Figures S1-S16 and Table S1 and S2, which depict additional results (PDF) The authors declare the following competing financial interest(s): W.Z. is a co-founder of Velox Biosystems Inc., Baylx Inc., and Amberstone Biosciences Inc. J.L. has equity in Codiak BioSciences and holds the rights to multiple extracellular vesicle diagnostics and therapeutics patents.

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.

Myeloid-derived suppressor cells (MDSCs) are innate immune cells that acquire the capacity to suppress adaptive immune responses during cancer. It remains elusive how MDSCs differ from their normal myeloid counterparts, which limits our ability to specifically detect and therapeutically target MDSCs during cancer. Here, we sought to determine the molecular features of breast cancer–associated MDSCs using the widely studied mouse model based on the mouse mammary tumor virus (MMTV) promoter–driven expression of the polyomavirus middle T oncoprotein (MMTV-PyMT). To identify MDSCs in an unbiased manner, we used single-cell RNA sequencing to compare MDSC-containing splenic myeloid cells from breast tumor–bearing mice with wild-type controls. Our computational analysis of 14,646 single-cell transcriptomes revealed that MDSCs emerge through an aberrant neutrophil maturation trajectory in the spleen that confers them an immunosuppressive cell state. We establish the MDSC-specific gene signature and identify CD84 as a surface marker for improved detection and enrichment of MDSCs in breast cancers.

Coxsackievirus B (CVB) is a common enterovirus that can cause various systemic inflammatory diseases. Because CVB lacks an envelope, it has been thought to be inherently cytolytic, wherein CVB can escape from the infected host cell only by causing it to rupture. In recent years, however, we and others have observed that various naked viruses, such as CVB, can trigger the release of infectious extracellular microvesicles (EMVs) that contain viral material. This mode of cellular escape has been suggested to allow the virus to be masked from the adaptive immune system. Additionally, we have previously reported that these viral EMVs have LC3, suggesting that they originated from autophagosomes. We now report that CVB-infected cells trigger DRP1-mediated fragmentation of mitochondria, which is a precursor to autophagic mitochondrial elimination (mitophagy). However, rather than being degraded by lysosomes, mitochondrion-containing autophagosomes are released from the cell. We believe that CVB localizes to mitochondria, induces mitophagy, and subsequently disseminates from the cell in an autophagosome-bound mitochondrion-virus complex. Suppressing the mitophagy pathway in HL-1 cardiomyocytes with either small interfering RNA (siRNA) or Mdivi-1 caused marked reduction in virus production. The findings in this study suggest that CVB subverts mitophagy machinery to support viral dissemination in released EMVs. Coxsackievirus B (CVB) can cause a number of life-threatening inflammatory diseases. Though CVB is well known to disseminate via cytolysis, recent reports have revealed a second pathway in which CVB can become encapsulated in host membrane components to escape the cell in an exosome-like particle. Here we report that these membrane-bound structures derive from mitophagosomes. Blocking various steps in the mitophagy pathway reduced levels of intracellular and extracellular virus. Not only does this study reveal a novel mechanism of picornaviral dissemination, but also it sheds light on new therapeutic targets to treat CVB and potentially other picornaviral infections.

One Sentence Summary:We used single cell transcriptomics to identify the unique molecular features distinguishing myeloid-derived suppressor cells (MDSCs) from their normal, myeloid counterparts, which enabled us to reveal distinct transitory gene expression changes during their maturation in the spleen, and to identify novel cell surface markers for improved detection and isolation of MDSCs.Abstract: Myeloid-derived suppressor cells (MDSCs) are innate immune cells that acquire the capacity to suppress adaptive immune responses during cancer. It remains elusive how MDSCs differ from their normal myeloid counterparts, which limits our ability to specifically detect and therapeutically target MDSCs during cancer. Here, we used single-cell RNAseq to compare MDSC-containing splenic myeloid cells from breast tumor-bearing mice to wildtype controls.Our computational analysis of 14,646 single-cell transcriptomes reveals that MDSCs emerge through a previously unrealized aberrant neutrophil maturation trajectory in the spleen giving rise to a unique chemokine-responsive, immunosuppressive cell state that strongly differs from normal myeloid cells. We establish the first MDSC-specific gene signature and identify novel surface markers for improved detection and enrichment of MDSCs in murine and human samples. Our study provides the first single-cell transcriptional map defining the development of MDSCs, which will ultimately enable us to specifically target these cells in cancer patients. M-MDSCCD11b + CD14 + PBMC-Control PBMC-Treated I J H CD11b + /CD15 +