherapy.See related Spotlight by Cassatella, p. 725.

Microtubules and Dynein Regulate Human Neutrophil Nuclear Volume and Hypersegmentation During H. pylori Infection.

Neutrophils (also called polymorphonuclear leukocytes, PMNs) are heterogeneous and can exhibit considerable phenotypic and functional plasticity. In keeping with this, we discovered previously that Helicobacter pylori infection induces N1-like subtype differentiation of human PMNs that is notable for profound nuclear hypersegmentation. Herein, we utilized biochemical approaches and confocal and super-resolution microscopy to gain insight into the underlying molecular mechanisms. Sensitivity to inhibition by nocodazole and taxol indicated that microtubule dynamics were required to induce and sustain hypersegmentation, and super-resolution Stimulated Emission Depletion (STED) imaging demonstrated that microtubules were significantly more abundant and longer in hypersegmented cells. Dynein activity was also required, and enrichment of this motor protein at the nuclear periphery was enhanced following H. pylori infection. In contrast, centrosome splitting did not occur, and lamin B receptor abundance and ER morphology were unchanged. Finally, analysis of STED image stacks using Imaris software revealed that nuclear volume increased markedly prior to the onset of hypersegmentation and that nuclear size was differentially modulated by nocodazole and taxol in the presence and absence of infection. Taken together, our data define a new mechanism of hypersegmentation that is mediated by microtubules and dynein and as such advance understanding of processes that regulate nuclear morphology.

In vitro activity of tedizolid against linezolid-resistant staphylococci and enterococci.

The tedizolid MIC of 27 clinical isolates of linezolid-resistant staphylococci and enterococci was determined. Tedizolid MICs were ≥1µg/mL and were 4- to 32-fold lower than those of linezolid. Linezolid resistance mechanisms included G2576T 23S rRNA gene and rplC and rplD mutations.