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HTLV-1 type-A rarely causes lung disease in humans, whereas HTLV-1 type-C is more frequently associated with respiratory failure and premature death. We investigated the genetic basis of HTLV-1C morbidity by constructing a chimeric HTLV-1A/C oI-L encompassing the highly divergent type C orf-I. We demonstrate that systemic infectivity of HTLV-1A and HTLV-1A/C oI-L is equivalent in macaques, but viral expression in lungs is significantly higher in HTLV-1A/C oI-L infection. In addition, bronchoalveolar-lavage immune cell dynamics differs greatly with neutrophils and monocytes producing TNF-α in HTLV-1A/C oI-L , but producing IL-10 in HTLV-1A infection. Animals infected with HTLV-1A/C oI-L develops bronchiectasis at 10 months from infection, but at the same timepoint those infected with HTLV-1A do not. HTLV-1A/C oI-L expressed a 16 kDa fusion protein (p16C) via a doubly spliced, Rex-orf-IC, mRNA able to shield T-cells from efferocytosis, a monocyte function that mitigates inflammation via clearance of apoptotic cells. The Rex-orf-IC mRNA is expressed as more frequent in the lung of HTLV-1A/C oI-L than HTLV-1A infected animals. Since defective efferocytosis is associated with lung obstructive pathologies, the data raise the hypothesis that p16C may contribute to the lung morbidity observed in HTLV-1C infection. HTLV-1 type-C causes more severe disease than type-A, but the underlying reason is unclear. Here the authors show in a macaque model how type-C orf-I affects lung pathogenesis and the immune response to HTLV-1, providing a model to test viral targets for disease prevention.

Exposure to Bacillus Calmette-Guérin (BCG) or Canarypox ALVAC/Alum vaccine elicits pro- or antiinflammatory innate responses, respectively. We tested whether prior exposure of macaques to these immunogens protected against SARS-CoV-2 replication in lungs and found more efficient replication control after the pro-inflammatory immunity elicited by BCG. The decreased virus level in lungs was linked to early infiltrates of classical monocytes producing IL-8 with systemic neutrophils, Th2 cells, and Ki67+CD95+CD4+ T cells producing CCR7. At the time of SARS-CoV-2 exposure, BCG-treated animals had higher frequencies of lung infiltrating neutrophils and higher CD14+ cells expressing efferocytosis marker MERTK, responses correlating with decreased SARS-CoV-2 replication in lung. At the same time point, plasma IL-18, TNF-α, TNFSF-10, and VEGFA levels were also higher in the BCG group and correlated with decreased virus replication. Finally, after SARS-CoV-2 exposure, decreased virus replication correlated with neutrophils producing IL-10 and CCR7 preferentially recruited to the lungs of BCG-vaccinated animals. These data point to the importance of the spatiotemporal distribution of functional monocytes and neutrophils in controlling SARS-CoV-2 levels and suggest a central role of monocyte efferocytosis in curbing replication.

Human pluripotent stem cells (hPSCs) are inherently sensitive cells. Single-cell dissociation and the establishment of clonal cell lines have been long-standing challenges. This inefficiency of cell cloning represents a major obstacle for the standardization and streamlining of gene editing in induced pluripotent stem cells for basic and translational research. Here we describe a chemically defined protocol for robust single-cell cloning using microfluidics-based cell sorting in combination with the CEPT small-molecule cocktail. This advanced strategy promotes the viability and cell fitness of self-renewing stem cells. The use of low-pressure microfluidic cell dispensing ensures gentle and rapid dispensing of single cells into 96- and 384-well plates, while the fast-acting CEPT cocktail minimizes cellular stress and maintains cell structure and function immediately after cell dissociation. The protocol also facilitates clone picking and produces genetically stable clonal cell lines from hPSCs in a safe and cost-efficient fashion. Depending on the proliferation rate of the clone derived from a single cell, this protocol can be completed in 7–14 d and requires experience with aseptic cell culture techniques. Altogether, the relative ease, scalability and robustness of this workflow should boost gene editing in hPSCs and leverage a wide range of applications, including cell line development (e.g., reporter and isogenic cell lines), disease modeling and applications in regenerative medicine. This protocol combines microfluidic cell sorting with the CEPT small-molecule cocktail to minimize cellular stress and enable the robust single-cell cloning of human pluripotent stem cells in a high-throughput fashion.

A mucosal route of vaccination could prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication at the site of infection and limit transmission. We compared protection against heterologous XBB.1.16 challenge in nonhuman primates (NHPs) ~5 months following intramuscular boosting with bivalent mRNA encoding WA1 and BA.5 spike proteins or mucosal boosting with a WA1–BA.5 bivalent chimpanzee adenoviral-vectored vaccine delivered by intranasal or aerosol device. NHPs boosted by either mucosal route had minimal virus replication in the nose and lungs, respectively. By contrast, protection by intramuscular mRNA was limited to the lower airways. The mucosally delivered vaccine elicited durable airway IgG and IgA responses and, unlike the intramuscular mRNA vaccine, induced spike-specific B cells in the lungs. IgG, IgA and T cell responses correlated with protection in the lungs, whereas mucosal IgA alone correlated with upper airway protection. This study highlights differential mucosal and serum correlates of protection and how mucosal vaccines can durably prevent infection against SARS-CoV-2. Seder and colleagues show that mucosal adenoviral-vectored vaccine boosting durably prevents infection in nonhuman primates of the highly transmissible, heterologous XBB.1.16 strain of SARS-CoV-2.

The regulation of chromatin is important for transcription, DNA replication and DNA damage repair. In the presence of environmental stress, the cell mounts stress response pathways that involve changes in chromatin structure to promote cell survival. Set4 belongs to the Set3 subfamily of SET domain-containing proteins in yeast. Other members of this subfamily are budding yeast Set3, Drosophila melanogaster UpSET and the mammalian proteins MLL5 and SETD5. The defining features of this subfamily are the presence of a divergent SET domain and the presence of a PHD finger, with the exception of SETD5. Set4’s paralog, Set3 has been shown to work in a complex and catalyze histone deacetylation leading to gene repression through binding H3K4 methylation via its PHD finger. Set4 shares sequence similarity to fly UpSET and human MLL5, however, little is known about the biochemical or biological roles of Set4. We have shown a role for Set4 in promoting cell survival during oxidative stress by contributing to stress response gene expression programs. We also determined that Set4 regulates genes enriched near telomeres under normal and stress conditions. We also found that deleting Set4 disrupts the histone deacetylase complex occupancy at telomeres, thereby leading to changes in the chromatin landscape. Similar to other members of the Set3 subfamily, Set4 does not possess methyltransferase activity due to the inactive SET domain, therefore we hypothesize that it functions through directing other chromatin regulators, including histone deacetylases, to chromatin. Set4 has a PHD finger as well and we found that although other PHD fingers generally bind to methylated lysines on histone tails, Set4’s PHD finger does not bind to histones. However, we did find that Set4’s PHD finger is important for its chromatin localization and binds to nucleic acids in vitro. This suggests that Set4 may regulate gene expression through a mechanism independent of histone methylation. This work has defined a role for Set4 as a regulator of the telomeric chromatin landscape by working in conjunction with histone deacetylases and as a calibrator of stress defense pathways. The study of Set4 has expanded our understanding of the Set3 subfamily of SET domain-containing proteins in gene expression control during environmental stress response.

The yeast chromatin protein Set4 is a member of the Set3-subfamily of SET domain proteins which play critical roles in the regulation of gene expression in diverse developmental and environmental contexts. We previously reported that Set4 promotes survival during oxidative stress and regulates expression of stress response genes via stress-dependent chromatin localization. In this study, global gene expression analysis and investigation of histone modification status identified a role for Set4 in maintaining gene repressive mechanisms within yeast subtelomeres under both normal and stress conditions. We show that Set4 works in a partially overlapping pathway to the SIR complex and the histone deacetylase Rpd3 to maintain proper levels of histone acetylation and expression of stress response genes encoded in subtelomeres. This role for Set4 is particularly critical for cells under hypoxic conditions, where the loss of Set4 decreases cell fitness and cell wall integrity. These findings uncover a new regulator of subtelomeric chromatin that is key to stress defense pathways and demonstrate a function for Set4 in regulating repressive, heterochromatin-like environments.

The human cerebellum is a specialized brain region that is involved in various neurological and psychiatric diseases but has been challenging to study due its complex neurodevelopment and cellular diversity. Despite the progress in generating neural tissues from human induced pluripotent stem cells (iPSCs), an organoid model that recapitulates the key features of cerebellar development has not been widely established. Here, we report the generation of a 60-day method for human cerebellar organoids (hCBOs) that is characterized by induction of rhombomere 1 (R1) cellular identity followed by derivation of neuronal and glial cell types of the cerebellum. In contrast to forebrain organoids with multiple neural rosettes and inside-out neuronal migration, hCBOs develop a SOX2+ cerebellar plate on the outermost surface of organoids with outside-in neuronal migration, which is a characteristic hallmark of cerebellar histogenesis. These hCBOs produced various other cell types including granule neurons, Purkinje cells, Golgi neurons, and deep cerebellar nuclei. By using a glial induction strategy, we generate Bergmann glial cells (BGCs) within the hCBOs that not only serve as scaffolds for granule cells migration but also enhance electrophysiological response of the hCBOs. Furthermore, by generating hCBOs from patients with Friedreich's ataxia (FRDA), we revealed abnormal disease-specific phenotypes that could be reversed by histone deacetylase (HDAC) inhibitors and gene editing by CRISPR-Cas9. Taken together, our advanced hCBO model provides new opportunities to investigate the molecular and cellular mechanisms of cerebellar ontogenesis and utilize patient-derived iPSCs for translational research.

Waning immunity and continued virus evolution have limited the durability of protection from symptomatic infection mediated by intramuscularly (IM)-delivered mRNA vaccines against COVID-19 although protection from severe disease remains high. Mucosal vaccination has been proposed as a strategy to increase protection at the site of SARS-CoV-2 infection by enhancing airway immunity, potentially reducing rates of infection and transmission. Here, we compared protection against XBB.1.16 virus challenge 5 months following IM or mucosal boosting in non-human primates (NHP) that had previously received a two-dose mRNA-1273 primary vaccine regimen. The mucosal boost was composed of a bivalent chimpanzee adenoviral-vectored vaccine encoding for both SARS-CoV-2 WA1 and BA.5 spike proteins (ChAd-SARS-CoV-2-S) and delivered either by an intranasal mist or an inhaled aerosol. An additional group of animals was boosted by the IM route with bivalent WA1/BA.5 spike-matched mRNA (mRNA-1273.222) as a benchmark control. NHP were challenged in the upper and lower airways 18 weeks after boosting with XBB.1.16, a heterologous Omicron lineage strain. Cohorts boosted with ChAd-SARS-CoV-2-S by an aerosolized or intranasal route had low to undetectable virus replication as assessed by levels of subgenomic SARS-CoV-2 RNA in the lungs and nose, respectively. In contrast, animals that received the mRNA-1273.222 boost by the IM route showed minimal protection against virus replication in the upper airway but substantial reduction of virus RNA levels in the lower airway. Immune analysis showed that the mucosal vaccines elicited more durable antibody and T cell responses than the IM vaccine. Protection elicited by the aerosolized vaccine was associated with mucosal IgG and IgA responses, whereas protection elicited by intranasal delivery was mediated primarily by mucosal IgA. Thus, durable immunity and effective protection against a highly transmissible heterologous variant in both the upper and lower airways can be achieved by mucosal delivery of a virus-vectored vaccine. Our study provides a template for the development of mucosal vaccines that limit infection and transmission against respiratory pathogens.

The yeast chromatin protein Set4 is a member of the Set3-subfamily of SET domain proteins which play critical roles in the regulation of gene expression in diverse developmental and environmental contexts, although they appear to lack methyltransferase activity. The molecular functions of Set4 are relatively unexplored, likely due to its low abundance in standard growth conditions. We previously reported that Set4 promotes survival during oxidative stress and regulates expression of stress response genes via stress-dependent chromatin localization. In this study, global gene expression analysis and investigation of histone modification status has revealed a role for Set4 in maintaining gene repressive mechanisms within yeast subtelomeres under both normal and stress conditions. We show that Set4 works in a partially overlapping pathway to the SIR complex and the histone deacetylase Rpd3 to maintain proper levels of histone acetylation and expression of stress response genes encoded in subtelomeres. This role for Set4 is particularly critical for cells under hypoxic conditions, and the loss of Set4 decreases cell fitness and cell wall integrity in hypoxia. These findings uncover a new regulator of subtelomeric chromatin that is key to stress defense pathways and demonstrate a function for yeast Set4 in regulating repressive, heterochromatin-like environments.