Biology subjects

Deng, W.

Publications and source records attributed to Deng, W..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Mature and migratory dendritic cells promote immune infiltration and response to anti-PD-1 checkpoint blockade in metastatic melanoma

Immune checkpoint inhibitors (ICIs) have heralded a remarkable shift in cancer care, significantly extending survival for advanced cancer patients. However, despite their remarkable clinical successes, a substantial majority of patients fail to achieve a lasting response to ICIs. To address this challenge and gain insights into the complex landscape of the tumor microenvironment (TME), we conducted an extensive analysis using single-cell RNA sequencing (scRNA; [~]216K cells across 39 samples) and single-nucleus transposase-accessible chromatin sequencing (snATAC; [~]43K cells from 15 samples) in a metastatic melanoma cohort. This systematic approach delineates 14 distinct cell types and 55 cell subtypes, including the identification of 15 transcriptional hallmarks of malignant cells. Through correlation analysis of cell subtype proportions, we unveiled six distinct clusters associated with varying tumor responses. Particularly intriguing was the identification of the mature DC enriched in immunoregulatory molecules (mregDC) subtype exhibiting correlations with naive T and B cells, forming an anti-tumor program that underscores the importance of multiple immune cell types in mediating anti-tumor immunity. Notably, we found that mregDC abundance represents a good prognostic predictor of progression-free survival (PFS) in the context of ICI treatment, and when combined with the TCF7+/- CD8 T cell ratio, it reliably predicts patient PFS across treatments beyond ICI. We validated our findings using an independent cohort of 274 ICI-treated melanoma samples analyzed using tissue-level expression. We next compared mregDCs and conventional dendritic cell types 1 and 2 (cDC1 and cDC2) using transcriptome signature, differentiation trajectory, interactome, cytokine milieu, and epigenome landscape analyses. This comparative analysis shed light on the unique attributes of mregDCs within the TME. Finally, we investigated cell type/subtype-specific genes, pathways, immune response enrichment, and ligand-receptor interactions closely associated with the proportions of mregDCs within the TME. These molecular and cellular insights, with their critical roles in enhancing the immune response against cancer, offer valuable prospects for predicting the efficacy of ICI regimens, and potentially guiding the selection of rational combinatorial therapies.

cancer biology↗

bioRxiv2024

MIPDB: A maize image-phenotype database with multi-angle and multi-time characteristics

Plant phenomics has become one of the most significant scientific fields in recent years. However, typical phenotyping procedures have low accuracy, low throughput, and are labor-intensive and time-consuming. Large-scale phenotypic collection equipment, on the other hand, is pricy, rigid, and inconvenient. The advancement of phenomics has been hampered by these restrictions. Lightweight picture collection equipment can now be used to capture plant phenotypic data thanks to the development of deep learning-based image identification. For the purpose of training the model, this approach needs high-quality annotated datasets. In this study, we used a handheld camera to gather multi-angle, multi-time series images and an unmanned aerial vehicle (UAV) to create a maize image phenotyping database (MIPDB). Over 30,000 high-resolution photos are available in the MIPDB, with 17,631 of those images having been carefully tagged with point-line method. The MIPDB can be accessed by the general public at http://phenomics.agis.org.cn. We anticipate that the availability of this superior dataset will stimulate a new revolution in crop breeding and advance deep learning-based phenomics research.

plant biology↗

bioRxiv2024

The Genomes of Nematode-Trapping Fungi Provide Insights into the Origin and Diversification of Fungal Carnivorism

Nematode-trapping fungi (NTF), most of which belong to a monophyletic lineage in Ascomycota, cannibalize nematodes and other microscopic animals, raising questions regarding the types and mechanisms of genomic changes that enabled carnivorism and adaptation to the carbon-rich and nitrogen-poor environment created by the Permian-Triassic extinction event. Here, we conducted comparative genomic analyses of 21 NTF and 21 non-NTF to address these questions. Carnivorism-associated changes include expanded genes for nematode capture, infection, and consumption (e.g., adhesive proteins, CAP superfamily, eukaryotic aspartyl proteases, and serine-type peptidases). Although the link between secondary metabolite (SM) production and carnivorism remains unclear, we found that the numbers of SM gene clusters among NTF are significantly lower than those among non-NTF. Significantly expanded cellulose degradation gene families (GH5, GH7, AA9, and CBM1) and contracted genes for carbon-nitrogen hydrolases (enzymes that degrade organic nitrogen to ammonia) are likely associated with adaptation to the carbon-rich and nitrogen-poor environment. Through horizontal gene transfer events from bacteria, NTF acquired the Mur gene cluster (participating in synthesizing peptidoglycan of the bacterial cell wall) and Hyl (a virulence factor in animals). Disruption of MurE reduced NTFs ability to attract nematodes, supporting its role in carnivorism. This study provides new insights into how NTF evolved and diversified after the Permian-Triassic mass extinction event.

evolutionary biology↗

bioRxiv2024

Characterization of Genome-wide Phylogenetic Conflict Uncovers Evolutionary Modes of Carnivorous Fungi

Mass extinction has often paved the way for rapid evolutionary radiation, resulting in the emergence of diverse taxa within specific lineages. While the emergence and diversification of carnivorous nematode-trapping fungi (NTF) in Ascomycota has been linked to the Permian-Triassic (PT) extinction, the processes underlying NTF radiation remain unclear. Here, we conducted phylogenomic analyses using 23 genomes spanning three NTF lineages, each employing distinct nematode traps -- mechanical traps (Drechslerella spp.), three-dimensional (3-D) adhesive traps (Arthrobotrys spp.), and two-dimensional (2-D) adhesive traps (Dactylellina spp.), and one non-NTF species as the outgroup. This analysis revealed how diverse mechanisms contributed to the tempo of NTF evolution and rapid radiation. The genome-scale species tree of NTFs suggested that Drechslerella emerged earlier than Arthrobotrys and Dactylellina. Extensive genome-wide phylogenetic discordance was observed, mainly due to incomplete lineage sorting (ILS) between lineages ([~]81.3%). Modes of non-vertical evolution (i.e., introgression and horizontal gene transfer) also contributed to phylogenetic discordance. The ILS genes that are associated with hyphal growth and trap morphogenesis (e.g., those associated with the cell membrane system and cellular polarity division) exhibited signs of positive selection.

evolutionary biology↗

bioRxiv2024

Hairpin-locker mediated CRISPR/Cas tandem system for ultrasensitive detection of DNA without pre-amplification

Achieving ultra-sensitive detection of DNA is of paramount importance in the field of molecular analytics. Conventional amplification technologies such as polymerase chain reaction (PCR) currently play a leading role in ultrasensitive DNA detection. However, amplicon contamination common in these techniques may lead to false positives. To date, CRISPR-associated nucleases (type V & VI) with their programmable cleavage have been utilised for sensitive detection of unamplified nucleic acids in complex real samples. Nevertheless, without additional amplification strategies, the pM range sensitivity of such CRISPR/Cas sensors is not sufficient for clinical applications. Here, we established a hairpin-locker (H-locker) mediated Cas12-Cas13 tandem biosensing system (Cas12-13 tandem-sensor) for ultrasensitive detection of DNA targets. Without the need for any additional amplification reaction or device, this system is capable of detecting DNA at a notable 1 aM level (<1 copy/uL) sensitivity. In addition, the system was able to distinguish cancer mutations in colorectal cancer (CRC) mice. This is a significant advance for CRISPR/Cas biosensing technology offering simple, highly sensitive, and user-friendly diagnostics for next-generation nucleic acid detection. O_TBL View this table: org.highwire.dtl.DTLVardef@7fc32eorg.highwire.dtl.DTLVardef@1e716ceorg.highwire.dtl.DTLVardef@8a43e0org.highwire.dtl.DTLVardef@16e286dorg.highwire.dtl.DTLVardef@1202eb0_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONDNA and RNA oligos used in this study. C_TABLECAPTION C_TBL

molecular biology↗

bioRxiv2024

Scalable single-cell pooled CRISPR screens with conventional knockout vector libraries

Current methods for single-cell RNA profiling of pooled CRISPR screens are limited, either by indirect capture of single guide RNAs (sgRNAs) or by custom modification of plasmid libraries. Here, we present a direct sgRNA capture platform called Native sgRNA Capture and sequencing (NSC-seq) that enables single-cell CRISPR screens using common knockout plasmid libraries, facilitating genotype-phenotype mapping at multiple scales in vitro and in vivo. Additionally, we characterize sgRNA expression in three whole-genome knockout libraries, revealing a substantial subset of truncated (isoform) spacer reads. We provide this dataset as a reference of expressed sgRNA isoforms that may potentially have compromised CRISPR gene editing efficacy and precision.

genomics↗

bioRxiv2023

Respiration and growth of Paracoccus denitrificans R-1 with nitrous oxide as an electron acceptor

In the nitrogen biogeochemical cycle, the reduction of nitrous oxide (N2O) to N2 by N2O reductase, which is encoded by nos gene cluster, is the only biological pathway for N2O consumption. However, the capability and mechanisms of microbial N2O reduction are poorly understood. In this study, we investigated the ability to obtain energy for growth of Paracoccus denitrificans R-1 by coupling the oxidation of various electron donors to N2O reduction. This strain has strong N2O reduction capability, and the average N2O reduction rate was 5.10{+/-}0.11x10-9 mol{middle dot}h-1{middle dot}cell-1 under anaerobic condition at 30 using acetate as the electron donor in a defined medium. This reduction was accompanied by the stoichiometric consumption of acetate over time when N2O served as the sole electron acceptor and the reduction can yield energy to support microbial growth, suggesting that microbial N2O reduction is an electron transport process. Cu2+, silver nanoparticles, O2, and acidic conditions can strongly inhibit the reduction, whereas NO3- or NH4+ can promote it. Genomic analysis showed that the gene cluster encoding N2O reductase of P. denitrificans R-1 was composed of nosR, nosZ, nosD, nosF, nosY, and nosL, and nosZ, which was identified as clade I. The respiratory inhibitors test indicated that the pathway of electron transport for N2O reduction was different from that of the traditional electron transport chain for aerobic respiration. These findings suggest that modular N2O reduction by P. denitrificans R-1 is linked to the electron transport chain and energy conservation, and that dissimilatory N2O reduction is a form of microbial anaerobic respiration. IMPOETANCEIn the nitrogen biogeochemical cycle, the reduction of N2O to N2 by N2O reductase, which is encoded by nos genes, is the only biological pathway for N2O consumption. However, the capacity and mechanisms of microbial N2O reduction are poorly understood. We investigated the ability to obtain energy for growth of Paracoccus denitrificans R-1 by coupling the oxidation of various electron donors to N2O reduction. Our study showed that the nosZ type I bacterium, P. denitrificans R-1, can respire N2O as the sole electron donor. Thus, the modular N2O reduction process of clade I denitrifiers not only can consume N2O produced by themselves but can also consume the external N2O generated from non-denitrification biological or abiotic pathways under suitable conditions, which is critical for controlling the release of N2O from ecosystems into the atmosphere.

microbiology↗

bioRxiv2023

Pax37 gene function in Oikopleura dioica supports a neuroepithelial-like origin for its house-making Fol territory

Larvacean tunicates feature a spectacular innovation not seen in other animals - the trunk oikoplastic epithelium (OE). This epithelium produces a house, a large and complex extracellular structure used for filtering and concentrating food particles. Previously we have shown that several homeobox transcription factors may play a role in patterning the OE. Among these are two Pax3/7 duplicates that we named Pax37A and Pax37B. The vertebrate homologs, PAX3 and PAX7, are involved in developmental processes related to neural crest and muscles. In the ascidian tunicate Ciona robusta, Pax3/7 has been given a role in development of cells deriving from the neural plate border including trunk epidermal sensory neurons and tail nerve cord neurons as well as in neural tube closure. Here we have investigated the roles of Pax37A and Pax37B in the development of the OE using CRISPR-Cas9, analyzing scRNA-seq data from wild-type animals that were compared with scRNA-seq data from C. robusta. We revealed that Pax37B but not Pax37A is essential for the differentiation of cell fields that produce the food concentrating filter of the house: the anterior Fol, giant Fol and Nasse cells. Lineage analysis supports that expression of Pax37 is under influence of Wnt signaling and that Fol cells have a neuroepithelial-like transcriptional signature. We propose that the highly specialized secretory epithelial cells of the Fol region either maintained or evolved neuroepithelial features as do "glue" secreting collocytes of ascidians. Their development seems to be controlled by a GRN that also operates in some ascidian neurons.

developmental biology↗

bioRxiv2023

Quorum Sensing Modulates Bacterial Virulence and Colonization Dynamics During an Enteric Infection

Quorum Sensing (QS) is a form of cell-to-cell communication that enables bacteria to modify behaviour according to their population density. While QS has been proposed as a potential intervention against pathogen infection, QS-mediated communication within the mammalian digestive tract remains understudied. Using an LC-MS/MS approach, we discovered that Citrobacter rodentium, a natural murine pathogen used to model human infection by pathogenic Escherichia coli, utilizes the CroIR system to produce three QS-molecules. We then profiled their accumulation both in vitro and across different gastrointestinal sites over the course of infection. Importantly, we found that in the absence of QS capabilities the virulence of C. rodentium is enhanced. This highlights the role of QS as an effective mechanism to regulate virulence according to the pathogens spatio-temporal context to optimize colonization and transmission success. These results also demonstrate that inhibiting QS may not always be an effective strategy for the control of virulence.

microbiology↗

bioRxiv2023

Inhibition mechanism and antiviral activity of an α-ketoamide based SARS-CoV-2 main protease inhibitor

SARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an -ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (Mpro), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with a Ki of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease-inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 Mpro:RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the -ketoamide warhead; more extensive interactions are identified between bound RAY1216 and Mpro active site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials (https://clinicaltrials.gov/ct2/show/NCT05620160) to test real-world therapeutic efficacy against COVID-19.

biochemistry↗

bioRxiv2023

Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations - application to HIV-1 quasispecies

Pathogen diversity resulting in quasispecies can enable persistence and adaptation to host defenses and therapies. However, accurate quasispecies characterization can be impeded by errors introduced during sample handling and sequencing which can require extensive optimizations to overcome. We present complete laboratory and bioinformatics workflows to overcome many of these hurdles. The Pacific Biosciences single molecule real-time platform was used to sequence PCR amplicons derived from cDNA templates tagged with universal molecular identifiers (SMRT-UMI). Optimized laboratory protocols were developed through extensive testing of different sample preparation conditions to minimize between-template recombination during PCR and the use of UMI allowed accurate template quantitation as well as removal of point mutations introduced during PCR and sequencing to produce a highly accurate consensus sequence from each template. Handling of the large datasets produced from SMRT-UMI sequencing was facilitated by a novel bioinformatic pipeline, Probabilistic Offspring Resolver for Primer IDs (PORPIDpipeline), that automatically filters and parses reads by sample, identifies and discards reads with UMIs likely created from PCR and sequencing errors, generates consensus sequences, checks for contamination within the dataset, and removes any sequence with evidence of PCR recombination or early cycle PCR errors, resulting in highly accurate sequence datasets. The optimized SMRT-UMI sequencing method presented here represents a highly adaptable and established starting point for accurate sequencing of diverse pathogens. These methods are illustrated through characterization of human immunodeficiency virus (HIV) quasispecies. Author SummaryThere is a great need to understand the genetic diversity of pathogens in an accurate and timely manner, but many errors can be introduced during the sample handling and sequencing steps which may prevent accurate analyses. In some cases, the errors introduced during these steps can be indistinguishable from real genetic variation and prevent analyses from identifying true sequence variation present in the pathogen population. There are established methods which can help to prevent these types of errors, but can involve many different steps and variables, all of which must be optimized and tested together to ensure the desired effect. Here we show results from testing different methods on a set of HIV+ blood plasma samples and arrive at a streamlined laboratory protocol and bioinformatic pipeline which prevents or corrects for different types of errors that can arise in sequence datasets. These methods should be an accessible starting point for anyone wanting accurate sequencing without extensive optimizations.

molecular biology↗

bioRxiv2022

Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery

Formic acid (FA), an organic one-carbon source that is easily produced from CO2, has emerged as a promising CO2-equivalent feedstock for one-carbon biorefinery. However, developing efficient formatotrophs for an economically competitive FA utilization system remains a grand challenge. Here, we discovered that the gram-negative bacterium Vibrio natriegens has exceptional FA tolerance and metabolic capacity natively. This strain was then remodeled by rewiring the serine cycle and the TCA cycle which resulted in a non-native closed loop (S-TCA) for enhancing FA assimilation. The rational design generated a powerful metabolic sink that enabled rapid emergence of the evolved strains with further significantly improved performance in using FA as the major or sole carbon source. Furthermore, the introduction of a foreign indigoidine-forming pathway into the best-performing V. natriegens strain (S-TCA-2.0) led to production of 29.0 g{middle dot}L-1 indigoidine and consumption of 233.7 g{middle dot}L-1 formate within 72 h, achieving an order of magnitude higher formate consumption rate (3.2 g{middle dot}L-1{middle dot}h-1) than the reported highest level in microorganisms. This work represents a significant step towards the development of industrially viable microorganisms for FA biorefinery.

microbiology↗

bioRxiv2022

Subset-specific mitochondrial and DNA damage shapes T cell responses to fever and inflammation

Heat is a cardinal feature of inflammation. Despite temperature variability and dependence of enzymes and complexes, how heat and fever affect immune cells remains uncertain. We found that heat broadly increased inflammatory activity of CD4+ T cell subsets and decreased Treg suppressive function. Th1 cells, however, also selectively developed mitochondrial dysfunction with high levels of ROS production and DNA damage. This led Th1 cells to undergo Tp53-dependent death, which was required to minimize the accumulation of mutations in heat and inflammation. Th1 cells with similar DNA damage signatures were also detected in Crohns disease and rheumatoid arthritis. Fever and inflammation-associated heat thus selectively induce mitochondrial stress and DNA damage in activated Th1 cells that requires p53 to maintain genomic integrity of the T cell repertoire. One Sentence SummaryFever temperatures augment CD4+ T cell-mediated inflammation but induce differential metabolic stress and DNA damage in T cell subsets, with Th1 cells selectively sensitive and dependent on p53 to induce apoptosis and maintain genomic integrity.

immunology↗

bioRxiv2022

Extinction drives a discontinuous temporal pattern of species-area relationships in a microbial microcosm system

The species area relationship (SAR) can be a general law in ecology only if its formation mechanisms are clarified. Essentially, the SAR addresses the relationship between regional area and biodiversity, which is shaped by processes of speciation, extinction and dispersal. Since those three processes has temporal dynamics, we propose the hypothesis that the occurrence of SAR should also have temporal dynamics. Here, we designed independent closed microcosm systems, in which dispersal/speciation can be excluded/neglected, to reveal the role of extinction in shaping the temporal dynamics pattern of SAR. We find that extinction can shape SAR in this system independently. Due to the temporal dynamics of the extinction, SAR was temporally discontinuous. We also observe that small-scale extinctions modified community structure to promote ecosystem stability and shaped SAR, while mass extinction pushed the microcosm system into the next successional stage and dismissed SAR. Our result suggested that SAR could serve as an indicator of ecosystem stability; moreover, temporal discontinuity can be the explaination for many controversies in SAR studies.

ecology↗

bioRxiv2022

Gaq-PKD/PKCμ regulates the IkB transcription to limit the NF-kB mediated inflammatory response essential for early pregnancy

Decidualization, denoting the transformation of endometrial stromal cells into specialized decidual cells, is a prerequisite for normal embryo implantation and a successful pregnancy in human. Here we demonstrated that knockout of Gaq lead to an aberrantly enhanced inflammatory state during decidualization. Furthermore, we showed that deficiency of Gaq resulted in over-activation of nuclear factor (NF)-{kappa}B signaling, due to the decreased expression of NF{kappa}BIA, which encode the I{kappa}B protein and is the negative regulator for NF{kappa}B. Mechanistically, Gaq deficiency decreased the PKD/PKC phosphorylation levels, so leading to attenuated HDAC5 phosphorylation and thus its nuclear export. Aberrantly high level of nuclear HADC5 retarded histone acetylation to inhibit NF{kappa}BIA transcription during decidualization. Consistently, pharmacological activation of the PKD/PKC or inhibition of the HDAC5 signaling restored the inflammatory state and proper decidual response. Finally, we disclosed that over-active inflammatory state in Gaq deficient decidua deferred the blastocyst hatching and adhesion in vitro, and the decidual expression of Gq was significantly lower in women with recurrent pregnancy loss compared with normal pregnancy. In brief, we showed here that Gq as a key regulator of the inflammatory cytokines expression and decidual homeostasis in response to differentiation cues, which is required for successful implantation and early pregnancy.

cell biology↗

bioRxiv2022

STAT3 promotes RNA polymerase III-directed transcription by controlling the miR-106a-5p/TP73 axis

AbstractsDeregulation of Pol III products causes a range of diseases, including neural diseases and cancers. However, the pathways and factors that control Pol III-directed transcription have yet to be elucidated. Here, we show that STAT3 positively regulates the activities of Pol III-dependent transcription and cancer cell growth. RNA-seq analysis revealed that STAT3 inhibits the expression of TP73, a member of the p53 family. We found that TP73 is not only required for the regulation of Pol III-directed transcription mediated by STAT3 but also independently suppresses the synthesis of Pol III products. Mechanistically, TP73 can disrupt the assembly of TFIIIB subunits and inhibit their occupancies at Pol III target loci by interacting with TFIIB subunit TBP. MiR-106a-5p can activate Pol III-directed transcription by targeting the TP73 mRNA 3 UTR to reduce TP 73 expression. We show that STAT3 activates the expression of miR-106a-5p by binding to the microRNA promoter, indicating that miR-106a-5p links STAT3 to TP73 and Pol III transcription. Collectively, these findings indicate that STAT3 functions as a positive regulator in Pol III-directed transcription by controlling the miR-106a-5p/TP73 axis.

molecular biology↗

bioRxiv2022

Spatiotemporal landscape of SARS-CoV-2 pulmonary infection reveals Slamf9+Spp1+ macrophages promoting viral clearance and inflammation resolution

While SARS-CoV-2 pathogenesis has been intensively investigated, the host mechanisms of viral clearance and inflammation resolution are still elusive because of the ethical limitation of human studies based on COVID-19 convalescents. Here we infected Syrian hamsters by authentic SARS-CoV-2 and built an ideal model to simulate the natural recovery process of SARS-CoV-2 infection from severe pneumonia1,2. We developed and applied a spatial transcriptomic sequencing technique with subcellular resolution and tissue-scale extensibility, i.e., Stereo-seq3, together with single-cell RNA sequencing (scRNA-seq), to the entire lung lobes of 45 hamsters and obtained an elaborate map of the pulmonary spatiotemporal changes from acute infection, severe pneumonia to the late viral clearance and inflammation resolution. While SARS-CoV-2 infection caused massive damages to the hamster lungs, including naive T cell infection and deaths related to lymphopenia, we identified a group of monocyte-derived proliferating Slamf9+Spp1+ macrophages, which were SARS-CoV-2 infection-inducible and cell death-resistant, recruiting neutrophils to clear viruses together. After viral clearance, the Slamf9+Spp1+ macrophages differentiated into Trem2+ and Fbp1+ macrophages, both responsible for inflammation resolution and replenishment of alveolar macrophages. The existence of this specific macrophage subpopulation and its descendants were validated by RNAscope in hamsters, immunofluorescence in hACE2 mice, and public human autopsy scRNA-seq data of COVID-19 patients. The spatiotemporal landscape of SARS-CoV-2 infection in hamster lungs and the identification of Slamf9+Spp1+ macrophages that is pivotal to viral clearance and inflammation resolution are important to better understand the critical molecular and cellular players of COVID-19 host defense and also develop potential interventions of COVID-19 immunopathology.

immunology↗

bioRxiv2022

Head-to-head comparison of CCN4, DNMT3A, PTPN11, and SPARC as suppressors of anti-tumor immunity

BackgroundEffective communication between innate and adaptive immunity is essential for mounting an effective antitumor immune response. Emergent cancer cells likely secrete factors that inhibit this communication. To identify such factors, we applied an in vitro workflow that coupled a functional assay with proteomics to a syngeneic mouse model of melanoma known to be resistant to immunotherapies. Collectively, these in vitro results suggested CCN4, DNMT3A, PTPN11, and SPARC as secreted factors that potentially mediate immunosuppression and could potentially become easily accessible targets for novel immunotherapies. The objective of this study was to test for consistent clinical correlates in existing human data and to verify in vivo whether knocking out tumor cell production of these secreted factors improved immune-mediated control of tumor growth. ResultsAnalyses of available human data indicate that high CCN4 expression is associated with reduced survival in primary melanoma patients. High DNMT3A, PTPN11, and SPARC expression showed no association with overall survival. In addition, scRNAseq data from patients with melanoma confirmed CCN4 is expressed by the tumor cells, as opposed to other cells in the tumor microenvironment. An experimental system was created using a CRISPR/Cas9 approach to generate knock out cell lines for each of the four genes of interest using the B16F0 mouse melanoma cell line. In a immunocompetent C57BL/6, DNMT3A, PTPN11, and SPARC knockouts had no effect on overall survival compared to mice challenged with wildtype B16F0 cells while the CCN4 knockout significantly increased survival. This increase in overall survival was lost when the CCN4 knockout cells were injected into severely immunocompromised NSG hosts, indicating that CCN4s effect on the tumor microenvironment is immune mediated. A kinetic analysis leveraging a Markov Chain Monte Carlo approach quantified the various knockouts effect on cells intrinsic growth rate. The analysis shows CCN4 is the only knockout tested that decreased the net proliferation rate in immunocompetent mice compared to wildtype cells. ConclusionsThe results suggest that CCN4 is a mediator of immunosuppression in the melanoma tumor microenvironment and a potential collateral immunotherapy target.

cancer biology↗